nutrients

Diet and
Immune Function
Edited by
Elizabeth A. Miles, Philip Calder and Caroline E. Childs
Printed Edition of the Special Issue Published in Nutrients

www.mdpi.com/journal/nutrients
Diet and Immune Function
Diet and Immune Function

Special Issue Editors

Elizabeth A Miles
Philip Calder
Caroline E Childs

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Special Issue Editors
Elizabeth A Miles Philip Calder
University of Southampton University of Southampton
UK UK

Caroline E Childs
University of Southampton
UK

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MDPI
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4052 Basel, Switzerland

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Contents

About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Diet and Immune Function

Reprinted from: Nutrients 2019, 11, 1933, doi:10.3390/nu11081933 . . . . . . . . . . . . . . . . . . 1

Bethan Dalton, Iain C. Campbell, Raymond Chung, Gerome Breen, Ulrike Schmidt and
Hubertus Himmerich
Inflammatory Markers in Anorexia Nervosa: An Exploratory Study
Reprinted from: Nutrients 2018, 10, 1573, doi:10.3390/nu10111573 . . . . . . . . . . . . . . . . . . 10

Paulina Torres-Castro, Mar Abril-Gil, Marı́a J. Rodrı́guez-Lagunas, Margarida Castell,

Francisco J. Pérez-Cano and Àngels Franch
TGF-β2, EGF, and FGF21 Growth Factors Present in Breast Milk Promote Mesenteric Lymph
Node Lymphocytes Maturation in Suckling Rats
Reprinted from: Nutrients 2018, 10, 1171, doi:10.3390/nu10091171 . . . . . . . . . . . . . . . . . . 26

Lourdes Santiago-López, Adrián Hernández-Mendoza, Verónica Mata-Haro,

Belinda Vallejo-Córdoba, Abraham Wall-Medrano, Humberto Astiazarán-Garcı́a,
Marı́a del Carmen Estrada-Montoya and Aarón F. González-Córdova
Effect of Milk Fermented with Lactobacillus fermentum on the Inflammatory Response in Mice
Reprinted from: Nutrients 2018, 10, 1039, doi:10.3390/nu10081039 . . . . . . . . . . . . . . . . . . 42

Starin McKeen, Wayne Young, Jane Mullaney, Karl Fraser, Warren C. McNabb and
Nicole C. Roy
Infant Complementary Feeding of Prebiotics for the Microbiome and Immunity
Reprinted from: Nutrients 2019, 11, 364, doi:10.3390/nu11020364 . . . . . . . . . . . . . . . . . . . 55

Francesca Sassi, Cristina Tamone and Patrizia D’Amelio

Vitamin D: Nutrient, Hormone, and Immunomodulator
Reprinted from: Nutrients 2018, 10, 1656, doi:10.3390/nu10111656 . . . . . . . . . . . . . . . . . . 78

Nour Yahfoufi, Nawal Alsadi, Majed Jambi and Chantal Matar

The Immunomodulatory and Anti-Inflammatory Role of Polyphenols
Reprinted from: Nutrients 2018, 10, 1618, doi:10.3390/nu10111618 . . . . . . . . . . . . . . . . . . 92

Ga Young Lee and Sung Nim Han

The Role of Vitamin E in Immunity
Reprinted from: Nutrients 2018, 10, 1614, doi:10.3390/nu10111614 . . . . . . . . . . . . . . . . . . 115

Nina Wærling Hansen and Anette Sams

The Microbiotic Highway to Health— New Perspective on Food Structure, Gut Microbiota,
and Host Inflammation
Reprinted from: Nutrients 2018, 10, 1590, doi:10.3390/nu10111590 . . . . . . . . . . . . . . . . . . 133

Vinicius Cruzat, Marcelo Macedo Rogero, Kevin Noel Keane, Rui Curi and
Philip Newsholme
Glutamine: Metabolism and Immune Function, Supplementation and Clinical Translation
Reprinted from: Nutrients 2018, 10, 1564, doi:10.3390/nu10111564 . . . . . . . . . . . . . . . . . . 151

v
Silvia Maggini, Adeline Pierre and Philip C. Calder
Immune Function and Micronutrient Requirements Change over the Life Course
Reprinted from: Nutrients 2018, 10, 1531, doi:10.3390/nu10101531 . . . . . . . . . . . . . . . . . . 182

Joseph C. Avery and Peter R. Hoffmann

Selenium, Selenoproteins, and Immunity
Reprinted from: Nutrients 2018, 10, 1203, doi:10.3390/nu10091203 . . . . . . . . . . . . . . . . . . 209

Julio Plaza-Dı́az, Luis Fontana and Angel Gil

Human Milk Oligosaccharides and Immune System Development
Reprinted from: Nutrients 2018, 10, 1038, doi:10.3390/nu10081038 . . . . . . . . . . . . . . . . . . 229

Wiebke Alker and Hajo Haase

Zinc and Sepsis
Reprinted from: Nutrients 2018, 10, 976, doi:10.3390/nu10080976 . . . . . . . . . . . . . . . . . . 246

Mensiena B. G. Kiewiet, Marijke M. Faas and Paul de Vos

Immunomodulatory Protein Hydrolysates and Their Application
Reprinted from: Nutrients 2018, 10, 904, doi:10.3390/nu10070904 . . . . . . . . . . . . . . . . . . . 263

Marcelo Macedo Rogero and Philip C. Calder

Obesity, Inflammation, Toll-Like Receptor 4 and Fatty Acids
Reprinted from: Nutrients 2018, 10, 432, doi:10.3390/nu10040432 . . . . . . . . . . . . . . . . . . . 285

vi
About the Special Issue Editors
Elizabeth A Miles is Lecturer in Nutritional Immunology at the University of Southampton.
She has researched the influences of early nutrition and the maternal diet in pregnancy on offspring
health and development. Her early research demonstrated that the neonatal immune responses of
babies who subsequently developed allergic symptoms were different from babies who did not.
Dr. Miles’ research has also showed that the supplementation of healthy humans with omega-3
polyunsaturated fatty acids results in a dose-dependent increase in omega-3 fatty acids in plasma and
immune cells and decreases ex vivo immune and inflammatory mediator production. Furthermore,
increasing the intake of omega-3 fatty acids in pregnancy in families with an increased risk of allergic
disease results in a decrease in ex vivo production of mediators involved in the allergic response.
Dr Miles has published over 76 peer-reviewed research papers, 4 book chapters and 17 reviews based
on her research interests.

Philip Calder is Professor of Nutritional Immunology at the University of Southampton, UK. He is

an internationally recognized researcher on the metabolism and functionality of fatty acids with
an emphasis on the roles of omega-3 fatty acids and on the influence of diet and nutrients on the
immune and inflammatory responses. His research addresses both life course and translational
considerations. He has received many awards and prizes for his work including the prestigious
Danone International Prize for Nutrition (2016). Professor Calder was President of the International
Society for the Study of Fatty Acids and Lipids (2009–2012), Chair of the Scientific Committee
of the European Society for Clinical Nutrition and Metabolism (2012–2016) and President of the
Nutrition Society (2016–2019). He will be President of the Federation of European Nutrition
Societies (2019–2023). He was previously Editor-in-Chief of the British Journal of Nutrition and is
currently an Associate Editor of several journals, including Journal of Nutrition and Clinical Science.

Caroline E Childs is Lecturer in Nutritional Sciences at the University of Southampton. Her research
to date has focused on nutrients such as dietary fatty acids, probiotics and prebiotics and has assessed
the effect of nutrient interventions on outcomes such as tissue composition, immune function,
inflammatory status, immunosenescence and the gut microbiota. In 2010, she was named as one
of the top three young investigators in the category “Lipids and Nutrition” at the International
Society for the Study of Fatty Acids and Lipids conference. Dr Childs is Co-Chair of ILSI Europe’s
Nutrition, Immunity and Inflammation Task Force and a member of the ILSI Europe Expert Group
on Determinants of Immune Competence. Dr Childs is a Nutrition Society Ambassador for the
University of Southampton and Regional Representative for the Association for Nutrition. She is
an editorial board member of The Journal of Nutrition and sits on the Editorial Advisory Board of
Nutrition Bulletin.

vii
 nutrients
Editorial
Diet and Immune Function
Caroline E. Childs 1 , Philip C. Calder 1,2 and Elizabeth A. Miles 1, *
1 Human Development and Health, Faculty of Medicine, University of Southampton,
Southampton SO16 6YD, UK
2 NIHR Southampton Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust
and University of Southampton, Southampton SO16 6YD, UK
* Correspondence: [email protected]; Tel.: +44(0)23-8120-6925

Received: 9 August 2019; Accepted: 15 August 2019; Published: 16 August 2019

Abstract: A well-functioning immune system is critical for survival. The immune system must be
constantly alert, monitoring for signs of invasion or danger. Cells of the immune system must be able
to distinguish self from non-self and furthermore discriminate between non-self molecules which are
harmful (e.g., those from pathogens) and innocuous non-self molecules (e.g., from food). This Special
Issue of Nutrients explores the relationship between diet and nutrients and immune function. In this
preface, we outline the key functions of the immune system, and how it interacts with nutrients
across the life course, highlighting the work included within this Special Issue. This includes the
role of macronutrients, micronutrients, and the gut microbiome in mediating immunological effects.
Nutritional modulation of the immune system has applications within the clinical setting, but can
also have a role in healthy populations, acting to reduce or delay the onset of immune-mediated
chronic diseases. Ongoing research in this field will ultimately lead to a better understanding of
the role of diet and nutrients in immune function and will facilitate the use of bespoke nutrition to
improve human health.

Keywords: nutrition; immunity; macronutrients; micronutrients; microbiome; life course; probiotic;

prebiotic; inflammation

1. Overview of the Immune System

Broadly, cells of the immune system may be divided into those of the innate and those of the
adaptive immune response. The innate response is the first response to an invading pathogen. Cells of
the innate immune response include phagocytes (e.g., macrophages and monocytes), neutrophils,
dendritic cells, mast cells, eosinophils, and others. The innate response is rapid, but not specialised
and is generally less effective than the adaptive immune response.
The adaptive immune response has the ability to specifically recognise a pathogen and ‘remember’
it if exposed to it again. T cells are critical in antigen recognition and the co-ordination of the immune
response. T cells are present in an array of subtypes that coordinate different types of immune responses.
Broadly, they are divided into the cytotoxic T cells (bearing the CD8 receptor), which are involved
in direct killing of infected damaged cells and tumour cells, and the T helper cells. T helper (Th)
cells bear the CD4 receptor and are important in coordinating the responses of other immune cells.
There are a number of subtypes of Th cells, defined by the cytokines they produce. Initial studies
identified two subsets, the Th1 cells, which produced interferon gamma (IFN-γ) and interleukin (IL)-2
and were important in antiviral and cellular immune responses, and the Th2 subset producing IL-4,
IL-5, and IL-13 and involved in humoral (antibody) and anti-parasitic responses (but also in allergic
responses) [1]. It is now apparent that there are a number of other Th subtypes, which do not fall
into these categories. This includes Th17 cells, which produce IL-17A, IL-17F, and IL-22 and are
important in fighting extracellular pathogens (bacteria and fungi) [2]. There are also T regulatory cells

Nutrients 2019, 11, 1933; doi:10.3390/nu11081933 1 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 1933

(Treg), which are CD4-bearing T cells vital in maintaining immune tolerance to allow the immune
system to ignore non-harmful non-self (such as food, pollen, and environmental antigens such as
latex). Thus, the role of T cells is coordinating an appropriate immune response following immune
stimulation or challenge.
The other lymphocytes of the adaptive immune system are the B cells, which are responsible for
antibody or immunoglobulin (Ig) production. Like T cells, B cells respond specifically to an antigen.
They can differentiate into short-lived plasma cells, which produce Igs in the short term, or can become
long-lived plasma cells. Igs are pathogen-specific molecules, which help the immune system to recognise
and destroy pathogens. The B cells can differentiate into plasma cells, which produce one of five classes of
Ig (IgM, IgD, IgG, IgA, and IgE). Each class of Ig has a specialised role [3]. IgM is the first Ig expressed
during development, is often found as a multimeric molecule (e.g., pentameric), and can bind an antigen
to identify it for destruction by immune cells. IgD is found in low concentrations in the plasma and the
specialist role of IgD is not yet clear. IgG is the predominant Ig class and can persist for long periods.
It has important roles in antigen labelling, resulting in more effective removal. IgA can be found in the
serum (mostly as a monomer) and at mucosal surfaces (normally as a dimer). At the mucosal surface,
IgA protects against bacteria and or viruses, preventing infection. IgA also has an important role in
neutralising food antigens and helping to maintain immune tolerance to food antigens (preventing the
development of food allergy) [4]. IgE has a role in clearance of extracellular parasites (e.g., helminths) but
when produced inappropriately to innocuous environmental and food antigens, has an important role in
IgE-mediated allergy. B cells go through a process called class switching to set the class of Ig that the
plasma cells derived from them will produce. B cell class switching is controlled by the cytokines present,
particularly IL-4, IL-6, and IFN-γ secreted from Th cells [5].
T and B cells can specialise to become memory cells, which persist permanently or for very long
periods and are able to recognise the antigen if encountered again and elicit a rapid, pathogen-specific
immune response.
The effective deployment of the immune system against pathogens or harmful signals and the
swift resolution of the immune response is required for survival. The fighting of infection is only
one piece of the puzzle. A fulminating immune response is costly in terms of energy expended and
results in damage to the host tissues; thus, rapid and complete resolution of an immune response is
also key. Cytokines play a role in resolution of immune responses. IL-10, which is produced by a range
of immune cells including Tregs, has anti-inflammatory actions including suppressing inflammatory
cytokine production [6].
The instigation of an immune response and the activities of the immune cells results in inflammation
(seen as redness, swelling, and the feeling of heat and pain), which are signs of the damage to the
tissue going on whilst the immune system does its work. This is an expected outcome of an effective
immune response. Increasingly there is concern that modern lifestyle changes have resulted in the
promotion of ongoing, low-grade, whole-body (systemic) inflammation caused by immune and other
cells (e.g., adipocytes, the cells that store lipids in fat tissue) [7]. Such exposures may include diet
quality and quantity [8].

2. The Role of Nutrition in Immune Function

Adequate and appropriate nutrition is required for all cells to function optimally and this includes
the cells in the immune system. An “activated” immune system further increases the demand for
energy during periods of infection, with greater basal energy expenditure during fever for example.
Thus, optimal nutrition for the best immunological outcomes would be nutrition, which supports the
functions of immune cells allowing them to initiate effective responses against pathogens but also
to resolve the response rapidly when necessary and to avoid any underlying chronic inflammation.
The immune system’s demands for energy and nutrients can be met from exogenous sources i.e., the diet,
or if dietary sources are inadequate, from endogenous sources such as body stores. Some micronutrients
and dietary components have very specific roles in the development and maintenance of an effective

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immune system throughout the life course or in reducing chronic inflammation. For example, the amino
acid arginine is essential for the generation of nitric oxide by macrophages, and the micronutrients
vitamin A and zinc regulate cell division and so are essential for a successful proliferative response
within the immune system.
Undernutrition is well understood to impair immune function, whether as a result of food
shortages or famines in developing countries, or as a result of malnutrition arising from periods
of hospitalisation in developed countries. The extent of impairment that results will depend upon
the severity of the deficiency, whether there are nutrient interactions to consider, the presence of
infection, and the age of the subject [9]. A single nutrient can also exert multiple diverse immunological
effects, such as in the case of vitamin E, where it has a role as both antioxidant, inhibitor of protein
kinase C activity, and potentially interacting with enzymes and transport proteins [10]. For some
micronutrients, excessive intake can also be associated with impaired immune responses. For example,
supplementation with iron can increase morbidity and mortality of those in malaria endemic regions.
As well as nutrition having the potential to effectively treat immune deficiencies related to poor
intake, there is a great deal of research interest in whether specific nutrient interventions can further
enhance immune function in sub-clinical situations, and so prevent the onset of infections or chronic
inflammatory diseases.

3. Gut-Associated Lymphoid Tissue

The majority of immune cells within the human body are found within the gut-associated
lymphoid tissue (GALT), reflecting the importance of this immune tissue in maintaining host health.
In ingesting food, we expose ourselves to near constant and massive antigenic stimulation, and our
immune system must be able to provide strong and protective immunity against invasive pathogens,
while tolerating food proteins and commensal bacteria. In order to achieve this, the GALT contains a
variety of sensing and effector immune functions. Dendritic cells and M cells sample the gut content,
while plasma B cells within the lamina propria produce IgA, providing protection against pathogenic
organisms. Specialised immune regions known as Peyer’s patches, rich in immune cells, allow for
communication between immune cells resident within the GALT, propagation of signals to the wider
systemic immune system, and the recruitment or efflux of immune cells [11].
Within the gut lumen itself, the human gut microbiome will provide antigens and signals with the
potential to interact with resident and systemic immune cells. The composition of the gut microbiome
changes over the life course, in response to dietary components, and to environmental factors such
as antibiotic exposure. Dietary interventions targeted at the gut microbiome include probiotics
and prebiotics. Probiotics are defined as “live microorganisms, which, when consumed in adequate
amounts, confer a health benefit of the host” [12] while prebiotics, “a substrate that is selectively utilized
by host microorganisms conferring a health benefit” [13], tend to be non-digestible oligosaccharides
such as fructo-oligosaccharides and galacto-oligosaccharides. Provision of plant-based diets may
enhance the diversity of nutrients that reach the gut microbiome, with the indigestibility of plant cell
walls enabling peptides and lipids, which may otherwise have been absorbed in the upper digestive
tract to reach the microbiome [14]. There may be circumstances in which immune cells of the GALT
come into direct contact with nutrients or gut microbiota, such as in the case of reduced epithelial
integrity, or ‘leaky gut’ observed in both acute and chronic gut inflammation [15]. Such changes in gut
permeability may be influenced by micronutrient status such as that of vitamin D [16].
A number of nutrients and dietary interventions have demonstrated the capacity to improve
measures of gut health or to reduce gut inflammation. Protein hydrolysates have been demonstrated to
enhance barrier function and IgA production in animal models, and as a result may have applications
for incorporation within hypo-allergenic infant formula and clinical nutrition for those with conditions
such as inflammatory bowel disease [17]. Animal models of gut inflammation have identified that
providing probiotic bacteria can reduce inflammation, with reductions in proinflammatory Th1 and
Th17 cytokines such as IL-17 and IFN-γ, and enhanced production of inflammation resolving cytokine

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IL-10 [18]. Prebiotics can also enhance barrier function, in addition to their role as substrates for
bacterial metabolism [19]. Santiago-Lopez et al. have investigated the effect of fermented milk on a
murine model of inflammatory bowel disease [18] and demonstrated a reduction in serum IL-17 and
IFN-γ following fermented milk consumption when compared with the control group.

4. Immune Function Over the Life course

The developing foetus and neonates have an immature immune system, with poor antibody
production and a low proliferative response to challenge. In utero, the foetus can gain passive protection
from its mother via antibodies, which cross the placenta. This is the basis by which infants in the
UK are provided with early protection against whooping cough, with mothers offered vaccination in
their third trimester, in order to provide passive immunity to their infants until they reach the age of
infant vaccinations. While immature, the foetal immune system can produce antibodies, and allergens
can reach the developing foetus, and allergen-specific IgE can be detected in cord blood samples [20].
Another signature of the immaturity of the immune system in early life is the susceptibility of neonates
to infections, and the associated higher burden of morbidity and mortality.
The development of the immune system in early life will be influenced by both feeding practices and
environmental exposures. Breastfeeding provides further passive immunity to the infant, for example
via transfer of antibodies and cytokines. Breast milk components can also stimulate maturation of
the gut-associated lymphoid tissue, with breast milk known to be rich in bifidogenic oligosaccharides
and to contain its own unique microbiota. Human milk oligosaccharides (HMOs) are synthesised
from lactose in the mammary gland, and the specific HMO profile will vary between individuals and
across contexts and changes over the time course of lactation [21]. These HMOs have been found to
confer health benefits to infants by inhibiting the adhesion of microorganisms to the intestinal mucosa,
enhancing the production of short-chain fatty acids by bacteria within the microbiome, and inhibiting
inflammation [22]. Other immune active components of breast milk are also likely to be involved in
immune system maturation, with studies identifying that the growth factors epidermal growth factor,
fibroblast growth factor 21, and transforming growth factor-β2 can change lymphocyte phenotypes in
new-born rats when provided as supplements by oral gavage [23].
In infancy, diverse environmental factors will impact upon immune system development;
identified factors include pet ownership, antibiotic use, and the timing of introduction of foods [24].
The opportunity for introduction of prebiotic oligosaccharides during the introduction of foods has been
explored, with the suggestion that this could provide a unique opportunity to influence the developing
microbiome and thereby interact with the developing immune system [19]. These early years of life
are a critical period in the development of the immune system, particularly for T cell function, with the
thymus maturing and reaching its maximum size relative to body weight in infancy [25].
As we move through the life course towards later life, a decline in immune function is observed
among older adults. As was the case in infancy, older adults are more susceptible to infections, and have
more serious complications as a result than younger people. This declining immune function is known
as immunosenescence and reflects deterioration of both the acquired and innate immune systems [26].
Declining T cell function with age arises from thymic involution and decreased thymic output, resulting
in fewer naive T cells and more memory cells in the circulation [27]. Ageing is also associated with
increased inflammation in the absence of infection and has been found to predict hospitalisation
and death [28]. A number of micronutrient deficiencies have been identified as contributors to such
declining immunity, and so may provide opportunities for targeted interventions to restore immune
function [29].

5. Chronic Systemic Inflammation

Chronic systemic inflammation is a key underlying feature for a range of chronic
non-communicable disease conditions such as cardiovascular disease, stroke, and autoimmune
disorders such as rheumatoid arthritis. This chronic inflammation is positively correlated with aging

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and other co-morbidities (e.g., obesity, cardiovascular disease, insulin resistance). Interestingly, in a
study in healthy adults, increasing age was found to be a risk factor for chronic systemic inflammation,
independent of other risk factors such as body mass index, blood pressure, and blood lipid profiles [30].
The rising worldwide prevalence of obesity in children and adults is of grave concern. Obesity
and over nutrition are strongly associated with chronic inflammation, metabolic perturbation, and
higher risk for a number of chronic diseases including cardiovascular disease, stroke, type 2 diabetes
mellitus, and chronic liver disease. This metabolism-induced inflammation associated with obesity
is termed metaflammation, and the Western diet is a known risk factor [31,32]. The Western diet is
characterised by a diet high in sugar, trans and saturated fats, but low in complex carbohydrates, fibre,
micronutrients, and other bioactive molecules such as polyphenols and omega 3 polyunsaturated fatty
acids. The mechanisms by which the Western diet predispose individuals to metaflammation are still
under investigation. However, one mechanism which has been reported is the increased uptake of
lipopolysaccharide (LPS, a constituent of gram-negative bacterial cells walls), from microbes in the gut
because of increased gut leakiness. This LPS is sensed by cells of the innate immune system through
toll-like receptor 4 (TLR4). Activation of TLR4 by LPS will induce an inflammatory response by the
immune cells. Certain nutrients, notably long-chain omega 3 polyunsaturated fatty acids, can interfere
with TLR4 activation and, thus, can ameliorate this inflammatory signal. Rogero et al. describe the
relationship between obesity and inflammation and explores the immune pathway for this mechanism
and the anti-inflammatory roles of omega 3 fatty acids in this process [33].
Interestingly, in juxtaposition with the review by Rogero et al. on inflammation in obesity,
Dalton and colleagues report a study into systemic inflammation in individuals with the serious
psychological eating disorder, anorexia nervosa [34]. They show that in a severely undernourished
state, there are indications of systemic inflammation with an increased serum concentration of IL-6
when compared with healthy control participants. IL-6 is a classically inflammatory cytokine produced
by immune and other cells. Whether this inflammation is the result of the impact of undernutrition
or whether the clinical condition is the result of pre-existing inflammation is a matter that remains
to be determined. It has been shown that patients with clinical depression have increased systemic
inflammation suggesting that inflammation may have a bearing on mental health and wellbeing [35].
In contrast with the Western diet, the Mediterranean diet is rich in vegetables, fruit, nuts, legumes,
fish, and ‘healthy’ dietary fats. The Mediterranean diet is associated with a reduced risk of chronic
disease such as cardiovascular disease, cancer, and more recently Alzheimer’s disease [36]. A range of
bioactive compounds found in fruits and vegetables have been reported to offer one explanation for the
protective effect of diets rich in fruits and vegetables (e.g., Mediterranean diet) on the reduction of risk
for developing non-communicable diseases attributed to chronic inflammation (e.g., cardiovascular
disease). One family of molecules, which are known to have a role in regulation of inflammation
are the dietary polyphenols [37]. Yahfoufi et al. explain the mechanisms by which polyphenols
can be immunomodulatory and anti-inflammatory and explore the evidence for the role of dietary
polyphenols in reducing the risk of cardiovascular disease, some neurological diseases, and cancer [38].

6. Nutrition in the Clinical Setting

In clinical settings, acute inflammation may be a sudden, severe, and overwhelming process.
If not controlled, this severe systemic inflammation results in sepsis, culminating in multiple organ
failure and death. Sepsis is a major global cause of death killing approximately 6 million people per
year and is estimated to be the cause of 30% of neonatal deaths [39]. In this Special Issue of Nutrients,
the role of zinc in sepsis is discussed [40]. Zinc is known to be an important micronutrient for the
immune system. It has a role as a cofactor with both catalytic and structural roles in many proteins [41].
Even a mild deficiency in zinc has been associated with widespread defects in both the adaptive and
innate immune response [42]. During sepsis, zinc homeostasis is profoundly altered with zinc moving
from the serum into the liver. Alker and Haase consider this phenomenon and the implications for
therapeutic options to improve outcomes in patients presenting with sepsis [40].

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Nutrients 2019, 11, 1933

Selenium is a trace element that, like zinc, has critical functional, structural, and enzymatic roles,
in a range of proteins. Poor selenium status is associated with a higher risk for range of chronic diseases
including cancer and cardiovascular disease [43]. In addition to critical roles in many non-immune
tissues within the body, selenium is important for optimal immune function. Avery and Hoffman
explain the role of selenium in immunobiology and the mechanisms by which selenoproteins regulate
immunity. The evidence for the significance of selenium status in infectious diseases including human
immunodeficiency virus infection is reviewed [44].
Glutamine is a nonessential amino acid that provides an important energy source for many cell types
including those involved in immune responses. It also serves as a precursor for nucleotide synthesis,
particularly relevant for rapidly dividing cells such as the immune cells during an immune response.
During infection, the rate of glutamine consumption by immune cells is equivalent or greater than that
for glucose. Glutamine has roles in the functions of a number of immune cells including neutrophils,
macrophages, and lymphocytes [45]. In catabolic conditions (e.g., infection, inflammation, trauma),
glutamine is released into the circulation, an essential process controlled by metabolic organs such as the
liver, gut, and skeletal muscles. Despite this adaptation, a significant depletion of glutamine is seen in the
plasma and tissues in critical illness, which has provided a rationale for the use of in clinical nutrition
supplementation of critically ill patients. How glutamine homeostasis is maintained and when and how
to utilise glutamine in the clinical setting is explored in a review by Cruzat et al. [45].
The vitamin D receptor (VDR) is a nuclear receptor that can directly affect gene expression [46].
The presence of VDR in the majority of immune cells immediately suggests an important role
for this micronutrient in immune cell activities [47]. Furthermore, vitamin D-activating enzyme
1-α-hydroxylase (CYP27B1), which results in the active metabolite 1 α,25-dihydroxyvitamin D3
(1,25(OH)2 D3 ), is expressed in many types of immune cells. Ligation of VDR by 1,25(OH)2 D3 can elicit
the production of antimicrobial proteins and influence cytokine production by immune cells [47,48].
Sassi, Tamone, and d’Amelio have reviewed the evidence for the role of the nutrient vitamin D in the
innate and adaptive immune systems [16].

7. Conclusions
In this Special Issue of Nutrients, the collected works provide a breadth of reviews and research
indicating the key influence of nutrients and nutrition on immune responses in health and disease and
across the life course. Nutrients may impact directly or indirectly upon immune cells causing changes
in their function or may exert effects via changes in the gut microbiome. A better understanding
of the role of nutrients in immune function will facilitate the use of bespoke nutrition to improve
human health.

Author Contributions: Conceptualization, E.A.M.; writing—original draft preparation, E.A.M. and C.E.C.;
writing—review and editing, E.A.M., P.C.C., and C.E.C.
Funding: This article received no external funding.
Acknowledgments: This article received no specific grant from any funding agency, commercial or
not-for-profit sectors.
Conflicts of Interest: C.E.C. is member of the ILSI Europe Expert Group on Determinants of Immune Competence
and Co-Chair of ILSI Europe’s Nutrition, Immunity and Inflammation Task Force. C.E.C. receives research funding
from HOST Therabiomics and honoraria to speak at an event organised by Yakult. P.C.C. has research funding
from Bayer, has received research study products from Christian Hansen, and acts as a consultant/adviser to BASF,
DSM, Cargill, Smartfish and Pfizer. E.A.M. has no conflicts of interest to declare.

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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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 nutrients
Article
Inflammatory Markers in Anorexia Nervosa:
An Exploratory Study
Bethan Dalton 1, *, Iain C. Campbell 1 , Raymond Chung 2,3 , Gerome Breen 2,3 , Ulrike Schmidt 1,4
and Hubertus Himmerich 1,4
1 Section of Eating Disorders, Department of Psychological Medicine, Institute of Psychiatry,
Psychology & Neuroscience, King’s College London, London SE5 8AF, UK; [email protected] (I.C.C.);
[email protected] (U.S.); [email protected] (H.H.)
2 MRC Social, Genetic, and Developmental Psychiatry Centre, Institute of Psychiatry, Psychology &
Neuroscience, King’s College London, London SE5 8AF, UK; [email protected] (R.C.);
[email protected] (G.B.)
3 National Institute for Health Research Biomedical Research Centre for Mental Health, Institute of Psychiatry,
Psychology & Neuroscience at the Maudsley Hospital and King’s College London, London SE5 8AF, UK
4 South London and Maudsley NHS Foundation Trust, Bethlem Royal Hospital, Monks Orchard Road,
Beckenham, Kent BR3 3BX, UK
* Correspondence: [email protected]; Tel.: +44-0207-848-0183

Received: 28 September 2018; Accepted: 23 October 2018; Published: 24 October 2018

Abstract: Inflammation has been suggested to play a pathophysiological role in anorexia nervosa
(AN). In this exploratory cross-sectional study, we measured serum concentrations of 40 inflammatory
markers (including cytokines, chemokines, and adhesion molecules) and brain-derived neurotrophic
factor (BDNF) in people with AN (n = 27) and healthy controls (HCs) (n = 13). Many of these
inflammatory markers had not been previously quantified in people with AN. Eating disorder
(ED) and general psychopathology symptoms were assessed. Body mass index (BMI) and body
composition data were obtained. Interleukin (IL)-6, IL-15, and vascular cell adhesion molecule
(VCAM)-1 concentrations were significantly elevated and concentrations of BDNF, tumor necrosis
factor (TNF)-β, and vascular endothelial growth factor (VEGF)-A were significantly lower in AN
participants compared to HCs. Age, BMI, and percentage body fat mass were identified as potential
confounding variables for several of these inflammatory markers. Of particular interest is that most of
the quantified markers were unchanged in people with AN, despite them being severely underweight
with evident body fat loss, and having clinically significant ED symptoms and severe depression and
anxiety symptoms. Future research should examine the replicability of our findings and consider the
effect of additional potential confounding variables, such as smoking and physical activity, on the
relationship between AN and inflammation.

Keywords: anorexia nervosa; inflammatory markers; inflammation; cytokines; chemokines;

adhesion molecules

1. Introduction
Anorexia nervosa (AN) is a serious psychiatric disorder characterised by low body weight due to
food restriction and weight-control behaviours, such as excessive exercise and self-induced vomiting,
together with an intense fear of weight gain and disturbed body perception [1]. Altered concentrations
of inflammatory markers, in particular cytokines, have been reported in people with AN [2,3].
Cytokines are cell signalling molecules produced by a range of cells (e.g., microglia, astrocytes) in the
brain and the periphery (e.g., by macrophages and T-lymphocytes) and are essential in coordinating
responses to infection [4]. In addition, changes in the circulating concentrations and production of

Nutrients 2018, 10, 1573; doi:10.3390/nu10111573 10 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1573

cytokines have been associated with a range of disease states, including obesity [5] and diabetes [6],
as well as depression [7], schizophrenia [8], and eating disorders (EDs) [2,3].
Research in AN has primarily focused on pro-inflammatory cytokines, which promote and up-regulate
inflammatory reactions [4]. Recent meta-analyses have concluded that the pro-inflammatory cytokines
tumor necrosis factor (TNF)-α and interleukin (IL)-6, are elevated in people with AN, compared to
healthy individuals (for reviews see: [2,3]). However, few studies have quantified the concentrations
of cytokines in other categories, such as T-helper (TH )-1, TH 2, and anti-inflammatory cytokines (e.g.,
IL-10), the latter of which play an immunomodulatory role by reducing inflammation [9]. An example
of one such cytokine yet to be measured in people with AN is TNF-β, which is produced by TH 1
cells. TNF-β performs a variety of important roles in immune regulation [10,11], but has also been
implicated in the regulation of the commensal gut microbiota [12–14], which appears to be involved
in the pathology of AN [15–17]. Additionally, a number of cytokines implicated in other disorders,
such as depression and obesity, are yet to be measured in AN. One example is IL-17, a TH 17 cytokine
that has been reported to predict treatment response in people with depression [18], and seems to
be involved in the pathophysiology of schizophrenia [19] and the molecular and cellular effects of
antipsychotics [20].
Chemokines are a subcategory of smaller cytokines known to induce chemotaxis, with some
also having a homeostatic function in relation to haematopoiesis, immune surveillance, and adaptive
immune system responses [21,22]. The chemokines RANTES, monocyte chemoattractant protein
(MCP)-1, and fractalkine have been measured in two studies in people with AN [23,24]. Similarly,
adhesion molecules, which mediate the binding of cells in the immune system [25], have been measured
in one study in a sample of people with AN [26]. Circulating concentrations of vascular cell adhesion
molecule (VCAM)-1 have been reported to be elevated in people with AN compared to healthy
participants, but intercellular adhesion molecule (ICAM)-1 did not differ between the groups.
Cytokines and chemokines impact several biological domains implicated in the pathophysiology
of AN, including the modulation of neurotransmitter systems, neuroendocrine functioning, and neural
plasticity [27–31]. For example, in the depression literature, it has been hypothesised that elevated
pro-inflammatory cytokine levels may lead to symptoms of depression, partly via their disruption of
growth factor production, e.g., brain-derived neurotropic factor (BDNF) [32] and vascular endothelial
growth factor (VEGF)-A [33], which has a subsequent effect on adult neurogenesis [28,34]. Disruption
to these biological processes can then lead to alterations in mental state, including affect, learning and
memory, and behaviour (e.g., depressive-like behaviours) [28,35].
A number of factors, including body mass index (BMI), age, medication, and smoking status,
have been reported to influence cytokine concentrations [36]. These may be potential confounding
factors in studies of the role of cytokines in AN, particularly given the low weight seen in AN,
the tendency for research in EDs to focus on adolescents and young adults [2], and research indicating
that people with EDs report higher rates of smoking than healthy controls [37]. Previous studies
have not assessed the potential impact of depression symptoms on cytokine concentrations in AN.
A pro-inflammatory profile has been identified in people with depression [38] and the comorbidity
between AN and unipolar depression is of significant clinical relevance, as approximately 40% of
people receiving treatment for AN also suffer from depression [39]. Therefore, it is unclear as to
whether the alterations observed in cytokine concentrations are due to the AN or symptoms of
comorbid disorders, such as depression.
Few studies have considered a broad range of cytokines and other markers involved in
inflammatory processes and their potential role in the biological profile of AN. Therefore, in this
exploratory cross-sectional study, we measured a variety of inflammatory markers in a sample of AN
participants and healthy controls (HCs) to determine whether these markers are altered in AN. Several
of these inflammatory markers have not been previously quantified in people with AN. A secondary
objective was to test for the effects of potential confounders on concentrations of the inflammatory

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markers, including age and BMI, and explore the effect of current symptom severity on markers of
inflammation in people with AN.

2. Materials and Methods

2.1. Participants and Study Design

Between 2010 and 2013, 55 participants with AN (outpatients n = 27; day-patients n = 10; inpatients
n = 18) and 30 HCs were recruited as part of the “Relationship between Overactivity, Stress and Anxiety
in Anorexia Nervosa” (ROSANA) study [40,41]. Female adults with a primary diagnosis of AN
(restricting or binge-eating/purging type) and a BMI < 17.5 kg/m2 were recruited within the first four
weeks of treatment for AN via Specialist Eating Disorder Services in and around London. HCs (n = 30)
were recruited via an e-mail circular to students and staff at King’s College London. Exclusion criteria
for HCs were a history of or current mental health disorder, including EDs, and the presence of physical
illness, which were assessed in the initial research session using a specially designed record form
and the research version of the Structured Clinical Interview for Diagnostic and Statistical Manual of
Mental Disorders (DSM)-IV Axis I Disorders [42], described below. All participants provided informed
consent before study participation. The study was conducted in accordance with the Declaration
of Helsinki and the study received ethical approval from the South East London Research Ethics
Committee (REC ref: 09/H0807/4).

2.2. Measures

2.2.1. Demographic Characteristics and Screening

A specially designed record form was used to collect demographic data and additional information
relating to the inclusion and exclusion criteria described above, e.g., the presence of medical conditions.
To determine smoking status, participants were asked if they smoked and if so, to report the number
of cigarettes smoked per day. For AN participants, illness duration (in this case, time since diagnosis)
was also recorded. All participants completed the research version of the Structured Clinical Interview
for DSM-IV Axis I Disorders [42], a validated structured interview, to confirm diagnosis in the AN
participants and identify a history of and/or current mental health problems in the HCs.

2.2.2. Anthropometry
Height and body weight were measured, and from these measurements, BMI (kg/m2 ) was
calculated. Body composition was also measured using a portable and non-invasive Inbody S10
machine (Inbody Co., Ltd., Seoul, Korea) which uses the Bioelectrical Impedance Analysis (BIA)
measurement method. Following the input of height and weight details, this machine provides data
on muscle and fat, bone mineral content, intracellular and extracellular water, protein, and minerals.
The calculations used to do this are based on the assumption that the body is a cylindrical-shaped
conductor. Resistance is low in lean tissue (as it contains the majority of intracellular and extracellular
fluid and is thus a good conductor of electrical current), and fat mass is high in resistance as it is does
not contain any water (and thus does not conduct electrical current). Based on the assumption that
impedance (resistance) is proportional to total body water, predictive equations then determine total
body water, total body fat, and lean tissue mass. Given that adipose tissue has been implicated in the
genesis of cytokines and produces certain pro-inflammatory cytokines (e.g., IL-6), we focused on the
association between inflammatory markers and body fat percentage and did not include other body
composition parameters in our analyses.

2.2.3. Eating Disorder Behaviours and General Psychopathology

ED symptoms were assessed using the Eating Disorder Examination-Questionnaire (EDE-Q) [43].
This questionnaire has 36-items assessing ED symptoms and behaviours over the previous 28 days.

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Nutrients 2018, 10, 1573

A global score can be calculated, and items can also be categorised and scored into the following four
subscales: restraint, eating concern, weight concern, and shape concern. Related psychopathology
was assessed using the Depression Anxiety Stress Scale 21-Version (DASS-21) [44]. This is a 21-item
questionnaire measuring symptoms of general psychopathology over the previous seven days. As well
as a total score, a score for the three subscales—depression, anxiety, and stress—can be calculated.
Additional measures related to physical activity were also collected and findings are reported
elsewhere [40,41].

2.2.4. Inflammatory Markers

Blood samples were collected, and serum was stored at −80 ◦ C prior to use. Serum was thawed at
room temperature and the concentrations of 42 inflammatory markers were quantified simultaneously
using multiplex ELISA-based technology provided by the Meso Scale Discovery V-PLEX Human
Biomarker 40-Plex Kit and a customised human duplex kit assaying BDNF and interferon (IFN)-α,
following the manufacturer’s instructions (Meso Scale Diagnostics, LLC., Rockville, MD, USA). Cases
and controls were randomised across batches, and plates were scanned on the Mesoscale Scale
Discovery MESO Quickplex SQ 120 reader at the MRC SGDP Centre, Institute of Psychiatry, Psychology
& Neuroscience, King’s College London. The inflammatory markers measured in the 40-plex array
were: basic fibroblast growth factor (bFGF), C-reactive protein (CRP), Eotaxin, Eotaxin-3, Fms-like
tyrosine kinase (Flt)-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), ICAM-1, IFN-γ,
IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A,
interferon γ-induced protein (IP)-10, MCP-1, MCP-4, macrophage inflammatory protein (MIP)-1α,
MIP-1β, placental growth factor (PlGF), serum amyloid A (SAA), thymus and activation-regulated
chemokine (TARC), tyrosine kinase (Tie)-2, TNF-α, TNF-β, VCAM-1, VEGF-A, VEGF-C, and VEGF-D.
The assay for macrophage-derived chemokine (MDC) was not included in the current panel due to
quality control issues.

2.3. Statistical Analysis

Thirty-two AN and 14 HC participants had available blood samples. One HC participant
was excluded from the analyses due to having a BMI below 18.5 kg/m2 , i.e., in the underweight
range. Five AN participants were excluded from analyses due to reported autoimmune and/or
inflammatory diseases. Therefore, the current cross-sectional analyses are based on a sample consisting
of 27 participants with a diagnosis of AN and 13 HCs.
Standard curves were used to determine absolute quantities (pg/mL) of each inflammatory
marker. All statistical analyses were performed in Stata 15 [45].

