Best Practice & Research Clinical Gastroenterology

Vol. 18, No. 2, pp. 387 –404, 2004

doi:10.1053/ybega.2004.449, available online at [Link]

12

Mucosal immune responses

David W. K. Acheson* MD, FRCP

Stefano Luccioli MD
Food and Drug Administration, Center for Food Safety and Applied Nutrition, DHSS/FDA/CFSAN, 5100 Paint Branch
Parkway, Mail Code HFS 6, Room 2B-003, College Park, MD 20740-3835, USA

The host gastrointestinal tract is exposed to countless numbers of foreign antigens and has
embedded a unique and complex network of immunological and non-immunological
mechanisms, often termed the gastrointestinal ‘mucosal barrier’, to protect the host from
potentially harmful pathogens while at the same time ‘tolerating’ other resident microbes to
allow absorption and utilization of nutrients. Of the many important roles of this barrier, it is
the distinct responsibility of the mucosal immune system to sample and discriminate between
harmful and beneficial antigens and to prevent entry of food-borne pathogens through the
gastrointestinal (GI) tract. This system comprises an immunological network termed the gut-
associated lymphoid tissue (GALT) that consists of unique arrangements of B cells, T cells and
phagocytes which sample luminal antigens through specialized epithelia termed the follicle
associated epithelia (FAE) and orchestrate co-ordinated molecular responses between immune
cells and other components of the mucosal barrier. Certain pathogens have developed ways to
bypass and/or withstand defence by the mucosal immune system to establish disease in the
host. Some ‘opportunistic’ pathogens (such as Clostridium difficile) take advantage of host or
other factors (diet, stress, antibiotic use) which may alter or weaken the response of the
immune system. Other pathogens have developed mechanisms for invading gastrointestinal
epithelium and evading phagocytosis/destruction by immune system defences. Once cellular
invasion occurs, host responses are activated to limit local mucosal damage and repel the
foreign influence. Some pathogens (Shigella spp, parasites and viruses) primarily establish
localized disease while others (Salmonella, Yersinia, Listeria) use the lymphatic system to enter
organs or the bloodstream and cause more systemic illness. In some cases, pathogens
(Helicobacter pylori and Salmonella typhi) colonize the GI tract or associated lymphoid structures
for extended periods of time and these persistent pathogens may also be potential triggers for
other chronic or inflammatory diseases, including inflammatory bowel disease and malignancies.
The ability of certain pathogens to avoid or withstand the host’s immune assault and/or utilize
these host responses to their own advantage (i.e. enhance further colonization) will dictate the
pathogen’s success in promoting illness and furthering its own survival.

Key words: mucosal immunity; intestinal epithelial cells; food-borne pathogens; gut-associated
lymphoid tissue; follicle associated epithelia.

* Corresponding author. Tel.: þ1-301-436-1910; Fax: þ1-301-436-2633.

E-mail address: [Link]@[Link] (D.W.K. Acheson).

1521-6918/$ - see front matter Q 2003 Elsevier Ltd. All rights reserved.
388 D. W. K. Acheson and S. Luccioli

INTERACTION OF BACTERIAL PATHOGENS WITH THE HOST

MUCOSAL IMMUNE SYSTEM

Introduction

The mucosal immune system is located in a variety of different anatomical sites in which
the external environment comes into contact with the human host and has a complex
role to play in protecting the host. In the gastrointestinal (GI) tract, this system has the
important role of preventing the uptake of agents that will cause harm, while at the
same time allowing the passage of nutrients from the intestinal lumen into the systemic
system. This chapter focuses on the key cellular mechanisms of the intestinal mucosal
system and on strategies or other factors that allow intestinal pathogens to bypass
these mechanisms. A brief overview of the components of the intestinal immune
barrier is provided, and this is followed by a discussion of the ways in which certain of
the more common intestinal pathogens interact with the mucosal immune system and
how some have developed self-serving mechanisms to enhance their pathogenic
mission to infect the human host.

The intestinal mucosal barrier

The intestinal mucosal barrier has an enormous surface area of the order of 400 m2 and
comprises a variety of cellular and non-cellular elements.1 Structurally, the barrier is
formed by an epithelial cell lining with a complex array of agents on its luminal surface,
and by organized lymphoid tissues designed to assist in the protective function against
harmful foreign antigens (Table 1).
While consisting of only a single layer of columnar cells, the epithelial barrier must
serve the balanced function of providing a physical deterrent while at the same time
allowing the uptake of important nutrients. Typical intestinal epithelial cells have
microvilli on their apical surfaces with a filamentous brush border glycoclayx at the
tips.1 While this anatomy helps to prevent penetration by foreign antigens, these cells
simultaneously express MHC class II receptors (MHC II) to facilitate antigen
presentation to immune cells as needed. These cells can also recognize certain highly
conserved microbial structures by way of surface toll-like receptors (TLRs), and initiate
cellular signalling responses on their own.2 Moreover, cells of this villous epithelium
produce a variety of functional molecules, such as defensins3, trefoil factors4, and
mucins, that help protect the human host.4 Mucins are the principal components of
mucus, which lines the surface epithelium throughout the intestinal tract.5 Microbes of
all types (bacteria, viruses, protozoa) become trapped in the mucus layer and are
expelled from the intestine by peristalsis. Other important components of mucus
include various proteolytic enzymes which serve not only to facilitate digestion of
polypeptides but also to alter/diminish the immunogenic properties of these peptides
because peptides of less than 8 –10 amino acids are poor immunogens.6 The villous
epithelial cell and mucus barrier and their secreted molecules, although critical
components in mucosal first-line defence, are the subject of another chapter in this
volume and will not be discussed in detail here.
Interspersed with the villous epithelial monolayer is the follicle-associated
epithelia (FAE), which overlies a vast network of organized mucosa-associated
lymphoid tissue (MALT).7 This specialized epithelia is distributed throughout the
intestinal tract as part of the gut-associated lymphoid tissue (GALT) and is found in
more organized fashion in areas reflecting a high presence of foreign materials
 Mucosal immune responses 389

Table 1. Elements of the gastrointestinal mucosal barrier.

