RESEARCH ARTICLE crossmark

Histamine H2 Receptor-Mediated Suppression of Intestinal

Inflammation by Probiotic Lactobacillus reuteri
Chunxu Gao,a,b,d Angela Major,d David Rendon,c Monica Lugo,b,d Vanessa Jackson,b,d Zhongcheng Shi,b,d Yuko Mori-Akiyama,b,d
James Versalovica,b,d
Departments of Molecular Virology and Microbiology,a Pathology and Immunology,b and Obstetrics and Gynecology,c Baylor College of Medicine, Houston, Texas, USA;
Department of Pathology, Texas Children’s Hospital, Houston, Texas, USAd

ABSTRACT Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflamma-
tion in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated
immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intes-
tinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc
gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal
cytokine (interleukin-6 [IL-6] and IL-1␤ in the colon) gene expression. Suppression of acute colitis in mice was documented by
diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [18F]fluorodeoxy-
glucose ([18F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis
depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-
containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of L-histidine to histamine
by hdcⴙ L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon.
IMPORTANCE Probiotics are microorganisms that when administered in adequate amounts confer beneficial effects on the host.
Supplementation with probiotic strains was shown to suppress intestinal inflammation in patients with inflammatory bowel
disease and in rodent colitis models. However, the mechanisms of probiosis are not clear. Our current studies suggest that sup-
plementation with hdcⴙ L. reuteri, which can convert L-histidine to histamine in the gut, resulted in suppression of colonic in-
flammation. These findings link luminal conversion of dietary components (amino acid metabolism) by gut microbes and
probiotic-mediated suppression of colonic inflammation. The effective combination of diet, gut bacteria, and host receptor-
mediated signaling may result in opportunities for therapeutic microbiology and provide clues for discovery and development of
next-generation probiotics.

Received 6 August 2015 Accepted 9 November 2015 Published 15 December 2015

Citation Gao C, Major A, Rendon D, Lugo M, Jackson V, Shi Z, Mori-Akiyama Y, Versalovic J. 2015. Histamine H2 receptor-mediated suppression of intestinal inflammation by
probiotic Lactobacillus reuteri. mBio 6(6):e01358-15. doi:10.1128/mBio.01358-15.
Editor Margaret J. McFall-Ngai, University of Hawaii

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Copyright © 2015 Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license,
which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to James Versalovic, jamesv@bcm.edu.

T he incidence and prevalence of pediatric and adult inflamma-

tory bowel diseases (IBDs) have steadily increased with time
during recent decades (1–3). Immunomodulatory treatment
among the Firmicutes have been described in patients with IBD
(4). Relative deficiencies of specific intestinal microbes may con-
tribute to lack of microbe-derived anti-inflammatory factors in
strategies, including corticosteroids, immunosuppressants, and humans. For example, one commensal bacterial species, Faecali-
anti-tumor necrosis factor (anti-TNF) medications continue to bacterium prausnitzii, which can suppress human cytokine pro-
dominate the approach to amelioration of chronic intestinal in- duction, is present in reduced amounts and often undetectable in
flammation. Established pharmacological agents target activation patients with Crohn’s disease (5). In addition, manipulation of the
of immune cells and cytokine-mediated signaling pathways in hu- gut microbiome might result in new therapeutic strategies. For
man cells. As one example, the proinflammatory cytokine TNF example, probiotic combinations including lactobacilli and bifi-
has been a primary target in IBD therapeutics for more than a dobacteria can suppress inflammation when administered to pa-
decade, and delineation of druggable targets in the innate immune tients with acute and chronic pouchitis (6). The question remains
system has resulted in dramatic improvements in induction and whether and how host intestinal microbes (e.g., probiotics) con-
maintenance therapy of these chronic inflammatory diseases. tribute to chronic intestinal inflammation.
However, insufficient attention to targets within intestinal mi- Lactobacillus reuteri is a commensal intestinal firmicute and
crobes may limit progress in IBD therapeutics and may limit our probiotic that is widely prevalent in the gastrointestinal tracts of
understanding of the systems biology of chronic inflammation. diverse avian and mammalian species (7). L. reuteri is generally
The intestinal microbiota are dominated by the phyla Firmic- recognized as safe (GRAS) and is considered to be a beneficial
utes and Bacteroidetes, and differences in bacterial composition microbe that has been used globally as a probiotic for approxi-