2.3.1. Cross-Sectional Comparisons

For demographics and clinical characteristics, group comparisons were assessed using t-tests or
Mann-Whitney U-tests depending on the distribution of the data. Log-transformation of inflammatory
marker values was conducted due to the presence of outliers and non-normal distributions; however,
data remained non-normal for most of the measured molecules. Therefore, Mann-Whitney U-tests
were employed to compare concentrations of inflammatory markers between the AN and HC groups.
IFN-α was only detected in three samples (two HC and one AN); the results are therefore not presented.

2.3.2. Exploratory Regression Analyses

Absolute quantities of the inflammatory markers (pg/mL) were log-transformed to allow for
regression analyses. To identify marker-specific confounders, linear regressions were performed
for each log-transformed inflammatory marker (as the dependent variable) with each potential
confounding variable (age, BMI, percentage fat mass) separately in the whole sample. To test the
effect of illness severity on inflammatory marker concentrations, we performed linear regressions in
the AN participants, with the log-transformed inflammatory marker as the dependent variable and

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illness duration or ED symptoms, as measured by the EDE-Q, as the independent variable. To test
the effect of general psychopathology on inflammatory marker concentrations, linear regressions
in the AN participants, with the log-transformed inflammatory marker as the dependent variable
and the total DASS-21 score as the independent variable, were conducted. For both sets of analyses,
studentized residuals greater than ±3 standard deviations were deemed to be outliers and were
removed, and assumptions were tested and met.
The level of significance was set at p < 0.05, and as this was an exploratory study, levels of
significance were not adjusted for multiple testing.

3. Results
Demographic, anthropometric, and clinical characteristics of the AN participants and HCs are
presented in Table 1. All participants were female. Mean age did not significantly differ between the
AN and HC groups (U = 144, z = −1.36, p = 0.1735). Seven participants with AN reported being a
current smoker, with an average of 9.14 ± 5.90 cigarettes smoked per day. As expected, AN participants
had lower BMI (t (38) = 7.88, p < 0.001) and percentage body fat (U = −22, z = 3.63, p = 0.0003) scores,
and higher EDE-Q scores (global score: U = 85.5, z = −4.87, p < 0.001) than HCs. The EDE-Q global
score for the AN participants was greater than the commonly used clinical cut-off score of 4 e.g., [46,47].
AN participants also reported greater depression, anxiety, and stress than HCs on the DASS-21 (total
score: U = 92.5, z = −4.67, p <0.001). Proposed cut-off scores [44] suggest that the level of severity that
AN participants reported was severe for depression, anxiety, and stress.

Table 1. Demographic, anthropometric, and clinical characteristics for AN participants and HCs.

Demographic, Anthropometric, Healthy Controls Anorexia Nervosa

p Value
and Clinical Characteristics (n = 13) (n = 27)
Demographics
Age (years) (mean ± SD) 25.54 ± 4.52 31.48 ± 11.40 0.1735
Current smoker (n) 0a 7
Anthropometrics
BMI (kg/m2 ) (mean ± SD) 20.88 ± 1.68 15.33 ± 2.25 <0.0010
Body fat (%) (mean ± SD) 17.08 ± 6.05 b 7.76 ± 6.07 0.0003
Clinical characteristics
Duration of diagnosis, years (mean ± SD) 11.64 ± 11.54 c
AN-R/AN-BP (n) 12/15
Current outpatient/inpatient (n) 16/11
EDE-Q Global (mean ± SD) 0.66 ± 0.70 4.20 ± 1.27 <0.0010
EDE-Q Restraint (mean ± SD) 0.62 ± 0.88 4.04 ± 1.77 <0.0010
EDE-Q Eating Concern (mean ± SD) 0.26 ± 0.48 3.82 ± 1.28 <0.0010
EDE-Q Weight Concern (mean ± SD) 0.74 ± 0.83 4.13 ± 1.59 <0.0010
EDE-Q Shape Concern (mean ± SD) 1.02 ± 0.85 4.82 ± 1.24 <0.0010
DASS-21 Total (mean ± SD) 13.85 ± 13.89 72.30 ± 33.32 <0.0010
DASS-21 Depression (mean ± SD) 3.54 ± 5.43 24.59 ± 13.73 <0.0010
DASS-21 Anxiety (mean ± SD) 3.08 ± 4.94 19.48 ± 11.84 0.0001
DASS-21 Stress (mean ± SD) 7.23 ± 5.20 28.22 ± 10.69 <0.0010
a n = 6 missing; b n = 1 missing; c n = 3 missing. Abbreviations: HCs—healthy controls; AN—anorexia nervosa;

SD—standard deviation; BMI—body mass index; AN-R—anorexia nervosa restricting type; AN-BP—anorexia
nervosa binge-eating/purging type; EDE-Q—Eating Disorder Examination—Questionnaire; DASS-21—Depression
Anxiety and Stress Scales-21 Version.

3.1. Inflammatory Markers

3.1.1. Cross-Sectional Comparison

The median concentrations (pg/mL) of the quantified inflammatory markers, along with the
interquartile range, and minimum and maximum values, for HCs and AN participants are shown in
Table 2. Median serum levels of TNF-β (U = 76, z = 2.87, p = 0.004) and VEGF-A (U = 102, z = 2.12,
p = 0.030) were lower in AN participants than in HCs. Median concentrations of IL-6 (U = 92, z = −2.41,
p = 0.016), IL-15 (U = 85, z = −2.61, p = 0.009), and VCAM-1 (U = 106, z = −2.01, p = 0.032) were found
to be higher in AN participants compared to HCs. No other inflammatory parameters were found to
differ between groups.

14
 Table 2. Median serum concentrations (pg/mL), with interquartile range (IQR), minimum and maximum values, of inflammatory markers for HCs and
AN participants.

Inflammatory Healthy Controls Anorexia Nervosa

p Value
Marker N Median IQR Min Max N Median IQR Min Max
a. Concentrations significantly lower in AN compared to HCs
Nutrients 2018, 10, 1573

BDNF 13 17,375.24 7862.72 10,526.40 21,884.09 27 12,799.75 5947.87 7792.55 23,003.72 0.0315
TNF-β 13 0.86 0.32 0.43 53.22 27 0.60 0.19 0.16 1.22 0.0041
VEGF-A 13 471.96 220.76 205.98 749.41 27 288.82 155.41 133.70 973.73 0.0338
b. Concentrations significantly higher in AN compared to HCs
IL-6 13 0.38 0.29 0.10 1.11 27 0.49 0.90 0.20 3.35 0.0159
IL-15 13 2.54 0.39 1.91 3.18 27 2.90 0.81 1.57 5.24 0.0090
VCAM-1 13 612,378.30 92,337.87 402,297.20 739,682.80 27 709,059.60 220,832.10 434,057.50 1,069,997.00 0.0448
c. Concentrations not significantly different between groups
bFGF 13 11.01 10.73 2.17 21.13 27 12.13 13.39 1.15 65.25 0.3334
CRP 13 332,422.10 1,497,240.00 38,821.00 4,878,443.00 27 342,975.80 5,408,764.00 39,716.48 37,500,000.00 0.9424
Eotaxin 13 208.55 61.81 136.51 285.80 27 175.47 123.38 101.36 511.08 0.8511
Eotaxin-3 13 20.55 6.48 9.95 112.91 27 15.15 14.22 5.14 60.87 0.1058

15
Flt-1 13 61.83 11.64 27.93 86.21 27 68.69 28.37 11.99 117.06 0.1224
GM-CSF 13 0.15 0.21 0.02 0.88 22 0.19 0.17 0.01 0.35 0.8779
ICAM-1 13 658,988.00 183,427.10 517,283.10 861,717.20 27 701,224.80 244,044.70 415,274.60 977,044.00 0.1224
IFN-γ 13 3.94 1.64 2.11 6.84 27 4.64 5.72 2.11 64.28 0.1448
IL-1α 13 1.13 1.32 0.41 3.51 27 1.01 0.64 0.44 6.00 0.4105
IL-1β 9 0.20 0.33 0.04 1.86 21 0.19 0.19 0.01 4.07 0.7343
IL-2 6 0.12 0.24 0.03 0.43 13 0.15 0.16 0.01 0.85 0.9301
IL-4 13 0.08 0.07 0.01 0.20 25 0.05 0.05 0.00 5.24 0.1481
IL-5 12 1.09 0.87 0.43 3.45 24 1.29 1.01 0.07 4.39 0.7626
IL-7 13 13.66 6.11 8.76 20.13 27 12.58 6.80 5.82 26.20 0.8966
IL-8 13 33.51 62.10 8.44 503.23 27 23.09 92.94 5.80 388.41 0.4271
IL-10 13 0.18 0.10 0.06 0.41 27 0.28 0.26 0.04 4.40 0.3120
IL-12/IL-23p40 13 117.69 49.83 72.15 250.50 27 92.00 50.59 38.72 220.89 0.0806
IL-12p70 11 0.18 0.09 0.01 0.53 25 0.19 0.25 0.01 1.06 0.7572
IL-13 9 2.95 3.25 1.53 10.34 17 2.44 3.13 0.76 6.35 0.2692
IL-16 13 160.52 58.89 99.73 291.95 27 183.59 194.22 127.93 463.24 0.1702
 Table 2. Cont.

Inflammatory Healthy Controls Anorexia Nervosa

p Value
Marker c. Concentrations not significantly different between groups
IL-17A 13 1.78 1.94 0.36 3.85 27 1.91 1.51 0.63 43.83 0.9539
IP-10 13 110.93 57.70 75.89 175.65 27 115.99 108.88 72.71 596.43 0.3191
Nutrients 2018, 10, 1573

MCP-1 13 208.55 126.78 140.94 406.88 27 191.53 77.31 121.51 345.28 0.3480
MCP-4 13 142.79 80.25 54.04 212.93 27 120.65 83.26 53.61 290.55 0.6133
MIP-1α 13 25.01 7.73 16.36 56.15 27 23.02 15.10 15.68 66.68 0.8624
MIP-1β 13 102.15 62.83 34.70 280.41 27 81.06 44.32 41.10 271.72 0.3334
PlGF 13 3.38 1.57 1.91 6.06 27 3.85 1.81 1.71 6.21 0.6754
SAA 13 2,683,488.00 1,218,569.00 318,141.50 13,200,000.00 27 2,681,116.00 9,488,702.00 525,220.00 164,000,000.00 0.8061
TARC 13 365.16 319.52 168.47 690.47 27 370.33 378.45 68.69 7680.34 0.7617
Tie-2 13 5847.41 2784.74 4065.41 8455.58 27 6212.88 3028.74 2440.01 10,077.48 0.7617
TNF-α 13 1.59 0.50 0.91 2.70 27 1.64 1.08 0.61 2.95 0.8966
VEGF-C 13 574.92 119.71 242.97 739.49 27 449.26 243.10 277.52 852.72 0.2919
VEGF-D 13 790.06 358.08 383.69 1126.91 27 757.22 413.20 458.68 1906.49 0.8061
Abbreviations: IQR—interquartile range; min—minimum; max—maximum; AN—anorexia nervosa; HC—healthy controls; BDNF—brain-derived neurotrophic factor; TNF—tumor
necrosis factor; VEGF—vascular endothelial growth factor; IL—interleukin; VCAM-1—vascular cell adhesion molecule-1; bFGF—basic fibroblast growth factor; CRP—C-reactive protein;
Flt-1—Fms-like tyrosine kinase-1; GM-CSF—granulocyte-macrophage colony-stimulating factor; ICAM-1—intercellular adhesion molecule-1, IFN-γ—interferon- γ; IP-10—interferon

16
γ-induced protein-10; MCP—monocyte chemoattractant protein; MIP—macrophage inflammatory protein; PlGF—placental growth factor; SAA—serum amyloid A; TARC—thymus and
activation-regulated chemokine; Tie-2—tyrosine kinase-2.
Nutrients 2018, 10, 1573

3.1.2. Identification of Marker-Specific Confounding Variables

The full findings of the regressions assessing age, BMI, and percentage fat mass as potential
confounders of the serum concentrations of inflammatory markers across all participants are shown in
Table S1. Age significantly influenced the serum concentrations of Eotaxin-3 (F(1, 37) = 5.68, p = 0.0224),
IFN-γ (F(1, 33) = 7.91, p = 0.0082), MCP-1 (F(1, 38) = 8.87, p = 0.0050), MIP1-α (F(1, 38) = 4.48, p = 0.0408),
SAA (F(1, 38) = 6.99, p = 0.0119), and TNF-α (F(1, 38) = 5.07, p = 0.0302). BMI significantly predicted
concentrations of IL-4 (F(1, 34) = 4.73, p = 0.0367), IL-6 (F(1, 38) = 9.48, p = 0.0039), IL-10 (F(1, 37) = 6.67,
p = 0.0139), IL-12/IL-23p40 (F(1, 38) = 10.97, p = 0.0020), IL-15 (F(1, 38) = 9.54, p = 0.0037), TNF-β
(F(1, 35) = 7.45, p = 0.0098), and VEGF-C (F(1, 37) = 6.17, p = 0.0177). Percentage fat mass significantly
influenced IL-6 (F(1, 38) = 8.83, p = 0.0052), IL-12/IL-23p40 (F(1, 37) = 10.05, p = 0.0031), TNF-β
(F(1, 35) = 4.93, p = 0.0331), and VEGF-C (F(1, 38) = 5.00, p = 0.0315). Age, BMI, and percentage fat mass
were not found to be associated with concentrations of any of the remaining inflammatory markers.

3.1.3. Effect of Illness Severity

Full results from the regressions determining the effect of illness severity, in relation to illness
duration, ED symptoms, and general psychopathology, on serum concentrations of the inflammatory
markers in AN participants only are shown in Table S2. Illness duration of AN participants significantly
predicted serum concentrations of IL-4 (F(1, 18) = 15.82, p = 0.0009), IL-12/IL-23p40 (F(1, 21) = 6.70,
p = 0.0172), MCP-1 (F(1, 21) = 6.40, p = 0.0194), and VEGF-A (F(1, 22) = 5.55, p = 0.0278). Severity of ED
symptoms in AN participants, as measured by the EDE-Q, predicted serum concentrations of IP-10
(F(1, 23) = 12.60, p = 0.0017) and PlGF (F(1, 25) = 4.44, p = 0.0454). Level of general psychopathology
(symptoms of depression, anxiety, and stress, as measured by the DASS-21) significantly predicted
Eotaxin (F(1, 25) = 4.64, p = 0.0410), IL-7 (F(1, 25) = 4.60, p = 0.0419), IL-8 (F(1, 25) = 10.08, p = 0.0040),
IP-10 (F(1, 25) = 6.06, p = 0.0211), MCP-1 (F(1, 25) = 5.43, p = 0.0282), and TARC (F(1, 22) = 9.18,
p = 0.0062) concentrations. No other inflammatory markers were significantly associated with illness
duration or severity of ED and general psychopathology symptoms.

3.2. Brain-Derived Neurotrophic Factor

Median serum levels of BDNF were reduced in AN participants compared to HCs (U = 101,
z = 2.15, p = 0.0320). Concentrations of BDNF were significantly influenced by BMI (F(1, 38) = 13.86,
p = 0.0007) and percentage body fat mass (F(1, 38) = 7.08, p = 0.0114), but not by age, as shown in
Table S1. Illness duration, ED symptom severity, and general psychopathology symptoms were not
associated with serum concentrations of BDNF (see Table S2).

4. Discussion

4.1. Summary of Findings

We measured a range of markers involved in inflammatory processes, including cytokines,
chemokines, acute-phase reactants, and cell adhesion molecules, in people with AN and HCs. Median
concentrations of BDNF, IL-6, IL-15, TNF-β, VCAM-1, and VEGF-A were found to differ between
AN and HCs, with IL-6, IL-15, and VCAM-1 being elevated in AN, and BDNF, TNF-β, and VEGF-A
being reduced, compared to HCs. No other inflammatory markers differed between AN participants
and HCs. Our exploratory analyses also identified potential confounding variables of these markers,
including age, BMI, and percentage fat mass, which should be considered in future studies. Illness
severity, both with respect to ED and general psychopathology symptoms, predicted concentrations of
certain inflammatory markers that were not found to differ between AN participants and HCs. Of the
markers with significant cross-sectional differences, illness duration predicted serum concentrations
of VEGF-A.

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Nutrients 2018, 10, 1573

4.1.1. Inflammatory Markers

Given that the AN patients in this sample are seriously unwell, as evidenced by their low BMI,
the loss of fat mass, and the presence of clinically significant ED and severe depression and anxiety
symptoms, the finding that most inflammatory markers (n = 33) were unchanged may seem somewhat
surprising. However, it has been proposed that the functioning of the immune system is relatively
preserved in AN, despite malnutrition [48]. For example, extensive evidence suggests that patients
with AN tend to be free from infectious diseases, and rarely have colds and/or flu (at least until
very advanced stages of the disease) [48,49], which is in contrast to the heightened risk of infection
observed in those with typical malnutrition. A recent in vitro study of immune parameters in people
with AN reported that despite reductions in some immune cell populations, AN is also associated with
enhanced antioxidant potential and anti-inflammatory status, in comparison to HCs [50]. This may
contribute to the preservation of immune system functioning reported in AN. However, it needs
to be considered that data derived from in vitro methods may not reflect how cells would respond
in vivo. An additional contributing factor could be a relatively sustained/unaffected gut barrier
function in AN [51], in spite of the distinct alterations of the gut microbiome and low gut microbiome
diversity reported in AN patients [15]. A ‘leaky gut’ is associated with low-grade inflammation
(i.e., leaking of bacteria and/or their components from the gut into circulation is thought to elicit an
inflammatory response) [52,53]; therefore, sustained gut permeability will reduce the likelihood of an
inflammatory response.
To the best of our knowledge, this is the first time serum concentrations of IL-15, TNF-β,
and VEGF-A have been measured and were found to be altered in AN patients. Therefore, we have
discussed the potential significance of these findings in more detail.
IL-15, a T cell growth factor, has been suggested to be involved in the modulation of serotonergic
transmission [54,55], which may underlie the depressive symptoms and sleep disturbances that
are often present in people with AN, and fits with existing evidence of serotonergic alterations in
AN [56,57]. Elevations of IL-15 levels have also been reported in patients with schizophrenia [58],
which is interesting in light of the possible genetic overlaps between AN and schizophrenia that have
been identified in genome-wide association study (GWAS) data [59]. IL-15 has also been implicated
in cross-talk between fat and muscle and reported to have an anabolic role, i.e., decreasing fat and
increasing muscle mass [60]. It is unclear, therefore, why it is increased in the anorectic group given
that they show a severe loss of body fat. It may, for example, be raised as a way of trying to maintain
muscle mass in the ill state.
TNF-β (also known as lymphotoxin-α) has several roles in immune regulation [10] and is
involved in the regulation of cell survival, proliferation, differentiation, and apoptosis [11]. A TNF-β
polymorphism (+252G/A) has been proposed to increase the risk of developing schizophrenia [61].
TNF-β also has a role in maintaining lipid homeostasis, which is potentially important in AN. However,
what is perhaps of most interest is that it is involved in regulating intestinal microbiota [12–14],
especially as the gut microbiome has been implicated in the pathophysiology of AN e.g., [15].
VEGF-A induces angiogenesis, vasculogenesis, and endothelial cell growth, and also influences
vascular permeability, similar to VCAM-1 [62]. Altered levels of VEGF-A have also been found in
patients with depression [63] and schizophrenia [64]. Lastly, VEGF-A has been suggested to enhance
adult neurogenesis and hippocampus-dependent learning and memory [33], which may be important
in both responsivity to illness and in relation to therapy.
Taken together, changes in IL-15, TNF-β, and VEGF-A could potentially contribute to the
development of AN symptoms, such as low mood and disturbed sleep, as well as its clinical
consequences, such as impaired learning and memory. Furthermore, they provide a link between
the biological pathophysiology of AN with depression and schizophrenia, which are clinically- and
genetically-related psychiatric disorders. This association may be suggestive of inflammatory markers
being indicators of transdiagnostic symptoms, such as low mood, rather than specific psychiatric
diagnoses. Also, such a biological association between these disorders could have clinical and

18
Nutrients 2018, 10, 1573

therapeutic relevance. For example, we could consider the question as to whether antipsychotics,
such as olanzapine, which is approved for the treatment of schizophrenia and alters cytokine
production [65], might help patients with AN [31,66]. This may also be of particular interest in
AN as olanzapine has been shown to alter the gut microbiome, which could additionally contribute to
weight gain [67–70].
The findings of increased IL-6 and VCAM-1 serum concentrations in our sample of people with
AN compared to HCs are of less novelty, but indicate the reliability of our findings, as these results
have also been reported previously.
As described, there are already a number of studies that have consistently identified an association
between AN and the pro-inflammatory cytokine IL-6 [2]. IL-6 is an inducer of the acute-phase response,
which has been shown to have suppressive effects on food intake [71] and inhibit adipogenesis [72].
Our results replicated the findings of increased concentrations of IL-6 in people with AN, as compared
to HCs.
Víctor et al. [26] previously reported increased VCAM-1 serum levels in patients with AN.
VCAM-1 is a cell adhesion molecule with a key role in leukocyte recruitment from blood into tissue
and is thus important for cellular immune response [73]. Because of its wide distribution in human
tissues and organs, VCAM-1 has been implicated in the development of a variety of pathophysiological
states in the brain and in the body periphery, including autoimmune diseases, cardiovascular disease,
and infections [74].
It is unclear why these particular inflammatory markers are altered in people with AN compared
to HCs. However, there are a number of potential factors which may contribute to these alterations,
including stress and neuroendocrine functioning, genetics, the gut microbiota, early life stress,
and negative health behaviours (e.g., disturbed sleep, altered diet, smoking) [75].

4.1.2. Brain-Derived Neurotrophic Factor

The reduced serum concentrations of BDNF in AN participants compared to HCs is consistent
with previous findings [76]. BDNF is a neurotrophin implicated in both central and peripheral
nervous system development. It is well-established that BDNF and cytokines cross-regulate each other.
Certain pro-inflammatory cytokines can suppress the expression of BDNF [34] and BDNF-dependent
synaptic plasticity [32]. It is thought that the detrimental effect of pro-inflammatory cytokines on
neuroplasticity may be mediated by BDNF [34]. Taking together the evidence in AN of elevated
pro-inflammatory cytokines [2,3], reduced concentrations of BDNF and VEGF-A [76], and reduced
hippocampal volumes [77,78], a key area for adult neurogenesis, it could be hypothesised that this
mechanism may be at play in AN, as proposed in the depression literature [34,79]. This hypothesis
would be consistent with the high prevalence (approximately 40%) of depression in patients with
AN [39].

4.1.3. Marker-Specific Confounding Variables

Our exploratory regression analyses found that BMI significantly predicted BDNF, IL-4, IL-6,
IL-10, IL-12/IL-23p40, IL-15, TNF-β, and VEGF-C concentrations. BDNF, IL-6, IL-12/IL-23p40, TNF-β,
and VEGF-C were also significantly predicted by percentage fat mass. Previous studies assessing IL-6
in AN have failed to control for BMI in cross-sectional analyses. Therefore, we cannot be certain that
the alterations we identified in concentrations of BDNF, IL-6, IL-15, and TNF-β are attributable to AN,
rather than simply BMI or percentage fat mass. We also identified age as a significant confounder
of Eotaxin-3, IFN-γ, MCP-1, MIP1-α, SAA, and TNF-α. Therefore, studies in larger samples with
adequate power should ensure that BMI, and other confounding variables, such as age and percentage
fat mass, are incorporated into analyses as covariates to further explore this relationship. Research is
also needed to consider the effect of additional potential confounding variables on the relationship
between AN and inflammation. Physical activity, for example, may be particularly pertinent in AN
patients, given that excessive exercise is often a key feature of the disorder.

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Nutrients 2018, 10, 1573

4.2. Strengths and Limitations

This study is the first to measure several inflammatory markers in patients with AN, including
IL-15 and TNF-β, identifying several alterations in inflammatory markers in AN that warrant future
research in larger samples. We also assessed illness severity, in terms of illness duration; ED
symptoms, including psychological and anthropometric measures; and associated psychopathology
(e.g., depression and anxiety), in our participants. Few previous studies have included such
variables [2], which in the current study have allowed us to explore the relationship between illness
severity and inflammatory markers. In addition, for the HCs, the median values of a number of the
assessed markers are similar to those observed in previous studies, suggesting that our HC group is a
valid comparison group.
Several limitations should be noted. The sample is small, which limits the power in this study and
due to the exploratory nature, we did not correct for multiple testing, thus increasing the likelihood of
receiving a significant result. Additionally, while the AN and HC groups did not differ in mean age,
it must be mentioned that the AN group had a larger age range (18–67 years) than the HCs (20–36 years).
The AN sample included both inpatients and outpatients; differences between these treatment settings
in the opportunity to engage in ED behaviours, such as calorie restriction, self-induced vomiting,
and excessive exercise, may effect cytokine concentrations e.g., [80,81]. As BIA measurements of body
composition are influenced by fluid and electrolyte status (for which there are known imbalances in
AN and these parameters are reported to be particularly affected in early refeeding), the accuracy
of BIA measurements of body composition may be limited, particularly in the AN group [82,83].
For practical reasons, it was not possible to ensure blood samples were drawn at a specific time of
day and as cytokine production and release is reported to occur in a circadian manner, it is possible
that some natural variations may have occurred [84]. As previously described, the inflammatory
markers measured in the current study can be affected by a number of pre-analytical factors [36],
including age, BMI, smoking, and medication. We did not have data on medication for all participants;
however, research has shown that antidepressant and antipsychotic medication can influence cytokine
concentrations and production [20,85]. As these medications are prescribed to AN patients to target
comorbid features of AN, such as depression and/or to induce weight gain [31], it would be important
to identify medication status and incorporate this into analyses as a potential covariate. However,
the limited power in this study precludes more complex analyses, such as incorporating several
confounding variables or considering the effect of nominal confounders, such as smoking status,
on inflammatory markers. Overall, future investigations of inflammatory markers in AN need to ensure
that such confounders are assessed and reported, and if possible, accounted for in statistical analyses.

5. Conclusions
This exploratory study measured a broad range of inflammatory markers, many of which had
not been previously assessed in AN. IL-15, VEGF-A, and TNF-β, for the first time, were shown to be
altered in people with AN in comparison to HCs. Previous findings regarding an elevation of IL-6 and
VCAM-1 and a reduction in BDNF in AN participants were replicated. We also considered age, BMI,
and percentage fat mass as potential confounding variables of concentrations of the inflammatory
markers. Our findings suggest that future research should include covariates in analyses of this
relationship to explore whether this may account for some of the group differences in inflammatory
markers observed in the current study. Finally, given that these inflammatory markers function as part
of a complex network, future studies in larger samples should consider developing a composite score
of cytokine concentrations.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/11/1573/

s1: Table S1: Findings from the linear regressions of potential confounders—age, BMI, percentage fat mass
(independent variable)—on log-transformed values of inflammatory markers (dependent variable) in the whole
sample; Table S2: Findings from the linear regressions of illness severity (independent variable) on log-transformed
values of inflammatory markers (dependent variable) in AN patients only.

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Nutrients 2018, 10, 1573

Author Contributions: Conceptualization, G.B., U.S., and H.H.; Data curation, B.D.; Formal analysis, B.D.;
Funding acquisition, I.C.C., G.B., U.S., and H.H.; Investigation, B.D., I.C.C., R.C., and U.S.; Methodology, I.C.C.,
R.C., and U.S.; Project administration, U.S.; Supervision, I.C.C., G.B., U.S., and H.H.; Writing–original draft, B.D.;
Writing—review & editing, B.B., I.C.C., R.C., G.B., U.S., and H.H.
Funding: The ROSANA study was supported by an National Institute for Health Research (NIHR) Programme
Grant for Applied Research (RP-PG-0606-1043). The current study was funded by a studentship awarded to Bethan
Dalton by the Department of Psychological Medicine, King’s College London (KCL) and the Institute of Psychiatry,
Psychology and Neuroscience (IoPPN), KCL. Iain Campbell, Raymond Chung, Gerome Breen, and Ulrike Schmidt
receive salary support from the NIHR Mental Health Biomedical Research Centre at South London and Maudsley
NHS Foundation Trust and KCL. Ulrike Schmidt is supported by an NIHR Senior Investigator Award. The views
expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health
and Social Care. The APC was funded locally by monies supplied by Hubertus Himmerich.
Acknowledgments: We thank the participants of the ROSANA study and the research team who collected the
data between 2009 and 2013.
Conflicts of Interest: The authors declare no conflict of interest.

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 nutrients
Article
TGF-β2, EGF, and FGF21 Growth Factors Present in
Breast Milk Promote Mesenteric Lymph Node
Lymphocytes Maturation in Suckling Rats
Paulina Torres-Castro 1,2 , Mar Abril-Gil 1,2 , María J. Rodríguez-Lagunas 1,2 , Margarida Castell 1,2 ,
Francisco J. Pérez-Cano 1,2, * and Àngels Franch 1,2
1 Physiology Section, Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science,
University of Barcelona, 08028 Barcelona, Spain; [email protected] (P.T.-C.);
[email protected] (M.A.-G.); [email protected] (M.J.R.-L.); [email protected] (M.C.);
[email protected] (À.F.)
2 Nutrition and Food Safety Research Institute (INSA·UB), 08921 Santa Coloma de Gramenet, Spain
* Correspondence: [email protected]; Tel.: +34-934-024-505

Received: 6 August 2018; Accepted: 24 August 2018; Published: 27 August 2018

Abstract: Breast milk, due to its large number of nutrients and bioactive factors, contributes to
optimal development and immune maturation in early life. In this study, we aimed to assess the
influence of some growth factors present in breast milk, such as transforming growth factor-β2
(TGF-β2), epidermal growth factor (EGF), and fibroblast growth factor 21 (FGF21), on the immune
response development. Newborn Wistar rats were supplemented daily with TGF-β2, EGF, or FGF21,
throughout the suckling period. At day 14 and 21 of life, lymphocytes from mesenteric lymph nodes
(MLNs) were isolated, immunophenotyped, and cultured to evaluate their ability to proliferate
and release cytokines. The main results demonstrated that supplementation with TGF-β2, EGF,
or FGF21 modified the lymphocyte composition in MLNs. At day 14, all supplementations were able
to induce a lower percentage of natural killer (NK) cells with the immature phenotype (CD8+ ), and
they reduced the CD8αα/CD8αβ ratio at day 21. Moreover, the cytokine pattern was modified by
the three treatments, with a down regulation of interleukin (IL)-13 secretion. These results showed
the contribution of these growth factors in the lymphocytes MLNs immune maturation during the
neonatal period.

Keywords: growth factors; breast milk; immunonutrition; cytokines; lymphocytes

1. Introduction
At time of birth, the intestine is immature, not only anatomically, but also metabolically and
immunologically [1–3]. Intestinal development is a key process in early life because it includes
important functions related to growth and survival. It is important to develop mechanisms to digest
and absorb nutrients in an efficient way [4]. Moreover, the intestine begins hosting the gut microbiota
and establishing appropriate host immune responses against pathogens [2]. The intestinal maturation
process can be conditioned through synergy of several factors, such as genetics, microbial colonization,
and nutrition [1–3].
Breast milk is the gold standard to feed the newborn because it includes a rich number of
components, which are essential for optimal growth and development [5]. It also contains a high
number of bioactive factors, which participate in the immune maturation process of infants [2,6].
Among these components, immunoglobulins (Igs), cytokines, and growth factors (GFs) have an
important role [7–9]. In this sense, the effects of several GFs present in maternal milk that promote
intestinal maturation have been described, although their impact on immune development is still
unclear [9].

Nutrients 2018, 10, 1171; doi:10.3390/nu10091171 26 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1171

On the one hand, the transforming growth factor-β (TGF-β) family members have multifunctional
actions involved in maintaining intestinal homeostasis, regulating inflammation, and allergy
development [10–12]. TGF-βs act on different types of leukocytes, to control the initiation and
resolution of immune responses through the recruitment, activation, and survival of cells [12,13].
TGF-β2 is the predominant isoform present in human and rat breast milk; it reaches the neonatal
intestine where, at birth, endogenous production is low and increases towards weaning [3,10,12].
In infants, breast milk TGF-βs play an important role in developing immune response and promoting
oral tolerance development [12].
On the other hand, one of the most abundant GFs in breast milk is the epidermal growth factor
(EGF), the concentration of which is 500 times more than other GFs present in breast milk [14].
Its concentration is very high in colostrum and it decreases significantly during suckling, both in
human and rodent milk, suggesting that EGF plays a role in the promotion of early neonatal intestinal
growth [2,15]. In fact, EGF is a key intestinal regulator in protecting intestinal barrier integrity, essential
for the absorption of nutrients and the exclusion of pathogens, in both humans and animals [14,16,17].
EGF is a polyfacetic molecule, which acts by regulating different processes, such as cell growth,
survival, migration, apoptosis, proliferation, and differentiation. In early life, milk EGF seems to be
one of the crucial components involved in the prevention of necrotizing enterocolitis (NEC) [1,14].
EGF and TGF-β together with the immunosuppressive interleukin (IL)-10, also present in breast
milk, are involved in the functional development of the gastrointestinal mucosa, tolerance acquisition,
and inflammation downregulation in damaged intestinal cells [6,7].
In recent years, new components present in breast milk have been discovered. This is the case
of the fibroblast growth factor 21 (FGF21), which belongs to the hormone-like subgroup within the
FGF superfamily, and has been found in rodent and human milk [16]. The FGF21 present in milk,
seems to be involved in local actions in the neonatal intestine. It is a highly active pleiotropic factor,
involved in multiple aspects of metabolism through a variety of mechanisms, where it regulates both
the expression and activity of digestive enzymes, and the synthesis and release of several intestinal
hormone factors [16,18]. All these effects make FGF21 a good candidate to be studied, due to its
possible contribution to neonatal intestinal function.
Overall, because TGF-β2, EGF, and FGF21 are biologically active factors found in breast milk
and are suggested to be involved in neonatal intestine maturation; our hypothesis is that they could
also have an important role in the immune development process in early life. Therefore, the main
objective of the current study was to ascertain whether the effect of a daily supplementation with
these compounds could promote immune response development, during the suckling period in rats.
The effects of the supplementations on mesenteric lymph nodes (MLNs), an inductor site of the gut
associated lymphoid tissue (GALT), were studied specifically during and at the end of the suckling
period, in terms of lymphocyte phenotypic composition and on the immune functionality, such as their
lymphoproliferative and cytokine production abilities.

2. Materials and Methods

2.1. Animals
Pregnant Wistar rats (G15) were obtained from Janvier Labs (Le Genest-Saint-Isle, France), and
were individually housed in cages under controlled conditions of temperature and humidity in a
12:12 h light:dark cycle with access to food and water ad libitum. The pregnant rats were monitored
daily and allowed to deliver naturally. The day after birth was reported as day 1. The studies were
performed in accordance with institutional guidelines, for the care and use of laboratory animals, and
were approved by the Ethical Committee for Animal Experimentation (CEEA) at the University of
Barcelona (UB) and Catalonia Government (CEEA-UB Ref. 220/15, UB/DAAM 8521).

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Nutrients 2018, 10, 1171

2.2. Dietary Supplementation

The suckling pups were unified in litters of nine pups per mother and were randomized into
four groups, formed by three litters each (n = 27 pups/group). This sample size was required for each
group, as previous studies have demonstrated the remarkable role of variability among litters [19].
The Appraising Project Office’s program, from the Universidad Miguel Hernández de Elche (Alicante)
was used for such estimation, to detect statistically significant differences among groups, assuming
there was no dropout rate and a type I error of 0.05 (two-sided).
Besides the reference group (REF group), three supplemented groups based on nutritional
intervention were created: the transforming growth factor-β2 (TGF-β2), epidermal growth factor
(EGF), and the fibroblast growth factor 21 (FGF21). All animals were identified daily, weighed, and
supplemented by oral gavage with a volume of 10 mL/kg/day during the suckling period, from
day 1 to day 21 of age. The suckling pups were separated from their mothers 30 min before oral
administration, to allow gastric emptying. All daily handling was done at the same period of the day,
to avoid modifications in biological rhythms. All actions were performed as described in previous
studies in the group, References [19,20].
TGF-β2, EGF, and FGF21 groups were supplemented with recombinant human TGF-β2,
recombinant rat EGF, and recombinant human FGF21 (all from Peprotech® , Rocky Hill, NJ, USA).
The products were reconstituted according to the manufacturer’s recommendations.
The dose of TGF-β2 was 35 μg/kg/day, which was based on the amount of TGF-β2 found in the
last lactation rat milk (62 ng/mL), and the milk intake by pups within 4−14 days of age [3]. The dose
of EGF was 100 μg/kg/day, which had been demonstrated to be effective as a treatment in a rat model
of NEC [21]. Finally, the dose of FGF21 was 5 μg/kg/day, an amount that was established in relation
to TGF-β2, which has been found in a 1:10 ratio FGF21:TGF-β2 [10,16]. The REF group received a
matched volume of the vehicle used for the GFs administration (1% bovine serum albumin (BSA) in
phosphate buffer saline (PBS)).

2.3. Measurement of Growth and Development

Body weight was registered daily throughout the study. Two end points were established, at day
14 and at the end of the suckling period at day 21. At these times, prior to sacrifice, the pups were
anesthetized with intramuscular ketamine (90 mg/kg; Imalgene® , Merial, Barcelona, Spain) and
xylazine hydrochloride (10 mg/kg, Rompun® , Bayer, Barcelona, Spain); and body length (nose-anal)
was measured. These data allowed the calculation of morphologic variables, such as the body mass
index (BMI, g/cm2 ) and Lee index, for assessing obesity in rats ((g1/3 /cm) × 1000).

2.4. Sample Collection and Processing

Once anesthetized, MLNs and small intestine (SI) were obtained through a ventral laparotomy.
The SI was weighed, measured, and divided into three equal length portions. Gut washes (GWs) were
obtained from the distal SI. Briefly, the intestine was flushed with cold PBS and cut into 5 mm pieces.
The tissue was incubated with PBS (10 min, 37 ◦ C, shaking), centrifuged (538 g, 10 min, 4 ◦ C), and later,
supernatant was collected and stored at −20 ◦ C until Igs quantification.

2.5. Quantification of Intestinal IgA and IgM by ELISA

The IgA and IgM content were quantified in GWs from day 21 of study, by ELISA Quantitation
Set (Bethyl Laboratories, Inc., Montgomery, MD, USA), as previously described in Reference [19].
GWs were diluted at 1:20 (IgA) and 1:10 (IgM). Data were expressed as μg/g of tissue.

2.6. Lymphocyte Isolation from Mesenteric Lymph Nodes

The MLNs were placed in complete culture medium, containing Roswell Park Memorial Institute
(RPMI 1640, Sigma-Aldrich, St. Louis, MO, USA) 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis,

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Nutrients 2018, 10, 1171

MO, USA), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin streptomicin (PenStrep;
Sigma-Aldrich, St. Louis, MO, USA), and 0.05 mM 2-β-mercaptoethanol (Merck, Darmstadt, Germany).
MLNs lymphocytes were obtained in sterile conditions by passing the tissue through a cell strainer
(40 μm, BD Biosciences, San Diego, CA, USA). The cell suspensions were centrifuged (538 g, 10 min,
4 ◦ C), and the pellet was resuspended with complete RPMI medium. The cell counts and viabilities
were determined using an automated cell counter, after staining the cells with trypan blue (CountessTM ,
Invitrogen, Madrid, Spain), following usual laboratory procedures, as described in Reference [22].
The lymphocytes were immediately used to characterize their phenotype, and to determine their
ability to proliferate and secrete cytokines.

2.7. Mesenteric Lymph Node Cells Stimulation and Proliferation Assay

MLNs cell activation was performed in sterile conditions, using 96-well tissue culture plates
(TPP® , Trasadingen, Switzerland), which were pre-incubated with 200 μL/well of monoclonal
antibodies (mAbs) anti-CD3 (10 μg/mL, BD Biosciences, San Diego, CA, USA) and anti-CD28
(2 μg/mL, BD Biosciences, San Diego, CA, USA) (2 h, 37 ◦ C, 5% CO2 ). Then, MLNs lymphocyte
suspensions (5 × 104 cells/mL) were added to each well. A total of eight wells were used for
each sample: four pre-incubated with the mitogenic mAbs (stimulated cells, SC) and four without
pre-incubation (non-stimulated cells, NSC). The plates were incubated for 48 h, at 37 ◦ C and 5% CO2 .
Four hours before the end time of the incubation, 5-bromo-2’-deoxyuridine (BrdU, 20 μL/well, Merck,
Darmstadt, Germany) was added to measure its incorporation as an indicator of DNA synthesis in a
colorimetric immunoassay.
The plates were centrifuged (210 g, 5 min) and supernatants were collected and stored at −80 ◦ C,
until cytokine analysis. After fixation, the anti-BrdU mAbs and peroxidase conjugated anti-BrdU mAbs
and 3,3’,5,5’-tetramethylbenzidine (TMB) were added, following the manufacturer’s recommendations
of the BrdU Cell Proliferation Assay Kit (Merck, Darmstadt, Germany). The absorbance (Abs) was
measured at 450 nm (Multiskan MS, Labsystems, Helsinki, Finland). Data were expressed as a
proliferation rate as follows: Proliferation rate = (A/B), where, A = ((Abs-SC − Abs-NSC)/Abs-NSC)
supplemented group , and B = ((Abs-SC − Abs-NSC)/Abs-NSC) reference group .