Luminal/epithelial Function

Epithelium: glycocalyx, villi Innate immune response

Antigen presentation
Block penetration of ingested antigens
Defensins Antimicrobial peptides
Trefoil factors Protection from a variety of deleterious agents (bacterial
toxins4, chemicals, and drugs); provide restitution after
mucosal injury
Mucus/mucins Block penetration of ingested antigens
Proteases: pepsins, pancreatic enzymes Breakdown of ingested antigens
Secretory-IgA (s-IgA) Block adhesion to epithelial surface by pathogens
Gastric acid (pH) Breakdown of ingested antigens
Bile acids Breakdown of ingested antigens
Intestinal peristalsis Block penetration of ingested antigens
Indigenous microflora Competitive inhibition22
Direct: competition for essential nutrients and bacterial receptor
sites; creation of restrictive physiological environments;
secretion of antibiotic-like substances
Indirect: chemical modification of bile salts and dietary fats,
induction of protective Ig responses, stimulation of peristalsis
GALT-associated
IgA, IgGa, IgMa (serum) Clear antigens penetrating gastrointestinal barrier/
systemic immunity
Assist in opsonization and phagocytosis of antigens
Lymphoid follicles Lamina propria (LP) Clear antigens penetrating gastrointestinal barrier
Intraepithelial cells (IEL) Innate and acquired immune responses
Mesenteric lymph nodes (MLN) Phagocytosis and antigen presentation
a
Activates complement.

and microorganisms. These include the Peyer’s patches in the distal small intestine,
the palatine tonsils or pharyngeal mucosa of Waldeyer’s ring and the appendix.8 The
GALT contains important regulatory cells of the mucosal immune system such as
lymphocytes, which are equipped to organize and mount rapid, selective, and potent
immune responses against harmful foreign pathogens, and phagocytes, which play a
role in the sampling, presentation, and destruction of pathogens. The anatomy of this
network facilitates close association between these cells and antigens or microbes
from the lumen2,7, and these interactions are important in differentiating between
harmful or beneficial food antigens and/or pathogens through a process involving
oral tolerance. Tolerance is a necessary mechanism because the strong protective
immune responses to fight off invasion by pathogens could equally lead to food
allergy or other chronic disease when directed towards dietary proteins or
indigenous flora.2,6 Modulation of certain anti-inflammatory cytokine pathways, such
as IL-10 and TGF production, and/or interactions between T regulatory cells and
MHC II-expressing antigen-presenting cells (APCs), appear to favour tolerance.9
Some pathogens have developed strategies to enhance tolerance and therefore avoid
immune responses by the host.
390 D. W. K. Acheson and S. Luccioli

Table 2. Distinctive features of the intestinal mucosal epithelium.

Villous epithelia Follicular-associated epithelia (FAE)

Polarized and tight junction formation Yes Yes

Basolateral membrane Non-indented Indented
Apical brush border Present Reduced
Membrane glycocalyx Present Reduced
Clathrin-coated membranes No Yes
Membrane-associated hydrolases Present Little to none
Mucus production Present Little to none
Paneth cells within epithelia Present Little to none
MHC class II Yes No
IgA transporting Yes No

The FAE is specialized for uptake of antigens and microbes from the lumen. The cells
of FAE are distinctive compared with villous epithelia (Table 2) and their physiological
characteristics facilitate close surface contact with luminal antigens/pathogens.7 One of
the most striking and unique features of the FAE is the presence of M cells. M cells
possess a pocket in the basolateral membrane that pushes against the apical surface and
provides a docking site for special subpopulations of lymphocytes to remain close to
the luminal surface.7 The apical surface of the M cell has microfolds interspersed with
clathrin-coated microdomains which mediate endocytosis of ligand-coated particles,
macromolecules and viruses. Also, expression of unique surface glycosylation patterns,
such as sialyl Lewis A antigen, may enhance their interaction with and engulfment of
microbes.10 Thus, M cells are specially designed for transepithelial cell transport and for
delivering foreign antigens and microorganisms to the underlying GALT. At the same
time, however, these same features make this intestinal epithelium more easily
accessible to pathogens.
This gut-associated lymphoid tissue (GALT) comprises four distinct lymphoid
compartments: the Peyer’s patches and other lymphoid follicles associated with the
FAE; the lamina propria; intraepithelial lymphocytes (IELs); and mesenteric lymph nodes
(MLN).11 Lymphoid follicles are characterized by aggregates of immature B cells and
CD4 (þ ) helper T cells sitting within specific pockets of the M cell7 and resemble lymph
nodes without the afferent lymphatics. Thus, these lymphoid aggregates come into
contact solely with antigens from the gut lumen and serve as inductive sites for
intestinal immune responses.8 Another unique feature of these lymphoid tissues is the
propensity for IgA production. The production of immunoglobulin typically requires T
cell help, and specific interactions between the CD4 (þ ) T cells and dendritic cells
found within the dome surrounding these follicles lead to secretion of transforming
growth factor-b (TGF-b) which favours B cell class switching to IgA.12 B cells then
migrate out of the follicles into the surrounding mucosa and release secretory IgA
(s-IgA) into the gut lumen where it functions mainly to bind pathogens and prevent their
attachment to epithelial cells.13 IgA may also remove antigens from the subepithelial
space by binding free antigens while it is being transported from the basolateral surface
to the apical epithelial cell surface and secreted into the gut lumen.14 In these capacities,
IgA has a role in the tolerance function of the gut as well.2
The lamina propria (LP) represents the basement membrane layer of cells residing
below the FAE and around lymphoid follicles. The LP contains an enormous number of
 Mucosal immune responses 391