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mately 2 decades. L. reuteri has been reported to suppress proin- with TNBS in the absence of L. reuteri, expected results with in-
flammatory cytokines in intestinal epithelial cells (8) and mono- creased weight loss, Wallace scores, and serum SAA concentra-
cytes (9) and intestinal inflammation in different rodent models tions were observed (Fig. 1B to D), consistent with acute colitis. In
(8, 10–13). However, the underlying mechanisms are still not contrast, administration of L. reuteri 6475 in late-exponential-
clear. A recent pangenomic study showed that human-derived phase growth attenuated colitis as indicated by significant reduc-
clade II L. reuteri strains contained a complete chromosomal hdc tions in weight loss, Wallace scores, and SAA concentrations com-
gene cluster (genes hdcA, hdcB, and hdcP) and the genetic capacity pared with colitis-positive controls lacking L. reuteri 6475 (Fig. 1B
to convert L-histidine to histamine, and this clade could also sup- to D).
press human TNF production in vitro. In contrast, clade VI Ameho scores included assessment of histologic inflammation
strains, which lacked the hdc gene cluster in their bacterial chro- so that a comprehensive evaluation of colitis could be performed.
mosomes, failed to suppress human TNF production in vitro in TNBS instillation (sans L. reuteri) caused necrosis extending
the absence of histamine generation (14). Histamine is considered deeply into the muscularis propria, whereas L. reuteri 6475 admin-
to be a primary candidate immunomodulin, or immunomodula- istration in TNBS-treated mice yielded mild or prominent muco-
tory compound, produced by this Lactobacillus species. Inactiva- sal or submucosal inflammation with preservation of intact mus-
tion of histidine-to-histamine converting capacity by mutagenesis cularis mucosae and muscularis propria (Fig. 1E and F). These
of the histidine decarboxylase gene (hdcA) diminished the ability results were consistent with previous findings (19). Other mor-
of hdc-positive L. reuteri strains to suppress production of human phological comparisons yielded reduced colon lengths in mice
TNF in vitro (9). The question whether histidine metabolism, par- challenged with TNBS compared with L. reuteri 6475-treated mice
ticularly the production of histamine by hdc⫹ L. reuteri, may con- (see Fig. S1C and D in the supplemental material) and healthy
tribute to the anti-inflammatory effects of this species in vivo de- control mice receiving PBS only, consistent with previous findings
serves investigation as a possible gateway to deepening our (22, 23).
understanding of microbiome-mediated intestinal immuno- To extend the findings related to colitis suppression by hdc⫹
modulation (15, 16). L. reuteri, we introduced a second clade II hdc⫹ L. reuteri strain,
In the present study, we investigated the mechanisms of intes- ATCC PTA 4659 (L. reuteri 4659), into the same mouse colitis
tinal immunomodulation by probiotics in a mammalian host. model. Similarly, L. reuteri 4659 diminished the weight loss phe-
Clade II L. reuteri strains can serve as model microbes of the hu-
notype, reduced Wallace scores, and decreased SAA concentra-
man gut microbiome, and we explored the relative importance of
tions, compared with control mice receiving MRS medium only
L-histidine metabolism by hdc⫹ L. reuteri and whether histamine
(see Fig. S2 in the supplemental material).
could represent a key signal modulating intestinal immune re-
PET imaging demonstrates the ability of L. reuteri 6475 to
sponses. We demonstrated that microbiome supplementation
suppress intestinal inflammation. In order to visualize anti-
with hdc⫹ L. reuteri in a trinitrobenzene sulfonic acid (TNBS)-
inflammatory effects of L. reuteri 6475, combined computed to-
induced mouse model of colitis resulted in improvement of over-
mography (CT)-positron emission tomography (PET) imaging
all health status, reduction of colonic inflammation, and suppres-
was applied to the TNBS colitis model. Healthy control mice,
sion of proinflammatory cytokine production. Both the histidine-
to-histamine converting enzyme, histidine decarboxylase, and colitic mice, and L. reuteri-treated mice were subjected to live-
dietary L-histidine must be present for probiotic L. reuteri to ame- animal imaging prior to euthanasia (Fig. 2A). [18F]fluorodeoxyg-
liorate colitis in this mouse model. Luminal conversion of the lucose ([18F]FDG) has been applied to mouse colitis studies and

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amino acid L-histidine to histamine and signaling via histamine was used as the tracer compound because immune cells have been
H2 receptor (H2R) are required for maximal suppression of colitis shown to increase glucose uptake and phosphorylation after im-
by L. reuteri. mune activation (24). During colitis, FDG uptake by activated
lymphocytes and other immune cells in the colon can be measured
RESULTS by comparing relative intensities of tracer signals by anatomic
hdcⴙ L. reuteri attenuates colonic inflammation in vivo. L. reu- location. In healthy control mice, FDG signals were mostly de-
teri clade II strain 6475 (L. reuteri 6475) was isolated from a Finn- tected in the mouse bladder and upper chest region. These sites
ish mother’s breast milk sample (8) and has been used commer- were proposed to be body sites where increased glucose uptake
cially as a probiotic. This hdc-positive strain suppresses human and metabolism in healthy states have been documented (25).
TNF production by myeloid cells (9). The TNBS-triggered acute Trace amounts of FDG signals were shown in pericolonic areas in
colitis mouse model (17) was selected for evaluation of colitis the abdomen, indicating low glucose uptake in the colons of
suppression by intestinal lactobacilli. Adult (8-week-old) female healthy mice (Fig. 2B). In colitic control mice, FDG intensities in
BALB/c mice were fed L. reuteri 6475 (see Fig. S1A in the supple- the mouse abdomen adjacent to the colon were significantly in-
mental material) by daily orogastric gavage following acclimatiza- creased (Fig. 2B), indicating increased glucose uptake surround-
tion and at least 5 days prior to TNBS instillation (Fig. 1A). The ing or within the colonic mucosa during colitis. L. reuteri 6475
severity of colitis was evaluated 2 days after TNBS instillation by administration lowered FDG intensity in the mouse colon com-
weight loss (overall health status), Wallace and Ameho scores pared to colitic controls (Fig. 2B). FDG intensity was also shown in
(macroscopic and microscopic colonic injury) (18–20) (see three-dimensional images (see Videos S1 to S3 in the supplemen-
Fig. S1B), and serum amyloid A (SAA) protein concentrations tal material). FDG signal changes in different mouse groups were
(biomarker of mucosal inflammation) (21). In colitis-negative further confirmed by the FDG standardized uptake value (SUV)
control mice that received phosphate-buffered saline (PBS) only, quantified blindly in the region of interest (ROI) (colon) in each
L. reuteri 6475 maintained a healthy baseline without any evidence mouse using Inveon Research Workplace software (Fig. 2C), in-
of colitis. In colitis-positive control mice that were challenged dicating attenuation of colonic inflammation by L. reuteri.