2.8. Quantification of Cytokine Secretion by Mesenteric Lymph Node Lymphocytes

Supernatants collected after the cell stimulation process described above, were used for the
quantification of IL-2, IL-4, IL-10, IL-13, IL-17A, interferon (IFN)-γ, and tumor necrosis factor
(TNF)-α concentration using a ProcartaPlex® Multiplex Immunoassay, according to the manufacturer’s
instructions (eBioscience, San Diego, CA, USA) and as previously described in Reference [19].
Each analyte’s concentration was detected using the MAGPIX instrument (Luminex Corp., Austin,
TX, USA), in the facilities of the Scientific and Technological Centers of the University of Barcelona
(CCiT-UB), and the results were analyzed using xPONENT® 4.2 software (Luminex Corp., Austin,
TX, USA). The limits of detection were as follows: 2.10–8600 pg/mL for IL-2; 0.85–3500 pg/mL
for IL-4; 14–55,700 pg/mL for IL-10; 3.17–13,000 pg/mL for IL-13; 2.61–2675 pg/mL for IL-17A;
4.35−17,800 pg/mL for IFN-γ; and 3.08−12,600 pg/mL for TNF-α.

2.9. Immunofluorescence Staining and Flow Cytometry Analysis

Lymphocytes from MLNs (5 × 104 cells) were immunophenotyped by multiple
immunofluorescence staining technique. Mouse anti-rat mAbs conjugated to fluorescein isothiocyanate
(FITC), phycoerythrin (PE), peridinin-chlorophyll-a protein (PerCP), allophycocyanin (APC) or
APC-cyanine(Cy)7 were used, as in previous studies [19]. For cell subset differentiation, five different
mAbs panels were used; Panel 1: TCRαβ/NKR-P1A/CD8α; Panel 2: CD8α/CD8β/TCRγδ; Panel
3: αE integrin/CD62L/CD8α/CD4; Panel 4: CD45RA/TLR4/CD8α/CD4/CD25; and Panel 5:
CD25/CD4/Foxp3. With the first panel, NK cells (NKR-P1A+ TCRαβ− ), natural killer T (NKT)
cells (NKR-P1A+ TCRαβ+ ), and TCRαβ+ cells (TCRαβ+ NKR-P1A− ) could be differentiated, which

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Nutrients 2018, 10, 1171

in combination with the TCRγδ+ cells (obtained from Panel 2) constituted the total of T cells.
B cells (CD45RA+ ) were identified with Panel 4. The mAbs used in this study are detailed in the
Supplementary Materials (Table S1).
Briefly, cells were incubated with a mixture of 10 μL of saturating concentrations of each mouse
anti-rat mAbs in PBS pH 7.2, containing 2% FBS and 0.1% sodium azide (Merck, Darmstadt, Germany),
at 4 ◦ C in darkness for 20 min. For T reg evaluation, an intracellular staining was performed. For that,
cells previously labeled with anti-CD4-PE and anti-CD25-FITC mAbs, were fixed/permeabilized
using a specific buffer kit (eBioscience, San Diego, CA, USA). Then, intracellular staining with
anti-Foxp3-APC mAb was carried out, under the same conditions as extracellular staining. After
washing, all stained cells were fixed with 0.5% paraformaldehyde (Panreac, Barcelona, Spain) and
stored at 4 ◦ C in darkness, until analysis by flow cytometry. For each sample, a positive control
staining using each isotype matched mAbs and a negative control without staining were included.
Analyses were performed in a Gallios™ Cytometer (Beckman Coulter, Miami, FL, USA) in the
CCiT-UB. Data were assessed by FlowJo® version 10 software (Tree Star Inc., Ashland, Covington,
KY, USA). Results were expressed as percentages of positive cells in the lymphocyte population,
selected according to their forward- and side-scatter characteristics (FSC/SSC) using previous studies
protocols [19], or in a selected population. The gating strategy was specific for each panel used, but
overall, markers (e.g., CD8α in Panel 1, αE integrin, and CD62L in Panel 3) were evaluated in specific
gate subsets (e.g., NK, NKT, or T cells in Panel 1, and CD8 or CD4 cells in Panel 3).

2.10. Statistical Analysis

Statistical analyses were performed using the IBM Social Sciences Software Program (SPSS,
version 22.0, Chicago, IL, USA). Levene’s and Shapiro−Wilk tests were applied to assess variance
equality and normal distribution, respectively. When the results demonstrated equality of variance
and normal distribution, a one-way analysis of variance (ANOVA) test was performed. Nonparametric
tests were carried out when normal distribution and equality of variance did not exist. Specifically,
Kruskal–Wallis and Mann–Whitney U tests were used to assess significance for independent samples.
Significant differences were established at p < 0.05. Data in the text, tables, and figures are expressed
as the mean ± standard error of the mean (S.E.M).

3. Results

3.1. Animal Growth

Body growth was assessed in the three groups receiving supplementation (TGF-β2, EGF, and
FGF21), and compared with those receiving the vehicle (REF). Body weight increased during suckling
period in the REF group (p < 0.05) and the supplementations did not affect this growth pattern
(Supplementary Materials, Figure S1). With respect to morphometric variables and SI growth, there
were no differences among the studied groups at any age considered (Table 1).

Table 1. Morphometric variables and small intestinal growth.

BMI Lee Index SI Relative Weight SI Relative Length

√
(g/cm2 ) (( 3 g/cm) × 1000) (%) (%)
REF 0.365 ± 0.009 332.9 ± 3.473 3.51 ± 0.05 118.29 ± 5.49
TGF-β2 0.373 ± 0.010 336.9 ± 3.844 3.59 ± 0.14 114.34 ± 4.28
Day 14
EGF 0.340 ± 0.007 327.0 ± 2.845 3.48 ± 0.06 122.64 ± 2.91
FGF21 0.371 ± 0.009 337.4 ± 4.044 3.18 ± 0.06 105.59 ± 5.31
REF 0.404 ± 0.010 Ψ 319.5 ± 2.551 Ψ 4.15 ± 0.24 Ψ 89.57 ± 3.12 Ψ
TGF-β2 0.404 ± 0.009 Ψ 320.8 ± 3.225 Ψ 4.25 ± 0.09 Ψ 85.65 ± 2.20 Ψ
Day 21
EGF 0.404 ± 0.007 Ψ 322.9 ± 2.574 4.38 ± 0.07 Ψ 88.34 ± 2.20 Ψ
FGF21 0.420 ± 0.011 Ψ 329.0 ± 4.053 4.78 ± 0.17 Ψ 89.97 ± 4.01 Ψ
BMI: body mass index. SI: small intestine. TGF-β2: transforming growth factor-β2. EGF: epidermal growth factor.
FGF21: fibroblast growth factor 21. Results are expressed as mean ± standard error of the mean (S.E.M) (n = 9).
Ψ p < 0.05 vs. day 14 at same group.

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Nutrients 2018, 10, 1171

All groups increased BMI, SI relative weight, and SI relative length with age (p < 0.05, day 21 vs.
day 14). Regarding the Lee index, only the animals from the REF group and those supplemented with
TGF-β2, showed significant differences associated with age (p < 0.05), but the supplementation with
EGF and FGF21 did not display such a significant age difference.

3.2. IgA and IgM Concentration in Gut Wash

Intestinal secretory IgA and IgM were determined at day 21 of life. At that age, the pups’ intestinal
levels were not entirely influenced by maternal breast milk, as happens earlier in life when breast milk
(very rich in IgA) is the only source of food. Nevertheless, none of the interventions (TGF-β2, EGF,
and FGF21 supplemented groups) showed significant differences regarding IgA or IgM, compared to
the REF group (Supplementary Materials, Figure S2).

3.3. Mesenteric Lymph Node Lymphocytes Composition

The proportion of the main lymphocyte subsets in MLN was established during (day 14) and at
the end of (day 21) suckling period. On both days, most of the MLN cells (~75%) were T cells, the
majority of them being TCRαβ+ cells and only ~3% TCRγδ+ cells; B cells counted for about 15–18%,
NK and NKT cells represented subpopulations with less than 3% of total lymphocytes, and Treg cells
were ~4% (Table 2).

Table 2. Main lymphocyte subsets in mesenteric lymph nodes.

Reference TGF-β2 EGF FGF21

T cells (%) 75.90 ± 1.80 77.90 ± 1.39 74.36 ± 1.88 79.60 ± 1.50
T TCRαβ+ (%) 72.90 ± 1.98 75.24 ± 1.29 71.22 ± 1.94 75.90 ± 1.20
T TCRγδ+ (%) 3.00 ± 0.20 2.66 ± 0.20 3.14 ± 0.23 3.70 ± 0.40
Day 14
NK (%) 1.63 ± 0.30 1.22 ± 0.08 1.35 ± 0.12 1.42 ± 0.17
NKT (%) 1.15 ± 0.14 1.18 ± 0.20 0.94 ± 0.05 0.98 ± 0.06
B cells (%) 17.65 ± 1.65 16.19 ± 1.40 19.62 ± 1.23 15.86 ± 1.07
T cells (%) 74.60 ± 1.36 75.86 ± 1.10 75.29 ± 1.20 76.41 ± 0.69
T TCRαβ+ (%) 71.57 ± 1.44 73.28 ± 1.14 72.93 ± 1.26 73.10 ± 0.77
T TCRγδ+ (%) 3.07 ± 0.15 2.58 ± 0.15 2.36 ± 0.13 *,Ψ 3.31 ± 0.13
Day 21
NK (%) 0.95 ± 0.07 Ψ 0.79 ± 0.06 Ψ 0.77 ± 0.08 Ψ 1.05 ± 0.07
NKT (%) 1.57 ± 0.07 Ψ 1.55 ± 0.11 1.39 ± 0.21 1.63 ± 0.18 Ψ
B cells (%) 14.67 ± 0.71 14.30 ± 0.64 12.95 ± 1.19 Ψ 13.23 ± 0.62
Results are expressed as mean ± S.E.M (n = 9). * p < 0.05 vs. reference (REF) group at same age; Ψ p < 0.05 vs. day 14
at same group.

Although at 14 days there were no differences among the supplemented groups, some differences
could be seen at 21 days (Table 2). At the end of the suckling period, only the EGF supplementation
showed a significant decrease in the percentage of TCRγδ+ cells, compared to the REF group (p < 0.05).
Moreover, the REF and groups supplemented with TGF-β2 and EGF decreased the NK cell proportion,
in comparison to the same group at 14 days (p < 0.05). The animals from the REF and FGF21 groups
increased the percentage of NKT cells with age, and only those from the EGF group were able to
decrease the proportion of TCRγδ+ and B cell percentages at day 21 (p < 0.05 vs. day 14).
The percentage of CD8+ cells and the CD8αα/CD8αβ ratio from MLNs lymphocytes, were
determined as indicators of immune maturation (Figure 1). The proportion of CD8+ cells did not show
significant differences, either associated with age (day 14 vs. day 21) or to any of the supplementations,
compared to the REF group (Figure 1a). However, regarding the results from CD8αα/CD8αβ ratio, the
EGF supplementation induced a significant increase at 14 days (p < 0.05 vs. REF, Figure 1b). At day 21,
in all supplemented groups, the CD8αα/CD8αβ ratio had decreased when they were compared to
their respective group at day 14, but no differences among the interventions and the REF groups were
found (Figure 1b).

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Nutrients 2018, 10, 1171

5() 7*)β (*) )*)

(a) (b)



&'FHOOV

&'αα/&'αβ



 
Ψ Ψ
Ψ




 
   

Figure 1. Proportion of CD8+ cells and ratio CD8αα/CD8αβ in mesenteric lymph nodes at 14 and
21 days of life. (a) % CD8+ cells; (b) The ratio CD8αα/CD8αβ was calculated as the quotient of
the percentages of CD8αα cells and CD8αβ cells in CD8+ cells (%). Results are expressed as mean
± standard error of the mean (S.E.M) (n = 9). Statistical differences: * p < 0.05 vs. reference group
(REF group) at same age; Ψ p < 0.05 vs. same group at day 14. TGF-β2: transforming growth factor-β2.
EGF: epidermal growth factor. FGF21: fibroblast growth factor 21.

Further analysis of CD8+ and CD8− subsets revealed some changes associated with the
supplementations (Figure 2).
Regarding the TCRαβ+ subsets (CD8+ and CD8− , respectively, Figure 2a,b), no changes due
to supplementation were found either at 14 or at 21 days, with respect to the REF group. Only, the
supplementation with FGF21 decreased the proportion of TCRαβ+ CD8− (~8%), when values from
day 21 were compared to day 14 (Figure 2a, p < 0.05). The TCRγδ+ CD8− cell percentage (Figure 2c),
increased due to supplementation with EGF and FGF21 at 14 days, with only the latter achieving
statistical significance, but these changes were not observed at the end of the suckling period (day 21).
However, both interventions showed a 50% decrease in the TCRγδ+ CD8− cell proportion associated
with age (p < 0.05, day 21 vs. day 14). Moreover, the TCRγδ+ CD8+ cell proportion in the EGF group
(Figure 2d), was lower than the REF group at day 21 (p < 0.05). In relation to the NKT population (CD8-
and CD8+ subsets), only the NKT CD8− subset showed changes associated with age for all groups
(Figure 2e), but not due to the supplementations.

32
Nutrients 2018, 10, 1171

5() 7*)β (*) )*)

(a) (b)




7&5αβ &'
7&5αβ + &' ψ 






 

 

 
   
(c) (d)



7&5γδ&'

7&5γδ&'




 
ψ
ψ 



 
   
(e) (f)

ψ 
 ψ
1.7&'

ψ ψ 
1.7&'







 

 
   
(g) (h)
 


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1.&'



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 ψ ψ
 ψ





 
   

Figure 2. Percentages of CD8+ and CD8− lymphocyte subsets in mesenteric lymph nodes at 14 and
21 days of life. (a) TCRαβ+ CD8− ; (b) TCRαβ+ CD8+ ; (c) TCRγδ+ CD8− ; (d) TCRγδ+ CD8+ ; (e) NKT
CD8− ; (f) NKT CD8+ ; (g) NK CD8- ; and (h) NK CD8+ . Results are expressed as mean ± S.E.M (n = 9).
Statistical difference: * p < 0.05 vs. REF group at same age; Ψ p < 0.05 vs. same group at day 14.

However, NKT CD8+ cell proportions were not affected (Figure 2f). Results from the NK cell
population showed that in supplemented groups, there was a decrease in NK CD8- cell proportions
related to age (Figure 2g, p < 0.05, day 21 vs. day 14), and that only supplementation with TGF-β2
induced a significant decrease compared with the REF group at 21 days (Figure 2g, p < 0.05 vs. REF).
The phenotype of the intestinal NK cells based on CD8 expression, could be considered as immature
(NK CD8+ ) or more mature (NK CD8− ). The three supplementations were able to decrease the
proportion of NK cells expressing CD8 at 14 days (Figure 2h), which reached similar values to those
from reference 21-day-old rats. Thus, only the REF group decreased its percentage of NK CD8+ cells at
21 days, with respect to day 14 (Figure 2h).

33
Nutrients 2018, 10, 1171

The lymphocytes’ commitment to the mucosal compartment were studied by means of the
proportion of cells expressing two adhesion molecules of importance in the intestinal homing; thus, the
total percentage of cells bearing the selectin CD62L and the αE integrin were determined (Figure 3).

Figure 3. Surface expression of αE integrin and CD62L selectin, in mesenteric lymph nodes at 14 and
21 days of life. (a) CD62L/αE integrin surface expression at 14 days, (b) CD62L/αE integrin surface
expression at 21 days, (c) CD62L+ cells (%) and (d) αE+ cells (%). Results are expressed as mean ±
S.E.M (n = 9). Statistical difference: Ψ p > 0.05 vs. same group at day 14.

Figure 3a,b show the molecular pattern of αE/CD62L on day 14 and 21. The CD62L molecule was
expressed on both days in high proportion of cells (~60%). Although the percentage of CD62L+ cells
increased significantly with age in all studied groups (Figure 3a–c, p < 0.05), none of the supplemented
groups induced significant differences, when they were compared to the REF group at the same age.
The αE+ cells were present in a proportion lower than 5% at both studied times (Figure 3b–d) and
were not modified by any GFs administration (Figure 3d).
The percentages of integrin αE+ cells and selectin CD62L+ cells in CD8+ , CD4+ , and B cells were
further studied (Table 3). None of the supplemented groups showed significant differences, compared
to the REF group at the same age. All studied groups increased CD62L+ CD8+ and CD62L+ CD4+
percentages with age (p < 0.05, day 21 vs. day 14). The REF group increased (~15%) αE+ B cells
subset proportion, with respect to the same group at 14 days, whereas all supplemented groups
decreased (~20%) this subset with age (p < 0.05). In addition, the supplementation with EGF and
FGF21, decreased the percentage of αE expression in CD4+ cells with age. Although there was a
tendency to increase with age, the proportion of CD62L+ in B cells in all groups, it was only significant
in the TGF-β2 group (p < 0.05).
At the end of the suckling period, activated CD4 cells (Foxp3− CD25+ CD4+ cells) and Treg cells
(Foxp3+ CD25+ CD4+ cells) were also studied, and no changes were found due to diets (day 21), with
all groups together having a mean percentage of 1.22 ± 0.12 and 3.81 ± 0.11, respectively.

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Nutrients 2018, 10, 1171

Table 3. Surface expression of the αE integrin and the CD62L selectin in CD4+ , CD8+ , and B cells in
mesenteric lymph nodes.

14 Days
Reference TGF-β2 EGF FGF21
CD8+ 2.75 ± 0.72 2.92 ± 0.49 2.94 ± 0.52 2.88 ± 0.44
%αE CD4+ 2.81 ± 0.39 3.07 ± 0.45 4.46 ± 0.34 3.80 ± 0.26
B cells 15.98 ± 1.61 18.33 ± 1.39 18.92 ± 2.50 20.32 ± 3.12
CD8+ 65.27 ± 1.56 62.38 ± 3.47 59.25 ± 2.52 59.21 ± 3.55
%CD62L CD4+ 59.55 ± 1.76 55.41 ± 2.36 53.61 ± 1.37 53.65 ± 2.40
B cells 48.43 ± 2.77 46.6 ± 2.05 38.77 ± 2.08 37.29 ± 4.78
21 Days
Reference TGF-β2 EGF FGF21
CD8+ 3.84 ± 0.48 2.98 ± 1.04 3.70 ± 0.48 3.97 ± 0.66
%αE CD4+ 2.83 ± 0.75 2.61 ± 0.52 1.94 ± 0.18 Ψ 2.52 ± 0.30 Ψ
B cells 18.43 ± 1.83 Ψ 13.03 ± 1.55 Ψ 14.95 ± 1.69 Ψ 15.75 ± 1.16 Ψ
CD8+ 71.49 ± 1.38 Ψ 72.10 ± 1.93 Ψ 70.78 ± 2.19 Ψ 72.88 ± 2.37 Ψ
%CD62L CD4+ 66.02 ± 1.74 Ψ 68.24 ± 1.64 Ψ 66.66 ± 0.42 Ψ 66.80 ± 1.22 Ψ
B cells 56.79 ± 2.25 60.92 ± 2.17 Ψ 60.82 ± 3.92 57.61 ± 2.37
Results are expressed as mean ± S.E.M (n = 9). Ψ p < 0.05 vs. day 14 at same group.

3.4. Proliferation and Cytokine Production by Mesenteric Lymph Nodes Cells

To determine the functional capacity of MLN lymphocytes, we studied their proliferative response
and their ability to secrete cytokines on days 14 and 21. With regard to the proliferation of MLNs
cells on days 14 and 21, GFs supplementations were not able to significantly modify such lymphocyte
function induced by anti-CD3 and anti-CD28 mAbs (Supplementary Materials, Figure S3). However,
cytokine production was influenced by GFs supplementation (Table 4).
The pattern of cytokines released from MLNs cells at day 14, showed a high secretion of IFN-γ,
followed by IL-10. In the conditions tested, the secretion of IL-2, IL-4, IL-13, IL-17A, and TNF-α was
lower than 90 pg/mL. IL-10/TNF-α (anti-inflammatory/pro-inflammatory cytokines) and IFN-γ/IL-4
(Th1/Th2) ratios were also calculated. No changes in the cytokine pattern at day 14 were detected
by GFs supplementation. However, at day 21, some changes appeared due to supplementations and
due to age. IL-2 production was 5–10 times higher due to age in the REF, TGF-β2, and EGF groups
(p < 0.05 day 21 vs. day 14). IL-4 levels were ~50% lower in the EGF group at day 21, than those at
day 14 (p < 0.05). The groups that received EGF and FGF21 for 21 days, decreased the IL-10 content
~1.5 times with respect to their values at day 14 (p < 0.05). IL-13 secretion was lower at day 21 with
respect to day 14, in the EGF and FGF21 groups (p < 0.05), and the three GFs groups showed lower
values than those present in the REF group (p < 0.05). The lower levels of IFN-γ production at day
21 compared to day 14 were only significant in the REF group, which accounted for a 50% reduction
(p < 0.05). Moreover, TNF-α release decreased (~30%) with age in the REF, TGF-β2, and FGF21 groups,
whereas supplementation with EGF increased TNF-α production (p < 0.05 vs. REF group).
The IL-10/TNF-α ratio decreased according to age in the EGF and FGF21 groups, and in the EGF
group, values were significantly lower than the REF group (p < 0.05).

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Nutrients 2018, 10, 1171

Table 4. Cytokine production by mesenteric lymph node cells after in vitro stimulation.

14 Days
(pg/mL) Reference TGF-β2 EGF FGF21
IL-2 42.71 ± 5.96 39.24 ± 5.66 50.45 ± 6.66 56.72 ± 11.04
IL-4 89.82 ± 13.00 57.14 ± 7.81 82.65 ± 12.47 74.97 ± 12.18
IL-10 879.82 ± 239.40 688.01 ± 145.14 1450.87 ± 296.29 1326.99 ± 217.06
IL-13 25.38 ± 5.64 17.53 ± 3.03 31.91 ± 4.28 26.18 ± 4.03
IL-17A 71.98 ± 26.92 70.22 ± 20.16 60.12 ± 13.61 50.82 ± 9.53
IFN-γ 10323.26 ± 2391.46 8302.75 ± 1964.09 9329.17 ± 1662.87 7882.51 ± 2810.34
TNF-α 8.76 ± 0.98 9.48 ± 0.68 9.19 ± 1.16 7.40 ± 0.25
IL-10/TNF-α 115.00 ± 33.11 79.71 ± 21.80 170.87 ± 43.10 178.05 ± 29.25
IFN-γ/IL-4 110.35 ± 20.65 195.32 ± 61.39 124.85 ± 37.55 103.80 ± 26.93
21 Days
(pg/mL) Reference TGF-β2 EGF FGF21
IL-2 390.57 ± 141.49 Ψ 383.80 ± 89.21 Ψ 244.79 ± 80.37 Ψ 270.16 ± 114.54
IL-4 70.52 ± 10.51 52.21 ± 7.69 48.52 ± 1.91 Ψ 66.60 ± 8.80
IL-10 705.45 ± 63.95 514.70 ± 55.30 508.26 ± 61.43 Ψ 542.36 ± 78.82 Ψ
IL-13 19.51 ± 2.44 12.92 ± 0.74 * 9.91 ± 1.24 *,Ψ 9.83 ± 1.65 *,Ψ
IL-17A 59.97 ± 7.14 46.91 ± 4.03 52.30 ± 10.7022 41.24 ± 11.77
IFN-γ 4231.26 ± 365.41 Ψ 4488.50 ± 416.14 6900.74 ± 1129.78 4595.11 ± 1058.87
TNF-α 6.63 ± 0.11 Ψ 6.55 ± 0.20 Ψ 10.02 ± 0.40 * 5.91 ± 0.38 Ψ
IL-10/TNF-α 106.47 ± 9.73 80.13 ± 9.55 50.48 ± 5.72 *,Ψ 95.64 ± 17.03 Ψ
IFN-γ/IL-4 64.25 ± 9.61 111.99 ± 25.23 147.25 ± 27.9166 81.50 ± 25.37
Results are expressed as mean ± S.E.M (n = 9). * p < 0.05 vs. REF group at same age; Ψ p < 0.05 vs. day 14 at
same group.

4. Discussion
At birth, the immune system is immature, as evidenced by a poor antibody production and low
proliferative response of immune cells. In addition, there are mucosal immune impairments, such
as low intestinal IgA content, reduced number of B lymphocytes in the intestinal mucosa, and few
intestinal T cells [23]. Immune development is driven, among other factors, by components of breast
milk. It is known that growth factors, such as TGF-β2 and EGF, regulate the immune response in early
life and confer protective effects against gut mucosal inflammation by enhancing oral tolerance [1,2,7].
FGF21, which is also present in maternal milk, is a less-studied component and could be involved
in such effects as well [16]. The current study aimed to evaluate whether supplementation with
TGF-β2, EGF, or FGF21 had a role in immune maturation in early life. A rat pup model was used to
evaluate the effect of a daily supplementation with TGF-β2, EGF, or FGF21 during suckling on the
GALT, particularly in the MLNs, which is an inductor site of intestinal immune response. We assessed
the immune maturation by functions, such as lymphoproliferation, cytokine production ability, and
establishing lymphocyte MLNs composition.
Regarding growth, the body weight of pups was not modified due to supplementation with
any of the GFs studied. These results were in line with other investigations, in which rats receiving
either TGF-β2 [10] or EGF [24,25] during the first two weeks of life did not change their body weight.
Likewise, although a study showed that the body weight of FGF21-knockout mice compared to
wild-type 3-month-old mice was not different [26], its physiological role as a weight regulating factor
was discussed [18]. Overall, it seems that the tested GFs, under the conditions we used, did not have a
key role in the growth of neonates.
Intestinal length and weight are useful tools for evaluating the primary impact of a nutrient
on the maturation of the rat small intestine [27]. The GFs supplementation in the current study did
not affect these variables. However, it has been reported that suckling rats receiving intraperitoneal
administration of EGF (100 μg/kg) for only two days, increased stomach and intestinal weights [24];
and that rat pups receiving oral administration of EGF through formula at concentrations exceeding

36
Nutrients 2018, 10, 1171

the reported concentrations of EGF in rodent milk, enhanced intestinal growth (weight and length) [15].
Thus, in agreement with our results, only high doses of these compounds seem to be able to modify
intestinal growth.
Although no impact on the body and intestinal growth due to any of these GFs was observed,
some effects of TGF-β2, EGF, or FGF21 on immune variables have been shown to be specific; and
therefore, the influence of each GF tested on MLN lymphocyte maturation will be discussed separately.
Transforming growth factor-β (TGF-β) has a wide range of biological activities and among
them, it has an important role in cell proliferation and differentiation. TGF-β acts as a cytokine
having predominantly suppressive effects on the growth of T and B lymphocytes. In this study,
the supplementation of suckling rats with TGF-β2 did not have a significant influence on unspecific
MLNs proliferative cell response. This result contrasted with a report showing that suckling rats
receiving a whey-enriched TGF-β formula, down-regulated the MLNs lymphocyte proliferative
response to specific antigen after being sensitized [28]. On the other hand, we observed that MLNs
cells after TGF-β2 supplementation did not modify the changes in IL-2 and TNF-α secretion associated
with age, but attenuated IL-13 production at day 21 with respect to reference animals. The attenuation
on IL-13 production, a Th2 cytokine linked to allergic processes [29–31], agrees with results showing
that Brown Norway rat pups receiving a formula with TGF-β2 between 4 and 18 days of life shifted the
immune response from a Th2 type towards a Th1 profile [13]. Likewise, it is known that low levels of
IL-13 in colostrum and mature milk are associated with less eczema in early life [32]. Thus, our results
suggested that early TGF-β2 intake could play an important role in the prevention of Th2 mediated
alterations, such as allergy.
In the current study, the TGF-β2 supplementation was already able to modulate MLN lymphocyte
composition in suckling rats. It decreases the proportion of the immature phenotype of the intestinal
NK cells (NK CD8+ ) on day 14 at levels observed later (day 21); which indicated a positive action
on the intestinal immune maturation. This developmental pattern, decreasing CD8 expression in the
NK cell surface with age, has previously described in rat neonatal intraepithelial lymphocytes (IEL)
and MLN cells [19,27]. In line with this, we found that, although no changes in total CD8+ cells were
observed due to TGF-β2 supplementation, a lower CD8αα/CD8αβ ratio at day 21, compared to day
14 appeared. This ratio has been described to be reduced according to age in healthy MLNs cells
from suckling rats [19], thus our results may suggested a promotion of the intestinal immune system
maturation. This contrasts with results in BALB/c mice pups, showing higher CD8+ T cell proportions
when their lactating mothers were treated with mAbs, against TGF-β twice a week from delivery until
weaning [33]. On the other hand, we did not find changes in the MLNs Treg (CD4+ CD25+ Foxp3+ ) cell
proportion. However, some authors have suggested that oral tolerance induced by breastfeeding could
depend on TGF-β signaling from breast milk, which would be able to up-regulate Foxp3+ cells [33].
Regarding the intestinal humoral immune response, it is known that IgA is poorly produced
by the neonate mucosal immune system [1,28], and that TGF-β1 and TGF-β2 from mammalian milk
are able to stimulate the synthesis of mucosal IgA [28,33]. Thus, TGF-β acting in synergy with IL-10,
can promote IgA production and oral tolerance induction [28]. However, our results did not show
significant changes in the intestinal IgA and IgM content of the suckling rats. This lack of effect could
be due to the fact that breast milk contains IgA and IgM, which are transferred to the pups, and then
these milk antibodies in the intestine would mask the pups’ own levels. For this reason, intestinal IgA
and IgM assessment cannot be a good marker of nutritional supplementation at this level.
Epidermal growth factor (EGF) is a peptide that modulates a variety of biological responses, such
as cell proliferation and differentiation [14]. It is known that it has a clear effect on epithelium, where it
accelerates maturation and stimulates cell proliferation [34–36]. However, in the current study, we have
not detected any effect of the EGF supplementation on the proliferative response of MLNs cells in the
suckling pups. There is evidence that EGF prevents and reduces the incidence and severity of NEC
by modulating important transcription factors for cytokine regulation [37], and it is known that the
balance of pro-inflammatory and anti-inflammatory cytokines may play a key role in the development

37
Nutrients 2018, 10, 1171

of NEC [17]. This effect may be attributable to a down-regulation of pro-inflammatory IL-18 and to
an increase of anti-inflammatory IL-10 at the site of injury [17,37]. Here we found that, 21-day EGF
supplementation increased TNF-α and decreased IL-13 production by MLNs cells. The changes in these
two cytokines, contained in maternal milk [32,38], could be associated with positive effects. Indeed,
decreased IL-13 can be useful in the prevention of allergy, as stated for TGF-β2 [29–31]. Moreover,
EGF supplementation kept the levels of TNF-α on day 21 at the same level as that on day 14, which
could play in favor of its own effects because it is known that EGF receptor (EGF-R) is up-regulated by
TNF-α [39]. Regarding MLNs cell composition, EGF supplementation shares some maturate effects
with TGF-β2, such as the induction of lower levels of NK CD8+ cells and CD8αα/CD8αβ ratio.
Finally, focusing on fibroblast growth factor 21 (FGF21), recent studies showed that its function is
not limited to the regulation of metabolism, but that it is also involved in the protection of multiple
physiological processes, such as oxidation, inflammation, atherosclerosis, and aging processes [40].
A recent study demonstrated that, in the spleen of mice with collagen-induced arthritis (CIA), FGF21
reduces the expression of inflammatory cytokines, such as IL-17, TNF-α, IL-1β, IL-6, IL-8, and MMP3,
whereas IL-10 levels were increased, compared to PBS treated CIA mice [41]. In line with this, we have
not found any significant difference with respect to non-supplemented animals on MLNs inflammatory
cytokines, but the FGF21 supplementation decreased IL-13 at 21 days, as we also found with the other
GFs, suggesting, therefore, its role in preventing allergic events.
It is known that FGF21 is present in breast milk and does not contribute to systemic levels in
mouse neonates, but appears to act locally on the mouse neonatal intestine [16]; and it is unknown
whether this factor plays an important role in the maturation of the immune system. We found that
suckling rats receiving FGF21 promoted maturation of the early life intestinal immune system by
accelerating the decrease in the proportion of NK CD8+ cells and the decrease in the CD8αα/CD8αβ
cell ratio in MLNs, as well as TGF-β2 and EGF. This is the first time that the immunomodulatory effect
of FGF21 has been demonstrated in early life.

5. Conclusions
In conclusion, our study demonstrated that supplementation with TGF-β2, EGF, or FGF21 during
the suckling period had an immunoregulatory effect. Although some specific effects appeared, the
three growth factors were able to modulate similar aspects of MLN cells, such as promoting lymphocyte
maturation, as observed by increasing NK cells with a more mature phenotype (CD8− ) and reducing
IL-13 production, which could be useful in avoiding allergic processes. Further studies should be
carried out to establish the effect of the supplementation of TGF-β2, EGF, or FGF21 on the response of
suckling pups in other parts of the intestinal immune system, such as in the intestinal epithelium or
even at the systemic level.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/9/1171/

s1, Figure S1: Growth curve of all studied groups during the suckling period (from 1 to 21 days of life), Figure
S2: IgA and IgM concentration in gut wash from 21-day-old animals after the different nutritional interventions,
Figure S3: Proliferation of mesenteric lymph nodes lymphocytes at 14 and 21 days of life, Table S1: List of
monoclonal antibodies (mAbs) used for immunophenotyping the mesenteric lymph nodes (MLN) cells.
Author Contributions: M.J.R.-L., M.C., F.J.P.-C., and À.F. conceived and designed the experiments; P.T.-C. and
M.A.-G. performed the experiments; P.T.-C., F.J.P.-C., and À.F. analyzed the data and wrote the manuscript. F.J.P.-C.
and À.F. have primary responsibility for the final content. All the authors reviewed and approved the final version
of the manuscript.
Funding: This study was supported by a grant from the Spanish Ministry of Economy, Industry and
Competitiveness (AGL2013-48459-P). Paulina Torres-Castro holds a fellowship from the National Secretary
of Higher Education, Science, Technology and Innovation of Ecuador (SENESCYT-DMPF-2015-1666-CO).
Acknowledgments: The authors thank Lidia Marín-Morote for their help with the laboratory work. The authors
also thank J. Comas and his laboratory technicians from the Scientific and Technological Centres of the University
of Barcelona (CCiT-UB) for their assistance in the cytometry service.
Conflicts of Interest: The authors declare no conflict of interest.

38
Nutrients 2018, 10, 1171

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 nutrients
Article
Effect of Milk Fermented with
Lactobacillus fermentum on the Inflammatory
Response in Mice
Lourdes Santiago-López 1 , Adrián Hernández-Mendoza 1 , Verónica Mata-Haro 2 ,
Belinda Vallejo-Córdoba 1 , Abraham Wall-Medrano 3 , Humberto Astiazarán-García 4 ,
María del Carmen Estrada-Montoya 1 and Aarón F. González-Córdova 1, *
1 Laboratorio de Química y Biotecnología de Productos Lácteos, Centro de Investigación en Alimentación y
Desarrollo A. C. (CIAD), Carretera a La Victoria Km. 0.6, Hermosillo, Sonora 83304, Mexico;
[email protected] (L.S.-L.); [email protected] (A.H.-M.); [email protected] (B.V.-C.);
[email protected] (M.d.C.E.-M.)
2 Laboratorio de Microbiología e Inmunología, Centro de Investigación en Alimentación y Desarrollo
A. C. (CIAD), Carretera a La Victoria Km. 0.6, Hermosillo, Sonora 83304, Mexico; [email protected]
3 Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de
Ciudad Juárez, Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, Mexico;
[email protected]
4 Laboratorio de Patología Experimental, Centro de Investigación en Alimentación y Desarrollo A. C. (CIAD),
Carretera a la Victoria Km. 0.6, Hermosillo, Sonora 83304, Mexico; [email protected]
* Correspondence: [email protected]; Tel./Fax: +52-662-289-2400

Received: 12 July 2018; Accepted: 6 August 2018; Published: 8 August 2018

Abstract: Currently, the effect of fermented milk on the T-helper 17 response in inflammatory bowel
diseases (IBDs) is unknown. The aim of the present study was to evaluate the effect of milks
fermented with Lactobacillus fermentum on the Th1/Th17 response in a murine model of mild IBD.
Exopolysaccharide (EPS), lactic acid (LA), and total protein (TP) contents and bacterial concentration
were determined. Male C57Bl/6 mice intragastrically received either raw (FM) or pasteurized (PFM)
fermented milk before and during a dextran sulfate infusion protocol. Blood, spleen, and colon
samples were collected at Weeks 6 and 10. IL-6, IL-10, and TNFα were determined in serum, and IL-17,
IL-23, and IFNγ were determined in intestinal mucosa and serum. The FM groups did not differ in cell
concentration, LA, or TP content (p > 0.05); FM-J28 had the highest EPS content. Spleen weight and
colon length did not differ among the FM groups (p > 0.05). In the FM-J20 and PFM-J20 groups, IL-17
and IFNγ decreased, and the IL-10 concentration was enhanced (p < 0.05) at Week 6. IL-6, TNFα, IL-23,
and IFNγ did not differ in serum and mucosa (p > 0.05), and IL-17 was lowest in FM-J28 and FM-J20.
Therefore, FM appears to potentially play a role in decreasing the Th17 response. However, further
studies are needed to elucidate the FM-mediated anti-inflammatory mechanisms in IBD.

Keywords: fermented milk; Th1/Th17 response; inflammatory process

1. Introduction
Inflammatory bowel diseases (IBDs) are characterized by chronic and uncontrolled inflammation
in the intestinal mucosa. Different factors have been evidenced to affect the immune system at
the mucosal level [1]. For example, inflammatory mediators such as cytokines play an important
role in the adaptive immune response at the intestinal mucosal level. The modulation of biological
cellular functions may initiate downstream signaling pathways and mediate cellular proliferation and
differentiation [2]. In particular, Th1 and Th17 cells have been implicated in the development of IBD.
Th1 is characterized by the presence of interferon-γ (IFNγ) and Th17 by the presence of interleukin

Nutrients 2018, 10, 1039; doi:10.3390/nu10081039 42 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1039

(IL)-17, IL-21, and IL-22 [3]. Cytokines such as IL-6, TGF-β, and IL-23 promote the development of Th17
cells in IBD [4]. One study related Th1 and Th17 responses to the pathogenesis of IBD and suggested
that Th1 cells may enhance the production of Th17 cells. In this previous study, a higher concentration
of Th17 vs. Th1 cells was reported in a colitis model with CBirl TCR transgenic mice, which are
immunodominant susceptible to flagellin microbiota [5]. In contrast, another study examining the
cytokine profiles of Th1 and Th17 in a colitis model found that IL-4 and IL-10 levels were enhanced
while IL-17 levels were reduced [6].
In another study, the pathogenic action of IL-23 was demonstrated. In IL-23R-deficient mice,
reduced Reg3b protein expression in intestinal mucosa was shown to directly affect antimicrobial
activity. In addition, IL-23-dependent Reg3b triggers an influx of IL-22, regulating the number of
neutrophils in the lamina propia and restoring IL-22 secretion. This finding is important given the role
of IL-23 in neutralizing the Th17 response in IBD [7].
Moreover, the administration of probiotics was shown to possibly modulate the inflammatory
process through the Th1-Th2-Th17 response. Zheng et al. [8] reported that Bifidobacterium breve and
Lactobacillus rhamnosus reduce Th17 and increase the Th2 cell subset in human peripheral blood
mononuclear cells. In addition, the active components of probiotics were shown to be responsible
for enhancing the numbers of CD4 + FoxP3 + regulatory T cells in mesenteric lymph nodes and
for decreasing the cytokines tumor necrosis factor-α (TNFα), IFNγ, and IL-10 in Peyer’s patches
and the large intestine. In another study, probiotics were shown to play an important role in the
down-regulation of the nuclear factor kappa B (NFkB) pathway in RAW 264.7 cells to prevent TNFα
expression in a lipopolysaccharide-induced model [9].
Several additional studies have documented the regulation of the immune response in IBDs by
administering probiotics [10–14]. However, few studies have documented the effects of fermented
milk on the Th17 response, which regulates various inflammatory processes at the intestinal level.
In one case, Dahi-fermented milk containing a probiotic reduced myeloperoxidase (MPO) activity and
TNFα, IL-6, and IFNγ levels [14]. Meanwhile, milk with fermented Lactobacillus rhamnosus GG reduced
colonic inflammation and injury and stimulated the activation of epidermal growth factor receptor
(EGFR) and protein kinase B (Akt), which may be attributed to the release of p40 and p75 proteins
during fermentation [15].
These latter studies demonstrated the potential role of milk fermented with probiotics on the
inflammatory process. However, the effect of fermented milk on the Th1/Th17 response has not
been reported. The aim of the present study was to evaluate the effect of milk fermented with
Lactobacillus fermentum (J20 and J28) on the Th1/Th17 response in a murine model of inflammation.

2. Materials and Methods

2.1. Preparation of Fermented Milk

The Lactobacillus fermentum strains J20 and J28 were cultured in MRS broth (De Man, Rogosa
and Sharpe, Difco) for 12 h at 37 ◦ C. Posteriorly, to elaborate the fermented milk, the strains
were sub-cultured twice in commercial milk (1% v/v) and incubated for 24 h and 12 h at 37 ◦ C.
Then, commercial skimmed milk was inoculated (3% v/v) with the 12-h cultures and incubated for
48 h at 37 ◦ C. Finally, the fermented milks (FMs) were placed in a cold water bath or submitted to
heat treatment (75 ◦ C, 15 min) to obtain pasteurized fermented milk (PFM), which was subsequently
submersed in a cold water bath to inactivate bacteria. The samples were stored at 4 ◦ C. A control pH
treatment was prepared with acidified milk (AM) by adding 800 μL of lactic acid (~90%, Sigma-Aldrich,
Mexico City, Mexico) to skimmed milk to obtain a similar lactic acid concentration as FM.