terminally differentiated B cells, probably more than any other organ in the body, as well
as T cells, dendritic cells (DCs), macrophages, mast cells and polymorphonuclear
leukocytes.8 Although antigen presentation also occurs, this compartment provides
important effector sites for preventing the entry and systemic spread of pathogens
across the intestinal cell barrier and for destroying pathogens when invasion occurs.
Also, in co-operation with the underlying MLN and vascular endothelium, they serve as
important sites for cellular expansion and differentiation within GALT.8
The LP lymphocytes are largely IgA-secreting plasma cells of the B cell lineage and
memory T effector cells. T cell effector functions include two types of helper T cells
(TH), mostly of the CD4 (þ ) lineage, based on their pattern of cytokine secretion. This
T cell population also includes a significant percentage of CD8 (þ ) cells which have
cytotoxic functions but which may also elaborate effector responses as well.
Macrophages, DCs and epithelial cells sample and engulf antigens to present to T
cells.2 The differentiation into cytokine-producing cells is dependent on specific cellular
interactions with antigen and on the cytokine milieu.15 TH1 cell responses occur in
environments where IL-12 is released by these antigen-presenting cells (APCs). IL-12 is
a known inducer of natural killer (NK) cell cytotoxic function16 and induces T cells to
produce interferon-gamma (IFN-g). IFN-g is important for cell-mediated responses and
for killing intracellular pathogens by phagocytes. In particular, IFN-g upregulates
production within macrophages of reactive oxygen intermediates (ROI) and reactive
nitrogen intermediates (RNI) which have important antimicrobial properties.16 Other
important cytokines in this cascade are IL-1 and TNF-a, which activate macrophages,
and IL-8, which recruits other phagocytes such as neutrophils. TH2 cells traditionally
respond to multicellular pathogens and/or are produced abnormally to exogenous
environmental proteins. They secrete a cytokine repertoire of IL-4, IL-5 and IL-13 to
induce B cell activation and differentiation and recruitment of eosinophils and mast cells
to tissues.15 A third effector cell (TH3), referred to as the T regulatory cell, has also
been identified and responds to immunosuppressive cytokines in the monitoring of oral
tolerance.2,6,9
Populations of intraepithelial lymphocytes (IELs), predominently cytotoxic T cells,
inhabit the interdigitating spaces between epithelial cells above the basement
membrane. NK-like lymphocytes have also been detected in these spaces.17 To date,
the role of IELs in immune defence remains to be fully elucidated. However, because of
their location and the observation that most cells are cytotoxic, IELs are believed to
play an important role in innate defence and tumour surveillance in the gut. Recent
immunohistological studies in mice have elucidated regional and quantitative differences
within the small intestine between intraepithelial lymphocytes (IELs) and lamina propria
lymphocytes (LPLs).18 These studies also highlighted differences between the T cell
receptor (TCR) gd and TCR ab lymphocyte populations.18 The functional significance
of the unusually high percentage of gd T cell populations, both at baseline and following
certain infections, in the gut is not fully understood.13,19 Mice deficient in gd T cells
generally have greater difficulty with immune regulatory function than with ability to
clear intracellular pathogens.20 Also, the observation that gd T cells, as opposed to ab
T cells, respond directly to certain bacterial antigens without antigen presentation
suggests that these cells also are involved in innate defence against pathogens.21
The MLN and the vascular endothelium also serve important functions in the GALT.
After ingestion of foreign antigen, DCs and macrophages often migrate to MLN and
present antigen to MLN T cells. These interactions aid MLN B cell differentiation into
primarily IgA-producing plasma cells and induce a systemic response to the foreign
antigen. The plasma cells are released into the bloodstream and then eventually ‘home’
392 D. W. K. Acheson and S. Luccioli

back through the gut vascular endothelium to populate the LP, where they are needed
to release IgA into the gut lumen. Gut endothelial cells, which release and recognize
these homing factors, thus serve the important functions as ‘gatekeepers’ for
recruitment of immune cells to the infected or damaged mucosal sites.8 Furthermore,
killing and removal of pathogens also occur in the MLN. Pathogens able to bypass
immune defences and reach these targets have an optimal chance to spread systemically
through the bloodstream or lymphatics.