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L. reuteri administration increased microbial hdc gene ex- is important for the anti-inflammatory activity of L. reuteri 6475.
pression in the intestine. In order to explore whether hdc genes Dietary deficiency of L-histidine results in weight loss in mice (see
contribute to the anti-inflammatory effects of L. reuteri in vivo, Fig. S4A in the supplemental material), as other studies have
the relative abundances of hdc genes and mRNA were determined shown (26), so weight loss was not used as a primary parameter to
by quantitative PCR (qPCR) in mice gavaged with L. reuteri 6475. evaluate colitis severity in this study. To exclude the possibility
Our results demonstrated that L. reuteri 6475 administration sig- that the diets per se contributed to colitis development, groups of
nificantly increased the relative abundance of bacterial hdcA genes mice were fed one of the three diets, and none of these mice
in the mouse gut microbiome (Fig. 3A). In terms of mRNA, both developed colitis without TNBS instillation (see Fig. S4B). In
hdcA and hdcP gene expression was significantly increased in co- summary, L-histidine provides the substrate for intestinal
lonic luminal contents of mice receiving L. reuteri (Fig. 3B and C). L. reuteri to generate histamine in the presence of active histi-
These studies indicate a correlation between elevated hdc gene dine decarboxylase.
expression via L. reuteri administration and the effect of colitis Activation of H2R is required for anti-inflammatory effects
attenuation. By enhancing the ability of the intestinal microbiome of microbial histamine. Histamine is a biogenic amine that exerts
to convert L-histidine to histamine, the gut microbiome was capa- various pathophysiological functions via four receptors (H1R,
ble of suppressing intestinal inflammation. H2R, H3R, and H4R) (27). The predominant histamine receptors
Inactivation of the L. reuteri histidine decarboxylase gene in the intestinal epithelium are H1R and H2R (28). H2R was
diminished its ability to suppress intestinal inflammation. To shown to be expressed in the intestinal epithelium of humans,
further investigate the importance of the bacterial histidine decar- simians, and mice (28, 29). Immunohistochemical studies show
boxylase gene with respect to intestinal immunomodulation, we H2R expression in the BALB/c mouse small intestine and colon,
explored the anti-inflammatory effects of hdcA within an isogenic with relatively high intensities in the villi and crypts (Fig. 6A). It
mutant and compared with wild-type L. reuteri 6475 using the has been reported previously that H1R activation results in pro-
same TNBS colitis model. The hdcA gene encodes histidine decar- inflammatory effects such as interferon (IFN) production and
boxylase, and the hdcA mutant derived from L. reuteri 6475 does Th1 cell proliferation, while H2R activation appears to suppress
not generate histamine from L-histidine (9). The L. reuteri 6475 inflammation (27, 30, 31). So, we hypothesized that microbe-
hdcA mutant strain was shown to be deficient in terms of colitis derived histamine binds and activates H2R in the intestinal epi-
suppression in the TNBS colitis mouse model. The hdcA mutant thelium, thereby mediating its anti-inflammatory effects.
yielded diminished effects in terms of weight loss, Wallace scores, To determine whether H2R activation was required for L. reu-
and SAA concentrations (Fig. 4), suggesting that bacterial histi- teri’s capacity to attenuate colitis in the TNBS model, ranitidine,
dine decarboxylase mediates anti-inflammatory effects via hista- an H2R-specific antagonist, was used to block H2R activation in
mine generation. To exclude the possibility that TNBS may ad- the mouse gut. In the TNBS colitis experiment, addition of rani-
versely affect the function of L. reuteri in the mouse intestine, we tidine (100 mg/kg of body weight) to mice receiving hdc⫹ L. reuteri
performed additional in vitro experiments and found that TNBS by orogastric gavage diminished the anti-inflammatory effects of
did not affect the survival or proliferation of the L. reuteri (wild- L. reuteri 6475, whereas blocking H1R with its specific antagonist
type or mutant) strain (see Fig. S3A and B in the supplemental pyrilamine lacked such effects (Fig. 6B to D). These findings sup-
material). port the proposition that L. reuteri 6475 attenuates colitis via an
Dietary L-histidine enables hdcⴙ L. reuteri to suppress intes- H2R-dependent signaling mechanism. To exclude the possibility
tinal inflammation. In addition to experiments with different that ranitidine or pyrilamine may have adversely affected the