2.2. Characterization of Fermented Milk

Bacterial cell concentration, lactic acid (LA) content, and total protein (TP) content were
determined in the FMs and PFMs. The cell concentrations were determined at 48 h of fermentation

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by counts on plates of MRS agar. The LA and TP contents were determined by AOAC techniques
2000. The titratable acidity was expressed as percent LA titrated in 10 mL of fermented milk, using
NaOH (0.1 N) and phenolphtalein as the indicator. TP was quantified by the Kjeldahl method using a
nitrogen-to-protein conversion factor of 6.25.

2.3. Determination of Exopolysaccharide

The exopolysaccharides (EPS) were precipitated from the supernatant of FM at 48 h of
fermentation. Samples were centrifuged (3600× g, 60 min, 10 ◦ C), and the supernatants were recovered.
Afterwards, trichloroacetic acid solution (20% v/v, Sigma-Aldrich) was added to the supernatants,
which were incubated for 2 h at 4 ◦ C. Precipitated proteins were removed by centrifugation (3600× g,
60 min, and 10 ◦ C). Next, the supernatants were treated with two volumes of cold ethanol, followed by
12 h of incubation at 4 ◦ C. The EPS were recovered by centrifugation and posteriorly suspended in
1 mL of milli-Q water. The total EPS content was estimated for each sample by the phenol-sulfuric
method using glucose as the standard [16].

2.4. Animal Study

Seventy mice C57Bl/6 (weight 30 g, six weeks old) were obtained from BIOINVERT (Mexico City,
Mexico). The mice were randomly allocated into seven groups (n = 10/group) using a simple
randomization procedure (computerized random numbers) based on their initial weight to obtain
statistically equal groups (ANOVA, p > 0.05). Intestinal chronic inflammation was induced by the
administration of dextran sulfate sodium (DSS). The mice were divided into the following treatment
groups: (1) negative control (water); (2) DSS group; (3) AM + DSS (AM); (4) FM-J20 + DSS (FM-J20);
(5) PFM-J20 + DSS (PFM-J20); (6) FM-J28 + DSS (FM-J28); and (7) PFM-J28 + DSS (PFM-J28). Half of
the animals from each group were tested in a model of mild inflammation (six weeks) to evaluate
the course of inflammation. The other mice were tested up to the end of the experimental period
to evaluate chronic/systemic inflammation. Mice were housed in a controlled environment (22 ◦ C,
12 h/12 h light/dark cycle) and fed a conventional diet and water ad libitum. This study, including the
corresponding animal experiment, was approved by the Bioethics Committee of the Research Center for
Food and Development (CIAD for its Spanish acronym), Hermosillo, Sonora, Mexico (CE/002/2015).
Mice were intragastrically fed 800 μL/day/mouse of either FM or water (control groups) for 10 weeks.
To induce chronic inflammation, mice were intragastrically fed with 3% (w/v) DSS (40 kDa; Sigma-Aldrich)
dissolved in sterile water. Mice were treated with four cycles of DSS. For each cycle, mice were fed with
200 μL/day/mouse for seven days; subsequently, between each cycle, mice drank water normally for
seven days [6] (Figure 1). Body weight and water and food consumption were recorded daily. Half of
the animals per group were euthanized at Week 6, while the rest of the mice underwent two additional
rounds of DSS administration and milk feeding and were euthanized at Week 10.
The body weight initial ranged from 27.2 ± 4.9 g to 30.0 ± 3.1 g. The body weight gain was
4.2 ± 9.2 g and was highest for water group and lowest for the LFJ28 group.

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Figure 1. Experimental design. FM + DSS, PFM + DSS, and AM experimental groups of mice
received daily treatments from Week 1 to Week 10. Mice received DSS in Week 2, 4, 6, and 9.
*DSS administration period

2.4.1. Serum, Organ, and Mucosa Collection

After mice were euthanized (n = 5), the spleen and colon were removed. Spleens were weighed,
and the colon lengths were measured. Blood samples were collected by cardiac puncture. The blood
samples were allowed to clot at room temperature for 30 min before centrifugation (3500 rpm, 5 min,
4 ◦ C), and the serum was collected and stored at −80 ◦ C until further analysis.
For the mucosal collection, segments of the distal colon were removed from mice, cut open
longitudinally, and mixed with 1 mL of fresh RPMI 1640 medium (Sigma-Aldrich) supplemented
with penicillin–streptomycin (1%). Then, the samples were incubated at 37 ◦ C for 24 h. The culture
supernatants were harvested by centrifugation (3600× g, 10 min, 10 ◦ C), and stored at −20 ◦ C until
being assayed [17].

2.4.2. Quantification of Cytokines

Murine IFNγ minimum detectable dose (MDD 1.8 pg/mL), IL-17 (MDD, 5 pg/mL), IL-22, and
IL-23 (MDD 2.28 pg/mL) were evaluated in samples of serum and mucosal tissues by ELISA (R&D
System). Serum samples were also used for quantification of IL-6 (MDD 1.4 pg/mL), IL-10 (MDD
6.8 pg/mL), TNFα (MDD 0.9 pg/mL), IFNγ (MDD 0.5 pg/mL), and IL-17 (0.8 pg/mL) (CBA kits, R &
D Systems, San Jose, CA, USA) by flow cytometry (BD FACSCantoTM II, San Jose, CA, USA).

2.4.3. Histological Analysis

A histological examination of distal colon samples was performed by triplicate observations of
three samples from the distal colon of each animal. Specifically, 0.5 cm of each sample were fixed in 10%
buffered formalin, dehydrated in ethanol, paraffin embedded, and continuously sliced. Then, samples
were deparaffinized, rehydrated, and stained with hematoxylin and eosin staining. The samples
were evaluated under a 10× light-fluorescent microscope (LEICA DM2000, Leica Mycrosystems Inc.,
Chicago, IL, USA) to observe morphological changes to colonic tissue and the imagens were processed
in the LEICA V2 program.

2.5. Statistical Analysis

EPS, LA, TP, cell concentration, spleen weight, and colon length were analyzed by one-way ANOVAs.
Differences among means per group were analyzed using Tukey–Kramer tests and were considered
significant when p < 0.05. The cytokine values were analyzed using the non-parametric Kruskal–Wallis test;
the data were presented as medians and considered significant when p < 0.05. The cytokine concentrations
were determined by ELISA using ELISAanalysis.com software; the concentrations from CBA kits were
analyzed using the BD FACSArrayTM bioanalyzer (Becton Dickinson, San Jose, CA, USA) Statistical
differences were analyzed in the Minitab V.16.1.1. package.

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3. Results and Discussion

Several studies have reported that an anti-inflammatory response may be attributed to the
presence of LA bacteria and their cell components [18]. Additionally, other components, such as EPS
released during the fermentation process [19] and LA present in the matrix of FM [20], may generate
an anti-inflammatory response. Therefore, cell concentration, LA, EPS, and TP content from both FMs
were analyzed and are described in Table 1. Notably, several bacteria are known to synthesize EPS; the
EPS are associated with the cell surface or are present as slime. Some studies have reported on the role
of EPS in protecting against desiccation and toxic compounds. In addition, an important characteristic
of probiotics is the presence of EPS, which allows adhesion to solid surfaces and biofilm formation [16].
Under fermentation conditions, it was previously suggested that EPS production was associated
with bacterial growth. In the present study, EPS production was determined at 48 h of fermentation,
which corresponded with the highest cell concentration (9 log CFU/mL) for both FMs. FM-J28 showed
the highest EPS concentration (p < 0.05). Some studies have described other conditions that may
influence EPS production, such as type of bacteria, carbon source, and temperature of incubation [21].
Moreover, it has since been suggested that EPS may enhance the anti-inflammatory effect [19].
We determined that Lactobacillus fermentum was able to produce EPS under these fermentation
conditions even though cell concentration, LA, and TP did not show significant differences (p > 0.05)
among the milks fermented with different strains or the milks fermented with different strains that
underwent pasteurization.

Table 1. Cell concentrations, total protein in fermented milk and metabolites derived from the
fermentation process.

Cell Concentration Exopolysaccharides

Fermented Milk Lactic Acid (%) Total Protein (%)
(Log CFU/mL) (mg/mL)
FM-J20 9.0 ± 0.01 a 0.72 ± 0.01 a 50.99 ± 0.10 a 2.80 ± 0.04 a
PFM-J20 ND 0.75 ± 0.03 a 61.00 ± 0.15 a 2.71 ± 0.09 a
FM-J28 9.04 ± 0.01 a 0.90 ± 0.01 a 79.59 ± 0.05 b 3.01 ± 0.05 a
PFM-J28 ND 0.87±0.01 a 77.30 ± 0.01 b 2.88 ± 0.07 a
ND = Not detected. The values are the means ± SD (n = 3). Different letters for a same parameter indicate statistical
differences (p < 0.05) among dietary groups. Statistical differences were established by one-way ANOVAs and
Tukey–Kramer Post Hoc tests. Raw (FM) and pasteurized (PFM) milks were fermented with Lactobacillus fermentum
strains J20 and J28.

3.1. Anti-Inflammatory Response

Previous studies have demonstrated that, after four or five cycles of DSS administration,
inflammation at the intestinal level may occur, affecting physiological parameters such as weight loss,
intestine length, and the weight of other organs [22]. In the present study, the spleen of the DSS group
had a significantly lower weight (p < 0.05) with respect to the water group at Week 6; however, it was not
different (p > 0.05) compared to the groups administered with either FM. Conversely, in the end assay,
the spleen weight did not significantly differ among groups (p > 0.05) (Figure 2). Furthermore, spleen
weight at Weeks 6 and 10 for each group presented statistical differences (p < 0.05); the highest weight
was found at Week 10 for the groups administered with DSS or FM + DSS.
The importance of the spleen as a secondary organ in the immune response may be highlighted.
It is one of the sites where the immune response may be regulated and also stores immunological
cells such as B and T cells, dendritic cells, and macrophages, which exert discrete functions [23].
Studies have demonstrated that, during inflammatory processes, spleen weight may increase because
of immune activation or infection, leading to the hyperactivity of the spleen [12]. In the present study,
the spleen weight increased in all treatments at Week 10, with exception of the water group; however,
these differences were not significant (p > 0.05), indicating that the effect of DSS and FM treatments
may be limited.

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Colon length did not differ significantly among all groups (p > 0.05) nor between Weeks 6 and 10,
which may indicate a low response to the inflammatory process during the study period.
The administration of DSS is known to reproducibly induce mild intestinal inflammation and
the development of ulcers in mice, increasing neutrophil counts in the intestine [24]. In this regard,
the capacity of Th17 cells to secrete IL-17 but not IFNγ or IL-4 has been described; these prior cells
play an important role in the mediation of the inflammation process and tissue destruction [25,26].
Therefore, it is important to know their functions and develop strategies that block their response at
local level, principally in IBDs [27].
The serum cytokine profiles at Week 6 showed a significant increase in IL-17 in the groups
subjected to DSS-induced inflammation with respect to the water group (p < 0.05) (Figure 3A).
Meanwhile, the concentration of IFNγ in the DSS group was enhanced compared to the water group at
Week 6, yet decreased by Week 10 (p < 0.05) (Figure 3B). The concentration of IFN-γ in serum samples
was enhanced at Week 6 for the DSS groups FM-J20 and PFM-J28. These values corresponded with an
enhancement in the Th1 response, as various reports have documented. The inflammation process,
as based on IL-17, was sufficient at Week 6; hence, by Week 10, the organisms were possibly able to
regulate the inflammation process. In our study, for example, the FM-J28 group at Week 6 showed
enhanced IL-17 but not IFN. This is a possible response mechanism to a low concentration of IFNγ.
Finally, IL-22 and IL-23 were not detected.

Figure 2. Effect of dextran sulfate sodium and fermented milk administration on spleen weight (A)
and colon length (B) at Weeks 6 (black) and 10 (grey). Bars with an uppercase letter indicate statistical
differences at Week 6 among treatments, and bars with a lowercase letter indicate statistical differences
at Week 10 according to the Tukey–Kramer test (p < 0.05). The values are the means ± SD (n = 5).

The groups administered with FM-J20 and PFM-J20 showed the lowest concentration of IL-17
(p < 0.05) at Weeks 6 and 10. FM-J20 and PFM-J20 differed in the presence of viable bacterial cells, yet
both FMs were able to decrease the concentration of IL-17, suggesting that different components from
FM are involved in this effect. However, no statistical difference was encountered at Week 10 with
respect to the DSS group (p > 0.05). Furthermore, IL-6 and TNFα did not show statistical differences
among treatments and the control (DSS and water) at Weeks 6 and 10 (p > 0.05) (Figure 3C,D).
However, the concentration of IL-10 cytokine did significantly differ (p < 0.05) (Figure 3E); the
groups administered with PFM-J20 and FM J28 presented the highest concentration at Weeks 6
and 10, respectively.
It was previously reported that the expression of the genes that encode for IL-6, IL-1β, IL-23A,
TGFβ, and STAT3 are involved in the differentiation of Th17 cells in a DSS-induced inflammation
model [28]. Furthermore, studies have demonstrated that Th17 cells require specific cytokines such as

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IL-23, which mediates the expansion of IL-17. On the other hand, low levels of IFNγ and IFNα may
enhance the gene expression of IL-17 and IL-17-producing cells generated by IL-23 stimulation [29].
The findings reported in the present work are in agreement with those of other studies.
For example, the administration of VSL#3 probiotics decreased TNFα and IL-6 and increased IL-10
serum levels in a DSS-induced colitis model of inflammation [10]. However, the concentration of
inflammatory cytokines in our study is minor compared with other studies, which may indicate that
the response following FM intake is lower in our inflammation model. Different compounds derived
from FM are responsible for the anti-inflammatory effect [30,31]. For instance, milk fermented with
Lactobacillus rhamnosus GG (LGG) was found to reduce colonic inflammation and injury and to also
stimulate the activation of EGFR and Akt pathways while suppressing cytokine-induced apoptosis.
This effect was attributed to the soluble proteins p40 and p75 present in milk fermented with LGG [15].
Furthermore, the immunoprotective effect of probiotic Dahi containing Lactobacillus acidophilus LaVK2
and Bifidobacterium bifidum BbVK3 on DSS-induced ulcerative colitis in mice was demonstrated to
significantly reduce TNFα, IL-6, and IFNγ cytokines in colonic tissue [14].

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Figure 3. Effects of DSS and fermented milk administration on: IL-17 (A); and IFNγ (B) in serum samples,
determined by ELISA; and on: IL-6 (C); TNFα (D); and IL-10 (E), determined by flow cytometry at
Weeks 6 and 10. Bars with an uppercase letter indicate statistical differences at Week 6 among treatments,
and bars with a lowercase letter indicate statistical differences at Week 10 (p < 0.05) according to the
Kruskal–Wallis test. The values correspond to medians ± interquartile ranges for n = 5.

Several reports have shown the different mechanisms of action involved in the anti-inflammatory
effect of probiotics, but few studies have demonstrated possible pathways at the cellular level.

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One possible mechanism of action may be related with cell viability or cell wall components, which
can be internalized into M cells, interact with dendritic cells, and then down-regulate the production
of the pro-inflammatory cytokines IL-1β, IL-6, IL-17, TNFα, and IFNγ [32,33]. Furthermore, bioactive
peptides have shown anti-inflammatory activity by down-regulating LPS-induced cytokine production
in monocytes cells via the NF-kB pathway [34].
Moreover, LA may induce different inflammatory responses in LPS-stimulated RAW 264.7 cells.
Some studies have reported its association with metabolic acidosis, which frequently complicates
sepsis and septic shock and may be deleterious for cellular function. In this study, the AM group
showed no statistical difference (p > 0.05) with respect to the control group and FM groups; however,
it is important to carry out further studies on LA and its role in inflammatory processes.
In the determination of cytokines in samples of intestinal mucosal (Figure 4), FM-J20, FM-J28,
and PFM-J28 showed statistical differences (p < 0.05) among treatments with respect to the controls
(DSS and water). However, IFNγ levels were not statistically different (p > 0.05), and IL-23 was not
detected at the mucosal level. These results correspond with those reported in other studies wherein
the presence of IFNγ inhibited IL-17 [5]. IL-23 is important for the expansion, stabilization, and
conditioning of the Th17 response; hence, the IL-23/17 axis may be considered as a hallmark and an
attractive probiotic therapeutic target in IBD [32]. The study by Jadhav et al. [14] showed the lowest
concentrations of TNFα, IL-6, and IFNγ in samples of colonic tissue from mice administered with Dahi
(fermented dairy) + DSS as well as a group administered probiotic + DSS.
This effect has also been observed in mice following the administration of milk fermented with
Bifidobacterium strains. In particular, these mice showed a lower concentration of TNFα in culture
supernatants, while IL-10 increased in the FM group and also reduced the histological score compared
to treatments with saline and unfermented milk [35]. A possible mechanism for the anti-inflammatory
effect may be related to the inhibition or modulation of the expression of several genes such as NF-kB,
RELB, and TNFα, which have been reported as active during inflammation processes [36].

Figure 4. The effect of DSS and fermented milk administration on IL-17 (A) and IFNγ (B) determined
by ELISA of colonic mucosa at Week 10. Different letters show statistical differences (p < 0.05) among
treatments according to the Kruskal–Wallis test. The values are medians ± interquartile ranges (n = 5).

The binding of IL-10 to its receptor triggers phosphorylation-dependent activation of the

transcription factor STAT3, which upregulates the gene expression of members of the SOCS family
and proinflammatory cytokines such as TNFα and macrophage inflammatory protein 2. However, this
process is possibly inhibited by the presence of bacteria or other components, such as EPS [37].

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Histological Analysis
The DSS inflammation process is characterized by histological findings such as edema, infiltration
of inflammatory cells into both the mucosa and submucosa, and destruction of epithelial cells [28].
Our results indicated that neither the administration of DSS or fermented milk affected the structure of
mucosa or submucosa (Figure 5). A low number of inflammatory cells infiltrated (Figure 5B) but did not
result in mucosal injury. Therefore, the histological analysis of samples confirmed that the inflammation
process was not extensive; this finding may suggest that the presence of pro-inflammatory cytokines
did not cause extensive changes. On the other hand, some studies have suggested that invasion of
inflammatory cells into the mucosa produces increased concentrations of inflammatory cytokines such
as TNFα, which then induce the expression of genes associated with inflammation [10]. In addition,
DSS may induce the expression of COX-2, an enzyme responsible for the formation of prostanoids [38]
that has been shown to be specifically induced in epithelial cells under IBD conditions [39].

Figure 5. Histological analysis of cross sections of colon tissue samples stained with hematoxylin–eosin
(10×): (A) water control group; (B) DSS group; (C) AM + DSS group; (D) FM-J20 + DSS group;
(E) PFM-J20 + DSS group; (F) FM-J28 + DSS group; and (G) PFM-J28 + DSS group.

These findings show that FM administration may prevent intestinal inflammation by stabilizing
mucosal immunity through different components or metabolites derived during the fermentation
process [13]. In particular, the present results show that the administration of FM could mediate the
Th1/Th17 response, but more studies are required to determine the possible metabolites involved
and the activation pathways. The interaction of derivative compounds from fermentation, such as
LA and EPS, as well as the presence of bacteria could result in an anti-inflammatory response that
stimulates IL-10 production and inhibits TNFα despite the interaction of TLR2 and TLR4 and activation
of NF-kB. For example, in one previous study, the presence of LA and Lactobacillus casei Shirota culture
supernatants suppressed phosphorylation and degradation of I-kB-α [40].

4. Conclusions
In the present study, several strategies to reduce inflammation in an IBD model employing
milk fermented with Lactobacillus strains were evaluated. The administration of milk fermented

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with Lactobacillus fermentum possibly decreased the inflammatory response at Week 6 because of
the metabolites or cell components present in this product. However, further studies are needed to
determine the modulation of Th1/Th17 by fermented milk. The findings in the present study show the
potential regulatory effect of FM on the inflammatory process, although this effect was minor, possibly
as a result of irregularity in the inflammatory process. Future studies are needed to establish an
adequate model of inflammation that allows the Th17 response to be evaluated considering the other
biomarkers involved in this response, including chemokines, transcription factors, and metabolites
released during fermentation, as well as cell differentiation, which may all be responsible for promoting
an anti-inflammatory response.

Author Contributions: A.F.G.-C., A.H.-M., V.M.-H., B.V.-C., A.W.-M., H.A.-G. and L.S.-L. designed the study.
V.M.-H. and L.S.-L. contributed to the flow cytometer analysis of the samples. L.S.-L. wrote the manuscript.
V.M.-H., A.H.-M. and A.F.G.-C. revised the manuscript. A.W.-M. contributed to the histological analysis. H.A.-G.,
B.V.-C. and M.d.C.E.-M. provided valuable scientific knowledge and advisory throughout the study.
Funding: This study was supported by the Mexican Council of Science and Technology (CONACyT; Mexico City)
research project 240338 CONACyT.
Acknowledgments: The authors would like to thank Alejandro Santos-Espinosa, Alejandro Epigmenio-Chavez,
Lilia María Beltrán-Barrientos, and Miguel Angel Rendón-Rosales for their technical assistance in this study.
The authors would also like to thank the Mexican Council of Science and Technology (CONACyT) for the graduate
scholarship provided to L. Santiago-López. Histological analyses were performed with equipment acquired with
funding from the Comprehensive Institutional Strengthening Program (PIFI) 2007-2008, 2909 5001-004-09
Conflicts of Interest: The authors declare that they have no conflicts of interest regarding the publication of
this paper.

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40. Watanabe, T.; Nishio, H.; Tanigawa, T.; Yamagami, H.; Okazaki, H.; Watanabe, K.; Tominaga, K.; Fujiwara, Y.;
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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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 nutrients
Review
Infant Complementary Feeding of Prebiotics for the
Microbiome and Immunity
Starin McKeen 1,2,3 , Wayne Young 1,2,3 , Jane Mullaney 1,2,3 , Karl Fraser 1,2,3 ,
Warren C. McNabb 2,3 and Nicole C. Roy 1,2,3, *
1 AgResearch, Food Nutrition & Health, Grasslands Research Centre, Private Bag 11008, Palmerston north
4442, New Zealand; [email protected] (S.M.); [email protected] (W.Y.);
[email protected] (J.M.); [email protected] (K.F.)
2 Riddet Institute, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand;
[email protected]
3 High-Value Nutrition National Science Challenge, Auckland, New Zealand
* Correspondence: [email protected]; Tel.: +64-6-3518-1101

Received: 7 January 2019; Accepted: 6 February 2019; Published: 9 February 2019

Abstract: Complementary feeding transitions infants from a milk-based diet to solid foods, providing
essential nutrients to the infant and the developing gut microbiome while influencing immune
development. Some of the earliest microbial colonisers readily ferment select oligosaccharides,
influencing the ongoing establishment of the microbiome. Non-digestible oligosaccharides
in prebiotic-supplemented formula and human milk oligosaccharides promote commensal
immune-modulating bacteria such as Bifidobacterium, which decrease in abundance during weaning.
Incorporating complex, bifidogenic, non-digestible carbohydrates during the transition to solid foods
may present an opportunity to feed commensal bacteria and promote balanced concentrations of
beneficial short chain fatty acid concentrations and vitamins that support gut barrier maturation and
immunity throughout the complementary feeding window.

Keywords: weaning; oligosaccharides; non-digestible carbohydrates; metabolites; gut barrier;

tolerance

1. Introduction
The strategic introduction of prebiotic compounds during weaning presents an opportunity to
promote infant health and to support development via balanced co-maturation of the gut microbiome
and host. Between 4 and 6 months of age, nutrient demands of growing infants surpass what is
provided by breastmilk or formula alone [1–4]. Complementary foods accompany and gradually
replace breastmilk and formula throughout the weaning period, providing essential nutrients to the
developing digestive system and modulating microbial colonisation [1,5–8]. The young immune
system is influenced by the gut microbiome and supported by metabolites produced during the
microbial fermentation of prebiotic compounds, leading to a tolerance for commensal microbes
and specific responses to pathogens [9–15]. Prebiotic compounds in breastmilk and supplemented
formulas promote commensal immune-modulating bacteria, such as Bifidobacterium, and beneficial
metabolites, such as short chain fatty acids (SCFAs) and vitamins [16–21]. Introducing non-digestible
starches through complementary foods may present an opportunity to promote commensal bacteria
and support microbial production of beneficial metabolites throughout the complementary feeding
window, with lasting effects on health [22–24].
Prior to weaning, the healthy infant gut microbiome is shaped by maternal factors, such as mode
of birth, environment, and first foods: breastmilk and infant formula [10,25–33]. The establishment of
microbial species changes dramatically throughout the first 2–3 years of life before stabilising at an

Nutrients 2019, 11, 364; doi:10.3390/nu11020364 55 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 364

adult-like composition [7]. While individual variations in taxonomic composition persist, analogous
genes consistently and predictably fill similar functional and metabolic niches as new foods are
introduced and formula or breastfeeding ceases [7]. Commensal species that colonise the immature
gut modulate gene expression of epithelial and immune cells and, in turn, are regulated by adaptive
and innate immune responses in the mucosal immune system [14,26,31,34–42].
Breastmilk and some types of prebiotic-supplemented formulas provide non-digestible
oligosaccharides (NDOs) to the gut microbiome, which exert a strong influence on the microbial
composition and metabolism [43]. The introduction of starchy foods such as cereals, porridges,
and pureed tubers is common practice due to the neutral tastes, smooth textures, and ease of
swallowing as oral coordination develops [44]. The role of these starches in the community dynamics
of the immature and unstable infant microbiome remains unknown.
Based on investigations into human milk oligosaccharides (HMOs) and NDOs, prebiotic whole
foods may support immunity and immune development through a variety of direct and indirect
mechanisms. While poorly characterised compared to oligosaccharides, starches may act as receptor
analogues to pathogens, reducing the quantity of enteric pathogens that reach the gut epithelium and
subsequent infection [45]. Starches also promote populations of bacteria of which some strains directly
interact with immunomodulatory factors in the gut mucosa [46]. These and other commensal bacteria
also ferment starches into metabolites such as SCFAs and vitamins, which have known benefits to gut
barrier integrity, immune-regulation, and immune response [47].
This review summarises the current body of knowledge on the complementary feeding of
prebiotic starches for the microbiome with a focus on the interactions of commensal species, microbial
metabolites, and the development of the gut barrier and immune system.

2. The Need to Complementary Feed

Complementary feeding is the necessary inclusion of solid foods alongside the milk-based diet of
infants during the transition to adult foods. The inclusion of solid foods is recommended to coincide
with sufficient oral maturation and an imbalance between the nutrient requirements of infants and
the nutritional provisions of breastmilk and formula, as demonstrated in Figure 1 [44]. Previously, it
was thought that the inclusion of solid foods in the diet was driven by an increase in the demand for
energy and protein between 4 and 6 months of age. However, Krebs and Hambidge (1986) found that
infants’ absorption of zinc from breastmilk is inadequate to meet factorial estimates of requirements
based on healthy growth curves [3]. Similarly, iron requirements increase with erythrocyte mass and
myoglobin in lean tissue from 4–12 months of age, surpassing the low concentrations (0.2–0.4 mg/L)
of highly bioavailable (50%) iron in breastmilk at approximately 6 months of age [48].

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Figure 1. The percent of nutrient requirements based on the recommended daily intakes (RDIs) [49]
that are met via average daily breastmilk consumption (750 mL from 0–6 months and 800 mL from
7–12 months) [50].

Timing of the introduction of solid foods has been investigated in both low- and high-income
countries. Delaying solids until 6 months of age was previously thought to be associated with
lower body mass index in high-income countries and with lower rates of allergy and decreased
water-borne diarrheal disease in low- and middle-income countries [51,52]. However, recent studies
in larger cohorts have challenged this assertion, proposing that individual oral maturation, nutrient
requirements, and environmental disease burden should determine when to introduce solids [4].
Results from the PIAMA (Prevention and Incidence of Asthma and Mite Allergy) cohort in the
Netherlands suggest that a short duration of breastfeeding (4 months or less) is associated with an
increased risk for being overweight during childhood rather than the early introduction of solid foods,
and the risk is not different between breastfed and formula-fed infants [53]. However, this study does
not report on the types of solid foods that were introduced, the duration of the overlap of breastfeeding
and solid feeding, or the potential mechanisms of metabolic programming.
In addition to nutritional provisions, breastmilk also provides non-nutritive and
immune-modulatory factors that impart significant benefits, even in partial concentrations or
shorter durations [12]. The health promoting properties of breastmilk include varying levels and
types of carbohydrates, non-digestible HMOs, immunoglobulins (IgG, IgM, and isoforms of sIgA),
amino acids, polyunsaturated fatty acids, monoglycerides, leuric acid, linoleic acid, cytokines,

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chemokines, soluble receptors, antibacterial proteins/peptides, and intact immune cells that are
governed by maternal Lewis blood type, secretor status, and phase of lactation [54]. HMOs have
received significant attention in infant nutrition for their ability to influence a variety of gut functions:
epithelial integrity, mucosal integrity, susceptibility to pathogenic infection, microbial community
structure, SCFA production, and vitamin synthesis. Over 2000 distinct HMO structures (Figure 2a)
have been identified, with significant variation between individuals and phase of lactation, but a 9:1
ratio of galactooligosaccharides (GOS):fructoligosaccharides (FOS) is typical [55,56]. Infant formulas
are continuing to develop based on an increasing understanding of the roles of each of these factors in
microbiome maturation, brain development, and immunity. Synthetic and plant-derived GOS, FOS
(Figure 2b), inulin, pectin, and β-glucans, either alone or in comparable ratios, are well characterised
and have been primary targets of infant formula additive research and product development [56,57].
Staged and follow-on formulas that vary in composition according to the recommended daily
allowances and the introduction of complementary foods are also increasingly recommended [2].

Figure 2. (a) The core structures of human milk oligosaccharides (HMOs), common modification
pathways, and an example of a complex HMO, connected by β1-3 and β 1-6 linkages that are resistant
to enzymatic cleavage by human-derived enzymes. (b) The structure of galactooligosaccharide (long
chain) and fructooligosaccharide (short chain), which are common prebiotic molecules in supplemented
infant formulas: β1-2, β1-4, and β1-6 linkages are resistant to enzymatic cleavage by human derived
enzymes. (c) A model of dietary starch, characterized by glucose molecules connected by α1-6 linkages
in a complex higher structure, which contributes to incomplete enzymatic cleavage by human enzymes.

Infant digestive systems are uniquely suited to digest macronutrients provided by breastmilk.
The intestinal epithelium of neonates has narrow villi and small crypts (Figure 3), which duplicate
and expand with age, a process which is influenced by components in breastmilk and host-microbe
interactions [58]. The expansion of the epithelial surface during weaning is necessary to accommodate

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the increasing nutrient load, but dysregulation of this process can lead to hyperplastic crypts, blunted
villi, inflammatory responses in the mucosa, and subsequent malabsorption of nutrients [59].
The enzymatic dynamics of infant digestion are poorly characterised due to wide variations
between individuals over time and infrequent investigations with replicated results [60]. Lactose, fatty
acids, and proteins are the most abundant macronutrients in milk, which are absorbed and utilised
predominantly in the small intestine [20]. Lipase and trypsin (lipid and protein digestive enzymes) are
present in concentrations comparable to adults and are sufficiently active at the less extreme pH (3.2) of
the infant gut. However, amylase secretion and activity are distinct in infants. Compared to lipid and
protein digestion, the ability to digest carbohydrates is limited to simple carbohydrates such as lactose
and sucrose, rather than complex carbohydrates, until weaning. At weaning, salivary α-amylase and
pancreatic α-amylase are present at reduced concentrations compared to that of adults [61]. However,
glucoamylase (also referred to as amyloglucosidase), a brush border enzyme in the small intestine
capable of cleaving α1,4-glycosidic bonds, is produced at 100–150% of adult concentrations at birth,
which may compensate for the otherwise minimal starch hydrolysis [62,63]. Non-digestible structures
such as HMOs and non-digestible carbohydrates (NDCs) resist complete enzymatic degradation
and pass to the large intestine where they become available as a nutrient source for the enteric
microbiota [64]. Breastfed infants also receive varying amounts of maternal amylase, as well as small
concentrations of up to 50 other digestive enzymes, through breastmilk [65]. During weaning, infants
that continue to consume breastmilk may have increased capacity to digest dietary starches compared
to those receiving formulas, but this and the subsequent interactions with enteric microbes has yet to
be investigated.

3. Gut Barrier Development

The epithelial barrier of the gut is formed by enterocytes, the primary absorptive cells with
crypts and villi, connected by tight junctions (TJ). The absorption of nutrients occurs transepithelially
(through) and paracellularly (between tight junction pores of ~4Å in healthy epithelia), requiring
the specificity and structural functionality of TJ proteins [66]. Compromised barrier integrity is
characteristic of inflammation and can lead to aberrant immune responses that have been implicated
in the development of allergies [67]. Apart from the histomorphological analysis of tissue biopsies,
epithelial integrity is measured in healthy infants by feeding non-digestible sugars, lactulose and
mannitol, and then measuring their ratios in urine over a multi-hour collection to indicate paracellular
sugar translocation into the bloodstream and subsequent excretion [68]. Faecal calprotectin has
also been used as an indicator of barrier integrity; however, this is unreliable and highly variable
among and within individuals and populations [68,69]. Faecal calprotectin levels are higher in infants
than in adults, possibly indicative of the immature gut undergoing cellular division, replication,
and differentiation, and are higher in breastfed infants than formula-fed infants [70]. Clinical
investigations with reliable measurements of barrier integrity in infants are rare, particularly those
that are sufficiently powered to understand the effects of foods and nutrients. To understand the
mechanisms by which nutrients, probiotics, and microbial metabolites may affect epithelial integrity,
in vitro experiments using single cell monolayers of Caco-2 cell lines are common and ex vivo tissue
microscopy from porcine and murine models provide further insights but are limited in their ability to
translate directly to humans.
HMOs and NDCs support barrier integrity by increasing TJs and promoting crypt and villus
differentiation (Figure 3). The effects of prebiotics on structural integrity are best understood for GOS,
which prevents loss of structure in Caco-2 monolayers when challenged by deoxynivanol (DON),
a mycotoxin that inhibits protein synthesis and increases paracellular permeability [71]. Additionally,
the stabilisation of claudin3, a TJ protein, and suppressed cytokine synthesis have been detected in
GOS-treated media [72]. Formulations with higher ratios of short chain molecules provide the most
protection to the epithelium, suggesting that complex resistant starch structures may not have direct,
non-microbial mediated benefits on the epithelium [73].

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Mucus Membrane
At the luminal surface of the enteric epithelium, the mucus layer provides a structural and
functional barrier that provides lubrication and separates the microbiota from epithelial cells while
allowing for the transport of nutrients and metabolites. Mucus is a complex heterogeneous suspension
matrix with high concentrations of high molecular weight glycoproteins called mucins, which are
secreted by goblet cells, and contains antimicrobial peptides, such as defensins [41]. Different types of
mucins have different roles in the lumen: secreted mucins form the mucus layer over the epithelium,
transmembrane mucins appear to be involved in signaling pathways, and some species of bacteria rely
on mucins as an energy source [40].
Several bacterial products, including lipopolysaccharides and flagellin on gram-negative bacteria
and lipoteichoic acids on gram positive bacteria, have been found to upregulate the mucin gene
expression and to stimulate mucin secretion [74]. Some probiotics, such as specific strains of
Bifidobacterium and Lactobacillus, successfully adhere to mucins and reach epithelial surfaces using
non-flagellar appendages called tight adherence pili, which influence immune responses [75,76].
This contributes to differences between the discarded microbiome identified in faecal collections and
the microbiome in the mucosa and at the epithelial surface [77,78]. Probiotic treatment, particularly
with Lactobacillus, has been shown to increase mucin and defensin secretion in murine models and
in vitro cell monolayers [79].
Prebiotics influence the composition of mucus by increasing the concentration of glycans [24],
decreasing the luminal pH, and increasing mucin glycosylation and sulphation [80], which protects
mucins from being degraded by host proteases and bacterial glycosidases (Figure 3) [81]. Mucus,
specifically secreted MUC-2, has also demonstrated immune-regulatory signals by interfering with the
expression of inflammatory cytokines but not tolerogenic cytokines by inhibiting gene transcription
through nuclear factor NF-κB (the nuclear factor kappa-light-chain-enhancer of activated B cells)
in dendritic cells (DCs) [82]. The mucus layer plays a significant role in microbial signaling,
cross-feeding, microbe-host interactions, and enteric immunity but can only be partially simulated in
in vitro experiments.

4. Establishment of the Microbiome and Immune System in the First Year of Life
Microbial community composition during the first year of life is dynamic, unstable, and susceptible
to perturbations [6,83]. The gut is the largest immune organ in the human body, containing
approximately 65% of immunologic tissues and up to 80% of the immunoglobulin-producing tissues
of the body [84]. During gestation, the foetal immune system is downregulated, making neonates
particularly susceptible to infection and aberrant immune responses. The epithelial barrier, mucosa,
and environmental conditions, such as pH, provide the majority of protection against pathogens in
the neonatal period (Figure 3) [85,86]. Healthy immune development in infants is characterised by
a transition from innate type 1 immunity, dominated by non-specific macrophages and neutrophils,
to adaptive type 2 immunity, characterised by specific T cells and B cells, which is fundamental
to the establishment of tolerance: the ability to distinguish between beneficial commensal bacteria
and harmful pathogens, leading to the appropriate scale and duration of responses to actual threats
(Figure 3) [87]. Spatial and temporal interactions between the microbiome, microbial metabolites,
and gut epithelial cells in the lumen, on the surface of epithelial cells, and in the interior components
of the gut-associated lymphoid tissue (GALT), such as DCs, modulate balanced immune development,
immune response, homeostasis, and disease (Figure 3) [88].

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Figure 3. A schematic of multiple mechanisms by which prebiotics modulate immune and gut
development. A. Prebiotics bind to pathogens as receptor analogues, preventing adhesion to the
epithelial surface and subsequent infection. B. Prebiotics promote populations of commensal microbes,
which outcompete pathogens for resources D, reducing infections. C. Prebiotics act directly upon the
epithelium promoting the mRNA transcription of proteins involved in barrier integrity. E. Commensal
microbes produce metabolites, such as short chain fatty acids (SCFAs), that decrease the lumen pH
and increase mucus F, increase TJ proteins and crypt and villi development G, and serve as an
energy source for enterocytes that form the epithelium H. In infants, the immature gut is susceptible
to allergy and pathogen translocation I through leaky gut barrier. J. Non-specific immune factors,
such as macrophages and neutrophils attack commensals and pathogens alike in poorly regulated
inflammatory responses. During immune development, dendritic cells K sample commensal microbes,
through Toll-Like Receptor (TLR) recognition, allowing for antigen specific immunoglobin production
L and promoting the differentiation of T and B cells M, resulting in improved tolerance to commensals
and targeted response to pathogens N.

4.1. Immune Ontogeny

Innate immunity favours Th2 responses and macrophage and neutrophil inflammatory activity,
which use specific classes of Toll-Like Receptors (TLRs), such as TLR4, which are capable of recognising
structurally conserved molecules on microbes [89]. As the immune system develops, additional
mechanisms of microbe recognition with increased specificity and response cascades develop: C-type
lectin receptors, pattern recognition receptors, TLR2, and TLR9 are expressed by immune cells,
such as DCs, in the mucosa and epithelium [90]. These immune cells can be both upregulated and
downregulated by exogenous factors, such as microbial metabolites of starch fermentation, and they
demonstrate cross-regulatory activity amongst themselves by way of immune factors and regulatory
cytokines [90].
Due to the poor specificity of the young immune system, commensal bacteria are rapidly killed
by macrophages. However, DCs can retain small numbers of live commensals for several days,
protecting them from innate immune responses while selectively inducing a protective IgA response
that protects against mucosal penetration by commensals [91]. Mesenteric lymph nodes restrict these
commensal-loaded DCs to the mucosal immune compartment, which allows for localised immune
responses while preventing more damaging systemic responses [91]. DCs express TLRs, which

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have been implicated in gut homeostasis and inflammatory responses characteristic of food allergies,
intestinal inflammation, and infections when poorly regulated [92]. Insufficient TLR exposure to
commensals, as found through antibiotic-mediated dysbiosis in murine models, is also correlated with
increased susceptibility to viral infections [93]. Infant TLR responses to commensal microbes differ
from responses in adults, demonstrating the impaired production of inflammatory mediators and
heightened production of inflammatory cytokines, such as IL-10 [85,87].
TLRs are susceptible to modulation by dietary starches in in vitro models. Different starch
structures bind differentially to TLRs, activating NF-κB, and activator proteins (AP-1), but the strong
immune-stimulating effects may also be attenuated by starch-exposed intestinal epithelial cells [94].
B2→1 fructans and High Maize 260 mainly stimulate TLR2, whereas Novelose 330 binds to TLR2
and TLR5 [95]. High Maize 260, which has a smaller average particle size of 12.8 μm, smooth surface,
and high degree of molecular order was found to have a stronger regulatory effect on epithelial cells
than Novelose 330, which has a larger average particle size of 46.6 μm and consists of destroyed and
convoluted granules due to the retrogradation process. Despite the attenuating activity, TLRs continue
to produce Th1 cytokines [94]. High-maize 260 is also more effective than inulin and sugar pectin in
reducing chemokine release in response to Sphingomonas paucimobilis infections in vitro [96]. In vivo,
the mucosal matrix is expected to drastically alter the exposure of epithelial cells to starch structures,
limiting the applicability of these findings to in vivo mechanisms.