Breakdown of mucosal barrier function

The mucosal barrier helps to maintain symbiosis between microbes residing in the gut
and the host animal, and the integrity of this barrier is regulated by a complex
network of physical, physiological, and immune factors which include dietary
influences, the host environment (which can be modified by age, external factors
such as antibiotics, and immune competency), and the indigenous microbial flora of
the gut.22 Modification and/or breakdown of these factors leads to ineffective
clearance or degradation of harmful ingested antigens and/or disruption of regulatory
cell function resulting in mucosal damage, increased gut permeability, and overgrowth
of harmful pathogens.
Diet not only may introduce carcinogens or toxins which may be directly toxic to
the gut mucosa but also provides nutrients which modify the physiological environment
and allow growth of certain bacteria in the gut. As an example, infant diets comprising
solely of breast milk are rich in acetic/propionic acids which induce an acidic
environment while diets comprised of infant formula have increased iso-(butyric,
valeric, caproic) acids favouring higher pH. Lower pH appears to inhibit growth of
certain E. coli and Vibrio cholerae strains, and this has been postulated as a reason for the
decreased incidence of intestinal symptoms or diseases in breast-fed compared to
formula-fed infants. Also, starvation leads to a gut environment with unconjugated bile
acids and decreased short-chain fatty acid concentrations which tend to inhibit
anaerobic bacterial growth while favouring growth of pathogenic coliform bacteria,
such as E. coli.23
Age or stage of gut development may dictate which pathogens colonize the gut
mucosa and contribute to disease. Infants tend to have a higher prevalence of
gastrointestinal disease, especially from enteric viruses, than adults. Developmental
immaturity of the infant gut plays a factor. Newborns typically lack IgA and IgM in
exocrine secretions until a few months of age, and s-IgA is found in low levels in the
saliva and gut following birth.11 These low antibody levels may provide an inefficient
barrier to microbes. Moreover, porous villous membranes, low basal acid output, and
immature proteolyic activity in the gut are additional factors which lead to altered
clearance of potentially harmful microbes.11 Infants are further colonized by a unique
repertoire of bacteria compared to adults, and this difference has been suggested to
make the infant more susceptible to botulism following ingestion of Clostridium
botulinum spores.22,24 At the same time, up to 90% of infants are colonized with
C. difficile but rarely develop disease from this organism.22
Antibiotics are beneficial for treating illness caused by certain enteric pathogens.
However, they may also contribute to disease by eliminating certain microbes from the
indigenous microflora which inhibit or suppress the growth of pathogenic microbes in
the gut. Moreover, prolonged use of antibiotics may favour colonization by antibiotic-
resistant strains of bacteria, which can pose serious consequences if these strains
become pathogenic. A prime example of the suppressive phenomenon of antibiotics is
 Mucosal immune responses 393

infection with C. difficile, leading to pseudomembranous colitis.22 Although spontaneous

cases of C. difficile colitis occur, most cases appear with use of antibiotics. Antibiotics
which preferentially eliminate anaerobes (such as clindamycin) compared to facultative
organisms tend to cause higher prevalence of infection. Also, it may take up to 30 days
for the suppressive effects of these antibiotics to wear off.25 C. difficile is non-invasive
and mediates a neutrophil-mediated colitis through elaboration of cellular toxins. One
of these toxins, toxin A, has been shown to interact directly with enteric neurons, and
the subsequent release of neuropeptides may contribute to gut hypermotility as well as
inflammatory responses.26
Disease states and/or pathogens are kept in check by competent cells or pathways of
the immune system. Immune competency can be altered by drugs (such as steroids),
age (infants and elderly individuals), acquired disease states (infections, malignancies),
stressors (trauma, surgery), and germline immunodeficiency. The end result is naı̈ve,
delayed or defective IgA, phagocytic, or T cell effector immune responses leading to
ineffective neutralization of pathogens and increased susceptibility to disease. IgA
represents a first line of defence by blocking the association of certain non-indigenous
microbes, namely, Salmonella spp and Vibrio cholerae, and enteric viruses, with the gut
epithelium13 and therefore reducing penetration of these pathogens into the lamina
propria. Phagocytes and T cells offer a second line of defence against cellular invasive
organisms and help to eliminate pathogens from cellular tissues. Also, interactions
between these cells and epithelial cells help to elaborate cytokines or other factors
which support the symbiotic relationship with the indigenous microflora. Immune
deficiency thus leads not only to acute microbial disease but, because of ineffective
measures to clear offending pathogens, favours chronic persistence of these pathogens
in diseased tissues, thus contributing to malignancies, inflammatory disorders, or other
systemic illness.2
The indigenous microflora also provides protection against infection from certain
pathogens. ‘Colonization resistance’ or ‘bacterial antagonism’22 represents the ability of
the host (mf) to interfere with colonization of the gastrointestinal tract by exogenous
microorganisms/pathogens (Table 1). Although the relationships between microbes in
the gut with the host and other microbes is discussed elsewhere, it is important to note
that interplay between microbes in the gut lumen leads to both direct and indirect
modes of protection from pathogens. Disruption of this interplay may not only affect
the integrity of the mucosal barrier but may also allow certain pathogens to overwhelm
or avoid mucosal immune responses.

Translocation of pathogens

Breakdown of the mucosal barrier network also facilitates the invasion or entry
through host cells by pathogenic organisms, some of which would not otherwise
normally cause disease. Although previously described for the microflora, the term
‘translocation’ refers to the process of migration of viable microbes from the lumen of
the gastrointestinal tract to extraintestinal locations such as the MLN, liver, spleen, and
bloodstream.25 Factors which favour translocation include bacterial overgrowth,
immune deficiencies and mucosal injury with loss of barrier integrity. Evidence of
increased translocation of pathogens has been observed in a variety of disease states25
and have been shown to be associated with cases of post-operative sepsis27 and with
spontaneous bacterial peritonitis in cirrhotic rats.28 The ability of a pathogen to
translocate efficiently through cells coupled with mechanisms to avoid immune
detection or destruction appears to provide a survival advantage for a pathogen
394 D. W. K. Acheson and S. Luccioli

and may account for the systemic spread of certain organisms. However, because
translocation is also routinely observed with non-pathogenic organisms, and
experimental introduction of pathogenic organisms past the GI mucosal barrier is
not always associated with disease29, some have disputed the importance of this
mechanism in inducing disease or pathogenesis.30
Bacterial overgrowth can occur from a variety of factors (described above) and
facilitates translocation. For some organisms, such as E. coli, the potential for
translocation is directly related to levels of organisms. Other microbes, such as
Pseudomonas spp and Gram-negative enterobacteria, are more efficient in translocating
across cells than are other bacteria in the gut such as obligate anaerobes (e.g.
Bacteroides spp). This may explain why these former organisms are more frequent
causes of opportunististic infections and sepsis in immunocompromised hosts. This
ability for translocation is independent of their ability to adhere to epithelial cells or of
host genetic factors25, but may be facilitated by defective host immune responses and
mucosal injury. Translocation routinely occurs across epithelial cell walls. However,
ingested agents (castor oil) and clinical conditions (shock) can cause direct damage to
tight junctions between epithelial cells, and this may then allow microbes to utilize
other portals of entry into the mucosa.25

Host –pathogen interactions

Many enteric pathogens have developed mechanisms to use the host immune system to
their benefit. These include bacterial, parasitic and viral agents that have evolved a
variety of strategies that facilitate their colonization, survival or ability to invade. In the
following sections a number of these mechanisms are discussed in relation to specific
common pathogens and how they have developed their own unique approach to
dealing with the host immune system.