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L. reuteri strains, the relative importance of dietary L-histidine as function of L. reuteri, in vitro assays were performed by adding
the substrate for histamine generation was evaluated by exposing ranitidine or pyrilamine to bacterial cultures, and neither com-
mice to different diets. BALB/c mice were randomly divided into pound affected the survival or proliferation of L. reuteri 6475 (see
three feeding groups: regular diet containing approximately 0.4% Fig. S3C and D in the supplemental material).
histidine from intact protein sources; defined diet with all essential L. reuteri administration affects cytokine gene expression in
amino acids, including 0.4% histidine; and a histidine-free diet the colon. To investigate the consequences of H2R-mediated anti-
derived from defined diet with all amino acids except histidine inflammatory effects by hdc⫹ L. reuteri, relative patterns of muco-
(see Table S1 in the supplemental material). sal gene expression of selected cytokines in the colons of colitis-
Mice in each feeding group were gavaged with the same negative, colitis-positive, and L. reuteri 6475-treated mice were
amount of L. reuteri 6475 or MRS medium only as the control evaluated by qPCR using glyceraldehyde-3-phosphate dehydro-
group. Mice receiving L. reuteri 6475 and dietary L-histidine (in genase (GAPDH) as the internal standard (Fig. 7). TNBS instilla-
regular mouse chow or amino acid defined diet) demonstrated the tion significantly increased expression of genes for proinflamma-
expected amelioration of colitis by reduced Wallace scores and tory cytokines interleukin-1␤ (IL-1␤) and IL-6 compared to
serum SAA concentrations (Fig. 5A and B). In contrast, when the healthy mice. L. reuteri 6475 treatment of TNBS-challenged mice
mice received a histidine-free diet, L. reuteri 6475 yielded dimin- reduced the relative amounts of mucosal IL-1␤ and IL-6 gene
ished effects on colitis attenuation, suggesting that histidine intake expression. Other cytokine genes (TNF, IL-10, IL-12, IFN, IL-17,

FIG 1 hdc⫹ L. reuteri attenuates colonic inflammation in vivo. (A) Time line of the mouse experiments. After 10 days of acclimatization, 8-week-old female
BALB/c mice received 5 ⫻ 109 CFU of bacteria in MRS or MRS medium only by orogastric gavage daily for 7 days. Acute colitis was induced by intrarectal
instillation of TNBS-ethanol before the sixth gavage, and colitis severity was evaluated in 2 days. (B to D) Weight loss (B), Wallace scores (C), and SAA
concentrations (D) of mice challenged with or without TNBS and gavaged with or without L. reuteri 6475. (E and F) Representative microscopic colonic images
(hematoxylin and eosin stained) (E) and Ameho scores (F) from mice in the healthy control group (MRS/PBS), the colitis control group (MRS/TNBS), and the
L. reuteri-treated group (L. reuteri 6475/TNBS).

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FIG 2 Detection of colitis attenuation by PET imaging. (A) Time line of the PET imaging experiments. Fasted (for 6 h) mice were anesthetized with isoflurane
and received 200 ␮Ci [18F]FDG by intraperitoneal (IP) injection. One hour later, these mice received 200 ␮l MD-Gastroview rectally immediately before scan
initiation. Computed tomography (CT) scanning was performed for 10 min followed by PET scanning for 20 min using the Inveon PET-CT multimodality
system. Mice were kept sedated during the scanning process by constant isoflurane inhalation. The images were recorded, and FDG standardized uptake values
(SUVs) were analyzed blindly using Inveon Research Workplace software. (B) Representative mouse images captured by PET-CT scanning in each group. The
color code bar represents FDG signal intensity. (C) Quantification of FDG signals in mouse colon using SUVs in different groups.

and IL-23) were examined, but no significant differences between pounds such as histamine. In the current study, hdc⫹ L. reuteri
colitic mice and L. reuteri-treated mice were detected. strains protected BALB/c mice in a TNBS-induced colitis model,
as indicated by improvement in overall health status and amelio-
DISCUSSION ration of the colitis phenotype. The colitis phenotype was evalu-
Probiotic lactobacilli modulate intestinal immune responses by ated by macroscopic and microscopic evaluation of colonic tissue,
luminal conversion of dietary amino acids into bioactive com- serum biomarker quantitation, mucosal cytokine gene expres-

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FIG 3 L. reuteri administration increases hdc gene expression in vivo. (A) Relative abundance of hdcA gene in mouse gut microbiome was significantly increased
in mice gavaged with L. reuteri 6475 compared to control mice gavaged with MRS. The relative abundance was determined by qPCR and normalized to the
bacterial housekeeping gene rpoB. (B and C) Both hdcA (B) and hdcP (C) gene expression levels were significantly increased in colonic luminal contents of mice
receiving L. reuteri compared to control mice receiving MRS. The relative gene expression was determined by qPCR and normalized to the bacterial housekeeping
gene rpoB. n ⫽ 6 per group. ****, P ⬍ 0.0001.

