ORIGINAL RESEARCH

published: 13 September 2017

doi: 10.3389/fphar.2017.00642

Lack of Histamine H4-Receptor

Expression Aggravates
TNBS-Induced Acute Colitis
Symptoms in Mice
Eva J. Wunschel, Bastian Schirmer, Roland Seifert and Detlef Neumann *
Institute of Pharmacology, Hannover Medical School, Hanover, Germany

Inflammatory bowel diseases (IBD) are a growing health problem worldwide, severely
affecting patients’ life qualities and life expectancies. Therapeutic options, which are
rare and focus on symptoms associated with the disease, suffer from increasing
numbers of patients refractory to the established strategies. Thus, in order to generate
Edited by:
new therapeutic regimens, the detailed understanding of the pathogenic mechanisms
Sabine Grösch, causing IBD is necessary. Histamine is an inflammatory mediator associated with IBD.
Goethe University Frankfurt, Germany Four histamine receptors are currently known of which the histamine H4 -receptor
Reviewed by: (H4 R) has been shown to possess a pro-inflammatory function in several experimental
Antonio Recchiuti,
Università degli Studi "G. d’Annunzio" models of inflammatory diseases, including dextran sodium sulfate (DSS)-induced colitis
Chieti – Pescara, Italy in mice. No single model reflects the complexity of human IBD, but each model
Melania Dovizio,
Università degli Studi "G. d’Annunzio"
provides valuable information on specific aspects of IBD pathogenesis. While DSS-
Chieti – Pescara, Italy induced colitis mostly relies on innate immune mechanisms, trinitrobenzene sulfonic acid
*Correspondence: (TNBS)-induced colitis rather reflects T-cell mechanisms. Consequently, an observation
Detlef Neumann made in a single model has to be verified in at least one other model. Therefore,
[email protected]
in the present study we investigated the effect of genetic blockade of H4 R-signaling
Specialty section: in mice subjected to the model of TNBS-induced acute colitis. We analyzed severity
This article was submitted to
and progression of clinical signs of colitis, as well as histopathologic alterations in the
Inflammation Pharmacology,
a section of the journal colon and local cytokine production. Genetic ablation of H4 R expression worsened
Frontiers in Pharmacology clinical signs of acute colitis and histological appearance of colon inflammation after
Received: 07 June 2017 TNBS application. Moreover, TNBS instillation enhanced local synthesis of inflammatory
Accepted: 30 August 2017
Published: 13 September 2017
mediators associated with a neutrophilic response, i.e., CXCL1, CXCL2, and interleukin-
Citation:
6. Lastly, also myeloperoxidase concentration, indicative for the presence of neutrophils,
Wunschel EJ, Schirmer B, Seifert R was elevated in cola of TNBS-treated mice due to the absence of H4 R expression.
and Neumann D (2017) Lack
Our results indicate an anti-inflammatory role of histamine via H4 R in TNBS-induced
of Histamine H4 -Receptor Expression
Aggravates TNBS-Induced Acute acute neutrophilic colitis in mice, thus questioning the strategy of pharmacological H4 R
Colitis Symptoms in Mice. blocked as new therapeutic option for patients suffering from IBD.
Front. Pharmacol. 8:642.
doi: 10.3389/fphar.2017.00642 Keywords: TNBS-induced colitis, histamine, inflammation, receptor, GPCR, mouse models