INTERACTION OF BACTERIAL PATHOGENS WITH THE HOST

IMMUNE SYSTEM
Helicobacter pylori

Helicobacter pylori is an excellent example of a pathogen which modulates the mucosal

immune system to produce disease and enhance its survival. It is a urease-producing
Gram-negative bacterium which preferentially colonizes the gastric and duodenal
mucosa in half of the world’s population.31 H. pylori has adapted to the harsh
environment of the stomach by producing urease, which helps to maintain pH
homeostasis within the bacterium and the outside environment, and by effective
storage and metabolism of iron, of which supply varies greatly.31 It adheres to gastric
epithelial cells by way of its BabA adhesin, binding to exposed fucosylated blood group
antigens. Another adhesin, SabA, has been shown to bind sialyl-dimeric-Lewis x.32 This
and other sialylated derivatives, which are adhesion and colonization targets for these
bacteria, are increased in gastric epithelial cells in response to inflammation. Thus,
specific adhesion to cellular receptors upregulated during inflammation may explain
how H. pylori thrives in gastric mucosa lesions. The sabA gene, which can be turned on
or off, may then dictate which organisms will bind to inflamed tissue and potentially lead
to chronic, persistent infections32 and subsequent gastritis, peptic ulcers, lymphoma,
and increased risk of gastric adenocarcinoma.33
 Mucosal immune responses 395

An interesting attribute of H. pylori is that, unlike the surrounding GI flora, it is able

to induce and modulate mucosal immune responses. Yet, these responses do not result
in eradication of the organism. As explained above, induction of inflammation could
provide a colonization advantage for the bacteria by providing a multitude of adhesion
sites. H. pylori has been shown to induce IL-8 production through engagement of a cag
pathogenicity island-encoded type IV secretion system with host cell receptors.34 – 36
This system also releases an effector molecule, cytotoxin associated antigen (CagA),
into cells which is believed to induce epithelial cell proliferation and scattering.37 IL-8 is
an important chemokine in the recruitment of polymorphonuclear cells (PMNs) to the
epithelial border. These PMNs then contribute to epithelial damage and further uptake
of organisms. H. pylori ‘hides’ in the gastric mucus and is protected from PMN injury.38
Moreover, bacterial urease has been shown to induce nitric oxide production in
macrophages leading to release of inflammatory mediators.39 H. pylori can also stimulate
a variety of specific B cell antibody and T cell responses with H. pylori-specific CD4þ
lymphocytes homing to the gastric epithelium.40
At the same time, however, H. pylori can down-regulate certain immune responses,
and this may offer selective advantages in its ability to infect the gastric mucosa
chronically. VacA, a vacuolating cytotoxin virulence factor produced by H. pylori, may
interfere with the ability APC’s to degrade and process antigen41 and, most recently, has
been shown to inhibit T cell activation through interference of the calcineurin-
associated IL-2 signalling pathway, in a similar fashion to that of the immunosuppressive
agent FK506.42 Moreover, cag genes may upregulate fas ligand (fasL), leading to ligand-
mediated T cell apoptosis.43 These anti-inflammatory mechanisms, coupled with the
evidence that human gastric epithelium lacks toll-like receptor-4 (TLR-4), which is part
of the protective innate immunity against organisms presenting LPS, such as H. pylori,
lends further support to the observation that the gastric epithelium is largely non-
responsive to H. pylori44 and thus allows chronic colonization by H. pylori in the
stomach. Once colonization is established, H. pylori tends to delete cag genes, thus
turning off the immune response.45

Escherichia coli

Many different types of E. coli have been linked with intestinal disease, including
enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic
E. coli (ETEC) and enteroaggregative E. coli (EAgg). While some of these types of E. coli
have very specific virulence factors, such as Shiga toxins produced by EHEC and heat
stable and labile toxins produced by ETEC, many other virulence factors are clearly
playing a role in pathogenesis. One possible pathogenic mechanism that is receiving
attention is the effect of E. coli bacterial products on intestinal lymphocytes. Recently
identified products of EPEC were found to inhibit cytokine production by human
peripheral blood and intestinal LP lymphocytes.46 Lysates obtained from Shiga toxin-
producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion
mutant all directly inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid
cells.47 Subsequently, a large gene (lifA) that encodes a toxin, lymphostatin, which
specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma
interferon production in response to a variety of stimuli, was identified in EPEC. This
gene shares significant homology with the catalytic domains of the large clostridial
cytotoxins.48 Shiga toxin-producing E. coli have a very similar gene (efa1) that is 97.4%
identical at the amino acid level to lymphostatin. Overall, these data indicate that
enteric bacterial products may alter immune homeostasis in the gastrointestinal tract
396 D. W. K. Acheson and S. Luccioli

through inhibition of regulatory cytokine production, which could then contribute to

bacterial pathogenesis.
A number of different types of pathogenic E. coli have also been shown to initiate the
production of a variety of chemokines from intestinal epithelial cells in vitro. For
example, Shiga toxins have been shown to induce multiple C-X-C chemokines at the
mRNA level, including IL-8, GRO-alpha, GRO-beta, GRO-gamma and ENA-78. IL-8,
GRO-alpha and ENA-78 mRNAs are induced and superinduced by Shiga toxin, and both
IL-8 and GRO-alpha protein are induced in a dose-response fashion.49,50 Further data
indicate that this induction is probably occurring via a ribotoxic stress response.51
Rather than providing a host protective response, the chemokine-induced influx of
PMNs actually leads to an increase in Shiga toxin uptake into the underlying tissues. The
precise mechanisms for this are unclear but it appears that in vitro transepithelial cell
migration of the PMNs in the luminal direction allows the Shiga toxin to cross in the
reverse direction.52 Thus, one could speculate that the influx of PMNs during acute
infection in vivo may actually favour uptake of Shiga toxins, the primary virulence factors
in the development of life-threatening EHEC complications such as haemolytic uraemic
syndrome, into cells.