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sion, and [18F]FDG live-animal PET imaging. The importance of type distributions (27). Histamine receptors are widely distrib-
dietary histidine and the bacterial enzyme histidine decarboxylase uted in the body, and the histamine type 2 receptor (H2R) appears
was established by mouse model studies. In the absence of dietary to be enriched in the mammalian gastrointestinal tract (28, 29).
L-histidine or a gut microbiome lacking histidine decarboxylase, H2R was first characterized in the human stomach as an impor-
colitis suppression by probiotic lactobacilli was reduced signifi- tant target for H2R blockers for treatment of peptic ulcer disease
cantly. Both the substrate amino acid and the enzymatic machin- by reduction of acid (HCl) secretion (35). In addition to the im-
ery, histidine decarboxylase, must be present in the intestinal mi- portance of H2R in gastric physiology, it is becoming apparent
crobiome in order to generate histamine as the bioactive that histamine may have an important immunoregulatory role in
compound. Histamine H2 receptor signaling in the intestinal ep- the intestine. H2R activation results in cAMP-mediated blockade
ithelium is required for probiotic L. reuteri-mediated immuno- of c-Raf and suppression of MAP kinase signaling by inhibition of
modulation and colitis suppression. Previously, L. reuteri was ERK phosphorylation (9, 27). Activation of H2R by histamine
shown to suppress H2R-mediated signaling by increasing cyclic suppressed IL-12 production by monocytes (36), IFN-␥ produc-
AMP (cAMP) production, protein kinase A (PKA) activation, and tion by macrophages (37), TNF secretion by mast cells (38), and
suppression of extracellular signal-regulated kinase (ERK) IL-12 release by immature dendritic cells (39). In vivo studies
(mitogen-activated protein [MAP] kinase) signaling (9). H2R- showed that histamine suppressed both Th1- and Th2-type re-
mediated signaling via cAMP production and protein kinase A sponses by H2R (30). Lactobacillus rhamnosus, which secretes his-
(PKA) activation, followed by inhibition of c-Raf and MEK/ERK tamine significantly, suppressed Peyer patch IL-2, IL-4, IL-5, IL-
MAPK signaling, was described in previous studies (9, 32–34). 12, TNF-␣, and granulocyte-macrophage colony-stimulating
The same microbe-derived biochemical compound (hista- factor (GM-CSF) secretion in wild-type but not H2R-deficient
mine) can yield different effects in the host depending on the mice (31).
specific type of histamine receptor. Four different G-protein- These results indicate that the net effect of luminal histamine
coupled histamine receptors have been described, and these re- may be immunosuppressive and anti-inflammatory in the mam-
ceptors differ based on downstream signaling pathways and cell malian gastrointestinal tract. Prior studies of the human metag-

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FIG 4 Inactivation of the L. reuteri histidine decarboxylase gene diminishes its ability to suppress intestinal inflammation. The anti-inflammatory effects of hdcA
within an isogenic mutant which does not produce histamine were compared with those of wild-type L. reuteri 6475 using the TNBS colitis model. The wild-type
strain attenuated colitis compared with the medium-control group (MRS/TNBS), whereas the hdcA mutant yielded diminished effects in terms of weight loss (A),
Wallace scores (B), and SAA concentrations (C).

enome reported that pathways of histidine biosynthesis (hista- by resulting in histamine generation only in intestinal regions en-
mine precursor) were diminished in patients with IBD relative to riched for histamine H2 receptors. Oral administration of hista-
healthy controls (40). These studies highlight the potential impor- mine may cause adverse outcomes, but the careful selection of
tance of microbiome-mediated histidine metabolism and hista- microbes that colonize specific areas of the small or large intestine
mine generation as a microbial mechanism for intestinal immu- may maximize histamine H2 receptor signaling in the intestinal
nomodulation. Recent evidence suggests that blocking H2R epithelium. Conversely, provision of L-histidine in the diet en-
signaling pathways in humans may result in adverse effects and ables luminal conversion and luminal histamine generation by
severe intestinal inflammation. Retrospective and prospective hdc gene cluster-positive microbes. Another consideration is the
clinical studies of newborns have documented significantly in- relative instability of histamine in vivo. Histamine is unstable in
creased incidence and mortality from necrotizing enterocolitis vivo and could be quickly metabolized by histamine
(NEC) following exposure to H2R antagonists (41–43). In addi- N-methyltransferase or diamine oxidase (45). Lower concentra-
tion to NEC, studies have reported an increased risk of exacerba- tions of histamine might be protective, whereas higher concentra-
tions in Crohn’s disease secondary to H2R blocker exposure (44). tions might be detrimental to epithelial protection from infection
The histamine receptor H2R appears to be the key receptor on the (27). The continuous production of small amounts of histamine
intestinal epithelium, involved in signaling and immunomodula- by the gut microbiome may result in suppression of intestinal
tion after binding microbiome-derived histamine. inflammation. With respect to bacterial genetics of histamine pro-
The consumption of hdc gene cluster-positive probiotics in the duction, the current study did not include an hdcA complemen-
presence of dietary histidine may maximize the potential benefits tation strain because antibiotic consumption by mice to maintain

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mines relieve respiratory tract symptoms by antagonizing H1R