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

INTRODUCTION membranous adenylyl cyclase activity (Morse et al., 2001; Leurs

et al., 2009). The biological function of H4 R remains elusive but
Inflammatory bowel diseases (IBD) are a global health concern accumulating evidence indicates a pro-inflammatory function of
with growing incidence, especially in industrialized countries H4 R in several (auto)inflammatory diseases like allergic asthma,
(Molodecky et al., 2012). There are mainly two clinical entities atopic dermatitis and rheumatoid arthritis (Beermann et al., 2012;
of IBD, namely ulcerative colitis (UC) and Crohn’s disease (CD), Cowden et al., 2014; Rossbach et al., 2016). Histamine has been
both characterized by chronic, relapsing inflammatory conditions shown to act as pro-inflammatory mediator in IBD (Bene et al.,
in the gastrointestinal tract. These two manifestations of IBD 2004; Neumann and Seifert, 2014) and blockade of H4 R has
differ in their clinical and histopathological presentation and, proven to ameliorate DSS-induced colitis in mice as well as in
from an immunological point of view, in the association of TNBS-induced colitis in rats (Varga et al., 2005; Schirmer et al.,
CD and UC with a dysregulation of a Th1 -type or a Th2 - 2015).
type immune response, respectively (Geremia et al., 2014). But In a previous study, we detected a pro-inflammatory effect of
nowadays this dichotomic viewpoint needs to be reconsidered: H4 R in DSS-induced acute colitis in BALB/cJ mice (Schirmer
Not only are there several published data indicating no clear et al., 2015). Because DSS- and TNBS-induced colitis differ in
Th1 /Th2 phenotype in UC and CD (Vainer et al., 2000; their immunologic pathomechanisms, we hypothesized that the
Rovedatti et al., 2009; Bernardo et al., 2012). But during the role of the H4 R depends on the model used for induction of
last few years it has become evident that, beneath the adaptive colitis. Therefore, in the present study the TNBS-induced acute
Th1 /Th2 immune response, constituents of Th17 response and colitis model was applied to H4 R knockout and wild-type mice in
mechanisms of innate immunity may play important roles in the order to elucidate a contextual function of the receptor.
pathophysiology of IBD, too (Geremia et al., 2014; Cătană et al.,
2015).
There are several different rodent models existing in order MATERIALS AND METHODS
to mimic specific features of human IBD. Because of their
relative simplicity, easy establishment, low costs and good Materials
reproducibility, chemically induced colitis models are currently If not stated otherwise, all chemicals were obtained from Sigma–
the most widely used models of IBD (Wirtz et al., 2007). When Aldrich (Munich, Germany).
administered directly into the colon, the haptenizing agent 2,4,6-
trinitrobenzene sulfonic acid (TNBS) elicits a Th1 -polarized
colonic immune response leading to wide-spread inflammation
Animals
Eight- to ten-week-old BALB/cJRj (WT) mice were purchased
in the colon, which recapitulates several features of human IBD
from Janvier Labs (Le Genest-Saint-Isle, France). H4 R knockout
(Morris et al., 1989; Elson et al., 1996; Camoglio et al., 2000).
mice (H4 R−/− ; strain: C.129HrH4 tm1Lex ) were generated by
Because of the Th1 -polarization, this model has often been
Lexicon Genetics (Woodlands, TX, United States) as described
associated with human CD (Jones-Hall and Grisham, 2014). In
by Hofstra et al. (2003) and backcrossed for more than 10
dextran sodium sulfate (DSS)-induced colitis, the animals are fed
generations onto the BALB/cJRj strain. Mice were housed in
with DSS in their drinking water, causing a direct toxic damage
the animal facility of Hannover Medical School (temperature:
of the mucosal epithelial lining with loss of barrier integrity (Ni
21◦ C ± 1; 14/10 h day/night cycle) with access to standard diet
et al., 1996; Dharmani et al., 2011). Due to the morphological
and drinking water ad libitum.
and symptomatic similarity, this model has been associated with
human UC (Jones-Hall and Grisham, 2014). In contrast to DSS,
TNBS does not directly damage the mucosal epithelium, but Induction of Colitis by TNBS and Animal
causes a delayed hypersensitivity reaction by haptenizing luminal Dissection
antigens (Elson et al., 1996; Wirtz et al., 2007). Therefore, TNBS- For induction of acute colitis 8- to 10-week-old mice (male and
induced colitis has been reported to focus on pathophysiologic female, weight: 20–25 g) were anesthetized (100 mg/kg ketamine
aspects of the adaptive immune response, whereas DSS-induced plus 10 mg/kg xylazine; i.p.) and then inoculated intra-rectally
colitis causes more of a dysregulation of the innate immune (i.r.) with TNBS (2 mg/mouse) dissolved in 100 µl 45% [v/v]
response (Wirtz et al., 2007). ethanol (EtOH) in PBS through a catheter inserted approx. 4 cm
One important inflammatory mediator involved in adaptive or proximal to the anus. Control mice were treated with 100 µl
innate immune responses is the biogenic amine histamine (2-(4- EtOH/PBS only. Mice were inspected for their health status every
imidazolyl)-ethylamine), which can transduce its signal via four 6 h. Three days after inoculation or when found with severely
currently known histamine receptors H1 R, H2 R, H3 R and H4 R, affected health status mice were killed with carbon dioxide and
all belonging to the superfamily of G protein-coupled receptors subsequent heart puncture. Caeca and cola were resected, washed
(GPCRs) (Seifert et al., 2013). The H4 R is the most recently with PBS to remove remaining feces, and the lengths of the
discovered histamine receptor and is expressed mainly on cola were documented. Thereafter, the cola were transversally
immune cells such as T-cells, eosinophils, mast cells and dendritic divided into three segments, proximal, medial, distal, and each
cells (Hofstra et al., 2003; Lundberg et al., 2011; Reher et al., 2012). segment was further longitudinally divided into 3 parts, the first
Upon ligand binding, H4 R couples to and activates Gi proteins comprising about 1/2 and parts two and three each about 1/4 of the
leading to intracellular calcium mobilization and inhibition of colonic circumference. The first part of each segment was fixed in