Shigella species (sonnei, flexneri, dysenteriae, boydii)

Shigella has evolved a number of mechanisms that facilitate its invasion of mucosal
epithelial cells of the colon and rectum. It bypasses apical epithelial cell defences by
utilizing M cells as a vehicle for entry; more invasive types are more efficient at crossing
M cells.35 Some species have also been shown to downregulate production of
bactericidal peptides by Paneth cells to avoid the innate immune response and facilitate
early infection.53 Although they encounter and are phagocytosed by DCs and
macrophages, these organisms secrete factors such as IpaB (S. flexneri) which, through
binding of caspase I, initiates phagosome lysis and allows the organism to escape into
the cytoplasm. Once in the cytoplasm, IcsA interacts with actin to allow movement of
the bacteria within the cytoplasm and to enter neighbouring epithelial cells by way of the
less-defensive basolateral membrane.45 Shigella invades neighbouring cells by utilizing a
type III secretion system35,36, and intracellular motility and cell-to-cell spread allows
more efficient epithelial invasion. Epithelial cells respond to infection by production of
IL-8 and other inflammatory molecules through activation of NF-kB.54 Although
destructive to mucosal cells, this response is necessary to clear the organism and
prevent bacteraemia. Moreover, in the more virulent strains of S. flexneri (and
S. dysenteriae45), one of two copies of the msbB gene is located on the virulence plasmid.
Activation of this gene acylates and thereby enhances lipid A expression, the major
immunostimulatory component of LPS. There is evidence that, in these organisms, LPS
is maximally stimulatory and induces intestinal leukocyte infiltration, which then leads
to further disruption of epithelial membranes and further bacterial invasion.
Bacterial mutants with inactivated msbB genes were neither able to invade nor
induce TNF-a production from epithelial cells.55

Salmonella enterica

Salmonella also utilize a type III secretion system to invade epithelial M cells and induce
apoptosis of phagocytic cells for survival.35 However, as opposed to Shigella, they often
remain intracellular and have evolved elaborate mechanisms to bypass immune
defences and cause enterocolitis (S. typhimurium) or systemic disease (S. typhi).
 Mucosal immune responses 397

S. typhimurium has been shown to adhere to murine M cells by long fimbriae. This leads
to ruffling of the cell membrane and macropinocytosis.35 These microbes may also
secrete effectors, SopE and SopE2, which interact with the actin cytoskeleton and allow
entire engulfment of the bacterium.45 Expression of Salmonella pathogenicity island 1
(spI 1) enhances invasiveness. However, SpI 1- deficient organisms may also invade cells
by binding to and entering CD18-expressing mononuclear cells.56 This may be a
potential pathway for systemic spread, as these cells could then carry the bacteria to
other tissues.
Once inside the cells, Salmonella is able to survive or withstand immune attack
through a number of mechanisms. Some strains have altered pathogen-associated
molecular patterns (PAMPs), such as hepta-acylated lipid A, resulting in low stimulatory
LPS, and/or lack flagellin, which makes these organisms less reactive. PAMPs are highly
conserved microbial structures which include lipopolysaccharide, peptidogylcans and
bacterial DNA which can initiate cellular signalling responses by interaction with innate
host receptors, such as TLRs.45 LPS is recognized by TLR4 and flagellin by TLR5.45,57
LPS expression may be further regulated by modification of signal transduction systems
that interfere with MHC II presentation of bacterial antigen45,58,59 S. typhimurium then
appears to become ‘trapped’ within the vacuolar compartment, presumably by
interfering with Nramp1, which is a phagosomal membrane protein that limits pathogen
replication by lowering the pH in the intravacuolar environment.16,60 Survival may also
depend on the bacterium’s ability to reduce NADPH oxidase and interfere with NO-
mediated ROI formation in vacuoles.45,60 Also, some organisms have been able to adapt
to antimicrobial peptides, such as defensins, through undefined mechanisms mediated
by a mig 14 gene.61 Although not entirely clear, S. typhimurium initiates the immune
response from its intracellular location. Effectors such as SipB, an analogue of SopB, can
induce apoptosis of macrophages and/or initiate NF-kB activation and release of pro-
inflammatory mediators leading to disease.62,63
S. typhi, the cause of typhoid fever, persists in a carrier state in 3– 5% of infected
individuals. Although the reasons for this are unknown, bacterial mutant studies have
shown that the persistence of bacteria may be due to decreased immunogenicity
factors such as lack of expression of immunogenic Agf A adhesive fibres and/or a
mutation in a housekeeping gene polynucleotide phosphorylase (PNPase), which is
involved in degradation of RNA and cold-adapted growth.31 Some species have been
shown to induce IL-10 from spleen and macrophages.31 This may not only suppress
inflammatory attack but may contribute to host tolerance of the microbe.