and, more recently, H4R (49). H3R is primarily neuronal, high-
lighting histamine’s role as a potential neurotransmitter. The
characterization of the relative distribution of histamine receptors
in the gastrointestinal tract and other body sites will facilitate a
more complete understanding of the biology of histamine in vivo.
This report highlights the potential importance of luminal
conversion and amino acid metabolism in the biology of the in-
testinal microbiome and host-microbe mutualism. Luminal con-
version of amino acids and different classes of nutrients effectively
links diet, the gut bacteria, and mammalian intestinal biology.
Bacterial amino acid decarboxylases have been reported to con-
vert glutamate to the neurotransmitter gamma-aminobutyric acid
(GABA) (50) and tyrosine to tyramine and phenylalanine to the
neuromodulatory compound phenethylamine (51). Differences
in the relative abundances of pathways involved in amino acid
metabolism present in the gut microbiome may contribute to dif-
ferent disease phenotypes in individuals genetically predisposed
to IBD or other immune-mediated conditions. Dietary amino ac-
ids are potential substrates for a variety of microbial amino acid
decarboxylases, and diverse compounds, including biogenic
amines, may be produced. These compounds, such as histamine,
may have important consequences for mucosal immunity or
functioning of the nervous system. Our study indicates that lumi-
nal conversion of an amino acid, L-histidine, to histamine by hdc⫹
L. reuteri activates H2R and yields anti-inflammatory effects in the
mouse colon. This study combined specific cellular elements of
the intestinal microbiome, the genes and enzymatic machinery
involved in luminal conversion, and the specific receptors in-
volved in receiving microbial signals. These studies could foster
the development of new probiotic therapies by facilitating the se-
lection of natural hdc gene cluster-positive strains (or strains with
any defined genetic feature contributing to immunomodulation)
combined with dietary elements (e.g., amino acids) or enabling
FIG 5 L-histidine deficiency diminishes anti-inflammatory effects. The anti-
inflammatory effects of L. reuteri 6475 were compared in mice fed with differ- genetic engineering of next-generation probiotics by defining spe-
ent diets using the TNBS colitis model. Mice fed a regular diet or an amino acid cific microbial genes involved in mitigation of intestinal inflam-
defined diet showed decreased Wallace scores (A) and plasma SAA concentra- mation. By defining mechanisms of microbiome-mediated im-

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tions (B) when receiving L. reuteri 6475 compared to the MRS medium con- munomodulation in the mammalian intestine, bacterial strains
trol. When mice were fed an L-histidine-deficient diet, L. reuteri 6475 showed
diminished anti-inflammatory effects in terms of Wallace scores and SAA
and microbial gene databases can be leveraged to identify next-
concentrations. generation probiotics and microbe-derived medicinal com-
pounds for the treatment of chronic inflammatory diseases.

MATERIALS AND METHODS

antibiotic resistance plasmids may profoundly affect gut micro-
Bacterial strains and microbiological culture conditions. L. reuteri
bial composition. Future generation of hdcA complementation ATCC PTA 6475 and its hdcA mutant as described previously (9) were
strains by recombineering (46) may remove the requirement for used to colonize the mice. L. reuteri ATCC PTA 4659, isolated from the
antibiotic selection. Such future genetic strategies would be help- breast milk of healthy Finnish women, was a gift from BioGaia AB (Stock-
ful to confirm whether histidine decarboxylase is essential for sup- holm, Sweden). All L. reuteri strains were cultured at 37°C in deMan,
pression of intestinal inflammation by probiotic L. reuteri. Rogosa, Sharpe (MRS) medium (Difco, Franklin Lakes, NJ) in an anaer-
Adverse effects of histamine are likely due to the preponder- obic workstation (MACS MG-500; Microbiology International, Freder-
ance of the H1 receptor in the airways (47) and upper gastrointes- ick, MD) supplied with a mixture of 10% CO2, 10% H2, and 80% N2.
tinal tract. When ingested orally, histamine may cause adverse Quantitative analysis of bacteria was performed by counting bacterial
reactions and symptoms such as pruritus, bronchoconstriction, CFU on an MRS agar plate per milliliter of bacterial culture relative to
airway inflammation, and allergic symptoms (48). For this reason, optical density at 600 nm (OD600) measured by a SmartSpec Plus spectro-
photometer (Bio-Rad Laboratories, CA).
the food and beverage industry has developed active programs to
Animals. Female BALB/c mice (45 days old) were purchased from Har-
screen for histamine in foodstuffs and contamination by land Laboratories (Houston, TX) and maintained under specific-pathogen-
histamine-generating bacteria. H1R is coupled to Gq/11 family free (SPF) conditions. Mice were kept under filter-top cages (5 mice per cage)
proteins, triggering downstream calcium mobilization with pro- and had free access to distilled water and Harlan rodent chow 2918 (default
inflammatory effects. In addition to H1R, H4R also appears to diet) or other diets as described in Table S1 in the supplemental material. All
contribute to respiratory disease symptoms. Classical antihista- mouse experiments were performed in an SPF animal facility according to an

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FIG 6 Activation of H2R is required for the anti-inflammatory effects. (A) Immunohistochemistry studies using H2R-specific antibody showed that H2R was
expressed in 9-week-old BALB/c mice, with high intensity in the villi and crypts (red arrows). (B to D) Mice that received pyrilamine (Pyr) did not show an effect
on the anti-inflammatory effects of L. reuteri 6475, while mice that received ranitidine (Ran) showed a diminished ability for L. reuteri 6475 to attenuate colitis,
as indicated by weight loss (B), Wallace scores (C), and plasma SAA concentrations (D).

Institutional Animal Care and Use Committee (IACUC)-approved mouse 5 ⫻ 109 CFU of bacteria in 0.2 ml MRS or MRS medium alone (medium
protocol at Baylor College of Medicine, Houston, TX. control) by orogastric gavage at a frequency of once per day for 7 days. In
Preparation of bacteria and administration to mice. L. reuteri strains selected experiments, pyrilamine or ranitidine at a dose of 100 mg/kg body
and culture conditions were as described above. Bacteria were harvested at weight was added in the bacterial medium to feed mice by orogastric
exponential phase (5.5 h in MRS medium with an initial OD600 of 0.03) gavage.
and centrifuged at 2,500 ⫻ g for 4 min, and bacterial pellets were resus- Induction of colitis by intrarectal instillation of TNBS. At 6 h before
pended in sterile MRS medium for animal feeding. All L. reuteri strains the six orogastric gavages mentioned above, mice preanesthetized by con-
were prepared freshly before administration to mice. Each mouse received stant isoflurane inhalation were challenged with 5% (wt/vol) TNBS in

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Gao et al.