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

4% (v/v) formaldehyde (Merck, Darmstadt, Germany) embedded transcribed for 30 min at 50◦ C into cDNA by means of
in paraffin, and processed for hematoxylin/eosin (H/E) staining. Maxima Reverse Transcriptase (Thermo Scientific). Cytokine-
The second and a third part of each segment was stored at −80◦ C specific sequences were quantified proportionately to GAPDH by
and in RNAlater (Thermo Fisher, Waltham, MA, United States), PCR using the customized Primer PCR Assay (Bio-Rad, Munich,
respectively, for protein and mRNA analyses. Germany).

Evaluation of Disease Activity Statistical Analysis

Mice were examined at 24 h intervals and a disease activity index Data are represented as single values and/or arithmetic mean
(DAI, adopted from Alex et al. (2009) ranging from 0 to 14 was of replicates ± SD for each parameter. Statistical analyses were
employed based on total body weight loss (0: no weight loss, performed with GraphPad Prism 6.07 using tests as indicated in
1: ≤ 5%, 2: ≤ 10%, 3: ≤ 15%, 4: > 15%), stool consistency the figure legends.
(0: normal, 2: soft, 4: diarrhea, 6: no defecation) and peranal
bleeding (0: no bleeding, 2: little bleeding, 4: massive bleeding/no Ethical Considerations
defecation). Animal housing and experimental procedures complied with the
German Animal Welfare Legislation and were approved by the
Histological Analysis Lower Saxony State Office for Consumer Protection and Food
H/E-stained tissue slices were analyzed in a blinded fashion by Safety (LAVES, AZ 33.14-42502-04-14/1670).
two independent researchers. A histological severity score was
calculated for each segment by evaluating the single parameters
severity of inflammation (0: normal, 1: mild, 2: moderate, 3: RESULTS
severe, 4: highly severe), hyperplasia of crypts (0: absent, 2:
The application of 2 mg/mouse TNBS in WT mice resulted in
present), degree of ulceration (0: no ulcers, 1: 1–2 ulcers involving
a fast onset of cumulative clinical colitis symptoms, reported
up to 20 crypts, 2: 1–4 ulcers involving 20 to 40 crypts, 3: any
as DAI. Already 1 day after inoculation of TNBS, the DAI
ulceration exceeding the aforementioned criteria), area of affected
reached a value which did not significantly increase at day
tissue (0: 0%, 1: ≤ 30%, 2: ≤ 70%, 3: > 70%), edema formation (0:
two and three. Colitis symptoms were virtually absent during
absent, 1: present), blood cell infiltration (0: absent, 1: present),
the whole observation period in WT mice treated with the
and changes in crypt architecture (0: 0%, 1: ≤ 30%, 2: ≤ 70%,
solvent EtOH/PBS (Figure 1A). The symptoms induced by
3: > 70%). The total maximum score thus sums up to 17 per
TNBS were clearly dose-dependent, since the application of
segment (Bleich et al., 2004). All ex vivo data reported in this
1 mg/mouse TNBS lead to significantly reduced severity of
manuscript refer to the medial colon segments since the tips of
symptoms (data not shown). In H4 R−/− mice, the symptoms
catheters used for TNBS administration reached this section after
induced by 2 mg/mouse TNBS were significantly more severe
i.r. insertion. Thus, in accordance with the flow of the luminal
as compared to those observed in WT mice. Moreover, the DAI
content of the colon, in the medial and proximal colon segments
progressively increased in H4 R−/− mice from day one until
substantial degrees of histopathological alterations due to TNBS
day 3, while the EtOH/PBS-treated H4 R−/− mice remained
application were found, while in the distal segments and in the
without symptoms (Figure 1A). These data indicate that the
caeca virtually no alterations could be observed.
absence of H4 R expression promotes TNBS-induced colitis and
were supported by the analysis of the survival rates of treated
Cytokine Measurements mice. All mice, WT and H4 R−/− , treated with EtOH/PBS
The frozen colon specimen were lysed at 4◦ C using a FastPrep24 survived the observation period. Some of the TNBS-treated mice,
device (MP Biomedicals, Santa Ana, CA, United States) and however, had to be euthanized due to severely impaired health
insoluble parts were removed by centrifugation (4◦ C, 10 min, conditions within the 3 days period. Of those, the number
10,000g). Total protein concentrations of the cleared lysates were of H4 R−/− mice (6/7) was significantly higher than that of
measured by BCA assay (Thermo Scientific). Concentration of WT mice (1/7) (Figure 1B). Lastly, also anatomical parameters
interleukin (IL)-6, IL-17, CXCL1 (KC), CXCL2 (MIP-2), and support the notion that lack of H4 R supports TNBS-induced
tumor necrosis factor (TNF) was measured in the colon lysates colitis: while in WT mice experimentally induced colitis does
using a customized multiplex magnetic Luminex Kit (R&D not lead to a reduction of the colon length, in H4 R−/− mice
Systems, Minneapolis, MN, United States). In the same samples, it is significantly reduced after TNBS treatment as compared to
myeloperoxidase (MPO) was quantified using a specific ELISA EtOH/PBS treatment (Figure 1C).
(R&D Systems). The histopathological analysis of the cola at the end of the
observation period revealed an elevated degree of derangements
mRNA Quantification in TNBS-treated WT mice as compared to their counterparts
The tissue specimen dedicated for mRNA analyses were lysed treated with the solvent EtOH/PBS (Figure 2A). The absence of
using the FastPrep24 device (MP Biomedicals) and the RNAs H4 R expression did not alter the histopathological appearance
were extracted using the Nucleospin RNA II kit (Macherey-Nagel, in solvent-treated mice, while TNBS-treated H4 R−/− mice were
Düren, Germany) essentially according to the manufacturer’s significantly stronger affected than the respective WT mice as
instructions. One microgram RNA of each sample were reverse detected by quantitative evaluation of the specimen (Figure 2B).