4.2. Microbiome Assembly

Pioneer species in the infant gut shape the early environment, which influences the dynamic
succession of subsequent microbes and immune cascades. Nutrients, digestive processes, gases,
and pH gradients throughout the gut modulate the microbial community, which in turn also influences
the characteristics of some of these attributes. Microbiota resembling maternal oral microbiota may
begin to colonise the infant gut in utero, for example low abundance commensal bacteria such as
Prevotella, Neisseria, and Escherichia Coli, which have been found through the sequencing of amniotic
fluids and placentas of preterm infants [97]. However, the mode of delivery is considered the first
major event confirmed to seed the infant microbiome with lasting colonisers [7].
Vaginally delivered infants are predominantly colonised by Bacteroides, Bifidobacterium,
Parabacteroides, and Escherichia/Shigella, several of which are obligate anaerobes. Infants delivered
by caesarean section are enriched with Enterobacter, Haemophilus, Staphylococcus, Streptococcus,
and Veilonella, which are associated with skin, oral, and environmental species [7], a larger proportion
of which are aerobic. The differences in microbial community structure and gene content (i.e.,
the metagenome) between caesarean- and vaginally-delivered infants gradually decrease over the first
year of life, but the differences in innate and adaptive immunity remain detectable for up to 2 years of
age. Caesarean-delivered infants have lower levels of IgA-, IgG-, and IgM-secreting cells, indicating
reduced adaptive immune responses, have lower levels of Th1 supporting chemokines, IFNy and
IL-8, and have decreased CD4+ T cell responses [12]. Caesarean-delivered infants, in particular those
who were born by elective caesarean delivery instead of emergency delivery, are at higher risk for
asthma, atopy, juvenile arthritis, and inflammatory bowel disease [98–100]. This effect is particularly
pronounced for developing obesity where any caesarean delivery has been associated with a 15%
increased risk for obesity, but there is a 30% increased risk in elective caesarean-delivered infants [101].
The risk for development of infectious diseases is not clear. Considering these differences, it is critical
that microbiome studies in infants consider the mode of delivery, and this will be strengthened by
accounting for differences between emergency and elective caesarean-delivered infants.
During the first several weeks of life, pioneer facultative anaerobic species, which have metabolic
flexibility in the presence of oxygen, shift the environment to favour obligate anaerobic species by
utilising oxygen to create a more anaerobic environment [102] and by reducing luminal substrates
through redox (oxygen)-dependent genetic pathways that produce metabolites, such as acetate,
which is often required or highly stimulatory for anaerobes [103]. The meconium of neonates is

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rich in facultative anaerobes such as E. Coli, but the faecal microbiota becomes more diverse with the
appearance of obligate anaerobes such as Bifidobacterium and Clostridium within the first week [104].
In a cohort of 19 healthy breastfed full-term Japanese infants, the averaged percentage of obligate
anaerobic bacteria in the gut progressed from 32% (1 day), 37% (7 days), 54% (1 month), 70% (3 months),
64% (6 months), to 99% at 3 years of age. Significant individual variations within this homogenous
cohort diminished by 3 years of age [105,106]. This study did not specify the delivery modes of
this cohort, and the consequent possibility of significant differences in the colonisation patterns of
facultative and obligate anaerobes.
The effects of breastmilk and formula feeding on the infant microbiome and immunity are a
popular topic of research. Breastfeeding has been associated with a decreased risk of necrotising
enterocolitis, infections, and diarrhoea in early life and with a lower incidence of inflammatory bowel
disease, type 2 diabetes, and obesity later in life compared to formula-fed infants [107]. Another
meta-analysis found no association between breastmilk consumption and allergy, asthma, high blood
pressure, or high cholesterol [108]. Considering the complexity of the immune-modulating factors
of breastmilk, the identifying characteristics of the microbiome that contribute to these benefits is
challenging. Bifidobacterium has consistently been found to exist in higher abundances in exclusively
breastfed infants, whereas Lactobacillus has been reported to be higher in formula-fed infants in some
studies [102,109], while at other times reported to be higher in breastfed infants [110]. Backhed et al.
associated exclusive breastfeeding with lower phylogenetic diversity dominated by Bifidobacterium
and Lactobacillus and lower relative abundances of Clostridiales and Bacteroides compared to mixed-fed
infants [7]. Some of these differences may persist throughout the weaning phase as breastmilk and
formula feeding continue with supplementation of solid foods.
In an effort to impart similar bifidogenic effects on formula-fed infants, the supplementation
of infant formula with prebiotics, or prebiotics and probiotics, has become common. A 9:1 ratio
of synthetic linear polymers of GOS:FOS is standard, but these prebiotics represent a simplistic
uniform version of the HMO structures found in breastmilk [20]. Abrahamse-Berkeveld et al. (2016)
found that a combination of short chain GOS (scGOS dp of 3–15), long chain FOS (lcFOS dp of 3–6),
and Bifidobacterium breve increased the abundance of Bifidobacterium from 48% to 60% of the total
bacterial species and reduced the percentage of Clostridium lituseburense/C. histolyticum from 2.6% to
2.0%. [46]. In an in vitro study, Leder et al. (1999) found that many different strains of Bifidobacterium
are capable of utilizing scGOS, but of the species analysed, only B. adolescentis can utilise lcFOS,
providing evidence of the selectivity between related commensal strains and prebiotic structures [111].
These investigations into the utilisation of HMOs and prebiotics in formula offer a starting point for
exploring the effects of prebiotics provided by whole complementary foods.
Oligosaccharides also provide additional protection against pathogenic infection by acting as
structural mimics of the pathogen binding sites that coat the surface of intestinal epithelial cells.
Pathogenic bacteria such as E. Coli bind to oligosaccharides in the lumen, reducing the pathogen load
available for adhesion to intestinal epithelial cells. In Caco-2 and human epithelial type 2 (Hep-2) cell
lines, purified GOS reduced adhesion by 70% and 65% respectively. This effect was dose-dependent
and reached a maximum at 16 mg/mL [45]. It is unclear if complex starches, such as resistant starch,
have the same effect.

4.3. Functional Transitions during Complementary Feeding

Investigations into the functional differences between modes of feeding at the metagenomic and
transcriptomic level are less common. Backhed et al. found differences in the relative abundance
of functional genes in the faecal microbiome of breastfed and formula-fed infant that accounted for
approximately 1.30% of the variation in KEGG Orthologs, which is substantial considering the expected
constitutive expression of most genes [7]. This study did not specify the types of formulas used in
this comparison, and the expression of genes during this dynamic age may be more facultative than
constitutive due to the inherent instability of the immature infant microbiome.

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The community structure and metabolic functions of the infant gut microbiota are strongly
influenced by dietary prebiotics. The bifidogenic nature of breastmilk is well-established and has
been attributed to HMOs [112]. HMO consumption has only been identified in select Bacteroides
(Bifidobacterium) and Lactobacillus species, and different species and subspecies have been found to
utilise different protein-substrate binding and enzymatic mechanisms to metabolise HMOs [113,114].
B. longum subsp. infantis, which is enriched in breastfed infants, express an overabundance
of proteins that transport HMO substrates into the cell, where they are broken up into their
constituent sugars before being metabolised. This limits the sugars that are available to other species
within the microbiota [115]. B. bifidum, however, relies on a set of diverse membrane-associated
extracellular glycosyl hydrolases, lacto-N-biosidase and endo-N-acetylgalactosaminidase [116], which
have comparable enzymatic affinities for HMOs but may release monosaccharides such as lactose,
fucose, and sialic acid into the lumen, which become available to other microbes [117].
Glycosylation patterns on HMOs influence the enzymatic activity that microbes employ. B. breve
has been found to have a preference for sialylated HMOs over neutral HMOs, engaging enzymes that
convert HMOs into multiple intracellular products, but it does not internalise the whole molecule [118].
B. longum has numerous genes for carbohydrate utilisation, including 30 glycosyl hydrolases that
are likely involved in HMO degradation, though adult strains have indicated a preference for plant
polysaccharides [119]. The transcriptomic analysis of B. longum SC596 when shifting from a neutral
HMO substrate to a fucosylated HMO substrate found the gene expression was altered to resemble
the intracellular import strategy of B. infantis, which may provide an example of the facultative gene
expression of infant microbiota in response to dietary factors [20]. A meta-transcriptomic analysis of
faecal samples from a single breastfed baby followed from birth to six months of age, during which
formula, dairy, and solid foods were introduced, found that the carbohydrate fermentation activity of
Bifidobacterium, based on β-galactosidase activity, decreases during weaning while that of the resident
Firmicutes increases, which corresponds with changes in relative abundance of major and minor
species [120].
At approximately 3 months of age, genes implicated in complex carbohydrate utilization are
enriched compared to meconium samples, which favour lactose/galactose and sucrose uptake and
utilisation based on a metagenomic analysis [6]. Just prior to introducing solid foods between 4
and 6 months of age, the gut microbiome derives energy through the degradation of simple sugars,
lactose-specific transport, and carbohydrate uptake, as is expected for a milk-based diet. However,
functional genes involved in plant-polysaccharide metabolism are present prior to the introduction
of complementary weaning foods [6]. By 12 months of age, the infant microbiome is highly enriched
with species and genes active in the degradation of complex sugars and starches [7]. For instance,
Bacteroides thetaiotaomicron, an anaerobic glycan degrading enzyme producer of the Bacteroidetes
phylum, can typically be detected at 12 months of age [6]. An additional study showed that the
increased abundance of Bifidobacterium and decreased abundance of Bacteroides and Clostridium in
breastfed infants compared to formula-fed and mixed-fed infants persists into the weaning phase [121].
Thompson et al. identified differences before and after the introduction of solid foods between
the microbiomes of exclusively breastfed and non-exclusively breastfed infants [122]. Veillonella,
Roseburia, and members of the Lachnospiraceae family appeared with the introduction of solids in
breastfed infants, whereas Streptococcus and Coprobacillus were identified after the introduction of
solids in non-exclusively breastfed infants [122]. Most notable of these findings was the increased
relative abundance of Bifidobacterium after the inclusion of solids in non-exclusively breastfed infants,
compared to a decreased relative abundance of Bifidobacterium after the inclusion of solids in exclusively
breastfed infants, which may reflect differential effects of dietary oligosaccharides and starches during
complementary feeding. Metabolic inferences using a PiCRUST analysis of this limited 16S dataset
showed that only 24 gene clusters encoding enzymes were overrepresented in exclusively breastfed
infants after the introduction of solid foods, including polysaccharide degradation, compared to 230
enzymatic gene clusters overrepresented in the non-exclusively breastfed microbiome, which were

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primarily involved in signal transduction regulatory systems [122]. This finding indicates differences
in metabolic plasticity between exclusively breastfed and non-exclusively breastfed infants, though
it is possible that the substantial immune factors in breastmilk have a stronger effect on which gene
clusters are overrepresented in this small cohort.
Human faecal microbiota may develop the capacity to degrade a specific type of starch (Type III
Resistant Starch) at weaning, as demonstrated in an in vitro fermentation study using faecal inoculum
collected from breastfed and formula–fed infants before and during weaning [123]. However, species
with the potential capacity to degrade starch have been found to be present at birth [6]. From a
metagenome perspective, microbial networks of infants at 4 months are drastically different to those at
12 months, but polysaccharide degradation has been found to be more pronounced after the cessation
of breastfeeding, rather than during the introduction of solid foods in breastfed infants [7]. It is
possible that the cessation of HMO substrates decreases the need for the expression of HMO-degrading
genes and reduces the competitive advantage of species selective for HMOs, allowing species with a
preference for polysaccharide substrates to assume a greater ecological niche. However, neither the
in vitro experiment nor the metagenomic analysis consider the nutrient availability and degradation
that occurs in the proximal regions of the large intestine prior to analysis of the faecal microbiome.
Starch degradation in the large intestine is a cooperative process that includes enzymatic starch
degradation into glucose, glycolysis leading to SCFAs and organic acids, and hydrogen production.
Starch binding capacity and enzyme specificity underpin the ability of amylolytic microbes to
metabolise starch structures [124]. The presence and function of cellulosomes, amylosomes, and starch
utilisation system gene clusters have been investigated in keystone species belonging to the Firmicutes
and Bacteroidetes families. Three broad classes of amylases have been identified in amylolytic bacteria
that hydrolyse starch into D-glucose: α-amylase for α-1,4 linkages, type 1 pullalanase for α-1,6
linkages, and amylopullalanases for α-1,4 and α-1,6 linkages [125]. Stable Isotope Probing (RNA-SIP),
which allows for the tracking of 13 C-isotope labelled carbon utilisation through metabolite production,
has identified complex trophic structures that implicate primary starch degraders, such as Ruminococcus
bromii, in downstream carbon utilization by microbes found in the infant gut such as Prevotella,
Bifidobacterium, and Eubacterium rectale [126]. The association of amylolytic enzymes with the cell wall
and the ability to stabilize large molecules for cleavage may indicate the function of a given microbe
within the trophic network [127,128]. For instance, extracellular protein complexes on Bacteroides
thetaiotamicron imports starch molecules for internal degradation, limiting the extracellular release of
mono and di-saccharides, compared to outer membrane protein complexes on Clostridium butyricum
which degrade starches outside of the cell before importing the mono- and disaccharides for subsequent
metabolism into SCFAs [47,129,130]. This variety of enzyme structures and systems points to the
metabolic flexibility, which may be increased during dietary transitions such as weaning, that the
microbiome utilises to maximise energy harvest.
Fermentation profiles vary by substrate structure, which changes throughout enzymatic
degradation. Short oligosaccharide chains, such as scFOS, are more rapidly fermented than long
oligosaccharide chains, such as inulin [131]. The rate of fermentation as measured by the SCFA
production was highest during the first 4 hours in a faecal inoculum provided with scFOS substrate,
whereas long chain inulin produced the most SCFA between 12–24 h [131]. Warren et al. (2018)
expanded upon these findings by comparing digested to non-digested starches from a range of
processed, un-processed, digested, and un-digested starch and resistant starch substrates. This study
found that microbiota are able to ferment amorphous and crystalline starches equally well, perhaps
attributable to the range of amylolytic enzymes found in the microbiome, and found no difference
in the fermentation rates of the digested versus undigested substrates [132]. Both the 16S rRNA
gene amplicon sequencing analysis of the inoculum and the SCFA analysis revealed differentiations
according to time-points depending upon the classification of the starch substrate [132].

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4.4. SCFAs
SCFAs are the primary class of microbial metabolites of starch degradation and are implicated in
immune regulation. SCFAs function as an energy source for the host epithelium and other microbes,
affect lipid metabolism, protect against infection, have anti-inflammatory properties, influence the
gut-brain axis, facilitate immune cell metabolic reprogramming, and regulate immune cell transcription
through epigenetic modifications [133]. SCFA production varies throughout the colon because of
substrate availability, population dynamics, and microbial cross-feeding [134]. The fermentation of
starch substrates by the gut microbial community is characterised by high acetate production, followed
by propionate and relatively less butyrate, though ratios are highly variable [132,135]. RNA-SIP
studies show that lactate is a precursor to both acetate and propionate and that acetate is precursor for
butyrate via both the Co-A transferase pathway and the butyrate kinase pathway [136]. For example,
Bifidobacterium adolescentis can degrade resistant starch leading to the byproducts lactate and acetate.
Actetate is, in turn, used by Eubacterium spp., Roseburia spp., and Coprococcus catus, resulting in the
production of butyrate. Faecalibacterium prausnitzii, an abundant butyrate producer in adults, has not
been detected in infants younger than approximately 2 years of age [137]. Figure 4 demonstrates a
simplified ecological network in which multiple species of bacteria commonly identified in infants
perform parts of the metabolic pathways leading to biosynthesis of SCFAs.


Figure 4. A simplified schematic of the biosynthesis of SCFAs by microbial species identified in human
infants. Organic acid metabolites are outlined, and SCFAs are highlighted in black boxes. Species
of bacteria found in the infant gut microbiome that are implicated in the corresponding pathway
are italicised.

SCFAs begin shaping the enteric environment with the introduction of breastmilk and formula.
Exclusive breastfeeding is associated with lower absolute concentrations of all SCFAs, except
lactate [105]. Ratios of SCFAs within total concentrations have been found to be variable: exclusively
breastfed infants are more likely to have higher proportions of acetate, while partially breastfed and
formula-fed infants are more likely to have relatively higher proportions of propionate, and exclusively
formula-fed infants are likely to have relatively higher proportions of butyrate [138]. However,
measuring SCFAs in faecal samples only provides an indicator of the balance between SCFA production
and absorption. Absorption is likely to vary with epithelial barrier integrity and maturity, which is
known to be influenced by other factors in breastmilk [58,139].
SCFAs modulate immune factors through multiple mechanisms. They increase the expression
of antimicrobial peptides excreted by epithelial cells; modulate the production of cytokines and

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chemokines; regulate the differentiation, recruitment, and activation of immune cells; and modulate
the differentiation of T lymphocytes [21]. Commonly cited anti-inflammatory properties of SCFAs
can be attributed to their ability to reduce the production and activity of pro-inflammatory cytokines
such as TNF-α and IL-12, often by modulating activity of neutrophils, DCs, and macrophages [140].
Alternatively, SCFAs increase the production of other cytokines, such as IL-18, which has been
implicated in the repair and maintenance of epithelial integrity [141].
Acetate is a minor energy source for gut epithelial cells, a major energy source for muscles
and brain tissue, has anti-inflammatory properties, decreases the pH of the colon, and is used
by cross-feeding species as a co-substrate to produce butyrate [139,142]. Numerous species of
Bifidobacterium readily produce acetate from starchy substrates. Anti-inflammatory properties of acetate
have been linked to the SCFA-dependent modulation of NF-κB in the COLO320DM adenocarcinoma
cell lines, to decreased IL-6 protein release from organ culture, and to decreased LPS-stimulated
TNFα from neutrophils. However, these dose-dependent effects are less pronounced for acetate than
propionate and butyrate [143]. Acetate has also been identified as an important metabolite by which
some subspecies of Bifidobacterium protect against infection, possibly by inhibiting the translocation of
toxins from the gut lumen to the bloodstream [144]. Several in vitro studies suggest that the benefits of
acetate are largely due to the enhanced epithelial integrity, which imparts protection from infection and
inflammation. For instance, B. longum infantis 157F, which is found in breastfed infants and metabolises
glucose to acetate, was found to protect against harmful protein translocation across a Caco-2 epithelial
barrier in an in vitro cell-culture experiment [144].
Propionate has been associated with health benefits most particularly in adults [145]. Similar to
acetate, propionate is a minor energy source for gut epithelial cells, decreases the pH of the colon,
is anti-inflammatory, and has immune modulating properties that in vitro studies of TER in Caco-2
cell lines suggest are linked to beneficial effects on epithelial barrier integrity [146]. Additionally,
propionate decreases liver lipogenesis, serum cholesterol levels, and colorectal carcinogenesis in other
tissues. Insulin sensitivity improvements and increased satiety in adults has also been correlated with
increased propionate levels [139,142,145]. These effects have not been investigated in weaning infants.
Butyrate is the preferred energy source for gut epithelial cells, meaning that little butyrate
reaches systemic circulation. Butyrate also decreases the pH of the colon, promotes epithelial
proliferation, prevents colorectal cancer cell proliferation, reduces oxidative stress, is anti-inflammatory,
and improves gut barrier function by stimulating the production of mucins, antimicrobial peptides,
and TJ proteins [139,142]. Gantois et al. found that butyrate also downregulates the expression
of virulence genes in Salmonella enterica and typhimurium [147]. Butyrate producing bacteria, such
as Eubacterium rectale, Roseburia spp, and Faecalibacterium prausnitzii frequently utilise acetate as a
substrate [148]. The effects of butyrate have been found to be paradoxical where low concentrations
of butyrate (2 mM) promote gut barrier function, characterised by increased TER and decreased
mannitol flux, but high doses (8 mM) may induce cell apoptosis and disrupt the intestinal barrier,
as is characteristic of necrotising enteric colitis [149]. One study identified the benefits of butyrate
to be characteristic of cellular differentiation because of the increased dome formation and alkaline
phosphatase activity [146], whereas another identified cell migration, as is needed for epithelial repair,
as a beneficial mechanism [149]. Both studies found that the effects were dependent on protein
synthesis and gene transcription but not the beta-oxidation or activation of adenosine 3’, 5’-cyclic
monophosphate [146,149].
Most investigations into SCFAs have focused on adult populations. In infants, SCFAs are
considered beneficial, but faecal measurements are inappropriate to use as an indicator of a healthy
microbiome due to its paradoxical effects at high concentrations and the importance of considering the
balance of SCFA utilisation by epithelial cells and absorption into the blood stream.

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4.5. Vitamins
Vitamins are an additional class of secondary metabolites produced by the microbiota with effects
on immunity. Commensal bacteria have the capacity to synthesise essential vitamins, particularly from
the B and K groups, the expression of which is distinct in infants compared to adults. The microbiota in
neonates demonstrate the enrichment of genes involved in the production of Vitamin K2, retinol, folate,
pyroxidal (B6), and biotin (B7), which are involved in bone, vision, tooth development, and glucose
conversion are upregulated in the neonatal microbiome. Genes involved in the transport of B12, iron,
hemin, and heme are also enriched in neonates but decline markedly with age, corresponding with
increased nutritional demands for iron between 4 and 6 months of age. Throughout the weaning
months, genes involved in the biosynthesis of thiamine (B1), pantothenate (B5), cobalamin (B12),
and lysine increase [150]. Methionine degradation and leucine and tryptophan biosynthesis increase
to reach levels comparable to mothers by 12 months of age [7].
It has been estimated that B vitamins are produced by 40–65% of human gut microbes, with
some microbes having the capacity to produce all 8 B-vitamins and some demonstrating pathways
that complemented those of other organisms [151]. These estimates were determined by aligning
metagenomes from the human gut microbiome to an annotated genome on the PubSEED platform [151].
Bifidobacteriales contained the most conserved pattern of B1, B3, and B7 in approximately 35% of the
genomes, whereas Bacteroidetes demonstrated biosynthetic pathways of all 8 vitamins present in
51% of the genomes [151]. Prevotellaceaes produce B2, B5, and B7; Lactobacillales either contain no
biosynthetic pathways, or were limited to B2; and Clostridiales produce only B12 [151]. The full folate
biosynthesis (B9) pathway is present in 43% of genomes, which is distributed in nearly all Bacteroidetes
genomes, in most Fusobacteria and Proteobacteria, and in partial pathways occuring in Actinobacteria and
Firmicutes [151]. Vitamin K in one of two forms is reported to be produced by Bacteroides, Enterobacter,
Veillonella, and Eubacterium lentum, though the bioavailability of bacterially-derived Vitamin K has not
been established [152]. How these genes are differentially expressed in the infant microbiome and in
response to specific types of complementary foods has yet to be explored.
The interactions between microbially-derived vitamins and immune cells are varied and poorly
characterised. Of the known pathways, B6 has been found to serve as a cofactor in immunomodulatory
pathways [153], B9 has been implicated in the maintenance of regulatory T cells [154], and B12 has been
found to augment CD8+ T lymphocytes and NK cell activity in deficient patients [35,155]. Interestingly,
the byproducts from vitamin synthesis pathways have also recently been implicated in immune cell
recognition: mucosa-associated invariant T cells, which produce IL-17 and IFN-γ, are activated in
response to microbe-derived products of the riboflavin biosynthetic pathway that are presented by
a monomeric major histocompatibility complex class 1 (MHC-1)-like related molecule (MR1) [156].
These MHC and MHC-like molecules are imperative to discriminate self from non-self, enabling
protective immunity [157].

5. Discussion and Conclusions

Complementary feeding merges neonatal nutrition with diverse childhood nutrition during
a window of high variability and instability in the microbiome. Starchy foods are common
complementary foods based on texture and palatability, but the health-promoting benefits of complex
starches during this window are unknown. Currently, no harmful effects or negative outcomes of
starch consumption during complementary feeding have been reported. However, certain common
complementary foods that contain starch, such as rice and wheat, also contain nutrient-binding
compounds such as phytic acid, which can be altered during processing [158]. Based on the evidence
that prebiotic HMOs and NDCs alter the microbial community structure and microbial metabolism
and promoted immunity and immune development, investigations into prebiotic complex starches
are warranted. However, the utilisation and fermentation of starch structures occurs in a complex
trophic network governed by keystone species, cross-feeding dynamics, host-microbe interactions,
and biogeography of the gut lumen that are particularly dynamic during the rapid growth and

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colonisation phase of complementary feeding. Identifying the interactions and characteristics of a

healthy infant gut that result in beneficial clinical outcomes remains challenging.
The mechanisms by which starch may contribute to immunity and immune development are
varied. While some oligosaccharides are known to act as receptor analogues for pathogens, thus
preventing adhesion to the epithelium and consequent infection, this effect has not been explored
for complex starch structures from whole foods. Starch also promotes populations of commensal
bacteria with direct immunomodulatory activity in the mucosa and at the intestinal epithelium.
These commensal bacteria also produce metabolites, including SCFAs, vitamins, and small molecules
that may beneficially alter the environment, support structural immunity at the epithelial barrier,
and promote balanced and appropriate immune responses to commensals and pathogens. Building
upon extensive research into HMOs and prebiotic-supplemented formulas by investigating transitions
to more complex starch structures may offer functional insights into the mechanisms that underpin
balanced microbiome-mediated immune development during the complementary feeding window
and facilitate the development and application of functional complementary foods.

Author Contributions: Conceptualisation, S.M., W.Y., and N.C.R.; resources, N.C.R. and W.C.M.;
writing—original draft preparation, S.M.; writing—review and editing, W.Y., K.F., J.M., N.C.R., and W.C.M.;
supervision, W.Y., K.F., W.C.M., and N.C.R.; project administration, N.C.R. and W.C.M.; funding acquisition,
N.C.R.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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Review
Vitamin D: Nutrient, Hormone,
and Immunomodulator
Francesca Sassi, Cristina Tamone and Patrizia D’Amelio *
Department of Medical Science, Gerontology and Bone Metabolic Diseases, University of Turin,
10126 Turin, Italy; [email protected] (F.S.); [email protected] (C.T.)
* Correspondence: [email protected]; Tel.: +39-011-6335533

Received: 24 September 2018; Accepted: 31 October 2018; Published: 3 November 2018

Abstract: The classical functions of vitamin D are to regulate calcium-phosphorus homeostasis and
control bone metabolism. However, vitamin D deficiency has been reported in several chronic
conditions associated with increased inflammation and deregulation of the immune system, such as
diabetes, asthma, and rheumatoid arthritis. These observations, together with experimental studies,
suggest a critical role for vitamin D in the modulation of immune function. This leads to the
hypothesis of a disease-specific alteration of vitamin D metabolism and reinforces the role of
vitamin D in maintaining a healthy immune system. Two key observations validate this important
non-classical action of vitamin D: first, vitamin D receptor (VDR) is expressed by the majority
of immune cells, including B and T lymphocytes, monocytes, macrophages, and dendritic cells;
second, there is an active vitamin D metabolism by immune cells that is able to locally convert
25(OH)D3 into 1,25(OH)2 D3 , its active form. Vitamin D and VDR signaling together have a
suppressive role on autoimmunity and an anti-inflammatory effect, promoting dendritic cell and
regulatory T-cell differentiation and reducing T helper Th 17 cell response and inflammatory cytokines
secretion. This review summarizes experimental data and clinical observations on the potential
immunomodulating properties of vitamin D.

Keywords: vitamin D; immune system; gut microbiota; autoimmune diseases; T cells

1. Introduction
The role of vitamin D in the regulation of calcium-phosphate homeostasis and in the control of
bone turnover is well known. Vitamin D status significantly affects skeletal health during growth and
in adult age, its deficiency during growth leads to rickets [1], whereas during adult age it is responsible
of osteomalacia and various degree of osteoporo-malacia [2]. Low vitamin D status increases bone
turnover, decreases bone density, and is associated with increased fracture risk. In addition to the
well-known effect on skeletal health in the last two decades evidence has been accumulated on the
pleiotropic effect of vitamin D other than on bone health thanks to the findings that vitamin D receptor
(VDR) and the vitamin D activating enzyme 1-α-hydroxylase (CYP27B1) are expressed in several cells
outside the bone and kidney, such as in the intestine, platelets, pancreas, and prostate [3]. Several cells
involved in the immune function express VDR and CYP27B1, this observation suggests that the active
form of vitamin D, 1,25(OH)2 D3 , is able to control the immune function at different levels. Previous
reviews on the role of vitamin D in the regulation of immune system have been published in recent
years [4,5]. Here we summarize the recent evidence sexploiting authors’ expertise in both experimental
and clinical fields.

2. Vitamin D Metabolism
Vitamin D enters the body trough dietary intake (about 20% of vitamin D3 is assumed with diet)
or is synthetized by the skin (80%) from 7-dihydrocholesterol following UVB exposure. Vitamin D3

Nutrients 2018, 10, 1656; doi:10.3390/nu10111656 78 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1656

becomes biologically active after hydroxylation in the liver by the enzymes cytochrome P450 2R1
(CYP2R1) and cytochrome P450 27 (CYP27A1) becoming 25(OH)D3 . The fully-active metabolite
1,25(OH)2 D3 is hydroxylated in the kidney by the enzyme CYP27B1, parathormone (PTH), and the
fibroblast growth factor 23 (FGF-23) control CYP27B1 synthesis and activity [6]. Synthesis of
1,25(OH)2 D3 is strictly regulated in a renal negative feedback loop: high levels of 1,25(OH)2 D3
and FGF-23 inhibit CYP27B1 and induce the cytochrome P45024A1(CYP24A1), which transforms
1,25(OH)2 D3 into the inactive form 24(OH)D3 [7].
In addition to the kidney, CYP27B1 is expressed by other cell types, including immune cells.
These cells produce 1,25(OH)2 D3 that has autocrine and/or paracrine effects, the high level produced
locally is thought to be responsible for immunomodulation. The regulation of CYP27B1 synthesis in
immune cells is different than the signals regulating kidney production of 1,25(OH)2 D3 . Inflammatory
signals, such as lipopolysaccharide (LPS) and cytokines, induce monocyte and macrophage production
of CYP27B1 [8–10]. These differences in the regulation of 1,25(OH)2 D3 production point to an
autocrine/paracrine effect as immunomodulatory.

3. Vitamin D Status
Vitamin D status is defined by the blood measurement of its hydroxylated form 25(OH)D3 ,
however, there is no common agreement on the threshold levels to identify desirable vitamin D level.
Guidelines from different scientific societies and different countries established 50 nM/L or 75 nM/L
to consider vitamin D sufficiency [11–13], however, it is generally accepted that 25(OH)D3 levels lower
than 50 nM/L are associated with bone metabolism alteration, increased risk of falls, and myopathy in
adults [14–18]. Experts in the field generally agree to maintain 25(OH)D3 between 20 and 125 nM/L in
order to obtain the certain skeletal effects without toxic effects. Recent literature raises the suspicion
that administration of a bolus of vitamin D3 higher than 50,000 UI may result in an increased risk of
falls and fractures [19,20]; moreover, the mortality related to 25(OH)D3 is a “U shaped curve” and
25(OH)D3 levels higher than 150 nM/L are associated with increased mortality [21].

4. Vitamin D and the Innate Immune System: Antimicrobial Activity

The innate immune system is the first defense against infection, it is required to rapidly fight
against invading pathogens. The innate immune system comprehends components both from the
host and resident microbes (microbiota). The host defense comprises physical barriers to infection
(as skin, mucous surfaces, mucus, and vascular endothelial cells), enzymes expressed by epithelial
and phagocytic cells (as lysozyme), antimicrobial peptides and proteins (as defensins, cathelicidins,
and others expressed by phagocytes), inflammatory humoral components (as complement and
opsonins), and cell receptors that rapidly recognize pathogens (as toll-like receptors) and cellular
components (as mast cells, dendritic cells, macrophages, neutrophils cells and natural killer).
Interaction between microbiota a vitamin D will be analyzed in the following paragraph.
Vitamin D is a well-known regulator of innate immunity, the first data on this topic have been
generated on the treatment of diseases caused by mycobacteria, such as tuberculosis and leprosy [22,23],
however, the mechanisms responsible for these actions have been elucidated in more recent years.
1,25(OH)2 D3 enhances the production of defensin β2 and cathelicidin antimicrobial peptide (CAMP)
by macrophage and monocyte keratinocytes increasing their antimicrobial activity [24–26]. Moreover,
1,25(OH)2 D3 increases chemotaxis, autophagy, and phagolysosomal fusion of innate immune
cells [27,28]. The exposition of human monocytes to pathogens, such as M. tuberculosis and others,
up-regulates the expression of CYP27B1 and of VDR, thus enhancing both the cell ability to produce
1,25(OH)2 D3 in the site of infection and to respond to this metabolite. However, macrophages are
heterogeneous, with different functions [29]. Macrophages formed after interleukin (IL)-15 stimulus
respond to vitamin D stimulus increasing their antimicrobial activity, whereas phagocytic macrophages
obtained after stimulus with IL-10 are weakly influenced by vitamin D levels regardless oftheir high
phagocytic activity [10,30].

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1,25(OH)2 D3 up-regulates CAMP not only by monocytes/macrophages, but also in other cells
participating in the innate immune system as first-barrier defenses, such as keratinocytes, epithelial,
intestinal, lung and corneal cells, and placenta trophoblasts (see for a comprehensive review Wei and
Christakos, 2015) [4].
Data in humans on infections other than mycobacterial have been generated on urinary and
respiratory infections and on sepsis. A predisposition to urinary tract infection in children with low
vitamin D levels due to the reduced production of CAMP and defensing β2 has been suggested
by association studies [31,32]. Additionally, in chronic obstructive pulmonary disease (COPD)
patients’ levels of CAMP and other antimicrobial peptides were associated with increased risk of acute
exacerbations [33]. Consistent with this datum treatment with 1,25(OH)2 D3 was effective in reducing
respiratory infections in asthma patients thanks to increased CAMP expression and inflammatory
cytokine modulation [34]. Data on the role of vitamin D status and vitamin D supplementation in
sepsis are also available both in pediatric and in adult patients: in pediatric patients a clear role
for 25(OH)D3 and CAMP was not demonstrated [35], whereas in adults lower levels of 25(OH)D3
were found in sepsis [36] and a high-dose of vitamin D3 increases circulating CAMP and reduces
inflammatory cytokines as IL-6 and IL-1β [37].
More recently data on a possible role of vitamin D in increasing resistance to HIV infection
have been published, in particular HIV-exposed seronegative individuals produced more CAMP in
oral-mucosa and peripheral-blood, and have higher CYP24A1 mRNA in vaginal-mucosa; CYP24A1 is
considered an indicator of high levels of 1,25(OH)2 D3 [38]. Low serum vitamin D has been associated
with HIV/AIDS progression and mortality [39].
1,25(OH)2 D3 is able to increase the production of other antimicrobial peptides, such as defensing
β2-4, this ability has been demonstrated both in vitro by monocytes stimulation [40,41] and in vivo in
pediatric patients’ blood [32].
Vitamin D is able to modulate innate immune system, also increasing the phagocytic ability on
immune cells [42,43] and by reinforcing the physical barrier function of epithelial cells. In particular
1,25(OH)2 D3 can enhance corneal [44] and intestinal [45] epithelial barrier function (Figure 1).
Taken together these data point to a role of vitamin D in defending the organism against pathogens
suggesting that vitamin D sufficiency has to be granted in patients affected by acute or chronic
infection. The ability of immune cells to hydroxylate 25(OH)D3 into its active form 1,25(OH)2 D3
suggests administrating vitamin D3 rather than hydroxylated metabolites to patients affected by
infections in order to allow the autocrine/paracrine function of 1,25(OH)2 D3 without overcoming local
hydroxylation and the feedback system.

5. Vitamin D and Microbiota: Increasing Host Defenses

The whole of the commensal, symbiotic, and pathogenic microorganisms living in different
areas of the human body has defined microbiota. Microbiota and the host have several relationships,
and the perfect balance between microbiota and the host is required for the development, maturation,
and properfunction of the immune system [46]. Several papers suggest that vitamin D is one of
the actors of the complex relationship between microbiota living in the gut (GM) and immune
system modulation. Vitamin D is responsible for the barrier function of the intestinal epithelium
and for the modulation of the bowel immune system, hence, low levels may be associated with
greater gut permeability and, consequently, with GM-induced metabolic endotoxemia that induces a
low-grade inflammation [47]. Moreover, vitamin D administration may influence GM composition,
and in vitro data demonstrate that vitamin D enhances macrophages’ ability to kill Escherichia coli. [48].
In animals with vitamin D depletion and the knockout of the VDR, the GM dysbiosis favors metabolic
disorders [49]. Other studies in mice demonstrated that VDR reduces the response to infection of the
intestinal epithelium [50].
Elegant studies in transgenic mice demonstrated that over-expression of VDR in the intestinal
epithelium induces resistance to colitis [51,52] and decreases mucosal inflammation suppressing

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epithelial cell apoptosis, boosting tight junction function [51,53]. On the other hand VDR selective
deletion in bowel favors a more severe form of colitis characterized by greater Th1 and Th17 mucosal
infiltration and inflammatory cytokines production [54]. In humans, observational studies suggest that
low levels of 25(OH)D3 are associated with increased risk of inflammatory bowel disease (IBD) [55–57]
and that high levels of 25(OH)D3 in these patients protect against Clostridium difficile infection [58].
The experimental data on the role of VDR in developing IBD have been confirmed by the finding of a
significant reduction of VDR expression (about 50%) in the colon epithelium in patients affected by IBD
with respect to healthy controls [51,53]. The reduction in VDR expression by IBD patients may explain
the different effect on GM composition of high oral dosages of vitamin D3 demonstrated in a small
cohort of patients affected by Crohn’s disease with respect to healthy controls [59], however, human
data on the effect of vitamin D supplementation on GM in IBD are still controversial, as other studies
did not confirm these results [60,61]. In the study by Luthold and coll. [61] dietary intake of vitamin D
and 25(OH)D3 were inversely correlated with Coprococcus and Bifidobacterium, however, thanks to their
ability to produce butyrate these bacteria are commonly considered as anti-inflammatory. A possible
explanation of these contradictory results may be the different effect of vitamin D on GM according
to the different gastro-intestinal tracts considered [62]. Recently, a double-blind placebo-controlled
study on patients affected by cystic fibrosis demonstrated that vitamin D insufficiency is associated
with different microbiota not only in the gut, but also in the airways, and that the administration of
50,000 IU of oral vitamin D3 weekly significantly affects microbiota composition [63]. Nevertheless,
the evaluation of clinical outcomes of microbiota change is still open.
Several data point to an effect of vitamin D on microbiota. Conversely, some recent reports suggest
that microbiota, per se, influences vitamin D metabolism mainly through FGF-23; germ-free (GF) mice
have low vitamin D and high FGF-23, whereas their colonization with bacteria results in increased
levels of tumor necrosis factor-α (TNF-α) and a decrease in FGF-23 with normalization of vitamin D
hydroxylated metabolites. Inhibition of FGF-23 in GF mice restores vitamin D metabolism without
bacterial colonization of the gut [64] (Figure 1).
The role of GM as an active player in the regulation of bone metabolism in humans is being
investigated more and more [46], and the role played by vitamin D is still under debate. Further studies
to clarify their interplay are needed.

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Figure 1. Effects of vitamin D on the innate immune system and gut microbiota. Abbreviations: EC,
enteral cells; GM, gut microbiota.