Yersinia species (enterocolitica, pseudotuberculosis)

Yersinia binds to integrins on the surface of M cells via an adhesin, Inv, and invades cells
through a ‘zippering’ process in which bacteria are enveloped by the cell membrane.35
Once in the Peyer’s patches, the organism remains largely extracellular and withstands
attack by phagocytes by secreting Yop effector molecules, which disrupt cytoskeletal
assembly and mediate pathogenicity.35 YopE disrupts actin filaments and the host
cytoskeleton and YopH helps the organism to resist opsonization and complement-
mediated phagocytosis.45 YopH further suppresses antigen-specific T cell activation and
IL-2 production through tyrosine phosphorylation of the T cell receptor (TCR) and
suppresses B cell receptor–mediated costimulatory pathways.64 These mechanisms
impair bacterial internalization.
Other Yop factors assist in bacterial survival and evasion of immune responses. YopP
(Y. entercolitica), equivalent to YopJ (Y. pseudotuberculosis), protects the organism from
398 D. W. K. Acheson and S. Luccioli

detrimental effects of IL-1, TNF, and IL-8 by inhibiting NF-kB and MAPK (mitogen-
associated protein kinase) signal transduction pathways.60,65,66 Also, an LcrV protein
induces IL-10 production by macrophages, thus exploiting the host cell’s production of
immunosuppressive cytokines to evade immune recognition.67 These organisms may
then disseminate to deeper lymphoid structures and cause mesenteric lymphadenitis in
addition to enterocolitis.35 Genes that encode effective mechanisms for the uptake of
ferrous iron and magnesium help the organism to resist phagocyte ROI and RNI60 and
survive in the nutrient-poor environment surrounding MLN and blood vessels.45

Listeria monocytogenes

Listeria is a food-borne pathogen that causes an especially important illness in

immunocompromised individuals such as infants and pregnant mothers. Bacterial
internalin A recognizes E cadherin host receptors, allowing for internalization.35 The
organism survives and proliferates in host phagocytes. A toxin, listeriolysin, disrupts the
endosomal membrane and allows the bacterium to escape into the cytosol, where it
modulates immune responses.60,68 Most noteworthy, Listeria activates NF-kB in
endothelial cells which results not only in production of IL-8 but in increased
expression of adhesion molecules, ICAM-1 and E selectin.69 This leads to recruitment
of more phagocytes to the area and allows for a continuous supply of host cells to
propagate disease. Of note, increased levels of g/d T cells have been observed in lesions
of Listeria-infected individuals. However, whether these cells modulate or propagate
Listeria infection is poorly understood.19

INTERACTION OF PARASITES WITH THE HOST IMMUNE SYSTEM

Major parasites of the gastrointestinal tract include protozoans (Giardia lamblia,

Cryptosporidium parvum, Entamoeba histolytica) and helminths (hookworms, nematodes).
Although these microorganisms have complex life cycles and an equally complex
interplay with host immune systems in various tissues, this review will look briefly at
specific examples of parasitic interactions with the gut mucosa.
Following systemic infection by the larva, adult helminth worms attach to intestinal
villi by burrowing through the mucosa with their buccal plates (hookworms,
Ancylostoma duodenale and Necator americanus)70 or by way of lectin binding protein
(nematodes).71 The worms then live and feed within the small intestinal lumen.70
Except in a few cases, helminth colonization results in a robust Th2-mediated response,
not unlike an allergic response, characterized by mast cell triggering and release of
proteases and potent vasodilatory mediators and by secretion of IL-4, IL-5, and IL-13,
which induce mucus production and increased peristalsis as well as promote IgE
production and recruitment of eosinophils.72 Certain nematodes may promote this
response by synthesis and secretion of prostaglandins.72 For reasons unknown,
hookworms are better able than most helminths to withstand proteolytic damage from
these immune responses and establish chronic infection in the host.70 Other helminths
utilize immunomodulatory mechanisms to accomplish this task. As examples, cystatins
from Brugia malayi interfere with MHC II-mediated antigen presentation and
onchocystatin from Onchocerca volvulus enhances IL-10 production by host cells.73,74
Also, helminths may secrete molecules which mimic immunosuppressive cytokines
such as TGF-b and macrophage migration inhibitory factor (MIF).72,75,76
 Mucosal immune responses 399

Upon ingestion, Giardia lamblia cysts are corrupted by gastric and pancreatic
enzymes in the small intestine and the resultant motile trophozoites attach to gut
mucosa by a sucking disk apparatus.77 This adherence process is facilitated by a trypsin-
activated binding lectin which adheres to the intestinal brush border membranes78 and
contributes to mucosal damage and release of further trypsin by mast cells. Infection
elicits a robust humoral and cellular host response. Despite these protective responses,
Giardia has adapted itself for survival in the harsh environment of the small intestine and
outside by processes of encystation and surface antigenic variation.79 The trophozoite,
which is susceptible to chemical and temperature changes, utilizes bile, carbohydrates,
and low oxygen tension for its growth, leading to eventual encystation. Encystation not
only renews the organism’s life cycle but also provides added protection from external
factors.77 The trophozoite’s survival, at the same time, is enhanced by variant-specific
surface proteins (VSPs) which are resistant to the detrimental effects of intestinal
proteases in the small intestine.80 These variations may also impair immunoglobulin
recognition and help to explain how these organisms avoid humoral immunity and, in
some cases, lead to chronic infections.
Like Giardia, encysted oocytes of Cryptosporidium parvum are degraded in the
duodenum and trophozoites interact with intestinal epithelial cells. Unlike Giardia,
however, the trophozoites reside at an unusual extracytoplasmic location at the luminal
surface of the cell and elicit mainly a CD4 (þ ) T cell response. Cellular invasion may be
enhanced by endogenous secretory phospholipase A(2), which is a known virulence
factor in other pathogens.81 This organism commonly infects immunocompromised
patients with HIV and T cell deficiencies, and has been described in patients with
mannose-binding lectin deficiency, suggesting that the innate mucosal defence plays a
role in immunity to Cryptosporidium as well.82
Entamoeba histolytica is a protozoan that may cause bloody diarrhoea and liver
abscesses. Cysts are ingested and degraded by intestinal and pancreatic enzymes into
eight trophozoites, which are haematophagous and colonize the colon.77 Invasion of
host cells is mediated by an adhesin, Gal/GalNAc lectin, which is not only toxic to
intestinal cells but mucolytic as well. This lectin may help the parasite to evade
complement-mediated assault as it mimics an inhibitor of the membrane attack
complex (MAC) of the complement cascade, CD 59.83 Cytotoxicity may also be
enhanced by amoebic pore-forming proteins, termed amoebapores, and by secretion of
cysteine proteinases.77 Likewise, bioamines and neuropeptides (serotonin and
substance P) found in amoebic lysates have been shown to enhance pro-inflammatory
prostaglandin production.84