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

FIGURE 1 | Lack of H4 R expression worsens TNBS-induced colitis. Wild type (WT) or H4 R-deficient (H4 R−/− ) BALB/cJ mice were treated intra-rectally with
2 mg/100 µl∗ mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH) at day 0. Mice were inspected for their health status every
6 h for a total of 3 days. (A) Every 24 h the disease activity of each single mouse was evaluated according to a scoring system which takes into account the body
weight, the stool consistency, and the degree of anal bleeding and assigns a numeric value to the respective status (Alex et al., 2009). The sum of the single values
of a mouse refers to as its disease activity index (DAI)-score, being the higher the worse is the health condition of a mouse. ∗ pWT; TNBS vs. H4 R−/− ; TNBS < 0.05
(Two-Way ANOVA), n = 7/group. (B) Mice which had to be euthanized due to severely impacted health status throughout the 3 days observation period were
recorded. On day 3 all remaining mice were killed for analysis. The relative numbers of surviving mice were plotted against the time. ∗ pWT; TNBS vs. H4 R−/− ;
TNBS < 0.05 (Log-rank (Mantle-Cox) test), n = 7/group. (C) The cola of the mice were prepared and their lengths recorded. ∗∗∗ <0.005 (One-Way ANOVA), TNBS:
n = 7/group; EtOH: n = 4/group.