6. Vitamin D and the Adaptive Immune System

The adaptive immune system or acquired immune system is the second defense against infection.
It is required to specifically fight against pathogens, is activated by exposure to pathogens, and unlike
the innate immune system it is able to learn about the pathogen and enhance the immune response
accordingly, thanks to an immunological memory. The adaptive immune system is composed of T and
B cells and is also responsible for autoimmune reaction.
25(OH)D3 suppresses adaptive immunity [4,65]. In experimental models it down-regulates
the immune responses mediated by T helper (Th) 1 cells, thus inhibiting the production
of pro-inflammatory cytokines, such as Interferon-γ IFN-γ, IL-6, IL-2, and TNF-α [66,67].
Although experimental studies in vitro and in animals have yielded encouraging results on the
immunomodulatory effect of 1,25(OH)2 D3 , the same cannot be said about human studies, and few
studies have confirmed the suppressive effect of vitamin D on Th1 cells and inflammatory
cytokine production in different diseases and spinal tuberculosis [68], uremia [69], and autoimmune
thyroiditis [70]; whereas others in IBD [71], dialysis [72], and rheumatoid arthritis [73] do not confirm
these results. These discrepancies may be due to the different diseases considered and also to the
different type of treatment administered, mainly 1,25(OH)2 D3 in vitro and in animals and vitamin
D3 in vivo in humans. Moreover, when considering administration of vitamin D3 different doses were
used in different studies. Therefore, it is almost impossible to compare the results.
It has been suggested that 1,25(OH)2 D3 acts as an immunomodulatory not only by suppressing
Th1 cells activation, but also modulating Th2 cells, T regulatory (Tregs) cells activity, and Th17 cells.
The majority of the in vitro studies assessing the effect of vitamin D on Th2 suggests that
1,25(OH)2 D3 upregulates Th2 cells activity [74–76]. Amongst immunomodulatory effects of vitamin D
its ability to suppress Th17 and increase Treg cells has been recently demonstrated [77–79]. Th17 cells
produce IL-17 and have been implicated in the pathogenesis ofseveral autoimmune diseases, some
experimental studies suggest that 1,25(OH)2 D3 suppresses Th17 formation and activity [67,80–83]
by blocking Nuclear Factor of Activated T-cells (NFAT) and Runt-related Transcription Factor 1

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(RUNx1) binding to the IL-17 promoter and inducing Forkhead box P3 (FOXP3) [81], and by inhibiting
RAR-related Orphan Receptor Gamma2 (RORγt) which is the transcription factor of IL-17 [84].
More recently our lab showed no effect of the administration of a high bolus of vitamin D3
(300,000 UI) in the modulation of Th subset in patients affected by early rheumatoid arthritis [73],
as well as a study on hemodialysis patients [72].
It has also been suggested that the administration of oral vitamin D3 increases Tregs function
in patients with type 1 diabetes mellitus [85], however, in other diseases, such as early rheumatoid
arthritis, this effect was not confirmed [73].
The overall effect of vitamin D on Th cells differentiation may be mediated by its effect on
dendritic cells, these cells are antigen-presenting cells (APCs), responsible for T cell differentiation
into an effector cell with pro- or anti-inflammatory properties, thus, modulation of APCs is crucial in
initiating and maintaining adaptive immune response and self-tolerance [86]. In vitro differentiation of
dendritic cells in the presence of 1,25(OH)2 D3 induces a “tolerogenic state” characterized by low levels
of inflammatory cytokines, such as IL-12 and TNF-α, with increased levels of the anti-inflammatory
IL-10, these cells induce the differentiation of Treg cells and induce apoptosis in the autoreactive T
cells [87–90] (Figure 2).
Taken together these data are not sufficient to prove a real role for vitamin D in the modulation
of adaptive immune system in humans, thus, the therapeutic use of vitamin D and its metabolites in
patients aiming to ameliorate the adaptive immune system is not sustained by sufficient data.

Figure 2. Effect of vitamin D on the adaptive immune system. Abbreviations: APC, antigen presenting
cell; IFN, interferon; IL, interleukin; Th1, T helper 1 cell; Th2, T helper 2 cell; Th17, T helper 17 cell;
TNF, tumor necrosis factor; Treg, T regulatory cell.

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7. Vitamin D and Autoimmune Diseases

Thanks to the evidences of immunomodulatory effect of vitamin D the role of vitamin D deficiency
and supplementation in autoimmune diseases has long been studied. Animal studies showed an
important role of 1,25(OH)2 D3 supplementation in the control of autoimmune diseases, such as
experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). In these
two conditions 1,25(OH)2 D3 prevents the initiation and reduces the disease progression [91–93].
Similarly, different mouse models of enterocolitis display a more severe phenotype during vitamin
D deficiency and reduced inflammation after administration of 1,25(OH)2 D3 (see for a review
Alhassan et al., 2017) [94]. Despite solid experimental evidence human studies are less convincing:
some epidemiological data link increasing latitude and consequent decrease sunlight exposure
with higher prevalence of multiple sclerosis [95–97], type I diabetes [98–100], and IBD [101]. It is
clear that such differences may be due to genetic and lifestyle factors other than 25(OH)D3 levels.
Other epidemiological data reinforcing the hypothesis of a link between sun exposure, vitamin D
synthesis, and the risk of developing multiple sclerosis stem from the observation that subjects born in
months associated with lower 25(OH)D3 level in the northern hemisphere (April) are at higher risk
of developing the disease, whereas patients born in October (higher vitamin D levels) are at lower
risk [102].
Some studies correlated vitamin D dietary intake and the prevalence of autoimmune diseases as
rheumatoid arthritis [103] and type 1 diabetes mellitus [104,105], however, the correct evaluation of
vitamin D intake is challenging as it is based on patient recall. To bypass the challenging measurement
of vitamin D intake and sun exposure, levels of 25(OH)D3 in the serum can be useful, and, indeed,
low levels of 25(OH)D3 in the serum of patients affected by autoimmune diseases with respect to
healthy controls have been found [106–112]. Nevertheless, these studies demonstrated a correlation
and not a causal relationship.
Intervention studies with different doses of vitamin D3 in autoimmune diseases lead to different
outcomes, recently we demonstrated that a bolus of vitamin D3 (300,000 UI) in patients affected by
early rheumatoid arthritis is effective in ameliorating general health, however, we found no effect
on disease activity nor on inflammatory markers and T cells subset [73]. In patients affected by
type 1 diabetes clinical intervention studies with vitamin D3 or hydroxylated analogs have been
disappointing, as no clinical study has demonstratedan effect of vitamin D in ameliorating glucose
metabolism and insulin secretion [113,114], however, in a small prospective trial in children with type 1
diabetes autoantibodies 1,25(OH)2 D3 administration decreased the serum glutamic acid decarboxylase
65 (GAD65) autoantibody, pointing to some immunomodulation of 1,25(OH)2 D3 [115].
In addition to autoimmune diseases vitamin D has also been implicated in the control of other
inflammatory conditions, such as cardiovascular diseases: in animal models vitamin D3 administration
reduces macrophage production of pro-inflammatory cytokines, and decreases atherosclerosis and
inflammation in the epicardial adipose tissue [116,117]. In humans an association between low
25(OH)D3 level and increased activation of inflammatory pathway in epicardial adipose tissuein
patients affected by coronary artery disease has been described [118]. Vitamin D deficiency has been
linked to cardiovascular disease not only by the modulation of inflammatory pathways, but also
through the modulation of endothelial function, the effect on arterial stiffness, and a possible beneficial
role on atherosclerotic plaque formation. However, this topic is beyond the scope of this review.
For further insight in the role of vitamin D in the pathogenesis of cardiovascular disease see the review
by Apostolakis and coll. [119].

8. Conclusions
In summary, several studies point to an important role of vitamin D as an immunomodulator,
and strong data demonstrate a role for 1,25(OH)2 D3 in increasing the ability of the innate immune
system to fight against pathogens, whereas data on the effect of 1,25(OH)2 D3 in the modulation of
acquired immune system are more controversial. There is no general consensus on the desired level of

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25(OH)D3 to achieve immunomodulatory effects, thus, there is no current indication for vitamin D3
supplementation in patients with infections and/or autoimmune diseases. Further studies are needed
to clarify the role of vitamin D as immunomodulator in humans.

Author Contributions: All three authors participated in the bibliographic search, discussion and writing of the
manuscript. The manuscript was finalized by P.D.
Funding: This research received no external funding
Conflicts of Interest: The authors declare that they have no conflict of interest.

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 nutrients
Review
The Immunomodulatory and Anti-Inflammatory Role
of Polyphenols
Nour Yahfoufi 1 , Nawal Alsadi 1 , Majed Jambi 1 and Chantal Matar 1,2, *
1 Cellular and Molecular Medicine Department, Faculty of Medicine, University of Ottawa,
Ottawa, ON K1H8L1, Canada; [email protected] (N.Y.); [email protected] (N.A.);
[email protected] (M.J.)
2 School of Nutrition, Faculty of Health Sciences, University of Ottawa, Ottawa, ON K1H8L1, Canada
* Correspondence: [email protected]; Tel.: +1-613-562-5406

Received: 30 September 2018; Accepted: 23 October 2018; Published: 2 November 2018

Abstract: This review offers a systematic understanding about how polyphenols target multiple
inflammatory components and lead to anti-inflammatory mechanisms. It provides a clear
understanding of the molecular mechanisms of action of phenolic compounds. Polyphenols regulate
immunity by interfering with immune cell regulation, proinflammatory cytokines’ synthesis, and
gene expression. They inactivate NF-κB (nuclear factor kappa-light-chain-enhancer of activated
B cells) and modulate mitogen-activated protein Kinase (MAPk) and arachidonic acids pathways.
Polyphenolic compounds inhibit phosphatidylinositide 3-kinases/protein kinase B (PI3K/AkT),
inhibitor of kappa kinase/c-Jun amino-terminal kinases (IKK/JNK), mammalian target of rapamycin
complex 1 (mTORC1) which is a protein complex that controls protein synthesis, and JAK/STAT.
They can suppress toll-like receptor (TLR) and pro-inflammatory genes’ expression. Their antioxidant
activity and ability to inhibit enzymes involved in the production of eicosanoids contribute as well to
their anti-inflammation properties. They inhibit certain enzymes involved in reactive oxygen species
ROS production like xanthine oxidase and NADPH oxidase (NOX) while they upregulate other
endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase, and glutathione (GSH)
peroxidase (Px). Furthermore, they inhibit phospholipase A2 (PLA2), cyclooxygenase (COX) and
lipoxygenase (LOX) leading to a reduction in the production of prostaglandins (PGs) and leukotrienes
(LTs) and inflammation antagonism. The effects of these biologically active compounds on the
immune system are associated with extended health benefits for different chronic inflammatory
diseases. Studies of plant extracts and compounds show that polyphenols can play a beneficial role in
the prevention and the progress of chronic diseases related to inflammation such as diabetes, obesity,
neurodegeneration, cancers, and cardiovascular diseases, among other conditions.

Keywords: polyphenols; immune system; inflammation; molecular mechanisms; nuclear factor

kappa-light-chain-enhancer of activated B cells (NF-κB); arachidonic acid; mitogen-activated
protein Kinase (MAPK); cytokines; oxidative stress; reactive oxygen species (ROS); cyclooxygenase
(COX); nitric oxide synthase (NOS); lipoxygenase (LOX); superoxide dismutase (SOD); inhibitor
of kappa kinase (IKK); extra-cellular signal regulated kinases (ERK); cancer; anti-inflammation;
anti-tumorigenic; chronic inflammatory conditions; macrophages; T helper 1 (Th1); Th17; Treg

1. Introduction
Numerous studies have attributed to polyphenols a broad range of biological activities including
but not limited to anti-inflammatory, immune-modulatory, antioxidant, cardiovascular protective
and anti-cancer actions [1–5]. Polyphenols are ubiquitously made by plants and are present either as
glycosides esters or as free aglycones [6]. More than 8000 structural variants exist in the polyphenol
family. Polyphenols are bioactive compounds found in fruits and vegetables contributing to their color,

Nutrients 2018, 10, 1618; doi:10.3390/nu10111618 92 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1618

flavor, and pharmacological activities [1]. They are classified according to their chemical structures
into flavonoids such as flavones, flavonols, isoflavones, neoflavonoids, chalcones, anthocyanidins, and
proanthocyanidins and nonflavonoids, such as phenolic acids, stilbenoids, and phenolic amides [7].
The majority of these molecules are metabolites of plants, they are made of several aromatic rings
with hydroxyl moieties [8]. Their chemical structures contribute to their classification into different
classes. Considering gastrointestinal digestion, some—but not all—polyphenols are absorbed in
the small intestine, for example, anthocyanins and the majority of remaining polyphenols except
flavonoids are usually stable; these later are unstable in the duodenum. Unabsorbed polyphenols must
be hydrolyzed first by digestive enzymes then glycosides with high lipid contents are absorbed by
epithelial cells [9,10].
In recent years, consumers prefer using natural food ingredients as additives because of their
safety and availability. Applications of phenolic compounds to multiple fresh perishable foods show
that they are worthy to be used as preservatives in foods and can be creditable alternatives to synthetic
food additives. In this sense, polyphenolic compounds start to substitute chemical additives in food.
Different methods like spraying, coating and dipping treatment of food are currently applied in food
technology preceding packaging as effective alternatives [11]. Grape seeds and olive oil polyphenols’
rich extracts can be used as food additives for their anti-oxidant properties [12]. Various polyphenols
like grape polyphenols demonstrate an efficient role as additives in fish and fish products for their
anti-oxidant properties in order to prevent lipid oxidation and quality deterioration of polyunsaturated
fatty acids [13]. In addition polyphenolic compounds like flavonols, p-coumaric, and caffeic acids can
be used as food preservatives for their antimicrobial activity [11].
Back to inflammation, continuous inflammation is known to be a major cause linked to
different human disorders involving cancer, diabetes type II, obesity, arthritis, neurodegenerative
diseases, and cardiovascular diseases [14,15]. Polyphenols derived from botanic origin have shown
anti-inflammatory activity in vitro and in vivo highlighting their beneficial role as therapeutic tools in
multiple acute and chronic disorders [16–20]. Accordingly, many epidemiological and experimental
researches have been studying the anti-inflammatory and immune modulation activities of dietary
polyphenols [15,21]. The ability of these natural compounds to modify the expression of several
pro-inflammatory genes like multiple cytokines, lipoxygenase, nitric oxide synthases cyclooxygenase,
in addition to their anti-oxidant characteristics such as ROS (reactive oxygen species) scavenging
contributes to the regulation of inflammatory signaling [22,23]. This review will discuss the
immunomodulatory effects of dietary polyphenols, their anti-inflammatory abilities, the different
mechanisms and pathways involved in reducing inflammation and their contribution to protect from
different chronic inflammatory diseases with a focus on their anti-cancer activity.

2. Polyphenols and Inflammation

The immune modulation effect of polyphenols is supported by different studies: some
polyphenols impact on immune cells populations, modulate cytokines production, and
pro-inflammatory genes expression [24,25]. For example, cardioprotective effects of resveratrol
present in red wine grape and nuts were mainly attributed to its anti-inflammatory properties.
In vivo and in vitro studies demonstrate that resveratrol can inhibit COX, inactivate peroxisome
proliferator-activated receptor gamma (PPARγ) and induce eNOS (endothelial nitric oxide synthase)
in murine and rat macrophages [26–28]. Likewise, a resveratrol analog, RVSA40, inhibits the
pro-inflammatory cytokines TNF-α (Tumor necrosis factor alpha) and IL-6 (interleukin-6) in RAW
(Murine macrophages cell line) 264.7 macrophages [29]. Another example is the non-flavonoid
curcumin found in turmeric plants and mustard. Curcumin was shown to reduce the expression
of inflammatory cytokines: TNF and IL-1, adhesion molecules like ICAM-1 (intercellular adhesion
molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in human umbilical vein endothelial
cells and inflammatory mediators like prostaglandins and leukotriens. It also inhibits certain
enzymes involved in inflammation like COX in mice (cyclooxygenase), LOX (lipoxygenase) in,

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human endothelial cells MAPK (mitogen-activated protein Kinase), and IKK (inhibitor of kappa
kinase). Moreover, curcumin downregulates NF-κB (nuclear factor kappa-light-chain-enhancer of
activated B cells) and STAT3 (signal transducer and activator of transcription) and reduces the
expression of TLR-2 (toll-like receptor-2) and 4 while, in vivo, it upregulates PPARγ (Peroxisome
proliferator-activated receptor gamma) in male adult rats [30–35]. Caffeic acid phenethyl ester
suppresses TLR4 activation and LPS-mediated NF-κB in macrophages, Quercetin was also shown to
inhibit leukotriens biosynthesis in human polymorphonuclear leukocytes [36,37]. COX2 expression is
also attenuated by ECGC (Epigallocatechin gallate) in colon cancer cell and androgen-independent
PC-3 cells of human prostate cancer, gingerol in and piceatannol (EGCG analog found in Norway
spruces) leading to NFκ B inactivation [30,38–40]. Furthermore, polyphenols, such as Gingerol and
Quercetin can activate the production of adiponectin known for its anti-inflammatory effects [30,39].
Similarily, EGCG blocks NFκ B activation in human epithelial cells and downregulates the expression
of iNOS (inducible nitric oxide synthase), NO (nitric oxide) production in macrophages resulting in its
immunomodulation [38,40,41]. A series of in vitro studies found that other polyphenols like oleanolic
acid, curcumin, kaempferol-3-O-sophoroside, EGCG and lycopene inhibit high mobility group box1
protein, an important chromatin protein that interacts with nucleosomes, transcription factors, and
histones regulating transcription and playing a key role in inflammation [35]. All of these examples
support the anti-inflammatory effects of polyphenols.
Polyphenols’ use is associated with a direct change in the count and differentiation of specific
immune cells. An increase in T helper 1(Th1), natural killer (NK), macrophages and dendritic cells (DCs)
in Peyer’s patches and spleen is associated with oral administration of polyphenols extracted from the
fruit date in male C3H/HeN mice [24]. In humans, the count of regulatory T cells (Treg or suppressor T
cells) characterized by the (CD4 + CD25 + Foxp3+) phenotype and involved in immune tolerance and
autoimmune control can be boosted by polyphenols [42–44]. In vivo, Epigallocatechin-3-gallate, found
in green tea and injected to Laboratory inbred strain (BALB)/c mice, rises the number of functional Treg
in spleens, pancreatic lymph nodes, and mesentheric lymph nodes [45]. Similarly, in vitro treatment of
Jurkat T cells with EGCG or green tea upsurges the expression of Foxp3 and IL10. Baicalin, a flavone,
extracted from Huangqin herb, induces Foxp3 expression in HEK 293 T cells and triggers functional
Treg from splenic CD4 + CD25− T cells [46]. Additionally, flavonoids show an agonistic effect of aryl
hydrocarbon receptor (AhR) and bind xenobiotic-responsive elements in promoter regions of certain
genes, including Foxp3 rising its expression [47].
Th1 and Th17 populations are also affected by polyphenols: EGCG reduces the differentiation of
Th1 and reduces the numbers of Th17 and Th9 cells in specific pathogen-free C57/BL6 female mice [48].
Other polyphenols like Baicalin show a reduction of Th17 differentiation in vitro and a diminution of
IL-17 expression [49].
Macrophages are affected by polyphenols as well. Macrophages are known to be a key
player in the inflammatory response. They initiate inflammation by secreting pro-inflammatory
mediators and cytokines like IL-6 and TNF-α [50]. Polyphenols repress macrophages by inhibiting
cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), thus they reduce the production of
TNF-α, interleukine-1-beta (IL-1-β) and IL-6 expression [51]. Chinese propolis [52] containing ferulic
acid and coumaric acid, an extract of Lonicera japónica Thunb) [53] or Kalanchoe gracilis [54] are a good
example in this case as per demonstrated by in vitro studies using RAW 264.7 cells.

3. Polyphenol and Cytokine Modulation

Cytokines are important mediators’ proteins, essential in networking communication for immune
system. Cytokines can be produced by lymphocytes (lymphokines), or monocytes (monokines) with
pro-inflammatory and anti-inflammatory effects. Cytokines with chemotactic activities are termed
chemokines. The equilibrum between pro-inflammatory cytokines (IL-1β, IL-2, TNFα, Il-6, IL-8,
IFN-γ . . . ) and anti-inflammatory cytokines (IL-10, IL-4, TGFβ) are thought to be an important
parameter in immune response homeostasis and inflammation underlining many disease [55]. In vivo

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and in vitro studies demonstrate that polyphenols affect macrophages by inhibiting multiple key
regulators of inflammatory response such as the inhibition of TNF-α, IL-1-β, and IL-6 [51].
In humans, consumption of bilberries is associated with a decreased inflammation score in
patients’ blood, reflected by decreasing serum levels of IL-6, IL-12, and high sensitivity C reactive
protein [56]. Moreover, clinical trials have shown the ability of polyphenol-enriched extra virgin olive
oil to reduce IL-6 and C-reactive protein expression in stable coronary heart disease patients [57].
In lipopolysaccharide (LPS)-treated BALB/c mice, a model system of inflammation olive
vegetation water show ability to inhibit the production of tumor necrosis factor-alpha usually
activated by inflammation [58]. Flavonoids, as well, play an important anti-inflammatory effect
by influencing cytokines’ secretion. Several flavonoids are found able to inhibit the expression
of various pro-inflammatory cytokines and chemokines like TNFα, IL-1β, IL-6, IL-8, and MCP-1
(monocyte chemoattractant protein-1) in multiple cell types such as LPS-activated mouse primary
macrophages, activated human mast cell line, activated human astrocytes, human synovial cells, and
human peripheral blood mononuclear cells [59–64]. In murine RAW 264.7 macrophages stimulated by
LPS, Chinese propolis as well as extract of Lonicera japónica Thunb (Caprifoliaceae) or Kalanchoe gracilis
demonstrated inhibitory effects on TNF-α, IL-1-β, and IL-6 [52–54]. Similarly, certain polyphenol
analogs, like curcumin analog EF31, have shown the ability to inhibit the expression and secretion of
TNF-α, IL-1-β, and IL-6 in mouse Raw 264.7 macrophages [65].
Likewise, reduction of the secretion of TNF-α and IL-6 without IL-1β modulation is observed
with extracts of chamomile, meadowsweet, willow bark, and isolated polyphenols such as quercetin
existing in these extracts in THP1 macrophages [66]. Extract of Cydonia oblonga inhibits TNF-α and
Interleukin 8 while it increases IL-10 and IL-6 in THP-1monocytes stimulated with LPS. The reduction
in TNF-α levels limits the acute inflammatory response [67,68]. Other cytokines like IFNγ might also
be inhibited by certain polyphenols. For example, kaempferol reduces the production of IFN-γ in a
dose-dependent manner in spleen cells and T cell lines [69].
Certain polyphenols exert their effects on the balance between pro- and anti-inflammatory
cytokines production such as quercetin and catechins, they enhance IL-10 release while they
inhibit TNFα and IL-1β [59,70]. Extract of Cydonia oblonga also inhibits the effects of TNF-α and
Interleukin 8 (IL-8) while it raises IL-10 in the same type of monocytes [67,68]. Modulation of
inflammatory cytokines is one of many common mechanisms by which polyphenols in general
exert their immunomodulatory effects.

4. Polyphenols, Inflammation, and Modulation of Different Signaling Pathways

4.1. NFκ B Signaling Pathway

NF-κB or nuclear factor kappa-light-chain-enhancer of activated B cells is a complex protein that
plays a key role in deoxyribonucleic acid (DNA) transcription, cytokine production and cell survival.
It controls immune, inflammation, stress, proliferation and apoptotic responses of a cell to multiple
stimuli [58].
The expression of a large number of genes involved in inflammation is controlled by NF-κB such
as COX-2, VEGF (vascular endothelial growth Factor), pro-inflammatory cytokines (IL-1, IL-2, IL-6,
and TNFα), chemokines (e.g., IL-8, MIP-1α, and MCP-1), adhesion molecules, immuno-receptors,
growth factors, and other agents involved in proliferation and invasion [71].
NFκ B is located in the cytoplasm, it exists as an inactive non-DNA-binding form. Iκ B proteins
(Iκ Bs), are inhibitors proteins that are associated with NFκ B resulting in its inactivation. Iκ Bs include
Iκ Bα, Iκ Bβ, Iκ Bγ, Iκ Bε, Bcl-3, precursors p100 and p105 [72]. Under stimulatory conditions, Iκ B
kinase (IKK) phosphorylate IκB proteins leading to successive ubiquitination, consequent degradation
of the inhibitory proteins and release of NFκ B dimer. This later can translocate into the nucleus and
prompts the expression of particular genes [72]. Different mechanisms regulate NFκ B activity as per
the accumulation and degradation of Iκ B, the phosphorylation of NFκ B, the hyper-phosphorylation

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of IKK, and the processing of NFκ B precursors [73–75]. Thus, the inhibition of NFκB can be of
a great benefit in controlling inflammatory conditions [76]. Several polyphenols modulate NFκ B
activation and reduce inflammation [77,78]. Quercetin blocks the nuclear translocation of p50 and
p65 subunits of NFκ B and represses the expression of pro-inflammatory associated genes, NOS and
COX-2 in RAW264.7 macrophages [79]. It inhibits the phosphorylation of Iκ Bα protein both in vitro
(using macrophages) and in vivo (using dextran sulfate sodium (DSS) rat colitis model) leading to
inactivation of the NFκ B pathway [80]. In human mast cells, quercetin prevents the degradation
of Iκ Bα, as well as the nuclear translocation of p65 resulting in reduction of TNFα, IL-1β, IL-6 and
IL-8 [63]. It can modulate chromatin remodeling, for example it blocks the recruitment of a histone
acetyl transferase called CBP/p300 to the promoters of interferon-inducible protein 10 (IP-10) and
macrophage inflammatory protein-2 (MIP-2) genes in primary murine small intestinal epithelial cell.
As a result, it inhibits the expression of these pro-inflammatory cytokines [81]. Quercetin can block
the activation of IKK, NFκ B, and it reduces the ability of NFκ B to bind DNA in microglia treated
by LPS and IFN-γ in mouse BV-2 microglia [82]. Luteolin, too, blocks NFκ B activation and inhibits
pro-inflammatory genes expression and the cytokines production in murine macrophages RAW 264.7
and mouse alveolar macrophages; it also inhibits IKKs in LPS-induced epithelial and dendritic cells [83].
In addition, in co-cultured intestinal epithelial Caco-2 and macrophage RAW 264.7 cells, luteolin
represses NF-k B activation and TNF-α secretion [84]. Likewise, Genistein represses LPS-induced
activation of NF-k B in monocytes and reduces the inflammation by inhibiting NF-k B activation
upon adenosine monophosphate activated protein kinase stimulation in LPS-stimulated macrophages
RAW 264.7 [83,85]. Galangin, as well, stops the degradation of Ik Bα and the translocation of p65 NF-k
B, repressing the expression of TNF-α, IL-6, IL-1β, and IL-8 in mast cell [86]. EGCG counteracts the
activation of IKK and the degradation of Iκ Bα and inhibits NFκ B in culture respiratory epithelial
cells and in vivo in male Wistar rats [87,88]. Furthermore, EGCG blocks DNA binding of NFκ B which
reduces the expression of IL-12p40 and iNOS in murine peritoneal macrophages [89,90]. Catechin and
epichatechin reduce NFκ B activity in PMA-induced Jurkat T cells. Flavonoids can modulate NFκ B
activation cascade at early phases by affecting IKK activation and regulation of oxidant levels or at late
phases by affecting binding of NF-κ B to DNA in jurkat Tcells [91]. Hydroxytyrosol, and resveratrol
inhibit NFκ B activation, and the expression of VCAM-1 in LPS-stimulated human umbilical vein
endothelial cells [92]. In summary, polyphenols can modulate NFκ B activation cascade at different
steps such as by affecting IKK activation and regulating of the oxidant levels or by affecting binding of
NF-κ B to DNA leading to an important anti-inflammatory effect responsible for their potential value
in treating chronic inflammatory conditions (Figure 1).

Figure 1. Potential points of action of polyphenols within inflammatory cascade. NF-κ B: nuclear factor
kappa-light-chain-enhancer of activated B cells; IKK: IkB-kinase; ERK: extracellular signal-related
kinases; JNK: c-Jun amino-terminal kinases; p38 (or p38-MAPK): p38-mitogen-activated protein kinase;
COX: cyclooxygenase; LOX: lipoxygenase; AA: arachidonic acid; PLA2: phospholipase A2; PGs:
prostaglandins; LTs: leukotriens. For references see the text.

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4.2. MAPK Signaling Pathway

The mitogen-activated protein kinases (MAPK) are a highly conserved family of serine/threonine
protein kinases. They play a key role in a range of fundamental cellular processes like cell
growth, proliferation, death and differentiation. They regulate gene transcription and transcription
factor activities involved in inflammation. Extracellular signal-related kinases, like (extracellular
signal-related kinases (ERK))-1/2, c-Jun amino-terminal kinases (JNK1/2/3), p38-MAP kinase (α, β, δ,
and γ), and ERK5 are different groups of MAPKs expressed in mammals. These are later activated by
MAP kinase kinases (MAPKK) which might be triggered by some MAPKK kinases (MAPKKK) [93].
MAPK, in its turn, cross-talks with other pathways such as NFκB, thus the complexity of the MAPK
signaling pathway and its interactions. Stress and mitogens activate MAPK signaling: For example,
ERK1/2 route is triggered by mitogens and growth factors while JNK and p38 cascade are stimulated
by stress [94–97]. Preclinical data propose an anti-inflammatory role of JNK and p38 cascades
inhibitors [98,99].
Polyphenols’ activity is specific, it depends on the cell types as well as the structure of the
polyphenol itself [100]. Polyphenols can block TNF α release by modulating MAPK pathway at
different levels of the signaling pathway. Luteolin reduces TNFα liberation by LPS-activated mouse
macrophages, it blocks ERK1/2 and p38phosphorylation [100]. In epithelial cells, luteolin, as well
as other polyphenols such as chrysin and kaempferol block TNFα triggered ICAM-1 expression by
inhibiting ERK, JNK and P38 [100,101]. Quercetin blocks the phosphorylation of ERK, JNK in THP-1
activated human monocytes, while in murine macrophages RAW 246.7 triggered by LPS it blocks the
phosphorylation and the activation of JNK/SAPK (stress activated protein kinases), ERK1/2, and p38
leading to a reduction in the transcription and expression of TNF-α expression [102]. EGCG reduces
inflammation in various cell types by exerting an anti-MAPK activity. It reduces IL-12 expression in
LPS-activated murine macrophages by prohibiting p38 MAPK phosphorylation [89,103]. In addition,
EGCG is found to play a protective role in autoimmune-induced tissue damage caused by Sjogren’s
syndrome: it protects human salivary glands from TNF-α induced cytotoxicity by acting on p38
MAPK1. In vivo, in female ICR mice, EGCG inhibits phorbol ester-induced activation of NFκB and
CREB (cAMP response element-binding protein—a cellular transcription factor) in mouse skin by
blocking the activation of p38 MAPK [104]. Polyphenols concentration plays as well a role in their
modulatory activities on signaling pathways: in human coronary artery endothelial cells, the activation
of the MAPKs pathways (p38, ERK1/2, and JNK) and the repression of the plasminogen activator
inhibitor by catechin and quercetin is time and dose dependent [105]. The ability of polyphenolic
compounds to block MAPK pathways (Figure 1) endowed these bioactive substances with therapeutic
potential to protect against inflammation.

4.3. Arachidonic Acid Signaling Pathway

Arachidonic acid (AA) is liberated by membrane phospholipids upon phospholipase A2 (PLA2)
cleavage. Cyclooxygenase (COX) or lipoxygenase (LOX) metabolize it and produce, respectively,
prostaglandins (PGs) and thromboxane A2 (TXA2) by COX, and hydroxyeicosatetraenoic acids and
leukotrienes (LTs) by LOX [106]. The COX family involves different members (COX1, COX-2, and
COX-3). COX-2 is responsible of the production of important quantity of prostaglandins, its expression
is triggered by lipopolysaccharide and pro-inflammatory cytokines [107]. The ability of polyphenols
to reduce the release of arachidonic acid, prostaglandins, and leukotrienes is considered one of their
most important anti-inflammatory mechanisms (Figure 1). Their action is mainly realized by their
ability to inhibit cellular enzymes, such as PLA2, COX, and LOX [21,108–111]. Quercetin blocks COX
and LOX in various cell types such as rat peritoneal leukocyte, murine leukocytes, and guinea pig
epidermis [110,112,113]. Similarly, red wine reduces COX-2 expression in old male F344 rats [114].
In LPS activated murine macrophages, green tea polyphenols not only suppress NF-κB and MAPK
pathways but also constrain the expression of COX-2 and the release of prostaglandin (PGE2) in
RAW 264.7 macrophages [115,116]. Equally, a reduction in the release of PGE2 is observed with

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other polyphenols, such as kaempferol in culture of LPS-stimulated human whole blood cells [117].
Extra virgin olive oil rich with more than 30 phenolic compounds inhibit 5-LOX in human activated
leukocytes reducing leukotriene B4 and suppresses eicosanoids production by animal and human
cells in vitro [118,119]. Finally, certain polyphenols show structural and functional similarities with
specific anti-inflammatory drugs. A phenolic compound—oleocanthal—demonstrates a natural
anti-inflammatory property and exhibits structural similarities to the ibuprofen (a well-known
anti-inflammatory drug). Oleocanthal—like ibuprofen—inhibits COX-1 and COX-2 activities in a
dose-dependent manner [120].

5. Polyphenols, Oxidative Stress, and Inflammation

Higher production of reactive oxygen species (ROS) is associated with oxidative stress and
protein oxidation [121]. In its turn inflammatory molecules and different inflammatory signals
(i.e., peroxiredoxin2) are triggered by protein oxidations [122]. Furthermore, overproduction of
ROS can prompt tissue injury that might initiates the inflammatory process [123–127]. Therefore,
the classical antioxidant actions of polyphenols undoubtedly contribute to their anti-inflammatory roles
by interrupting the ROS-inflammation cycle (Figure 2). Polyphenols are known for their antioxidant
activities; they scavenge a wide-ranging selection of ROS. Polyphenols can scavenge radicals and
chelate metal ions, for example quercetin chelates iron ion [128]. They also inhibit multiple enzymes
responsible of ROS generation [129]. In fact, free metal ions, as well as highly reactive hydroxyl radical
release, is increased by the formation of ROS. To the opposite, polyphenols are able to chelate metal
ions like Fe2+ , Cu2+ , and free radicals which lead to a reduction of highly oxidizing free radicals [130].

Figure 2. Key polyphenolic anti-oxidant actions in relation to anti-inflammation. Polyphenols scavenge

radicals, chelate metal ions, inhibit ROS production and promote ROS detoxification. On the right
panel ROS contribution to inflammation. ROS: reactive oxygen species; RNS: reactive nitrogen
species; NOX: NADPH oxidase; SOD: superoxide dismutase; GSH-PX: glutathione peroxidase; ERK:
extra-cellular signal regulated kinases; PI3K/AkT: phosphatidylinositide 3-kinases/protein kinase B;
EGCG: epigallactocatechine gallate.

Transition metal ions, like Fe+2 , Cu2+ , Co2+ , Ti3+ , or Cr5+ , results in OH• formation from
H2 O2 [131,132]. Curcumin is able to chelate transition metal (Cu2+ and Fe2+ ) ions. Alike, EGCG
and quercetin chelate Fe2+ (iron ion) [128]. Polyphenols like apocynin, reservatol, and curcumin
can inhibit NOX (NADPH oxidase) causing a reduction in the generation of O2 • during infections
consecutively in endothelial cells in THP1-monocytes [133–135]. Additionally, polyphenols can
attenuate the mitochondrial ATP synthesis by blocking the mitochondrial respiratory chain and
ATPase. As a result, ROS production is diminished. Curcumin [136], EGCG [137], phenolic acids [138],
capsaicin [139], quercetins [140], anthocyanins [140], and resveratrol analogs [141] inhibit xanthine
oxidase. Thus, they reduce ROS production. Polyphenols affect the activity of cyclooxygenase,

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lipoxygenase, and NOS (nitric oxide synthase) as per found in RAW 264.7 macrophes [142]. These
enzymes are known to metabolize arachidonic acid and their inhibition moderates the production
of key mediators of inflammation (prostaglandins, leukotrienes, and NO . . . ) [142]. Polyphenols
can also restrain LPS-induced iNOS gene expression in cultured macrophages, decreasing oxidative
harm [143]. Finally, they may act by upregulating endogenous antioxidant enzymes. In vivo, curcumin
can stimulate antioxidant enzymes like superoxide dismutase (SOD), catalase, and glutathione (GSH)
peroxidase (Px) which lead to ROS detoxification [144]. Likewise, EGCG rises SOD and GSH-Px
activities with augmented amount of cellular glutathione [145]. In conclusion, polyphenols exert
the anti-inflammatory action by different mechanisms: Radical scavenging, metal chelating, NOX
inhibition, tempering the mitochondrial respiratory chain, inhibition of certain enzymes involved in
ROS production, like xanthine oxidase and upregulation of endogenous antioxidant enzymes.

6. Polyphenols, Chronic Diseases and Cancer

Referring to the previously cited roles of polyphenols in maintaining tissue homeostasis by
targeting different signaling pathways and referring to their antioxidant, anti-inflammation, and
protection against pro-inflammation properties; polyphenols play a beneficial role in the prevention
and the process of chronic diseases related to inflammation.
Various polyphenolic compounds show protective actions in diabetes, obesity, neurodegeneration,
cancers, and cardiovascular diseases, among other conditions [30,146–154].

6.1. Polyphenols and Insulin Resistance

Polyphenols reduce insulin resistance. They promote glycolysis by activation of AMPK (AMP-
activated protein kinase) or inhibition of mTORC1 and PI3K/AkT in vivo (in rats), ex vivo (in rats’
muscles strips) and in vitro (in C2C12 myoblasts and HELA cells) [148,149,155,156]. Additionally,
AMPK activation by polyphenols increases glucose uptake by positively affecting eNOS imitating
muscle contraction and in vivo activity of insulin [148–150]. Similarly, it is found that polyphenols
lower insulin resistance by inhibiting PI3K/AkT and JNK of activation of the AMPK-SirT1-PGC1α axis
(i.e., gingerol and anthocyans, and their ability to protect from diabetes and reduce insulin resistance
using in vivo, ex vivo and in vitro studies [26–28,148,149,155]. In addition, polyphenols attenuate
glucose intake from carbohydrates by inhibiting rats’ α-glucosidase [157]. Lastly, polyphenols, like
falvonoids, can improve insulin secretion by reducing apoptosis of pancreatic β–cells [145].

6.2. Polyphenols and Inflammatory Cardiovascular Diseases (CVD)

Meta-analysis studies have reported that an intake of three cups of tea per day reduces CVD by
11% [151] while adequate intake of red wine is associated with 32% lower risk of cardiovascular disease
(CVD) [158]. Soy and cocoa flavonoids contribute to the prevention of CVD as per meta-analysis
of randomized controls trial [159]. Polyphenols exert their protective effects in CVD due to their
anti-hypertensive potentials. Resveratrol inhibits ACE (angiotensin converting enzyme) and PDE
(phosphodiesterase) and upregulates eNOS (endothelial NOS) resulting in a reduction in high blood
pressure as per multiple in vivo and in vitro studies [26–28,155,156,160]. In addition, flavanols and
flavonols exert their CVD prevention role by reducing the manifestations of age-related vascular injury.
They reduce nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by affecting MAPK
signaling and downregulating NF-κB in aged rats [161–163]. At the end, the antioxidant action of
polyphenols and their ability to suppress LDL oxidation leads to endothelium-protective activity [164].
Certain polyphenols like resveratrol and anthocyanins protect from CVD by multiple mechanisms;
they have (1) antihypertensive properties, they inhibit eNOS, and (2) inhibit NFκB mediated expression
of VCAM and ICAM expression as per previously discussed [39,165]. Polyphenols can also reduce
LDL oxidation or improve LDL/HDL ratio. For example, flavanones such as hesperetin in orange
juice reduce LDL/HDL ratio while quercetin inhibits LDL oxidation with elevated paraoxonase and
eliminate atherogenic lesions referring to in vitro and in vivo studies (using human male subjects) [166].

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6.3. Polyphenols and Inflammatory Neurological Diseases

Polyphenols show protective effects in neurological disease [152,153]. High flavonoid intake
can reduce by 50% dementia and aging. More precisely, it lowers the incidence of Parkinson’s and
delays the onset of Alzheimer’s disease as per different epidemiological studies [167–170]. EGCG
has neuroprotective properties due to its antioxidant (SOD, GSHPx) activities and cellular GSH
contents and ability to reduce ROS contents. Similarly, anthocyanins neuroprotective characteristics
are related to the improvement of oxidative stress and reduction of Aβ deposition [38,171,172]. Other
mechanisms of polyphenols protection in neurodegenerative diseases is modulation of neuronal and
glial signaling pathways [173]. Polyphenols can downregulate NF-κB related with iNOS generation
in glial cells [174–176]. Moreover, their ability to inhibit monoamine oxidase plays a positive role in
cognition, depression, and learning ability in vivo in male laca mice [172].

6.4. Polyphenols and Inflammatory Obesity

Polyphenols exert their anti-obesity effect by activation of AMPK (5’ adenosine
monophosphate-activated protein kinase) leading to a reduction of cholesterol, fatty acid synthesis, and
triglyceride formation by inhibiting HMG-CoA reductase and acetyl CoA carboxylase. Furthermore,
they can inhibit genes involved in adipocyte differentiation and triglyceride accumulation. They
block mTORC1 and repress specific signals associated with diminished levels of PPARγ and C/EBP
α/δ mRNA throughout adipogenesis (in an experimental model of sepsis) and in vitro [30,146].
They can improve energy expenditure, stop the maturation of preadipocytes into adipocytes and
increase the expression of adiponectin (a hormonal protein with a role in regulating glucose levels and
breaking down fatty acids). For example, capsaicin enhances the energy spending in adipose tissue.
Capsaicin diminishes intracellular triglycerides and improves brown adipose tissue thermogenesis.
Furthermore, in clinical studies, capsaicin is found able to increase satiety [30,177,178]. EGCG
inhibits MEK/ERK and PI3K/AKT pathways leading to inactivation of preadipocytes maturation by
downregulating the expression of different genes like PPARγ and C/EBPα that are associated with
adipogenesis [38,41,146,179]. Certain polyphenols can increase adiponectin such as gingerol and
curcumin in serum of human subjects based on randomized controlled trial [180,181].

