NEW & NOTEWORTHY Histamine-producing Lactobacillus reuteri can suppress development of inflammation-associated colon cancer in an established mouse model. The net effects of histamine may depend on the relative activity of H1R and H2R signaling pathways in the intestinal mucosa. Our findings suggest that treatment with H1R or H2R antagonists could yield opposite effects. However, by harnessing the ability to block H1R signaling while stimulating H2R signaling, novel strategies for suppression of intestinal inflammation and colorectal neoplasia could be developed.

Inflammatory bowel disease (IBD) is a well-known risk factor for the development of colorectal cancer. Prior studies have demonstrated that microbial histamine can ameliorate intestinal inflammation in mice. We tested the hypothesis whether microbe-derived luminal histamine suppresses inflammation-associated colon cancer in Apc min/+ mice. Mice were colonized with the human-derived Lactobacillus reuteri . Chronic inflammation was induced by repeated cycles of low-dose dextran sulfate sodium (DSS). Mice that were given histamine-producing L. reuteri via oral gavage developed fewer colonic tumors, despite the presence of a complex mouse gut microbiome. We further demonstrated that administration of a histamine H1-receptor (H1R) antagonist suppressed tumorigenesis, while administration of histamine H2-receptor (H2R) antagonist significantly increased both tumor number and size. The bimodal functions of histamine include protumorigenic effects through H1R and antitumorigenic effects via H2R, and these results were supported by gene expression profiling studies on tumor specimens of patients with colorectal cancer. Greater ratios of gene expression of H2R ( HRH2 ) vs. H1R ( HRH1 ) were correlated with improved overall survival outcomes in patients with colorectal cancer. Additionally, activation of H2R suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited chemokine gene expression induced by H1R activation in colorectal cancer cells. Moreover, the combination of a H1R antagonist and a H2R agonist yielded potent suppression of lipopolysaccharide-induced MAPK signaling in macrophages. Given the impact on intestinal epithelial and immune cells, simultaneous modulation of H1R and H2R signaling pathways may be a promising therapeutic target for the prevention and treatment of inflammation-associated colorectal cancer.

INTRODUCTION Discovered more than 100 years ago, histamine is an important signaling compound affecting a variety of biological processes, including neuronal activity, endothelial permeability, vascular tone and gastric acid secretion, inflammation, allergy, and development of cancer (14, 37). The pleiotropic effects of histamine are mediated by the potential activation of each of four histamine receptors (H1R, H2R, H3R, and H4R) present on mammalian cells. H1R, H2R, and H4R have been well described in human and mouse immune cells, cholangiocytes, hepatocytes, and endothelial cells (17, 71), whereas H3R is primarily expressed in neurons of the central nervous system (CNS) (21). Histamine influences myeloid cell differentiation (72), and H1R and H4R are considered to be the two histamine receptors involved in allergic inflammation (64). H2R is central in gastric acid production (8). Clearly, histamine is a biogenic amine synthesized endogenously by mammalian cells with important roles in basic physiologic processes. Previous studies have focused on the contrasting effects of histamine receptors with respect to inflammation and immune cell signaling (35). The effects of H1R and H2R in chronic inflammation and in inflammation-associated colon cancer are not fully understood. H1R activation regulates downstream pathways through intracellular calcium concentration, while H2R signals through cyclic AMP (cAMP) (15). Activation of H1R and H2R has been widely shown to yield opposing effects in multiple biological processes. For example, in human T-cell-mediated immune responses, H1R activation promotes Th1 polarization, while H2R activation suppresses Th1 polarization (46, 61). Distinct effects of H1R and H2R activation were also evident in terms of smooth muscle contraction. H1R and H2R antagonists, respectively, inhibit and exacerbate histamine-induced