FIGURE 2 | Lack of H4 R expression enhances histopathological findings of colonic inflammation. Wild type (WT) or H4 R-deficient (H4 R−/− ) BALB/cJ mice were
treated with 2 mg/100 µl∗ mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola were cut in three
sections, proximal, medial, and distal. Parts of the sections were fixed in formaldehyde, embedded in paraffin, cut into slices, and stained with hematoxylin/eosin.
(A) Representative micro-photographs of tissue slices of medial colon sections of each experimental group are demonstrated. (B) The tissue slices as demonstrated
in (A) were analyzed in a blinded fashion for pathological derangements as indicated above the single graphs applying a scoring matrix, i.e., the higher the score, the
worse the histological appearance. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 (One-Way ANOVA), TNBS: n = 7/group; EtOH: n = 4/group. (C) Representative
micro-photographs in enhanced magnification of sections obtained from cola of TNBS-treated mice.

In detail, TNBS treatment, as compared to instillation of the (Figure 2B). Two out of the seven mice in the TNBS-treated WT
solvent, induced a significant increase in all parameters analyzed group did not show histopathological derangements. Excluding
except hyperplasia of crypts in H4 R−/− mice, while in WT these two from the calculations as outliers, nevertheless, did not
mice these values were increased as well but without statistical alter the statistical significance of the differences between WT and
significance (Figure 2B). The comparison of TNBS-treated H4 R−/− mice (data not shown). TNBS-induced acute colitis is
WT and H4 R−/− mice revealed significant differences for the a local inflammatory reaction with a predominant involvement
parameters severity of inflammation and degree of ulceration of neutrophils (Campbell et al., 2016). Neutrophils represent the

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

FIGURE 3 | Lack of H4 R expression enhances mRNA expression of inflammation-associated cytokines and chemokines. Wild type (WT) or H4 R-deficient (H4 R−/− )
BALB/cJ mice were treated with 2 mg/100 µl∗ mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola
were cut in three sections, proximal, medial, and distal. From parts of the medial sections mRNAs were extracted and analyzed by RT-qPCR array. Relative
abundancies of individual mRNAs as indicated on the abscissa were calculated relative to the abundancy of the GAPDH mRNA. Reported are the values
comparative to those observed in solvent-treated WT mice, which were set to 1. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 (Student’s t-test), n = 4/group.

major cell type found in the inflammatory infiltrations in cola Thus, not only clinical symptoms but also histological and
of TNBS-treated WT and H4 R−/− mice (Figure 2C). A direct immunological parameters indicate that rectally applied TNBS
quantification of the neutrophilic infiltrates in order to compare induces a much more severe neutrophilic colitis in H4 R−/− mice
WT and H4 R−/− mice was not performed, but substituted by the as compared to WT mice.
more accurate surrogate parameter MPO concentration, which
represents a quantitative measure for not only the number of
infiltrated neutrophils but also their activation (see below). DISCUSSION
In order to more closely characterize the colitis reactions
occurring in WT and H4 R−/− mice, the expression of In several models of inflammatory diseases blockade of H4 R
inflammatory mediators, esp. those attracting and activating signaling provides a beneficial effect on inflammatory symptoms
neutrophils, was analyzed in colon tissues. In analyses on (Dunford et al., 2006; Cowden et al., 2010a,b, 2014; Gutzmer
the mRNA level a tendency to TNBS-induced expression et al., 2011; Hartwig et al., 2015; Kovacova-Hanuskova et al.,
of CXCL1, CXCL2, IL-6, IL-1α, and IL-1β was observed 2015). Accordingly, also in models of inflammation of the gut,
in WT mice, while the expression of other genes such as H4 R was reported to possess a pro-inflammatory function (Varga
TNF and IL-17 was only marginally altered (Figure 3). Of et al., 2005; Schirmer et al., 2015). However, these two studies
those mediators induced by TNBS, the mRNA expression of were performed in two different models. The first one used
CXCL1, CXCL2, and IL-6 as well as of IL-1β and TNF were TNBS to induce colitis in rats and analyzed the involvement
affected by the absence of the H4 R, i.e., the expression was of H4 R by application of the selective antagonist JNJ7777120
enhanced in cola of TNBS-treated H4 R−/− mice as compared (Varga et al., 2005). The other study was performed using the
to cola obtained from WT mice (Figure 3). On the protein model of DSS-induced colitis in mice taking advantage of a
level, similar observations were made. The concentrations of genetic H4 R knockout model (Schirmer et al., 2015). Thus, a
CXCL1, CXCL2, and IL-6 were significantly enhanced in tissues series of parameters (species, colitis induction method, H4 R
obtained from TNBS-inoculated H4 R−/− mice as compared blockade method) differed profoundly between these two studies,
to that of WT mice (Figure 4). Moreover, also the pro- disabling the extrapolation of the results of the one study to
inflammatory mediators TNF and IL-17 and the neutrophil- the other. Moreover, no single model reflects the complexity of
specific enzyme MPO, indicative for a neutrophilic inflammation, human IBD, but each model provides valuable information on
were detected at significantly higher protein concentrations after specific aspects of IBD pathogenesis. Thus, only data obtained
TNBS treatment in cola of H4 R−/− mice than in cola of WT in several models will provide evidence whether or not a
mice. proposed strategy may be useful in treating human IBD. This