6.5. Polyphenols and Cancer

Clinical and epidemiological studies have reported that polyphenols have chemo-preventive
and anticancer efficacy [182–184]. Polyphenol compounds have the ability to inhibit the
proliferation of different types of cancer such as prostate, bladder, lung, gastrointestinal, breast,
and ovarian cancers [154]. For instance, quercetin, resveratrol, green tea polyphenols [185],
epigallocatechin-3-gallate [186], and curcumin [187] have demonstrated efficacy as anticancer
compounds. Several studies reported that polyphenols are able to prevent cancer initiation
(cyto-protective), progression, recurrence, and metastasis to distant organs (cytotoxic) as per different
epidemiological, in vitro, and in vivo studie [188–190]. However, a dichotomy exists between
polyphenols’ antioxidant effects in normal cells, and their potential pro-oxidant effects in cancer
cells [154,188].
Recent studies illustrated a direct correlation between ROS in intracellular signaling cascade
and carcinogenesis [191]. Oxidative stress targets proteins, lipids, and DNA/RNA causing changes
that increase the risks of mutagenesis. ROS/RNS (reactive nitrogen species) overproduction over a
prolonged period of time damages cellular structure and functions and causes somatic mutations
such as pre-neoplastic and neoplastic transformations that may lead to cell death by necrotic and
apoptotic processes [192]. Polyphenols compounds contain hydroxyl groups that donate their protons
to reactive oxygen species (ROS) [193]. Moreover, they reduce the activity of phase I enzymes, primarily
cytochrome P450 enzymes (CYPs), such as CYP1A1 and CYP1B1 which lead to prevent the formation
of reactive and carcinogenic metabolites in human bronchial epithelial cells [194]. They also can induce

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phase II enzymes that initiate the formation of polar metabolites which are readily excreted from the
body [195]. Certain dietary polyphenols such as flavonoids reduce cellular formation of ROS which
protects from the oxidation of DNA [193].
In addition to their anti-oxidant properties, pro-oxidant characteristic of polyphenols is important
in treating and preventing cancer. Pro-oxidant activity can be initiated by certain conditions such
as superoxide leakage [196]. The pro-oxidant activities of polyphenols in cancer cells can result in
inducing apoptosis [197], cell cycle arrest [198] and inhibiting the proliferation signaling pathways
(i.e., epidermal growth factor receptor/mitogen activated protein kinase, phosphatidylinositide
3-kinases/protein kinase B, as well as NF-kB) [199]. For example, polyphenols from apple are able
to inhibit the proliferation of human bladder transitional cell carcinoma (TCC, TSGH-8301 cells),
inducing G2/M cell cycle arrest, and promoting apoptosis [200]. In human papilloma virus-18-positive
HeLa cervical cancer cells, green tea polyphenols can induce cell cycle arrest at the subG1 phase and
apoptosis through caspases activation [201]. Flavonoids, such as quercetin, induce apoptosis in many
cancer cells such as leukemic U937 cell [202], prostate cancer cells [203], hepatic cancer cells [204],
among other types. A combination of quercetin with resveratrol and catechin inhibits breast cancer
progression in vitro and in vivo by inducing apoptosis in carcinogenic breast cells [205]. In addition,
polyphenols can reduce cancer metastasis such as quercetin [206,207].
Sufficient studies have reported that NF-κB signaling pathways are closely related to cancer
metastasis. Polyphenols can disrupt the metastatic potential of cancer by inhibiting NF-κB activity [208].
Curcumin is a good example [209–211] of decreasing cancer metastasis in mice by suppressing
NF-κB expression and down-regulating VEGF (vascular endothelial growth factor), COX-2, and
MMP-9 (matrix metallopeptidase-9) expression in tissues of the breast, brain, lung, liver, and
spleen [212,213]. Moreover, the strength of metastasis is associated to the epithelial-to-mesenchymal
transition (EMT) [214]. There is robust evidence that polyphenols compounds can modulate EMT
and its related signaling pathways [215]. For example, EGCG, a flavan-3-ol, induces apoptosis and
significantly reduces colony formation and cell migration in nasopharyngeal carcinoma (NPC) and
cancer stem cells (CSC) in different cell lines [216]. Luteolin and quercetin reverse the migration and
invasiveness of metastatic cells by reducing the expression of mesenchymal markers and transcriptional
factors on the cell membrane (i.e., twist, snail, and N-cadherin) and upregulating adhesion molecules
such as E-cadherin [217]. Thus, through variable mechanisms, polyphenols broadly downregulate
inflammation origination, progression, and evolution to cancers (Figure 3).

Figure 3. Anti-tumorigenic activities of polyphenols. MAPK: mitogen-activated protein kinase; NFκB:

nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K: phosphatidylinositide 3-kinase;
ERK: extracellular signal-related kinases; ROS: reactive oxygen species; COX: cyclooxygenase; EMT:
epithelial mesenchymal transition; HIF-1α: hypoxia-inducible factor 1-aplha.

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In order to emphasize on the beneficial health effects of polyphenols, different medications

containing polyphenols are FDA-approved as pharmaceutical drugs. Polyphenon® E, a standardized
green tea polyphenol preparation, is an FDA-approved medication to treat genital warts [218]. Another
significant event in the use of polyphenols as pharmaceuticals is the FDA approval of crofelemer
(a medication rich in oligomeric proanthocyanidin) to manage HIV associated non-infectious diarrhea.

7. Conclusions
In conclusion, the vast number of published studies proved the immunomodulatory role of
polyphenols in vivo and in vitro. Different underlying regulatory mechanisms are now well elucidated.
These data highlight the promising role of polyphenols in prevention and therapy of diseases with
underlining inflammatory conditions, including cancer, neurodegenerative diseases, obesity, type
II diabetes, and cardiovascular diseases. However, the role of polyphenols in modulating multiple
inflammatory cellular pathways should be further investigated. Many questions remain unanswered
about the usage of polyphenols in clinical setting. The role of the microbiota in degrading these
polyphenols should be further studied. The notion of bioavailability and its impact on biofunctionality
should also be revisited. It is generally believed that polyphenol activity is principally located in the gut
where their immunoprotective and anti-inflammatory activities are initiated and subsequently ensuring
systemic anti-inflammatory effects. Since different polyphenols can have multiple intracellular
targets, additional data is needed to determine the consequences of the interaction or the synergistic
effects between multiple polyphenolic compounds or polyphenols and commonly used medications.
Moreover, further in vivo and meta-analysis studies in humans are necessary to fully reveal the
mechanisms of action of polyphenols in several physiological conditions in order to produce important
insights into their prophylactic and therapeutic uses.

Author Contributions: N.Y. wrote all parts of the paper and prepared all Figures as well as the graphabstract
except cancer and polyphenol paragraph and Figure 3; N.A. wrote cancer and polyphenol paragraph and prepared
Figure 3; M.J. collected some papers and revised the Figures and their design; C.M. revised and guided the work.
Funding: This research received no external funding.
Acknowledgments: Special thanks to uOttawa libraries especially health sciences library and Morisset library
and the department of “Nutrition Sciences”.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
ROS Reactive oxygen species
COX Cyclooxygenase
NOX NADPH oxidase
SOD Superoxide dismutase
GSH Glutathione
Px Peroxidase
PLA2 Phospholipase A2
PGs Prostaglandins
LTs Leukotrienes
MAPK Mitogen-activated protein Kinase
IKK Inhibitor of kappa kinase
NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells
Th1 T helper 1
NK Natural killer
DCs Dendritic cells
ECGC Epigallocatechin gallate
Treg Regulatory T cells

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AhR Aryl hydrocarbon receptor

iNOS Inducible nitric oxide synthase
LPS Lipopolysaccharide
MCP-1 Monocyte chemoattractant protein-1
VEGF Vascular endothelial growth Factor
IL Interleukin
JNK c-Jun amino-terminal kinases
CREB cAMP response element-binding protein
AA Arachidonic acid
AMPK AMP-activated protein kinase
ERK Extra-cellular signal regulated kinases
ATP Adenosine triphosphate
CVD Cardiovascular disease
PI3K Phosphatidylinositide 3-kinases
Akt Protein kinase B
LDL Low density lipoprotein
HDL High density lipoprotein
RNS Reactive nitrogen species
PPARγ Peroxisome proliferator-activated receptor gamma
CYPs Cytochrome P450 enzymes
MMP-9 Matrix metallopeptidase-9
NPC Nasopharyngeal carcinoma
CSCs Cancer stem cells
EMT Epithelial-to-mesenchymal transition
FDA Food and drug administration
ICAM Intercellular adhesion molecule
VCAM Vascular cell adhesion molecule
HIV Human immunodeficiency diarrhea
HMG-CoA 3-Hydroxy-3-methyl-glutaryl-coenzyme A
VEGF Vascular endothelial growth factor
TLR Toll-like receptor
NO Nitric oxide
eNOS Endothelial nitric oxide synthase
PDE Phosphodiesterase
ACE Angiotensin converting enzyme

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 nutrients
Review
The Role of Vitamin E in Immunity
Ga Young Lee 1,2 and Sung Nim Han 1,2, *
1 Department of Food and Nutrition, College of Human Ecology, Seoul National University,
Seoul 08826, Korea; [email protected]
2 Research Institute of Human Ecology, Seoul National University, Seoul 08826, Korea
* Correspondence: [email protected]; Tel.: +82-2-880-6836

Received: 30 September 2018; Accepted: 29 October 2018; Published: 1 November 2018

Abstract: Vitamin E is a fat-soluble antioxidant that can protect the polyunsaturated fatty acids
(PUFAs) in the membrane from oxidation, regulate the production of reactive oxygen species (ROS)
and reactive nitrogen species (RNS), and modulate signal transduction. Immunomodulatory effects
of vitamin E have been observed in animal and human models under normal and disease conditions.
With advances in understating of the development, function, and regulation of dendritic cells (DCs),
macrophages, natural killer (NK) cells, T cells, and B cells, recent studies have focused on vitamin E’s
effects on specific immune cells. This review will summarize the immunological changes observed
with vitamin E intervention in animals and humans, and then describe the cell-specific effects of
vitamin E in order to understand the mechanisms of immunomodulation and implications of vitamin
E for immunological diseases.

Keywords: vitamin E; macrophages; T cells; dendritic cells; immunomodulation; infection

1. Vitamin E: Definition, Structure, Sources, and Functions

1.1. Definition and Structure

Vitamin E is the collective term for four tocopherols (α-, β-, γ-, and δ-tocopherols) and four
tocotrienols (α-, β-, γ-, and δ-tocotrienols) found in food. These forms have antioxidant activities,
but cannot be interconverted, and only α-tocopherol meets the human vitamin E requirement [1].
Tocopherols have a chromanol ring and a phytyl tail, while tocotrienols have a chromanol ring and an
unsaturated tail. The α-, β-, γ-, and δ- forms differ in the number and position of methyl groups on the
chromanol structure. Natural tocopherols have only RRR stereochemistry, but synthetic tocopherols
are mixtures of eight stereoisomers (RRR-, RSR-, RRS-, RSS-, SRR-, SSR-, SRS-, SSS-), because there are
three asymmetric carbon atoms (2R, 4’R, 8’R) present in the phytyl tail. The structures of tocopherols
and tocotrienols are shown in Figure 1.

Nutrients 2018, 10, 1614; doi:10.3390/nu10111614 115 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1614

Figure 1. The structures of tocopherol and tocotrienols.

1.2. Sources
The major dietary sources of vitamin E are vegetable oils. Nuts are good sources of vitamin
E as well [2]. Soybean, sunflower, corn, walnut, cottonseed, palm, and wheat germ oils contain
relatively higher amounts (more than approximately 50 mg vitamin E/100 g oil) of vitamin E than
other oils. The proportions of α-, β-, γ-, and δ-tocopherols vary depending on the oil type. Safflower
and sunflower oils are high in α-tocopherol, soybean and corn oils contain mainly γ-tocopherol,
and cottonseed oil contains similar proportions of α- and γ-tocopherols. Therefore, the types of oils
consumed through the diet affect the dietary intake levels of α-tocopherol. Vitamin E supplements are
quite popular and contribute considerably to vitamin E intake among some populations. Either natural
or synthetic forms of α-tocopherol are used as supplements.
Despite the relatively higher intake of γ-tocopherol from the diet than α-tocopherol, α-tocopherol
is the major form of vitamin E in the circulation because α-tocopherol transfer protein (α-TTP) has the
preferential binding affinity for α-tocopherol. α-TTP is involved in the transfer of α-tocopherol to the
plasma membrane [1].

1.3. Functions
Vitamin E is a major fat-soluble antioxidant that scavenges peroxyl radicals and terminates the
oxidation of polyunsaturated fatty acids (PUFAs). In the presence of vitamin E, peroxyl radicals react
with α-tocopherol instead of lipid hydroperoxide, the chain reaction of peroxyl radical production
is stopped, and further oxidation of PUFAs in the membrane is prevented [1]. Tocopheroxyl
radicals—produced from α-tocopherol and peroxyl radicals—are reduced by vitamin C or glutathione,
form tocopherol dimers, undergo further oxidation, or act as prooxidants. The antioxidant activity of
vitamin E may be responsible for the regulation of several enzymes involved in signal transduction
because the activity of signaling enzymes is regulated by the redox state.
Vitamin E inhibits protein kinase C (PKC) activity by increasing PKC-α dephosphorylation
through the activation of protein phosphatase 2A. The inhibition of PKC by vitamin E has been
reported in various cells, and consequently, the inhibition of platelet aggregation; reduced proliferation
of monocytes, macrophages, neutrophils, and vascular smooth muscle cells; and decreased superoxide
production in neutrophils and macrophages have been observed [3,4].
Vitamin E may directly bind to the enzymes involved in the generation of lipid mediators or to
the transport proteins involved in signal transduction. Vitamin E may affect the membrane protein

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Nutrients 2018, 10, 1614

interaction and translocation of the enzymes to the plasma membrane and therefore change the activity
of signal transduction enzymes [4].

2. Modulation of Immune Responses and Infectious Diseases by Vitamin E Supplementation

2.1. Immune Responses in Animals

Dietary interventions of vitamin E at supplemental levels have been shown to enhance
cell-mediated and humoral immune responses in various species of animals. Increased lymphocyte
proliferation, immunoglobulin levels, antibody responses, natural killer (NK) cell activity,
and interleukin (IL)-2 production have been reported with vitamin E supplementation (Table 1).

2.2. Immune Responses in Humans

In humans, many intervention studies have reported increased lymphocyte proliferation
in response to mitogenic stimulation, enhanced delayed type hypersensitivity (DTH) response,
increased IL-2 production, and decreased IL-6 production with vitamin E supplementation above
the recommended levels. However, some studies showed no difference or decreased lymphocyte
proliferation responses and decreased chemiluminescence. (Table 2). Differences in dose of vitamin E
supplementation used, magnitude of vitamin E level changes with supplementation, age of subjects,
and methodology (determination of antibody levels with or without specific vaccination) might have
contributed to the different results observed.

2.3. Infectious Diseases in Animals

The immunostimulatory effect of vitamin E has resulted in enhanced resistance against several
pathogens. Animal studies in which infectious disease models were used to test the effects of vitamin
E supplementation are listed in Table 3.
The mechanisms involved with protection against infectious agents were increased macrophage
activity and antibody (Ab) production for D. pneumoniae type 1 [5], and higher NK activity and Th1
response for influenza virus [6,7].

2.4. Infectious Diseases in Humans

In humans, the effects of vitamin E on the natural incidence of infectious diseases have been
determined in several studies (Table 4). Many studies provided evidence that the immunostimulatory
effects of vitamin E confer improved resistance to infections. However, the magnitudes of the effects
were rather small, and in some studies, positive effects were only observed in subgroups of subjects.

117
 Table 1. Modulation of immune responses by vitamin E in animal models.

Species Dosage and Duration Form of Vitamin E Used Results References

Chicks, female broiler (n = 100 mg/kg diet for 21 ↑Plasma IgM levels at day 21
DL -α-tocopheryl acetate Dalia et al. 2018 [8]
6/group, 6 replicate) days ↔Splenic expressions of TNF-α, IFN-γ, IL-2,
IL-10
250 IU/day from day 107
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Pregnant cows (n = 24/group) of gestation to day 21 of NA ↑IgG and IgA concentration in sow plasma Wang et al. 2017 [9]
lactation
Domestic cats (39 castrated male
225, 450 mg/kg diet for
and 33 intact female) (n = α-tocopherol ↑Lymphocyte proliferation (ConA, PHA) O’ Brien et al. 2015 [10]
28 days
8/group)
Young and old mice 500 mg/kg diet for 6 ↑Lymphocyte proliferation in old (ConA, PHA)
DL -α-tocotrienol Ren et al. 2010 [11]
(n = 11–13/group) weeks ↑IL-1β production in young
50, 200 mg/kg diet for
Young rats (n = 6/group) ↑Lymphocyte proliferation (ConA, LPS) Bendich et al. 1986 [12]
8–10 weeks
↑Lymphocyte proliferation (ConA, LPS)
500 mg/kg diet for 6 ↑DTH response
Old mice (n = 10/group) DL -α-tocopheryl acetate Meydani et al. 1986 [13]
weeks ↑IL-2 production
↓PGE2 production

118
↑Lymphocyte proliferation (ConA) in young
Young and old mice (n = 5/group) 500 IU (500 mg) for 9 DL -α-tocopherol acetate ↔Lymphocyte proliferation (ConA) in old Wakikawa et al. 1999 [14]
weeks
↑IFN-γ in young under restraint stress

50, 100, 250, 500, ↑Lymphocyte proliferation (>100 mg/kg diet,

Moriguchi et al. 1990
Young rats (n = 10/group) 2500 mg/kg diet for 7 DL -α-tocopheryl acetate ConA) (>250 mg/kg diet, LPS)
[15]
days ↑NK activity (>250 mg/kg diet)
585 mg/kg diet for 12 DL -α-tocopheryl ↑Lymphocyte proliferation (ConA, PHA) Sakai S & Moriguchi
Old rats (n = 5/group)
months nicotinate ↑IL-2 production 1997 [16]

125, 250, 500 IU (125, 250, ↑Lymphocyte proliferation (PHA, ConA,

Young calves (n = 8/group) DL -α-tocopheryl acetate pokeweed mitogen) Reddy et al. 1987 [17]
500 mg)/day for 24
weeks ↑Antibovine herpesvirus Ab titer to booster in
125 IU/day group
200 mg/kg diet for ↑Ab response
Young mice (n = 8/group) α-tocopheryl acetate Tanake et al. 1979 [18]
6–12 weeks ↑Helper T cell activity
↓IL-6 and PGEs (unstimulated) production by
500 mg/kg diet for 6 α-tocopherol acetate
Mice (n = 10/group) macrophages Beharka et al. 2000 [19]
months (Tekland, Madison, WI)
↓Nitric oxide production (LPS) by
macrophages
Ab, antibody; ConA, concanavalin A; IFN-γ, interferon-γ; LPS, lipopolysaccharide; PGE2 , prostaglandin E2 ; PHA, phytohemagglutinin; TNF, Tumor necrosis factor.
 Table 2. Modulation of immune responses by vitamin E in humans.

Form of Vitamin E
Subjects Age Amount and Duration of Supplementation Effects on Immune Function References
Used
Young (n = 5) and senior 4.6 ± 0.3 mg/100 mL of vitamin E-enriched
18–25, 35–57 α-tocopherol acetate ↑15LOX2, TNF-α expression Capo et al. 2016 [20]
athletes (n = 5) beverage 5 days/week for 5 weeks
D -α-tocotrienol
↑IL-4 (TT vaccine), IFN-γ (ConA)
D -γ-tocotrienol Mahalingam et al. 2011
Healthy women (n = 108) 400 mg TRF/day for 56 days
Nutrients 2018, 10, 1614

18–25
[21]
D -δ-tocotrienol
↓IL-6 (LPS)
D -α-tocopherol

Healthy men and women Radhakrishnan et al. 2009

20–50 200 mg/day for 56 days α-tocopherol ↔IL-4, IFN-γ production (ConA)
(n = 19, 34) [22]
↓Lymphocyte proliferation (PHA)
Adult males and young boys DL -α-tocopheryl
25–30, 13–18 300 mg/day for 3 weeks ↔DTH Prasad 1980 [23]
(n = 18) acetate
↓Bactericidal activity
Institutionalized adult males Harman and Miller 1986
24–104 200, 400 mg/day for 6 months α-tocopherol acetate ↔Ab development to influenza virus
and females (n = 103) [24]
↑Lymphocyte proliferation (ConA)
Healthy elderly males and DL -α-tocopheryl ↑DTH
≥60 800 mg/day for 30 days Meydani et al. 1990 [25]
females (n = 32) acetate ↑IL-2 production (ConA)
↓PGE2 production (PHA)
Eldery males and females DL -α-tocopheryl ↔Lymphocyte proliferation (ConA, PHA)
≥65 100 mg/day for 3 months De Waart et al. 1997 [26]
(n = 74) acetate ↔IgG, IgA levels

119
Healthy elderly males and
≥65 60, 200, 800 mg/day for 235 days DL -α-tocopherol ↑DTH and antibody titer to hepatitis B with 200, 800 mg Meydani et al. 1997 [27]
females (n = 88)
↑No. of positive DTH reaction with 100 mg
Healthy elderly males and DL -α-tocopheryl ↑dDiameter of induration of DTH reaction in a
65–80 50, 100 mg/day for 6 months Pallast et al. 1999 [28]
females (n = 161) acetate subgroup supplemented with 100 mg
↔IL-2 production
↓IFN-γ production
Healthy young adults
600 mg/day for 3 months40 mg/kg body
(n = 31) and premature 24–31 ↓Chemiluminescence Okano et al. 1990 [29]
weight for 8–14 days
infants (n = 10)
Cigarette smoker (n = 60) 33 ± 4 900 IU/day for 6 weeks ↓Chemiluminescence Richards et al. 1990 [30]
Prevented fish-oil-induced suppression of ConA
Healthy males (n = 40) 24–57 200 mg/day for 4 months all-rac-α-tocopherol Kramer et al. 1991 [31]
mitogenesis
↑DTH (maximal diameter) in 100, 200, 400 mg groups
Healthy elderly (n = 40) >65 100, 200, or 400 mg/day for 3 months DL -α-tocopherol Wu et al. 2006 [32]
↑Lymphocyte proliferation (ConA) in 200 mg group
Sedentary young and ↓IL-6 secretion
22–29, 55–74 800 IU (727 mg)/day for 48 days DL -α-tocopherol Cannon et al. 1991 [33]
elderly males (n = 21) ↓Exercise-enhanced IL-1β secretion
ConA, concanavalin A; DTH, delayed type hypersensitivity; IFN-γ, interferon-γ; 15LOX2, 15-lipoxygenase-2; PGE2 , prostaglandin E2 ; PHA, phytohemagglutinin; TRF, tocotrienol-rich
fraction; TT vaccine, tetanous toxoid vaccine.
 Table 3. Effects of vitamin E supplementation on infectious diseases in animal models.

Dose and Duration of Form of Vitamin Infection Organism and Results: Effects of Vitamin E
Subjects Age References
Supplementation E Used Route of Infection Supplementation
Mice BALB/c 100 mg/kg for 8 days before MRSA, inoculated onto Higher NK cytotoxicity
6 months δ-, γ-Tocotrienol Pierpaoli et al. 2017 [34]
(n = 3–6/group) MRSA-challenge superficial surgical wounds Higher IL-24 mRNA expression levels

Young and aged 1000-fold fewer bacteria in their lung

Age-associated higher production of
Nutrients 2018, 10, 1614

male mice 2, 22–26 500 mg/kg for 4 weeks prior to D -α-tocopheryl Streptococcus pneumoniae,
months infection proinflammatory cytokines (TNF-, IL-6) Bou Ghanem et al. 2015 [35]
C57BL/6 acetate intra-tracheally injected
(n = 6/group) were reduced
3-fold reduction in the number of PMNs
No difference in serum IgG or peripheral
Worm-free lambs 5.3 IU (3.56 mg)/kg BW for H. contortus L3 larvae, route
28–32 weeks D -α-tocopherol mRNA expression of IL-4 or IFN-γ De Wolf et al. 2014 [36]
(n =10/group) 12 weeks NA
Lower PCV, FEC, and worm burden

Deficient, Adequate Higher viral titre and ILβ, TNF-α,

Male mice RANTES in the brain with E deficiency
BALB/c At weaning (38.4 mg/kg diet), or DL -α-tocopheryl
HSV-1, intranasally No difference in expressions of IL-6, Sheridan & Beck. 2008 [37]
(n = 6–7/group) Supplemented (384 mg/kg acetate
diet) for 4 weeks TNFα, IL-1β, and IL-10 between
adequate and supplemented
Mice C57BL DL -α-tocopherol Influenza by nasal Lower viral titer
22 months 500 mg/kg diet for 8 weeks Han et al. 2000 [6]
(n = 6–9/group) acetate inoculation Higher IL-2 and IFN-γ production
Influenza A/PC/1/73
Mice, C57BL/6 DL -α-tocopherol

120
22 months 500mg/kg diet for 6 weeks (H3N2) by nasal Lower viral titre Hayek et al. 1997 [7]
(n = 4–9) acetate
inoculation
Mice, C57BL/6 160 IU/L liquid diet for 4, 8, 12, all-rac-α-tocopheryl Murine LP-BM5 leukaemia Restored IL-2 and IFN-γ production by
5 weeks Wang et al. 1994 [38]
(n = 6) 16 weeks acetate retrovirus by IP injection splenocytes following infection
1400 or 2800 mg orally once per Serum from vitamin E-supplemented
Calves, Holstein DL -α-tocopheryl Bovine rhinotracheitis
1d week, 1400 mg injection once calves inhibited the replication of bovine Reddy et al. 1986 [39]
(n = 7) acetate virus, in vitro
per week for 12 weeks rhinotracheitis virus in vitro
Mice, Swiss DL -α-tocopheryl Diplococcus pneumoniae type
4 weeks 180 mg/kg diet for 4 weeks Higher survival Heinzerling et al. 1974a [5]
Webster (n = 10) acetate I by IP injection
25 or 250 mg/kg bw orally for Pseudomonas aeruginosa,
Mice, BALB/C DL -α-tocopheryl
NA 4 days, starting 2 days before subeschar injection to Lower mortality rate Fang et al. 1990 [40]
(n = 25) acetate
burn injury burned mice
Vitamin E
Mice, BALB/C 4000mg/kg diet for 2, 4, or Listeria monocytogenes by IP
3 weeks injectable No difference in resistance Watson & Petro 1982 [41]
(NA) 14 weeks injection
(aqueous)
Rats,
180 mg/kg diet + 6000 IU DL -α-tocopheryl Mycoplasma pulmonis by
Sprague-Dawley 3 weeks Higher resistance to infection Tvedten et al. 1973 [42]
vitamin A/kg diet for 6 weeks acetate aerosol
(n = 6)
1000 IU orally, 300 mg/kg diet DL -α-tocopheryl Chlamydia by intratracheal Faster recovery (higher food intake and
Lambs (n = 10) NA Stephens et al. 1979 [43]
for 23 days acetate inoculation weight gains)
 Table 3. Cont.

Dose and Duration of Form of Vitamin Infection Organism and Results: Effects of Vitamin E
Subjects Age References
Supplementation E Used Route of Infection Supplementation

Turkey, No effect on mortality by vitamin E

500 mg/kg diet for 14 days
broadbreasted DL -α-tocopheryl supplementation alone
1 day before infection and 18–21 days Histomonas meleagridis, oral Schildknecht & Squibb 1979 [44]
white poults acetate Lower mortality and lesion score in
after infection
(n = 6) combination with ipronidazole
Nutrients 2018, 10, 1614

200 mg/pig per day for 59 days Improved weight gain and recovery rate
DL -α-tocopheryl Treponema hyodysenteriae,
Pigs (n = 6) NA before infection and 22 days No beneficial effect on appetite and Teige et al. 1982 [45]
after infection acetate oral
diarrhoea

Clostridium perfringens type Higher Ab titre

Sheep (n = 12) 300 mg/kg diet starting 2 DL -α-tocopheryl Fail to prove beneficial effect of vitamin Tengerdy et al. 1983 [46]
3–6 months D by IV injection after two
weeks before first vaccination acetate E on protection (none of the vaccinated
IM vaccinations
lambs died)
Natural occurrence of
clinical mastitis due to
740 mg/cow per day, duration DL -α-tocopheryl
Cows (n = 20) NA Streptococci, Coliform, Lower clinical cases of mastitis Smith et al. 1984 [47]
NA acetate
Staphylococci, Clostridium
bovis
Chicks, broiler 150 mg or 300mg/kg diet for 2 DL -α-tocopheryl Escherichia coli, orally and Lower mortality
1day Heinzerling et al. 1974b [48]
(n = 12–14) weeks before infection acetate post-thoracic air sac Higher Ab titre

121
300 mg/kg diet for 6 weeks,
Chicks, broiler DL -α-tocopheryl
1 day starting 3 weeks before first E. coli, post-thoracic air sac Lower mortality Tengerdy & Nockels 1975 [49]
(n = 10) acetate
infection
Chicks, Leghorn 300 mg/kg diet for 4 weeks DL -α-tocopheryl
1 day E. coli by IV injection Lower mortality Likoff et al. 1981 [50]
(n = 22) before infection acetate
Vitamin E;
100, 000 mg/t diet for 10 weeks,
Tompson-Hayward,
Pigs (n = 10) 6–8 weeks starting 2 weeks before E. coli by IM injection Higher serum Ab titre Ellis & Vorhies 1976 [51]
Minneapolis, MN,
infection
USA
Ab, antibody; FEC, fecal egg count; HSV, Herpes simplex virus; MRSA, IFN-γ, interferon-γ; IM, intramuscular; IV, intravenous; Methicillin-resistant Staphylococcus aureus; NK, natural
killer; PCV, packed cell volume; PMN, polymorphonuclear leukocyte, RANTES, regulated on activation, normal T cell expressed and secreted; TNF-α, tumor necrosis factor-α.
 Table 4. Effects of vitamin E supplementation on infectious diseases in humans.

Dose and Duration of Form of Vitamin Infection Organism and Route of Results: Effects of Vitamin E
Subjects Age References
Supplementation E Used Infection Supplementation
69% Lower incidence of pneumonia
among subgroups including participants
who smoked 5–19 cigarettes per day at
DL -α-tocopheryl
Male smoker 50–69 50 mg/d for median of 6 years Natural incidence of pneumonia baseline and exercised at leisure time Hemila et al. 2016 [52]
Nutrients 2018, 10, 1614

acetate
14% Lower incidence of pneumonia
among subgroups including participants
who smoked ≥20 cigarettes per day at
baseline and did not exercise
30 mg during pregnancy
HIV-infected (multivitamin form with 20 mg Natural incidence of malaria after Lower incidence of presumptive clinical
pregnant 25.4 vitamins B1, 20 mg B2, 25 mg B6, 100 NA having received malaria malaria, but higher risk of any malaria Olofin et al. 2014 [53]
Tanzanian women mg niacin, 50 μg B12, 500 mg C, prophylaxis during pregnancy parasitemia
and 800 μg folic acid)
Reduced glutathione (GSH) and
Patients with glutathione peroxidase, which are
900 IU (604.03 mg for D - or 818.18
HCV-related 54–75 α-tocopherol Natural incidence of cirrhosis significantly lower in cirrhotic patients Marotta et al. 2007 [54]
mg for DL-)/day for 6 months
cirrhosis (p < 0.05), were comparably improved by
vitamin E regimens
945 IU (634.23 mg)/day for 6 months
Patients with No difference in median log plasma Groenbak et al. 2006

122
18–75 with 500 mg ascorbic acid and 200 μg D -α-tocopherol Natural incidence of HCV
chronic HCV HCV-RNA [55]
of selenium
Fewer numbers of subjects with all and
Nursing home Natural incidence of respiratory upper respiratory infections
>65 200 IU/day for 1 year DL -α-tocopherol Meydani et al. 2004 [56]
residents infections Lower incidence of common cold
No effect on lower respiratory infection
Natural incidence of common cold Lower incidence of common cold
Male smokers 50–69 50 mg/day during 4-year follow-up α-tocopherol Hemila et al. 2002 [57]
episodes Reduction was greatest among older city
dwellers who smoked fewer than 15
cigarettes per day
No overall effect on the incidence of
DL -α-tocopheryl
Male smokers 50–69 years 50 mg/day for median of 6.1 years Natural incidence of pneumonia pneumonia. Hemila et al. 2004 [58]
acetate
Lower incidence of pneumonia among
the subjects who had initiated smoking
at a later age (>21)
Natural incidence and severity of
Non-institutionalized α-tocopherol No effect on incidence and severity of
>60 years 200 mg/day for median of 441 days self-reported acute respiratory tract Graat et al. 2002 [59]
individuals acetate acute respiratory tract infections
infections
HCV, hepatitis C virus.
Nutrients 2018, 10, 1614

3. Vitamin E and Immune Cells

The immunomodulatory mechanisms of α-tocopherol in immune cells are depicted in Figure 2.

Figure 2. Immunomodulatory effects of vitamin E on immune cells. Abbreviations: PGE2 ,

prostaglandin E2 ; COX2, Cyclooxygenase 2; NO, Nitric oxide; CD, Clusters of Differentiation; DCs,
Dendritic cells; IL-12, Interleukin-12; Ab, antibody; NK, Natural killer.

3.1. Macrophages
Macrophages, important effector cells in the innate immune response, serve as antigen presenting
cells (APC) and regulate NK cells and T cells by producing cytokines, reactive oxygen species (ROS),
reactive nitrogen species (RNS), and prostaglandins. Cytokines produced by T cells and other immune
cells can shift the macrophages into different populations with distinct physiologies [60].
The effects of vitamin E on prostaglandin (PG)E2 production by macrophages from the aged
have been suggested as one of the mechanisms by which vitamin E improves the age-associated
decrease in the T cell-mediated immune response [61]. In a co-culture experiment in which purified
T cells and macrophages from young and old mice were cultured together, T cells from young mice
showed suppressed proliferation and IL-2 secretion when cultured with macrophages from old mice.
When macrophages from old mice were pre-incubated with 10 μg/mL vitamin E for 4 h, co-cultures
of old macrophages and young T cells showed significant improvement in proliferation. Vitamin E
pre-incubation of old macrophages improved proliferation and IL-2 production in co-cultures of old
macrophages and old T cells [62]. Macrophages from old mice produced significantly higher levels of
PGE2 , which was due to higher cyclooxygenase (COX) activity. Macrophages from old mice expressed
higher levels of inducible COX2 protein and mRNA [63]. These increases in PGE2 synthesis and COX
activity were lowered by in vivo vitamin E supplementation [64]. Macrophages isolated from old
mice fed a diet containing 500 ppm vitamin E for 30 days produced lower amounts of PGE2 and
had lower COX activity than those from old mice fed a control diet containing 30 ppm vitamin E,
but the COX2 mRNA levels and protein expression of the control and supplemented groups did not

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differ. Thus, vitamin E’s effect on COX activity seemed to be through post-translational mechanisms
rather than through its effect at transcriptome or translational levels. In a subsequent study, it was
shown that vitamin E reduced COX activity in macrophages from old mice by decreasing peroxynitrite
production [65]. The inhibition of COX activity by vitamin E in old mice disappeared specifically with
the addition of a nitric oxide (NO) donor in the presence of a superoxide to elevate peroxynitrite levels
in the macrophage culture. There is a complex interplay between the nitric oxide synthase (NOS)
and COX pathways and NO increases COX2 activity, which seems to be due to the NO preventing
self-deactivation of COX by the superoxide as NO interacts with the superoxide [66].
In vivo supplementation of vitamin E (1500 IU D -α-tocopheryl acetate/day for 16 weeks)
in allergic asthmatic patients prevented the suppression of alveolar macrophage nuclear factor
(erythroid-derived 2)-like 2 (NRF2) activity after allergen challenge [67]. This study presented the
possibility of vitamin E’s protective role in allergies and asthmas through regulation of macrophage
NRF2 activity, but, further studies are needed to confirm the findings because of the small number of
patients (nine mild non-smoking allergic asthmatics) and the lack of appropriate controls.

3.2. Natural Killer Cells

NK activity seems to be related with vitamin E status. The NK activity of a boy with Shwachman
syndrome who had a severe vitamin E deficiency was low, but improved after eight weeks of 100 mg/d
α-tocopherol supplementation. When α-tocopherol supplementation was stopped, NK activity and
CD16+ CD56+ cells decreased. NK activity and CD16+ CD56+ cells were restored upon resuming
eight weeks of 100 mg/d α-tocopherol supplementation [68]. In 37 women aged 90–106 years old,
NK cell cytotoxicity was positively associated with plasma vitamin E concentration [69]. A two-week
supplementation of 750 mg vitamin E in colorectal cancer patients resulted in increased NK activity in
six out of seven patients. Vitamin E treatment did not result in changes in perforin expression or IFN-γ
production; therefore, mechanisms of improved NK activity by vitamin E could not be determined
from the study [70].
NO appears to be involved in the impairment of NK cell function. Co-culture of NK cells and
myeloid-derived suppressor cells (MDSCs) showed that NK cell cytotoxicity and IFN-γ were impaired
by MDSCs and that the inhibition of inducible nitric oxide synthase (iNOS) rescued the impairment
by MDSCs. Exposure of NK cells to NO by treatment with an NO producer caused the nitration of
tyrosine residues on CD16+ NK cells. These results suggested that MDSCs impair NK cell function
via the production of NO and the nitration of protein tyrosine residues [71]. Vitamin E might exert its
effects on NK cell function by modulating NO levels.

3.3. Dendritic Cells

Dendritic cells (DCs) are effective antigen-presenting cells that recognize pathogens and present
pathogen-derived antigens to T cells. The interaction of DCs with pathogen-associated molecular
patterns (PAMPs) or damage-associated molecular patterns (DAMPs) elicits the activation and
maturation of DCs. The increased expression of surface major histocompatibility complex (MHC)
molecules and co-stimulatory molecules and the increased production of cytokines occur with the
activation of DCs, which allows the effective induction of the T cell response [72–74]. DCs are also
involved in tolerance and autoimmunity. DCs might promote tolerance by the generation of Treg cells
and/or by the induction of T cell unresponsiveness. DCs might be involved in the pathogenesis of
autoimmune disease by promoting the priming or differentiation of self-reactive T cells [72]. Therefore,
understanding the regulation of DCs by vitamin E will provide insight into the mechanisms of vitamin
E’s immune response modulation and implications of vitamin E in immunological diseases.
Several studies have shown that vitamin E could regulate the maturation and functions of
DCs. Tan et al. [75] investigated the effects of α-tocopherol and vitamin C, alone or in combination,
on the phenotype and functions of human DCs generated from peripheral blood mononuclear cells
(PBMCs). During the differentiation of human PBMCs into DCs, various concentrations of α-tocopherol

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were treated in culture starting from day 2, cells were stimulated on day 5, and then the surface
phenotype was determined on day 6. The expression of human leukocyte antigen(HLA)-DR, CD40
CD80, and CD86 appeared to be increased with lower concentrations of α-tocopherol (<0.05 mM), but
the combination of vitamin E and C prevented DC activation, as the upregulation of surface markers
was not observed. DCs treated with 0.5 mM vitamin E and 10 mM vitamin C showed lower levels
of intracellular ROS and inhibition of the nuclear factor (NF)-κB, PKC, and p38 mitogen-activated
protein kinase (MAPK) pathways. When bone marrow-derived dendritic cells (BMDCs) from Balb/c
mice were treated with 500 μM of α-tocopherol for 2 h, upregulation of phosphorylated inhibitor of κB
(IκB) by lipopolysaccharide (LPS)-stimulation was suppressed. Vitamin E treatment for 24 h resulted
in a reduced number of CD11+ CD86+ cells and ROS-positive cells, lower production of IL-12p70
and TNF-α, and decreased transwell migration of BMDCs. These effects of vitamin E on BMDCs
were partly dependent on Klotho expression. Vitamin E treatment on BMDCs resulted in higher
Klotho transcript and protein levels, and silencing of Klotho by transfection of Klotho siRNA abolished
the inhibitory effects of vitamin E on IL-12p70 production, number of ROS-positive cells, and DC
migration [76]. Klotho is a membrane protein that has been shown to mediate calcium transport into
the cells; regulate intracellular signaling pathways such as p53/p21, cyclin adenosine monophosphate
(cAMP), PKC, and Wnt; and inhibit the NF-κB pathway [77]. Therefore, the upregulation of Klotho
by vitamin E could be one of the mechanisms by which vitamin E modulates NF-κB mediated DC
function and maturation. However, the level of α-tocopherol used for in vitro treatment (500 μM) was
high and, therefore, further research is needed to elucidate the physiological relevance of vitamin E
treatment on the expression of Klotho and its involvement in the modulation of DC function.
In vivo supplementation of α-tocopherol at 150, 250, and 500 mg/kg diet in allergic female mice
reduced the lung CD11b+ DCs and mRNA levels of IL-4, IL-33, thymic stromal lymphopoietin (TSLP),
eotaxin 1 (CCL11), and eotaxin 2 (CCL24) in allergen challenged pups. Furthermore, when BMDCs
from 10-day-old neonates born to a control female were treated with 80 μM α-tocopherol for 24 h,
the number of CD45+ CD11b+ CD11+ DCs and the number of CD45+ CD11b+ CD11c+ Ly6c− MHCII−
DCs were reduced. Maternal supplementation with α-tocopherol was effective in decreasing allergic
responses in offspring from allergic mothers by affecting the development of subsets of DCs that
are critical for allergic responses [78]. On the other hand, γ-tocopherol supplementation exerted an
opposite response in the same model. In vivo supplementation of γ-tocopherol at 250 mg/kg diet in
allergic female mice resulted in a higher number of lung eosinophils, a higher number of lung CD11c+
CD11b+ DCs, and higher levels of lung lavage CCL11 in the offspring [79].
Modulation of the immune response by vitamin E has been observed in animal and human
studies, and DCs play a critical role in bridging innate and adaptive immune systems and initiating
adaptive immune responses. Despite the importance of DCs’ role in adaptive immune responses
and in diseases such as autoimmune diseases, few studies have investigated the DC-specific effect of
vitamin E.