INTERACTION OF VIRAL PATHOGENS WITH THE HOST IMMUNE

SYSTEM

Human immunodeficiency virus (HIV)

HIV primarily infects CD4 (þ ) cells, macrophages, and DCs and may be introduced into
the gastrointestinal tract directly through the rectal mucosa or by way of the
bloodstream. The particular anatomy and functional characteristics of intestinal
lymphocytes allow the gastrointestinal mucosa to be an advantageous site for
replication and cytopathicity.85 In the gut lumen, HIV may pass through M cells or may
directly infect villous epithelia via antibody-mediated Fc receptor binding.85 Once
inside, HIV may bud from the basal compartment of these cells and come into contact
400 D. W. K. Acheson and S. Luccioli

with lymphocytes in the lamina propria, resulting in an accelerated loss of CD4 (þ ) cells
in this compartment.85,86 Because CD4þ cells in this compartment play an important
role in foreign antigen recognition and B cell differentiation to IgA-producing cells, this
may explain the observed decrease in IgA plasma cells and secretion leading to
opportunistic infections and malignancies.86 Also, because of the close relationship
between the epithelium and mucosal immunity, the small intestinal mucosal atrophy and
villous maturation defects observed in HIV patients may also be explained by this
breakdown of the mucosal barrier.85

Enteric viruses

Enteric viruses are a common cause of secretory diarrhoea in humans. Most viral
infections are self-limited, and epidemic as in the case of Norwalk virus, while other
cases (rotavirus), for unclear reasons, appear more commonly in infants and children.
Astroviruses are becoming increasingly identified causes.87 Despite the high numbers of
infections, relatively little is known about the true pathogenicity of these organisms.
Viruses infect villous epithelia with varying affinity. The VP4 spike protein, with or
without association with VP7, has been identified as a rotavirus attachment protein.
Following viral adherence, this protein is cleaved by proteases into VP5 and VP8 which
enhance infectivity.88 Moreover, rotavirus may utilize an enterotoxin, NSP4, to cause
calcium-mediated damage to villous cells leading to a cyclic-nucleotide-independent
secretory diarrhoea.88 These interactions with cells stimulate a robust IgA and IgG
humoral response in the host (geared to these aforementioned surface molecules)
which in many cases is sufficient to clear infection. NK cells, cytotoxic T cells and other
cytokine responses also appear to be involved, but it is unclear whether these factors
ameliorate or propagate disease symptoms.89

CONCLUSIONS

The mucosal immune system is a multifaceted and highly complex system that has to
safeguard the human host but also allow critical nutrient antigens to cross the mucosal
barrier. As our understanding of this system advances it becomes more evident that
many microbes have developed ways to circumvent or even utilize the immune system
in the intestinal tract. As illustrated in many of the examples above, different microbes
use different parts of the mucosal defence system to assist them in their goal of
colonization or survival. It is only because of many built-in redundancies in the system
that the vast majority of pathogens are inactivated or destroyed before they can cause
harm to the human host. But as soon as there is a breach in the system, such as mucosal
injury, or one part of it begins to fail, such as in HIV, the microbial invaders are soon
causing disease. As our knowledge of host – pathogen interactions continues to develop
we will be better able to find ways to deal with the breaches when they occur. While it
may be counter intuitive, there may be situations in which reducing the inflammatory
response, such as the influx of neutrophils, may actually protect the host from further
damage. Gaining a better understanding of these intricate host – pathogen interactions
will facilitate the development of targeted therapies that can be used to minimize
pathogen invasion or the uptake of specific bacterial toxins that lead to systemic
disease. We have made a great deal of progress in this regard but there are still many
 Mucosal immune responses 401

host – pathogen interactions in relation to the intestinal immune system that are poorly
understood.

Practice points
† the mucosal immune system offers many overlapping defenses against enteric
pathogens
† certain situations, such as the use of antibiotics, may increase the risk of
developing specific enteric infections
† various pathogens have developed ways to manipulate the mucosal immune
system to their advantage. The inflammatory response may sometimes be
contributing to the disease process in the intestine
† early recognition and appropriate treatment of life-threatening enteric
infections is important since the mucosal immune system may be overwhelmed

Research agenda
† gaining a better understanding of the molecular mechanisms by which enteric
pathogens gain entry to the underlying tissues. Specifically identifying adhesion
molecules whose blockade may reduce pathogen uptake
† understanding which components of the immune system may be contributing
to the disease process or the uptake of enteric pathogens or toxins from the
intestinal lumen
† developing new vaccines directed toward specific pathogens that gain entry via
the intestinal epithelial cell barrier
† identifying ways to manipulate the mucosal immune response to reduce
invasion or uptake of enteric pathogens or toxins

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