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

FIGURE 4 | Lack of H4 R expression enhances protein expression of inflammation-associated cytokines and chemokines. Wild type (WT) or H4 R-deficient (H4 R−/− )
BALB/cJ mice were treated with 2 mg/100 µl∗ mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola
were cut in three sections, proximal, medial, and distal. From parts of the medial sections proteins were extracted and analyzed by Luminex Array (cytokines,
chemokines) and ELISA (MPO). Analyte concentrations are reported relative to the total protein concentrations of the colon lysates. ∗∗∗ p < 0.005 (One-Way ANOVA),
TNBS: n = 7/group; EtOH: n = 4/group.

prompted us to analyze the involvement of H4 R in acute TNBS- a H4 R-selective antagonist. Since the immune systems of mice
induced colitis in mice using the genetic knockout (H4 R−/− ) and rats, including the mechanisms of inflammation, work pretty
model. much the same, the different species rather cannot account
Surprisingly, both clinical and histopathological examination for the contrary H4 R functions in the two models. Specific
of the TNBS-treated mice demonstrated not only the progressive microbiota and age of an animal both affect experimental
occurrence of symptoms indicative for experimental colitis in diseases, including colitis (Hansen et al., 2014). However,
WT mice, but, in comparison to these, also a severe aggravation these parameters, which cannot be compared between the two
in mice lacking H4 R expression. These data were supported studies, only gradually regulate the disease and, thus, also very
by the reduced mean survival time, a pathological parameter unlikely may account for the controversial differences observed.
valid for this model but not for human IBD, and the reduced Regarding the method of H4 R blockade, in response to the
colon length of TNBS-treated H4 R−/− mice as compared to genetic ablation of the receptor compensatory or other secondary
solvent-treated mice. Thus, in the TNBS-induced model, colonic mechanisms may have been evoked, which do not occur upon
inflammation seems to be suppressed by H4 R signaling, as application of a pharmacological antagonist such as JNJ7777120.
already described for the model of experimental autoimmune However, at least for a series of immunologic parameters,
encephalomyelitis (EAE) (Ballerini et al., 2013; Saligrama et al., naïve WT and H4 R−/− mice do not differ significantly
2013). from each other (Hartwig et al., 2015; Kloth and Neumann,
In contrast to the study by Varga et al. (2005) we analyzed unpublished results). In contrast, for the pharmacological activity
the contribution of H4 R to TNBS-induced acute colitis in of the H4 R-selective antagonist JNJ7777120 mouse strain-related
mice, but not in rats, and we inhibited H4 R function by differences have already been described in a model of acute
genetic ablation of H4 R expression, but not by application of skin inflammation (Coruzzi et al., 2012), which also may