3.4. T Cells
The effects of vitamin E on immune cells have been studied the most with T cells.
The dysregulation of immune function occurs with aging and the most significant changes are observed
in T cells. Age-associated changes in T cells include, but are not limited to, (1) defects in T cell receptor
(TCR) signal transduction such as a decrease in linker for the activation of T cells (LAT) phosphorylation
by zeta chain of T cell receptor associated protein kinase 70 (ZAP-70), (2) decreased intracellular influx
of calcium following stimulation, (3) diminished synapse formation, (4) diminished activation of the
mitogen activated protein kinase (MAP kinase) pathway, (5) decreased nuclear factor of activated
T-cells (NFAT) binding activity, and (6) a shift of the T cell population toward memory T cells [80]. As a
result, diminished production of IL-2 and reduced proliferative capacity of naive T cells are observed
and impaired T cell functions contribute to increased susceptibility to infectious diseases and poor
response to immunization.

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Vitamin E has been shown to increase the cell division and IL-2 producing capacity of naïve T
cells, increase the percentage of T cells capable of forming an effective immune synapse, and reverse
the age-associated defect in the phosphorylation of LAT in T cells from old animals [81–83].
In vitro pre-incubation with 46 μM vitamin E for 4 h increased proliferation and IL-2 production in
T cells purified from old mice stimulated with anti-CD3 and anti-CD28. Increased IL-2 production was
due to both an increase in the number of activation-induced IL-2+ cells and an increase in the level of
IL-2 accumulated per cell. Vitamin E specifically increased the naive T cells’ ability to progress through
the cell division cycle in old mice [81]. The gene expression profile of T cells isolated from young and
old mice fed a diet supplemented with 500 ppm vitamin E for four weeks provided evidence that
vitamin E influences cell cycle-related molecules at the gene expression level. Higher expression of
cell cycle-related genes Ccnb2, Cdc2, and Cdc6 was observed in stimulated T cells from old mice fed
the vitamin E-supplemented diet compared with those fed the control diet, which was not observed
in young mice [84]. Cyclin B2, encoded by Ccnb2, binds to cyclin-dependent kinase 1 (also known
as Cdc2) and regulates the events during both the G2 /M transition and progression through mitosis.
Cdc6 is a key regulator in the early steps of DNA replication, as the binding of Cdc6 to chromatin is
a necessary and universal step in the acquisition of replication competences [85]. These alterations
in the expression of cell cycle-related genes observed with vitamin E might contribute to vitamin E
improving the proliferative ability of old T cells.
Marko et al. [82] showed that pre-incubation of CD4+ T cells isolated from old T cells with 46 μM
vitamin E for 4 h increased the percentage of CD4+ T cells displaying effective immune synapses.
Redistribution of Zap70, LAT, Vav, and phospholipase Cγ (PLCγ) into immune synapse increased
significantly with vitamin E treatment. This change was confirmed with in vivo supplementation
of vitamin E. In old mice fed a diet containing 500 ppm vitamin E for eight weeks, LAT and Vav
showed significantly higher redistribution into the T cell/APC contact area when purified CD4+
T cells were stimulated with murine CD3ε hybridoma. In a subsequent study, it was shown that
vitamin E could reverse the age-associated defect in the phosphorylation of LAT on tyrosine 191 [83].
The phosphorylation of LAT is required for the recruitment of adaptor and effector proteins. Therefore,
it plays a pivotal role in the assembly of microcluster structures in the initiation of T cell activation
signals. This evidence suggests that vitamin E can modulate the early stages of T cell activation.
Vitamin E seems to modulate Th1 and Th2 responses. The polarization of CD4 T cells to T
helper (Th)1 or Th2 cells has implications for the protection against different pathogens (intracellular
vs. extracellular pathogens) and the development of different types of chronic diseases (inflammatory
vs. allergic diseases). PBMCs isolated from allergic donors treated with vitamin E (12.5–50 μM)
showed dose-dependent decreases in IL-4 production [86]. IL-4 mRNA levels in activated PBMCs
were downregulated by 25 μM vitamin E treatment. Jurkat T cells treated with 50 μM vitamin E
exhibited downregulation of IL-4 promoter activity, which might be related to vitamin E blocking the
interaction of transcription factors with PRE-1 and P1. In vivo supplementation of vitamin E enhancing
the Th1 response has been observed in mice infected with influenza virus and in colorectal cancer
patients [6,87]. In colorectal cancer patients, two weeks of supplementation with 750 mg vitamin E
led to an increased frequency of IL-2 producing CD4+ T cells and increased IFN-γ production [87].
In old mice infected with influenza virus, 500 ppm vitamin E supplementation for eight weeks prior
to infection lowered the viral titer in the lung, and this protective effect of vitamin E was associated
with the enhancement of Th1 response. IFN-γ production levels correlated negatively with viral
titer, and old mice fed a vitamin E-supplemented diet produced significantly higher levels of IFN-γ
and IL-2 [6]. The gene expression profile of T cells isolated from young and old mice fed a diet
supplemented with 500 ppm vitamin E for four weeks provided evidence that vitamin E influences the
Th1/Th2 balance at the gene expression level. The increase in IL-4 expression following stimulation
was lower in T cells from old mice fed the vitamin E-supplemented diet compared with those fed the
control diet, and the ratio of IFN-γ and IL-4 expression levels was significantly higher in the vitamin E
group than in the control group [84].

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Vitamin E can affect activation-induced cell death in T cells. In vitro treatment of primary human
T cells with 25 μM vitamin E suppressed CD95L expression and activation-induced cell death [88].
The reduction of CD95L mRNA levels and the proportion of CD95L-positive cells were related to the
suppression of NF-κB and AP-1 binding to the CD95L promoter target site by vitamin E. On the other
hand, α-tocopheryl succinate was shown to trigger apoptosis in Jurkat cells with caspase-activation
involved [89].

3.5. B Cells
Vitamin E supplementation has been reported to enhance humoral responses. Higher antibody
responses have been observed in animals and humans [19,27]. However, it is hard to differentiate
whether vitamin E’s direct effect on B cells or indirect effect through T cells contributes to higher
antibody responses.

4. Conclusions
Vitamin E has been shown to enhance immune responses in animal and human models and
to confer protection against several infectious diseases. Suggested mechanisms involved with these
changes are (1) the reduction of PGE2 production by the inhibition of COX2 activity mediated through
decreasing NO production, (2) the improvement of effective immune synapse formation in naive T
cells and the initiation of T cell activation signals, and (3) the modulation of Th1/Th2 balance. Higher
NK activity and changes in dendritic function such as lower IL-12 production and migration were
observed with vitamin E, but underlying mechanisms need to be further elucidated
Several considerations are warranted for the advancement in our understanding of vitamin E’s
role in immunity. For in vitro studies to support implications for the regulation of immunological
diseases, the physiological relevance of vitamin E levels used for treatment should be considered.
Different forms of vitamin E exert differential effects on immune cells. Cell-specific effects of vitamin
E provide valuable evidence regarding the immunomodulatory mechanisms of vitamin E, but the
interplay between immune cells should not be ignored, because interactions between immune cells are
critical in the regulation of immune function.

Author Contributions: Literature search and manuscript preparation were performed by G.Y.L. and S.N.H.
The manuscript was revised and finalized by S.N.H.
Funding: This work was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (grant number NRF-2018R1D1A1B07049178).
Conflicts of Interest: The author declares no conflicts of interests.

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 nutrients
Review
The Microbiotic Highway to Health— New
Perspective on Food Structure, Gut Microbiota,
and Host Inflammation
Nina Wærling Hansen 1 and Anette Sams 2, *
1 Molecular Endocrinology Unit (KMEB), Department of Endocrinology, Institute of Clinical Research,
University of Southern Denmark, DK-5000 Odense, Denmark; [email protected]
2 Department of Clinical Experimental Research, Glostrup Research Institute, Copenhagen University
Hospital, Nordstjernevej 42, DK-2600 Glostrup, Denmark
* Correspondence: [email protected]

Received: 28 September 2018; Accepted: 23 October 2018; Published: 30 October 2018

Abstract: This review provides evidence that not only the content of nutrients but indeed the
structural organization of nutrients is a major determinant of human health. The gut microbiota
provides nutrients for the host by digesting food structures otherwise indigestible by human enzymes,
thereby simultaneously harvesting energy and delivering nutrients and metabolites for the nutritional
and biological benefit of the host. Microbiota-derived nutrients, metabolites, and antigens promote the
development and function of the host immune system both directly by activating cells of the adaptive
and innate immune system and indirectly by sustaining release of monosaccharides, stimulating
intestinal receptors and secreting gut hormones. Multiple indirect microbiota-dependent biological
responses contribute to glucose homeostasis, which prevents hyperglycemia-induced inflammatory
conditions. The composition and function of the gut microbiota vary between individuals and
whereas dietary habits influence the gut microbiota, the gut microbiota influences both the nutritional
and biological homeostasis of the host. A healthy gut microbiota requires the presence of beneficial
microbiotic species as well as vital food structures to ensure appropriate feeding of the microbiota.
This review focuses on the impact of plant-based food structures, the “fiber-encapsulated nutrient
formulation”, and on the direct and indirect mechanisms by which the gut microbiota participate in
host immune function.

Keywords: carbohydrates; fiber; food structure; formulation; plant; microbiota; inflammation;

metabolism; nutrition guidelines

1. Introduction
The gut microbiota is a complex ecosystem residing in the gastro-intestinal (GI) tract and consists
of a diverse microbiotic community living in symbiosis with the host. A diverse microbiota is
considered a major positive regulator of the interdependent metabolic and immune function of the
host. The gut microbiota is shaped alongside the host immune system in a synergistic partnership [1].
Trillions of microorganisms participate in the maturation and regulation of immune function, energy
metabolism, and hormonal balance [2], including regulation of intestinal mucosal barriers, fermentation
of undigested nutrients, and synthesis of short chain fatty acids (SCFA) [3]. Furthermore, the microbiota
releases microbiota-derived antigens [4,5], vitamins [6,7], and other molecules, such as tryptophan
metabolites [8,9], that interact with the host biology.
The dynamic relationship between the microbiotic ecosystem and its host is emphasized by
the joint utilization of consumed nutrients. Diet is a shared substrate between the host and the gut
microbiota and the choice of diet affects host health both directly and indirectly by affecting the
abundance and composition of the microbiotic community [10,11].

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The structural organization of complex carbohydrates determines the site of digestion and
absorption in the GI tract [12], and hence the level of cross feeding [13], community organization,
and proliferation of specific microorganisms. Importantly, all plant cell walls consist of complex
carbohydrate structures that are digested selectively by microbiotic enzymes and not by host enzymes.
Thus, complex polysaccharides in plant cell walls function as both substrate for the microbiota and as
a sustained release delivery system of other plant-cell derived nutrients and biomolecules for both
microbiotic and host utilization. The structural properties of the cell wall vary between different plant
cells [14], and in theory determine the specific site of plant cell wall degradation and nutrient release.
Plant-based foods can be placed on a moving scale from whole foods (unrefined) to fully refined
foods, and currently no official distinction based on the degrees of refinement exists. Foods with added
refined nutrients (e.g., sugar, starch or oils) or any food, which has been structurally disrupted on the
macroscopic level are considered refined. The degree of refinement is thus determined by the degree of
macroscopic structural disruption (e.g., intact whole grain vs. ground whole grain vs. sifted flour) and
the degree of refined supplementation. When plant-based food structures are completely disrupted
requiring no microbiotic interaction for digestion we consider the food fully refined.
Microbiota composition varies greatly among different cultures and individuals [15,16],
and specific genera of bacteria are more abundant in healthy individuals as compared to individuals
in different disease states, as shown, for example, for Bifidobacterium and Lactobacillus [17–19], whose
functions include carbohydrate fermentation and vitamin synthesis [6,20,21]. The degradation and
utilization of complex carbohydrates rely on specific microbiotic enzymes secreted from the microbiota
in the lower intestine [22]. The fermentation process yields energy for microbiotic proliferation
and metabolites, e.g. SCFA [23] for regulation of inflammatory responses [24] and gut hormone
secretion [25] in the host. The latter is of great importance for glucose homeostasis and plays a major
role for the metabolic and inflammatory health of the host [26].
Microbiota composition is also closely connected to the integrity of our intestinal mucosal barrier.
Upon microbiota dysbiosis mucosal bacteria can impair epithelial function and cause increased gut
permeability with consequent immune dysregulation, leading to inflammatory disease states [27].
A number of disorders with an inflammatory component including obesity, inflammatory bowel
diseases (IBD), type 2 diabetes (T2D), colorectal cancer, and cardiovascular diseases have been linked
with dysbiosis [28–32]. This association presents the gut microbiota as a potentially modifiable factor
in the etiology of these conditions. It is therefore of great importance for public and individual health
to recognize that microbiota composition may be diversified within days or weeks upon introduction
of unrefined plant-based diets [16].
In clinical trials diets are often classified as low carbohydrate vs. high carbohydrate, and low
fiber vs. high fiber. These trials lack discrimination between refined and unrefined carbohydrates.
Acknowledging the degree of refinement would likely strengthen the reproducibility of diet
intervention studies for reasons discussed in the present review [33–35].
This paper analyzes the interplay between structures of plant-based foods or “fiber-encapsulated
nutrients”, the gut microbiota, and their joint impact on host nutrient bioavailability, immune function,
and metabolic function, and proposes a simple tool to select plant-based foods from a nutritional-
and microbiota-promoting perspective. The review provides a biological explanation of the health
superiority of structurally maintained and unrefined foods compared to nutrient-matched refined
foods, combining classic and current scientific knowledge within nutrition, pharmaceutical science,
microbiology, and plant biology. In addition, we add the perspective of “fiber as nutrient encapsulation”
to the well-established molecular benefits of fiber as microbiotic nutrient (prebiotic). This structural
perspective should be included in the current nutritional guidelines, which focus on nutrient and fiber
content rather than nutrient and fiber structure.

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2. Carbohydrates: Different Structures, Different Properties

The structural characteristics of dietary carbohydrates include the chemical composition,
physicochemical properties, and resistance to human digestive enzymes. Dietary polysaccharides
display a variety of linkages, branching, and polymerizations and must be broken down into their
corresponding monosaccharides (e.g., glucose, galactose, and fructose) before they can be absorbed
and metabolized by humans. This carbohydrate breakdown is initiated by α-amylase secreted by
salivary glands, continues with pancreatic amylase in the duodenum, and is completed by intestinal
enzymes (e.g., sucrase and lactase) located in the brush-border membrane of the enterocytes in the
small intestine [22]. Pancreatic amylase hydrolyzes α-linked glucose polymers such as starch and
glycogen, but humans lack the enzymes necessary to hydrolyze β-linked glucose polymers like those
present in resistant starches and dietary fibers found in plant structures [36].
Resistant starches include starch where (i) the granules are inaccessible to enzymes due to
conformation; (ii) the starch is retrograded or (iii) chemically modified. Importantly, starch may also
be digestion-resistant due to encapsulation in plant cell fiber matrices [37] and new perspectives on
this aspect are the main focus of this review.
Dietary fiber includes a large variety of carbohydrate polymers, e.g., xylans, β-glucans, fructans,
β-mannans, celluloses, hemicelluloses, and pectins [38]. Cellulose, hemicellulose, and pectin are
major components of plant cell walls, and starch [39] and inulin [40] are heavily represented in
plant carbohydrate storage. As such, the above-mentioned molecular carbohydrates are regarded as
prebiotics that may stimulate or alter the preferential growth of health-promoting bacterial species
already residing in the colon [41].
The microbiota encodes a broad spectrum of enzymes catalyzing the depolymerization and further
degradation of polysaccharides [22]. The structural properties of the carbohydrates are important
since the ability to utilize different substrates differs between bacterial species, for example, it has
been shown in vitro, that Roseburia spp. can utilize amylopectin starch and that Bifidobacterium spp.
cannot [42].
Plant polysaccharides can be separated based on origin (cereals and grains, fruits, vegetables
nuts, and legumes), and chemical compositions and physicochemical properties vary depending on
origin [14]. The physicochemical properties of plant polysaccharides include fermentability, solubility,
and viscosity, each of which impact site and degree of fermentation. Soluble fibers, such as short-chain
fructo-oligosaccharides and pectin are depolymerized by bacterial enzymes more proximally in the GI
tract, while fibers with a lower solubility, such as cellulose, are partially fermented in the distal colon
where transit time is slower and microbiotic density higher [12].
Simple carbohydrates, such as monosaccharides, disaccharides, and oligosaccharides can be
classified as host nutrition with direct utilization by host enzymes, whereas complex carbohydrate
polymers, such as plant polysaccharides and resistant starches are vital substrates for the microbiota.
Thus, a diet that includes a variety of unrefined plant structures can therefore promote proliferation
of a broader spectrum of bacteria with specific intrinsic properties. A diet rich in plant structure
variety also represents a diverse sustained release device for delivery of various nutrients and bioactive
molecules for metabolic and immunological stimulation, as described below.

3. Plant Cell Structure

The plant cell wall is the major difference between animal and plant cells. Except for the
cell wall, chloroplasts, and vacuoles, animal and plant cells share intracellular structures and
compartmentalization (Figure 1).

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Figure 1. Schematic illustration of an animal and a plant cell. The cell wall (mainly complex
carbohydrates), vacuoles (usually starch or lipid storage, depending on cell type), and chloroplasts
are plant-cell specific structures that serve as nutrients for the microbiota, whereas the cellular content
serves as signal molecules for host metabolic and immune regulation as well as nutrients for both
host and microbiota. An intact plant cell is defined as unrefined food whereas the degree of structural
disruption (and the supplementation of additives) defines the degree of refinement.

The most characteristic component found in all plant cell walls is the fiber, cellulose [43], which
consists of a collection of β-1,4-linked glucan chains that interact with each other via hydrogen
bonds to form a crystalline microfibril [44]. In addition to cellulose, the primary plant cell wall
contains two categories of matrix polysaccharides-pectins and hemicelluloses, and several proteins
and glycoproteins, including various enzymes and structural proteins [45].
Intracellular plant compartments and their relative ratio constitute the macro- and micronutrients
in the plant and the dispersion of macronutrients varies from plant to plant, changing during aging
with a larger proportion of protein and lipids during early growth [46]. In addition to macronutrients
and vitamins, the encapsulated plant cells also contain phytonutrients, such as polyphenols (flavonoids
and stilbenes), carotenoids, plant sterols, and poly-unsaturated fatty acids (PUFA), many of which
are used as nutraceutical ingredients and exhibit beneficial effects on metabolic and cardiovascular
parameters [47].
Although monomers from the cell wall degradation process are absorbed via host transporters,
most of the plant-cell wall functions as microbiota substrate [12]. Furthermore, the mixture of
macronutrients, vitamins, and phytonutrients encapsulated within the plant cell membrane have
a two-tier function, with part of their host-health-promoting effects also benefiting the microbiota.
In other words, the microbiota and the host share both host-enzyme-resistant carbohydrates and
fiber-encapsulated macro- and micronutrients.

4. Microbiota Composition
In human adults, five bacterial phyla have been reported to be dominant in the gut microbiota
of healthy individuals: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia [48],
with more than 90% of the species belonging to Firmicutes and Bacteroidetes. Representatives of the
other phyla comprise from 2 to 10% with great interpersonal variation [49]. Changes in the relative
abundance of the two dominant bacterial divisions, Bacteroidetes and Firmicutes, have been associated
with an increased BMI [50]. An overrepresentation of butyrate-producing species, such as Roseburia spp.
and Eubacterium spp. within the Firmicutes phylum affects the metabolic potential of the microbiota,
i.e., the microbiotic capacity to harvest energy from the diet and produce SCFA available for host
consumption [51].
In humans, an abundance and diversity in polysaccharide-fermenting bacteria, such as species
within the Bacteroidetes phylum, are inversely related to obesity and other metabolic disorders
with an inflammatory component [52–55]. Members of Bacteroidetes express very large numbers of
genes that encode microbiotic enzymes and readily can switch between different energy sources
in the gut depending on substrate availability. Within the Bacteroidetes phyla, two genera are

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particularly dominant and mutual antagonistic; Bacteroides and Prevotella [56]. The relative ratio
between these two genera varies, with the Bacteroides-driven type reported as dominant in individuals
with a higher intake of protein and animal fat, and Prevotella more common in individuals who consume
more carbohydrate-rich diets [57,58] These findings represent distinct community composition
types—so-called enterotypes—based on genus composition [59], suggesting that such compositional
differences both reflect dietary intake and determine an individual’s response to different diets [55].
Long-term dietary habits have considerable effect on the gut microbiota as evidence by differences
in microbiotic compositions across geographical and/or cultural-dependent diets [28,57,60]. Microbiota
composition also respond to short-term macronutrient changes; moreover, functional traits linking
enterotypes to diet have been identified [57,58]. Animal and human studies show that an acute dietary
switch results in dramatic shifts in the gut microbiota within the course of 24 h [16]. An animal-based
diet increases the abundance of bile-tolerant genera (Alistipes, Bilophila, and Bacteroides) and decreases
the level of Firmicutes species capable of fermenting dietary plant polysaccharides. Microbiotic activity
mirrors differences between herbivorous and carnivorous mammals, reflecting trade-offs between
carbohydrate and protein fermentation [61]. Analyses of the relative abundance of taxonomic groups
have shown that short-term animal-based diets have a greater impact on microbiotic community
structure compared to plant-based diets [16], suggesting perhaps a default microbiotic setting favoring
plant-based diets.
As mentioned above, bacterial species have varying abilities to degrade fibers from different
sources. The ability to degrade a wider variety of complex carbohydrates therefore forms a more
diverse bacterial community. Moreover, the biodiversity, function, and abundance of a microbiotic
community also depend on cross-feeding among members, which is the process where essential
metabolites synthesized by a subset of the community provide substrate for growth of another/other
bacterial species [62]. These microbiotic interactions create a complex pattern of interconnected
metabolisms and link members of different genera/phyla together in communities.
Biodiversity therefore contributes to the stability and resilience of the bacterial communities.
A high level of resilience ensures renewal and reorganization of the community if a species is
lost, whereas communities with low biodiversity are more susceptible to loss of individual species.
Highly diverse ecosystems most likely contain species with redundant functions, making the loss of
a single species tolerable, because other functionally redundant species can take over [63]. Evidently,
the microbiotic composition and community organization are reflective of dietary habits and affect
host metabolic and immune processes. The distinction between enterotypes with distinct fermentation
kinetics should therefore be considered an environmental factor that contributes to both health
and disease.

5. Biological Journey and Destiny of Complex and Simple Carbohydrates

Monosaccharides can be absorbed via transporters in the human GI tract whereas polysaccharides
that are not encapsulated by a fibrous plant cell wall, undergo digestion in the upper GI tract devoid
of the microbiota. The more complex the carbohydrate the more glycosidase activity is necessary for
the depolymerization. Thus, the more complex the plant cell wall (nutrient delivery device), the more
microbiota interaction is needed. As a result, more caloric harvesting takes place and a larger intestinal
area is subject to nutrient absorption, enteroendocrine stimulation, and immunological maturation.
The human gut has the potential to absorb monosaccharides via several transporters located
in the absorptive epithelium. Apical sodium-dependent glucose cotransporter 1 (SGLT1) [64],
glucose transporter 2 (GLUT2) [65], and glucose transporter 5 (GLUT5) [66] facilitate absorption
of glucose and fructose from the intestine, and basolateral GLUT2 and GLUT5 facilitate the basolateral
monosaccharide secretion into the host interstitium. Upon ingestion of a sugar-enriched meal,
a transient upregulation of the absorptive monosaccharide transporters is induced, perhaps via
apical recruitment of GLUT2 [67–69]. Thus, sugar ingestion promotes host absorption and bypasses
caloric harvesting by microbiota.

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Throughout the GI tract the level of glucose transporters is highly regulated by hexose availability
and insulin levels [70–73]. In addition, the expression of glucose transporters has been shown to be
increased in e.g., obesity and diabetes [74,75] and as a result, certain individuals experience a larger
bioavailability of monosaccharides.
Despite individual differences in host digestive enzyme- and nutrient transporter expression,
a general notion must be highlighted: The density and diversity of the microbiota increases with the
distance from the stomach. The more complex the carbohydrate, the longer the journey until complete
fermentation, and therefore the more likely that carbohydrates are utilized by the microbiota and not
by the host.

6. The Plant Cell Wall Viewed as a “Pharmaceutical-Nutrient Formulation”

When applying the drug delivery perspective to unrefined plant-based foods, it is evident that
the polysaccharides of plant-cell walls [76,77] are more than just molecular nutrition for the host and
microbiota [78]. Unrefined plant-based food structures can be interpreted as a slow release device for
nutrients and bioactive molecules in the human GI tract, which has important implications for intestinal
absorption and local biological actions. The “device” releases a complex pool of molecules with
nutritional value for the host and the microbiota, and with biological effects that trigger the endocrine
and immunological maturation of the host. The release of nutrients from the fiber-encapsulated plant
cell is thus dependent on microbiota-specific plant cell wall fermentation.
In pharmaceutical drug discovery and development processes, both pharmacokinetic and
pharmacodynamic properties are carefully optimized. Whereas the optimized drug molecule
determines the specific pharmacological action, the site of action and the duration of action are
heavily influenced by the pharmaceutical formulation of the drug molecule. For example, a specific
formulation can be used to target sustained or fast absorption from the GI tract. In other words,
the drug molecule needs a relevant formulation to reach the relevant site of absorption and to obtain
the desired absorption kinetic [79].
Different saccharides (sugars, starches, and fibers) are used as coating material in oral drug
formulations to direct drugs through the GI infrastructure and deliver the drug molecule at the desired
site of action or absorption [80]. Salicylic acid is, for example, formulated for fast or sustained systemic
absorption using different formulation principles. Different cellulose coatings have been used to
deliver the drug to distal parts of the gut for local IBD therapy [81,82]. Basically, the microbiota
ferments the coating material and releases the drug distally for sustained release (or local treatment).
The diversity in fibrous plant cell structures therefore serves as both a saccharide delivery system for
bacterial species and as a fiber-encapsulated delivery device of other molecular nutrients (e.g., amino
acids, fatty acids, vitamins, and other bioactive molecules). These nutrients are released from the
plant cell upon plant-cell-wall fermentation, playing a major role for both host and microbiota biology
(Figure 2). Indeed, most bacteria, even Lactobacillus and Bifidobacterium, express lipid and amino acid
transporters [83–86] and regulate the expression dependent on the specific environment.
The biological properties of the microbiota are not solely dependent on the carbohydrate-based
prebiotic content, but indeed also on the content of lipids, proteins, amino acids, and vitamins [87].
In vitro studies of bacteria emphasize the perspective that plant-based nutrients beyond fibers are
important for microbiotic function. For example, the growth and adhesion properties of specific
microbial strains are different in two different media (soy bean media and MRS media) [88] and the
growth of Lactobacillus and Bifidobacterium depends on the media protein content [89,90]. Therefore,
a diversity of media is needed to actually culture a diversity of human gut microbiota and study their
selective and community properties [91]. Peptides and lipids that are not fiber-encapsulated would
most likely only reach the microbiota in limited amounts due to upper-intestinal host absorption. Thus,
a major source of amino acids and lipids for the microbiota is formulated in plant cells.

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Figure 2. Illustration of interactions between plant cells, microbiota, and the host epithelium. Plant cells
are encapsulated by the fibrous cell wall, which is a microbiota-selective nutrient. The cell wall also
serves as a transport vehicle for the additional plant cell contents as well as a vehicle for nutrients to be
shared between the microbiota and the host upon release from its plant cell structure. The microbiota
secretes glycosidases, peptidases, and lipases, thereby releasing molecular nutrients for transport into
epithelial or bacterial cells.

If plant cells have been refined, this organization does not remain intact, and even if the fibers
reach the microbiota (e.g., fiber supplements), only limited amounts of amino acids and lipids will
reach the microbes.
Some bacterial genera e.g., Bacteroides [92] and Lactobacillus [93] secrete both peptidases and
glycosidases and the more distal fiber-fermenting species Bacteroides thetaiotaomicron also express
dipeptidylpeptidases (DPPs) [94,95]. Interestingly, the DPP4 antagonist vildagliptin, used for the
treatment of T2D, induces a higher relative abundance of Bacteroidetes, a lower abundance of Firmicutes,
and thus a reduced ratio of Firmicutes/Bacteroidetes in rats [96]. This is one among many examples of
anti-diabetic drugs that has recently been shown to induce secondary health promoting actions via the
microbiota [97].
Another pharmaceutical exploration of the microbiota is the study of bacterial peptide transporters
with the aim to identify new antibiotic targets in virulent bacteria. Since blockage of peptide
transporters is a potent antibiotic pathway, specific bacteria are dependent on their endogenous
peptide transporters and on amino acid fuel [98]. In both gram negative and gram positive virulent
bacteria, two distinct vital types of peptide transporters exist, proton-dependent peptide transporters
(PRT or POT) [99] and ATP-binding cassettes (ABC transporters) [100].
As in the case of amino acids, energy dependent fatty acid transporters are also present in both
outer and inner membranes of bacterial strains. Even Lactobacillus expresses amino acid and fatty

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acid transporters [83], which supports the bacterial ability and need to consume fatty acids slowly
released from plant cells during the microbiotic digestion of the plant cell wall. In specific bacteria,
the process requires the outer membrane-bound fatty acid transport protein FadL and the inner
membrane associated fatty acyl CoA synthetase (FACS) [101,102].
The diversity in fiber-coated plant cell structures also serves as delivery system for local acting
biologically active substances, e.g., taste receptor agonist for distal enterocytes and the microbiota [103],
and maturation agents for the immune system [104] which will be discussed below.

7. Pro-Inflammatory Effects of Refined Foods

The consumption of unrefined plant-based foods maintains host health by engaging the
microbiota. The immunomodulatory effects of unrefined plant-based foods act through the
host-microbiota symbiosis and circumvent the pro-inflammatory effects of refined foods. Mono-
and disaccharides in a fibrous sustained release formulation do not produce significant increase
in blood glucose because they are partially consumed by the microbiota. The microbiota unwraps
molecules from the fibrous formulation in a time-consuming process. In contrast, industrial refinement
causes a lack of molecular engagement with the microbiota and distal enterocytes, and polysaccharide
molecules are rapidly monomerized and absorbed with the risk of elevated blood glucose levels [26].
Mounting epidemiological evidence suggests that intake of refined sugars and starch is associated
with the development and maintenance of several diseases. T2D, for example, is driven by insulin
resistance and β-cell dysfunction and manifested by increased risk and severity of hyperglycemic
events. The condition is associated with elevated risk of autoimmune and inflammatory diseases,
including rheumatoid arthritis [105] asthma [106], psoriasis [107], and cancer [108,109]. Drugs or
dietary regiments that normalize hyperglycemia do not surprisingly have a therapeutic effect on
immune diseases, e.g., psoriasis [110–112] and rheumatoid arthritis [113].
For diabetics as well as healthy individuals there is a relationship between the ingestion
of refined sugars and the risk of developing both obesity [114] and autoimmune/inflammatory
diseases, e.g., rheumatoid arthritis [115], asthma [116,117], IBD [118], chronic kidney disease [119],
and atherosclerosis [114,120].
Hyperglycemia influences inflammation and immune function through multiple mechanisms.
Chemical reactivity of glucose and other reducing sugars (fructose, mannose, galactose) towards
proteins [121], lipids, and nucleic acids [122] is important. Non-specific stochastic protein glycations
during incidents of hyperglycemia have the potential to initiate and potentiate several inflammatory
and immunomodulatory responses, involving activation of pattern recognition receptors (PRR), e.g.,
RAGE (receptors for advanced glycation end-products) [123,124]. Also, hyperglycemia-induced
cytokine release [125], formation of methylglyoxal and other auto-oxidation metabolites, e.g., reactive
oxygen species (ROS) and free radicals [126] maintains inflammatory responses. The activity of
auto-oxidation metabolites is prevented, for example, by antioxidants, which reduce the oxidative
damage induced by glucose metabolites and ROS [127]. The fact that antioxidants are prevalent in
plant-based food and synthesized by the microbiota [128] adds to the anti-inflammatory effects of
unrefined foods.

8. Anti-Inflammatory Effects of Unrefined Foods

The anti-inflammatory effects of unrefined foods are particularly mediated by their contribution
to a high gut biodiversity, which as mentioned above, contributes to the stability and resilience of
the bacterial communities. In contrast, the loss of diversity can cause an imbalance between the
inflammatory and the immunoregulatory taxa present in a given microbiota. The effects of a complex
microbiotic community are ascribed to the additive and synergistic effects of the bacterial species in
a microbiotic community. An example of this is monocolonization of a variety of organisms, which
cannot reverse the hyper-IgE phenotype in germ-free (GF) mice. However, complex polycolonizations
restore normal IgE levels [129].

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Imbalances in immunological homeostasis can occur due to the loss of habitat-specific species and
symbionts, resulting in an invasion of opportunistic species not normally able to colonize that specific
habitat within the intestinal mucosa. Such a mismatch between species and habitat triggers a potentially
pathogenic host response [130]. The detection of pathogens by the host is achieved through the families
of PRR localized in the intestinal epithelium. PRR recognize conserved molecular structures known as
microbe-associated molecular patterns and induce production of innate effector molecules [131,132].
The most well-known PRR are the toll-like receptors (TLR), RAGE, and the NOD-like receptors
expressed on the intestinal epithelial cells. Activation of TLR by bacterial products promotes epithelial
cell proliferation, secretion of IgA into the gut lumen, and expression of antimicrobial peptides,
thereby establishing a microorganism-induced system of epithelial cell homeostasis and repair in the
intestine [132].
Bacterial translocation, e.g., infiltration of lipopolysaccharide (LPS) through the intestinal barrier,
can promote local and systemic immune responses via PRR. Monocolonization of GF mice by
Escherichia coli is, for example, sufficient to induce macrophage infiltration, and polarization towards
pro-inflammatory phenotype of immune cells in the adipose tissue [133], contributing to a state of
low-grade inflammation in the adipose tissue. Disruption of intestinal barrier integrity by viable
bacteria has been attributed to various intestinal inflammatory diseases such as IBD, celiac disease,
irritable bowel syndrome, and colorectal cancer [32] as well as to diseases in extra-intestinal organs,
such as the liver [134].
The major microbiota-derived SCFA, butyrate, acetate, and propionate are implicated in multiple
anti-inflammatory mechanisms [135]. Besides being used by colonocytes as a source of ATP, SCFA can
act as extracellular signaling molecules that activate cell-surface free fatty acids receptors (FFAR)
(G-protein-coupled receptors (GPCR)) or inhibit histone deacetylases (HDAC) [136]. FFAR are
differentially expressed on adipocytes, immune cells, and enteroendocrine L-cells. Depending on the
location of the receptor and the amount and type of SCFA, the response can have multiple, variable
downstream effects [3]. FFAR, expressed on immune cells, such as macrophages, dendritic cells,
and neutrophils will upon activation by butyrate inhibit the release of pro-inflammatory cytokines, or
stimulate differentiation of T regulatory (Treg) cells [136].
Activation of FFAR2/GPR43 by butyrate on enteroendocrine L-cells of the ileum and colon induces
production and secretion of intestinal peptide YY (PYY), glucagon-like peptide 1 (GLP-1) [25,137].
An abundant production of SCFA can therefore indirectly regulate blood glucose levels via
the insulinotropic effect of GLP-1 and induce satiety by the anorexigenic effect of PYY on the
hypothalamus [138]. In addition, enteroendocrine cells in the duodenum are dependent on
microbe-mediated mechanisms to express cholecystokinin (CKK), a gut peptide hormone responsible
for stimulating fat absorption. GF mice have exhibited a reduced number of these cells with impact on
their fat absorption [139].
SCFA can also act by inhibition of histone deacetylase (HDAC) activity. HDAC is related to
a suppression of malignant transformation and a stimulation of apoptosis of precancerous colonic
cells [140] as well as to the stimulation of epithelial production of retinoic acid (RA) [141]. RA is
involved in many physiological processes, including regulation of IgA [142] and the polarization of
specialized mucosal myeloid cells [143].
Specific phytonutrients entail several immunomodulatory functions. Some are mediated by a shift
in microbiotic composition favoring the abundance of specific bacteria with health promoting effects
or create occupancy resistance to enteric pathogens with pro-inflammatory potential [144]. Others are
receptor-mediated, e.g., genistein, a flavonoid compound present in legumes, which can positively
effect β-cell mass and mitigate T2D in mice [145,146].
In summary, the biodiversity of the microbiotic community is critical for both inter-microbiotic
interactions and host-microbe engagements. In a healthy and proper fed microbiota the inflammatory
and immunoregulatory species are in balance and the diverse bacterial communities act together to
produce metabolites with direct effect on host health.

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9. The Interaction between Food, Microbiota, and Host

In the sections above, we have discussed how ingestion of unrefined plant-based food structures
implies that the host shares calories, saccharides, amino acids, fatty acids, and bioactive molecules
with the microbiota, which in return shares SCFA, vitamins, antioxidants, and other biomolecules
with the host. The microbiota also stimulates mucosal immune function upon ingesting an unrefined
diet; a diverse microbiota will express a multitude of different antigens, thereby promoting intestinal
bacterial recognition and shaping intestinal immune function.
In contrast, refined foods lack the fiber-encapsulation with the result that the host absorbs most
nutrients in a microbiota-independent manner. Refined food is thus directly absorbed through the host
GI mucosa without feeding the microbiota and without immunological engagement in the distal gut
(Figure 3). Furthermore, when bypassing the microbiota, glucose is rapidly absorbed creating a risk of
high blood glucose and associated pro-inflammatory effects.

Figure 3. Schematic overview of interactions between food, microbiota, and host. Most of a refined diet
bypasses the microbiota, providing a high nutrient bioavailability for the host. In contrast, unrefined,
plant-based food structures provide a lower nutrient bioavailability for the host, using the difference
to develop and maintain a healthy microbiota. The healthy microbiota provides short chain fatty
acids (SCFA), antioxidants, and vitamins for the host along with direct immunomodulatory effects.
The high monosaccharide bioavailability in refined foods introduces a risk of hyperglycemia, which
cause numerous direct and indirect pro-inflammatory actions [26] (not shown in figure).

10. Implications for Nutritional Guidelines

The mechanisms summarized above underscore the need to consider the microbiota when
evaluating human development, nutritional needs, physiological variations, and the impact of diet.
Historically, the processing of foods occurred to increase the caloric bioavailability but given the

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growing prevalence of non-communicable diseases, insulin resistance, overweight, and obesity [147],
it is obvious that current nutritional guidelines need to be revisited to re-establish the endogenous
microbiota-induced health promotion. The current nutritional guidelines are mainly based on optimal
molecular nutrient content for host metabolism, while establishment of synergies between food, host,
and microbiotic community are absent.
Nutritional guidelines need to consider both the content and the structure of carbohydrates,
because the microbiotic breakdown of carbohydrates strengthens our microbiotic ally in support of
host health. Furthermore, it should also reflect that consumption of “fiber-formulated nutrients”
is essential to maintain a healthy diverse microbiota to balance our nutritional, immunological,
and metabolic health.
We have developed a simple tool to visualize healthy microbiota-engaging food choices (Figure 4).
This tool divides foods into four categories depending on their carbohydrate content and the degree of
refinement. Healthy, unrefined foods with low relative carbohydrate content (green box) should make
up the plant-based bulk of an adult basic diet. These foods interact with the microbiota. Refined foods
or foods with high relative carbohydrate content (yellow boxes) should be considered carbohydrate or
energy supplementation, while the “comfort” foods in the red box, which are both refined and high in
carbohydrate content, should be used sparingly considering their potentially negative health impact
and their lack of microbiotic health promotion.

Figure 4. Novel classification of plant-based foods. Plant-based nutrition classified by microbiotic

interaction potential and by the potential to induce hyperglycemia. The illustration classifies
plant-based foods by the level of refinement and carbohydrate content. The green box represents
basic foods for the habitual diet, interacting with our gut microbiota and providing both nutrients and
bioactive molecules to microbiota and host. Foods from the green box are health promoting through
interaction with the microbiota, and these foods introduce no risk of hyperglycemia. Foods from the
green box should be combined to meet the needs for protein and lipid. Food from the yellow boxes
can induce hyperglycemia in insulin resistant individuals or be consumed as energy supplementation
for children and physically active individuals with a higher energy demand and a low risk of insulin
resistance. The yellow square to the left positively interacts with the microbiota whereas the yellow
square to the right only have limited microbiota interaction. The red box contains foods, which are
high in carbohydrate content and highly refined, introducing the risk of high blood glucose to most
people, not only insulin resistant individuals. The contents in the red box should be considered comfort
foods with no health promoting effects and with a potential to compromise health.

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11. Conclusions
The increasing prevalence of non-communicable diseases with an inflammatory component has
led to the understanding of the gut microbiota as an intrinsic regulator of host immune responses.
There is a growing awareness of the role of microbiotic communities, microbiota-derived products,
and more particularly of the link between these components and disease states in humans. The ability
to target immune pathways relies on the understanding of both acute and long-term impacts on the
gut microbiota. The microbiotic communities can adapt to host diet in multiple ways by the interaction
among different bacterial species, loss or proliferation of specific species, and by interaction with the
host. Diet therefore presents itself as a microbiota-modulating factor and hence a health-modulating
factor. A revision of the current nutritional guidelines considering both the molecular content and
molecular structure of nutrients for insulin resistant and insulin sensitive individuals, could be valuable
for the restoration of beneficial bacteria and microbiota diversity, enabling a shift from disease to health
promoting states in the individual as well as in the general population.

Author Contributions: Conceptualization, A.S.; Writing-Original Draft Preparation, N.W.H and A.S;
Writing-Review & Editing, N.W.H. and A.S.; Visualization, N.W.H and A.S.
Funding: This research received no external funding.
Acknowledgments: We are grateful to Anne Stæhr Johansen, PhD, for her editorial assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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