FIG 7 L. reuteri administration affects cytokine gene expression in the colon. Gene expression of IL-1␤ (A) and IL-6 (B) in the colons of healthy mice, colitic
mice challenged with TNBS, and L. reuteri 6475-treated colitic mice was measured by reverse transcription-quantitative PCR using GAPDH as the internal
standard. n ⫽ 10 per group. The P values are indicated in the figure.

H2O (Sigma-Aldrich, St. Louis, MO) diluted with an equal volume of and FDG standardized uptake values (SUVs) were analyzed blindly using
absolute ethanol intrarectally (4 cm distal to the anus) via catheter (Brain- Inveon Research Workplace software. The MD-Gastroview appears as
tree Scientific, USA) at a dose of 100 mg/kg of body weight. Mice were kept radiopaque in the CT image, thus highlighting the colon clearly. A region
head down in a vertical position for 2 min after enema to ensure complete of interest (ROI) comprising the colon was extracted from the CT scan
retention of enema in the colon. Mice from respective control groups and transferred to the space of the PET scan. Tissues/organs (such as the
received PBS at a dose of 100 mg/kg of body weight instead of TNBS. bladder) other than the colon were excluded from the measurements, and
Tissue preparations. Mice were euthanized 48 h after colitis induc- the averages of SUVs of the remaining voxels were calculated to represent
tion. Blood samples were collected from mice via cardiac puncture in the severity of the inflammation.
blood sample collection tubes with K2-EDTA (Becton, Dickinson and Toxicity of TNBS, pyrilamine, or ranitidine against L. reuteri 6475.
Company, Franklin Lakes, NJ), centrifuged at 17,000 ⫻ g for 10 min at 4°C In growth curve assays, different concentrations (0 ␮g/ml, 2.5 ␮g/ml,
to isolate plasma, and stored at ⫺80°C until use. The gastrointestinal tract 25 ␮g/ml, or 250 ␮g/ml) of TNBS, pyrilamine, or ranitidine were added to
was carefully removed. Colons and ceca were excised, and colon lengths MRS medium inoculated with wild-type L. reuteri 6475 or hdcA mutant
were measured. Luminal contents in the colon were collected, flash frozen strains. The OD600s were measured at different time points. In bacterial
immediately in liquid nitrogen, and stored at ⫺80°C until use. killing assays, L. reuteri 6475 or hdcA mutant cultures were normalized to
Mouse intestines were fixed in 10% formalin, embedded with paraffin, an OD600 of 1 and then treated for 1 h with 250 ␮g/ml of TNBS, pyril-
and sectioned with a microtome at 5 ␮m. The sectioned tissues were used amine, or ranitidine. The cultures were then diluted in sterile MRS me-
for immunohistochemistry targeting H2R expression using specific anti- dium and plated on MRS agar to count CFU.
body (Alomone Labs, Jerusalem, Israel). In specific experiments, the co- Determination of the relative abundances of hdc genes and mRNA
lon tissue samples located precisely 2 cm above the anal canal were cut into in vivo. Total DNA from luminal contents in the colon was isolated using
two parts. One part was fixed overnight in 10% formalin and embedded in ZR Fecal DNA MiniPrep (Zymo Research, Irvine, CA) according to the
paraffin. After hematoxylin and eosin staining, slides were examined mi- manufacturer’s instructions. For total RNA extraction, luminal contents