bronchoconstriction in patients with mild asthma (53). These findings strongly suggest that histamine may have opposing effects depending on the specific histamine receptor that is activated. In the pathogenesis of inflammatory bowel disease (IBD), activation of MAP kinase (MAPK) and nuclear factor-κB (NF-κB) pathways are key events that enhance the production of proinflammatory cytokines, such as TNF and IL-6 (5, 12). Therefore, IBD medications have been developed that target specific cytokine signaling pathways (26). Histamine was detected in increased concentrations in the intestinal mucosa of IBD (55) and promoted ERK phosphorylation through H1R activation in human epidermal keratinocytes (45) and aortic endothelial cells (25). Although it is not clear whether H1R is involved in MAPK activation in intestinal epithelial cells, H1R antagonists, such as loratadine (56) and ketotifen (32), have been used to alleviate clinical symptoms in IBD. In contrast, studies have shown that administration of the H2R antagonist cimetidine resulted in more rapid death of mice in an intestinal infection model (24) and our previous study showed that H2R activation suppressed trinitrobenzene sulfonate-induced colonic inflammation in mice (20). Interestingly, H2R blockers also increased the frequency of hospitalization or surgery in patients with ulcerative colitis (34) as well as cancer risk, including intestinal cancers (50). Furthermore, the H2R agonist dimaprit was reported to reduce TNF production induced by LPS in mouse models of endotoxin shock and hepatitis (52). A link between chronic inflammation and colorectal cancer (CRC) is well recognized (4, 51, 66). Yang et al. (19) demonstrated antitumorigenic effects of histamine in histidine decarboxylase gene (Hdc) knockout mice that lacked the ability to synthesize endogenous histamine and yielded increased susceptibility to chemically induced colon cancer (72). Our group demonstrated that Lactobacillus reuteri-derived histamine suppressed tumor formation in the colons of Hdc knockout mice. Activation of H1R signaling promoted proliferation of cholangiocarcinoma-derived cells (16). Abrogation of H1R signaling with antagonists enhanced radiosensitivity, resulting in reduced viability of colon cancer cells. Furthermore, elevated expression of the HRH1 gene has been shown to be associated with poor survival for both lung cancer and B-cell lymphoma patients (70). Several other studies demonstrated that H1R activation suppressed cell proliferation in prostate cancer (67), melanoma (40), and leukemia (31) cells and promoted cell migration of cervical carcinoma cells (57). Altogether, the effects of histamine-receptor activation seem to be tissue specific, depending on relative distributions of histamine receptors in different tissue types (37). In addition to the crucial role in IBD, MAPKs are also critical mediators of signal transduction in cancer development. For example, ERK activation promotes intestinal tumorigenesis in ApcMin/+ mice (41) and results in increased proliferation of colon cancer cells in vitro (33). By contrast, H2R activation suppressed ERK phosphorylation in human monocytes (63) and promoted ERK phosphorylation in HEK293T cells (10). The precise mechanisms, however, by which histamine affects inflammation associated-intestinal tumorigenesis through H1R and H2R signaling pathways remain to be elucidated. In this study, we demonstrated that H1R signaling promoted intestinal tumor formation in vivo and proliferation of colorectal cancer-derived intestinal epithelial cells. By contrast, H2R signaling suppressed tumor growth in inflammation-associated colon cancer in mouse models. To explore the implications in human cancer, we found that an increased ratio of HRH2 vs. HRH1 gene expression was associated with improved survival rates of CRC patients. We attempt to delineate molecular mechanisms explaining how histamine regulates chronic intestinal inflammation via different receptors, H1R and H2R, and how this deeper understanding of intestinal histamine signaling may facilitate the development of new preventive strategies and therapeutics in the future. New therapies aimed at blocking H1R signaling while promoting H2R signaling may offer real promise as cancer therapeutic strategies.