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

account for the differences between the mice- and the rat-based affects the inflammatory reaction. Unfortunately, H4 R expression
studies. and function on colonic epithelial cells remain to be analyzed in
An even more straightforward comparison can be drawn detail yet.
between the present study and that using the DSS-induced acute Certainly, one major drawback of the present study is the
colitis model (Schirmer et al., 2015), which both took advantage rather low number of animals in the experimental groups. These
of the same colony of WT and H4 R−/− BALB/cJ mice. Thus, were calculated to be sufficient when planning the described
these two studies only differ specifically in the method of acute experiments in advance; however, the obtained data indicate that
colitis induction, i.e., by intrarectal TNBS application (this study) an enhanced number would have been needed. Unfortunately,
and by DSS feeding (Schirmer et al., 2015), indicating that due to legislation hurdles it is hardly possible to get a permission
the opposite findings regarding the function of H4 R can be to just enhance the number of animals of an already existing
assigned to the different methods. In both models, which were study, limiting the strength of the data’s interpretation.
driven over a rather short period of time, i.e., 3 days (TNBS) In summary, in the present study we demonstrate that lack
and 7 days (DSS), the observed symptoms and parameters are of H4 R expression worsens the outcome of TNBS-induced acute
primarily based on the induction of intestinal epithelial damage colitis in mice, indicating an anti-inflammatory function of H4 R
and acute inflammation. Due to the protocol used, the adaptive in this model. The cellular and molecular bases for this function
immune system cannot have become activated and therefore only are still enigmatic; however, our data question the general view
plays a minor role in these models, while in other models it is on H4 R-selective antagonists as potential new anti-inflammatory
undeniably important (van Lierop et al., 2010). This indicates therapeutics.
that the applied DSS and TNBS methods activate different
mechanisms, which both result in colitis symptoms. In support
of this hypothesis, differing profiles of cytokine expression were AUTHOR CONTRIBUTIONS
already detected in DSS- and TNBS-induced acute colitis (Alex
et al., 2009). The bases for the differences of H4 R function in Substantial contributions to the conception or design of the
DSS- and TNBS-induced colitis in mice, however, have still to be work: BS and DN. Substantial contributions to the conception
explored. or design of the work; or the acquisition, analysis: EW, BS, and
In the present study, the chemokines CXCL1 (KC) and CXCL2 DN. Substantial contributions to the conception or design of the
(MIP2) together with the cytokine IL-6 and the neutrophils work; or the acquisition, analysis, or interpretation of data for
enzyme MPO were abundantly expressed in the cola after the work: EW, BS, RS, and DN. The work of drafting: BS and
TNBS application, and their expression was enhanced in mice DN. The work of drafting it or revising it critically for important
lacking H4 R expression. In addition, in sera obtained from intellectual content: EW, BS, RS and DN. Final approval of the
TNBS-treated H4 R−/− mice higher levels of CXCL1, CXCL2, version to be published: EW, BS, RS, and DN. Agreement to be
and IL-6 were detected as compared to those from WT accountable for all aspects of the work in ensuring that questions
mice, albeit these differences were statistically not significant related to the accuracy or integrity of any part of the work
(Supplementary Figure S1). Whether this is a direct or an are appropriately investigated and resolved: EW, BS, RS, and
indirect effect of H4 R on the mediator-releasing cells has DN.
to be analyzed in the future. Nevertheless, the regulation
of the expression of the neutrophils attracting chemokines
CXCL1, CXCL2, and of IL-6 and MPO connects H4 R to FUNDING
the neutrophilic inflammatory reaction, and thereby to innate
immune mechanisms. Cellular sources for CXCL1, CXCL2, This work was supported by a grant of the Deutsche
and IL-6 are monocytes and macrophages, implying that these Forschungsgemeinschaft [NE 647/8-1 to DN].
cells express a functional H4 R. While functional expression
of H4 R in macrophages has already been demonstrated by
us and others (Cowden et al., 2013; Czerner et al., 2014), ACKNOWLEDGMENT
H4 R expression on monocytes currently is controversially
discussed (Gschwandtner et al., 2013; Dib et al., 2014; The authors thank the Institute of Experimental Pathology
Werner et al., 2014; Capelo et al., 2016). Nevertheless, at from Hannover Medical School for the help in preparing and
least in macrophages H4 R function is described as pro- evaluating histologic specimen for this study.
inflammatory, thus, probably cannot account for the anti-
inflammatory effect in TNBS-induced colitis. Another colonic
cellular source that releases CXCL1, CXCL2, and IL-6 upon SUPPLEMENTARY MATERIAL
activation is epithelial cells (Song et al., 1999; Ohtsuka et al.,
2001; Sutton et al., 2008). Thus, one can hypothesize that The Supplementary Material for this article can be found
histamine via the H4 R regulates the expression of neutrophils online at: http://journal.frontiersin.org/article/10.3389/fphar.
attracting/activating mediators by epithelial cells and thereby 2017.00642/full#supplementary-material

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Wunschel et al. H4 R Expression Ameliorates TNBS-Induced Colitis

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(2013). Molecular and cellular analysis of human histamine receptor subtypes. be construed as a potential conflict of interest.
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