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croscopically and scored blindly by two pathologists according to Ameho in the colon were resuspended in RNAlater (Ambion, Austin, TX) and
criteria (20). Another tissue fragment was stored in RNAlater (Ambion, transferred to 2.0-ml FastPrep tubes (MP Biomedicals, CA) prefilled with
Austin, TX) and flash frozen immediately in liquid nitrogen for cytokine 200 ␮l 0.1-mm lysing beads. The samples were homogenized with a
gene expression analysis. FastPrep-24 instrument (MP Biomedicals, CA) at 4.0 m/s for 20 s twice,
Colitis assessment. Colitis severity was assessed by weight loss, Wallace and the supernatants were collected for RNA isolation using the RNeasy
score, and serum amyloid protein A (SAA) concentrations as described be- minikit (Qiagen, USA). Methods for cDNA synthesis and qPCR are de-
low. Weight loss is calculated by the formula weight loss ⫽ [(w1 ⫺ w2)/w1] ⫻ scribed below. Relative DNA and mRNA quantities of hdc genes were
100%, where w1 represents mouse body weight before TNBS instillation normalized to the housekeeping gene rpoB (␤ subunit of bacterial RNA
and w2 represents mouse body weight at 48 h after colitis induction. Wal- polymerase).
lace score (see Fig. S1B in the supplemental material), which was used to Determination of cytokine gene expression in the mouse colon. To
quantify colonic injury macroscopically on the excised and longitudinally quantify the relative mRNA levels of IL-1␤, IL-6, IL-10, IL-12, IL-17,
opened colons, was given blindly by two trained technicians as described IL-23, tumor necrosis factor (TNF), and interferon (IFN), RNA was ex-
before (18). SAA concentrations in plasma were measured using enzyme- tracted from the colon samples mentioned above using the RNeasy mini-
linked immunosorbent assay (ELISA) kits from Alpco (Salem, NH), ac- kit (Qiagen, USA). One microgram of RNA was reverse transcribed to
cording to the manufacturer’s instructions. single-stranded cDNA using the stated protocol for SuperScript III reverse
PET imaging. PET-CT scanning was performed 48 h after colitis in- transcriptase (Invitrogen, Carlsbad, CA). Real-time PCR was performed
duction in selected experiments as described previously (24) with minor using the real-time PCR system (Stratagene). The PCR mixture (adjusted
modifications. Briefly, fasted (for 6 h) mice were anesthetized with isoflu- with H2O to a total volume of 20 ␮l) contained 1 ␮l template DNA, 10 ␮l
rane and received 200 ␮Ci [18F]FDG by intraperitoneal (i.p.) injection. 2⫻ FastStart Universal Probe Master (Rox) (Roche Applied Science), and
One hour later, these mice received 200 ␮l MD-Gastroview (Mallinckrodt 0.5 ␮l of the respective primers (10 ␮M each). All primers used in this
Inc., MO) rectally via a 3.5 French (F) catheter immediately before scan study were designed using the Universal ProbeLibrary Assay Design Cen-
initiation. Computed tomography (CT) scanning was performed for ter (Roche Applied Science, Indianapolis, IN) and are described in Ta-
10 min followed by PET scanning for 20 min using the Inveon PET-CT ble S2 in the supplemental material. Relative mRNA levels of target genes
multimodality system (Siemens). Mice were kept sedated during the scan- were normalized to the housekeeping gene glyceraldehyde-3-phosphate
ning process by constant isoflurane inhalation. The images were recorded, dehydrogenase (GAPDH).

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 L. reuteri-Derived Histamine and Colitis Attenuation

Statistical analysis. Biostatistical analyses were performed using nitzii is an anti-inflammatory commensal bacterium identified by gut mi-
GraphPad Prism (version 5) software (GraphPad Inc., La Jolla, CA). For crobiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A
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errors, and different groups were compared with the t test (two groups) or G, Poggioli G, Miglioli M, Campieri M. 2000. Oral bacteriotherapy as
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SUPPLEMENTAL MATERIAL flammation. Am J Physiol Gastrointest Liver Physiol 299:G1087–G1096.
http://dx.doi.org/10.1152/ajpgi.00124.2010.
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lookup/suppl/doi:10.1128/mBio.01358-15/-/DCSupplemental. W, Britton RA, Kalkum M, Versalovic J. 2012. Histamine derived from
Video S1, MOV file, 0.7 MB. probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and
Video S2, MOV file, 1.2 MB. ERK signaling. PLoS One 7:e31951. http://dx.doi.org/10.1371/
Video S3, MOV file, 0.7 MB. journal.pone.0031951.
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Figure S2, TIF file, 0.2 MB. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice.
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ACKNOWLEDGMENTS 558 –568. http://dx.doi.org/10.1128/AEM.70.1.558-568.2004.
12. Schreiber O, Petersson J, Phillipson M, Perry M, Roos S, Holm L. 2009.
This work was supported by the National Institutes of Health (R01 Lactobacillus reuteri prevents colitis by reducing P-selectin-associated
AT004326, UH3 DK083990, and U01 CA170930) and the National Insti- leukocyte- and platelet-endothelial cell interactions. Am J Physiol Gastro-
tutes of Health (National Institute for Diabetes and Digestive and Kidney intest Liver Physiol 296:G534 –G542. http://dx.doi.org/10.1152/
Diseases)-funded Texas Medical Center Digestive Diseases Center ajpgi.90470.2008.
(DK56338) (J.V.). 13. Preidis GA, Saulnier DM, Blutt SE, Mistretta T, Riehle KP, Major AM,
We thank Eamonn Connolly (BioGaia AB, Stockholm) for providing Venable SF, Barrish JP, Finegold MJ, Petrosino JF, Guerrant RL,
the L. reuteri strains, Toni-Ann Mistretta and Bhanu Priya Ganesh for Conner ME, Versalovic J. 2012. Host response to probiotics determined
assistance with data plotting and statistical analysis, and Coreen Johnson by nutritional status of rotavirus-infected neonatal mice. J Pediatr Gastro-
enterol Nutr 55:299 –307. http://dx.doi.org/10.1097/
for assistance with bacterial DNA and RNA extractions from luminal
MPG.0b013e31824d2548.
contents. We thank Texas Children’s Hospital for the use of the Small 14. Spinler JK, Sontakke A, Hollister EB, Venable SF, Oh PL, Balderas MA,
Animal Imaging Facility and especially Caterina Kaffes and. M. Waleed Saulnier DMA, Mistretta TA, Devaraj S, Walter J, Versalovic J, High-
Gaber for PET imaging. lander SK. 2014. From prediction to function using evolutionary
We disclose the following: J.V. receives unrestricted research support genomics: human-specific ecotypes of Lactobacillus reuteri have diverse
from BioGaia AB. The remaining authors disclose no conflicts. probiotic functions. Genome Biol Evol 6:1772–1789. http://dx.doi.org/

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