MATERIALS AND METHODS Mice. Apcmin/+ mice were maintained under specific pathogen-free conditions with a 12-h:12-h light-dark cycle in animal facilities at Baylor College of Medicine. Two-month-old male mice were randomly divided into three groups (n = 8–11 mice per group, 3–4 mice per cage) and were gavaged with 5 × 109 colony forming units of histamine-producing L. reuteri 6475, histamine negative L. reuteri 6475 (inactivated hdcA and unable to convert l-histidine into histamine), or control media (MRS without bacteria), daily for 7 days. Thereafter, mice were given two cycles of 1.5% DSS (36,000 to 50,000 molecular weight; MP Biomedicals, Solon, OH) in drinking water as follows: DSS for 5 consecutive days followed by 17 days of recovery, and a second cycle of DSS for 4 days followed by 18 days of recovery. During DSS treatment and recovery periods, the mice received either histamine-generating L. reuteri 6475, histidine decarboxylase (HdcA)-deficient L. reuteri 6475, or control media once every 3 days. At the end of the second recovery period, mice were euthanized, and tumors were counted and measured under a dissecting microscope by two individuals blinded to the mouse treatment groups. The intestinal tissues were fixed in formalin and embedded in paraffin (FFPE) for histologic evaluation. Dysplasia was counted throughout the colon on hematoxylin-eosin-stained sections. To study receptor-specific effects of histamine, Apcmin/+ mice were given pyrilamine (H1R antagonist, 50 mg/l) (65), cimetidine (H2R antagonist, 100 mg/l) (1), or omeprazole (proton pump inhibitor, 10 mg/l) (30) in drinking water (all drugs were fully dissolved in water at the designated concentrations) starting 7 days before DSS treatment 1 until completion of the second recovery period post-DSS treatment 2. Mice were then euthanized and processed as mentioned above. All procedures were approved by the Institutional Review Board at Baylor College of Medicine. Human cell lines. Human colorectal cancer-derived cell lines, including HCT116, Caco2, DLD1, LS174T and HT29 (all from ATCC, Manassas VA), were grown in DMEM containing 10% fetal bovine serum (GIBCO, Life Technologies, Carlsbad, CA) in a humidified 5% CO 2 atmosphere at 37°C. L. reuteri strains. L. reuteri 6475 and the isogenic hdcA mutant strain were prepared as described previously (63). Bacterial cells were cultured in MRS medium at 37°C in anaerobic conditions (20). Antibodies, plasmids, and chemicals. Antibodies are listed in Table 1. Recombinant plasmids were generated for ectopic expression of histamine-receptor genes. Full-length open reading frames of the wild-type mouse Hrh1 and Hrh2 genes were subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The constructs were confirmed by DNA sequencing. Transfections were performed with 1 μg of plasmid DNA per well (24-well plate) and 2 μl of FuGENE HD transfection reagent (Roche, Indianapolis, IN). Chemicals including pyrilamine maleate salt and cimetidine were purchased from Sigma-Aldrich (St. Louis, MO). Omeprazole was obtained from TCI America (Portland, OR). Dimaprit dihydrochloride was obtained from Biotrend (Destin, FL), and 2-pyridylethylamine dihydrochloride was purchased from Tocris (Minneapolis, MN). Table 1. Antibodies and their applications used in this study Antibody Name Company Clone Number or Catalog Number Applications and Dilutions H1R Santa Cruz Biotechnology P-20 Fluorescent staining: 1:200; immunoblot: 1:500 H2R Santa Cruz Biotechnology M-19 Fluorescent staining: 1:200; immunoblot: 1:500 Human H1R Elabscience ESAP13556 Fluorescent staining: 1:400; immunoblot: 1:1,000 PCNA Santa Cruz Biotechnology PC10 Fluorescent staining: 1:400 β-Actin Santa Cruz Biotechnology 11B7 Immunoblot: 1:500 Phospho-ERK Cell Signaling D13.14.4E Fluorescent staining: 1:300; immunoblot: 1:1,000 Phospho-p38 Cell Signaling D3F9 Immunoblot: 1:1,000 Phospho-JNK Cell Signaling 81E11 Immunoblot: 1:1,000 Phospho-p65 Cell Signaling 93H1 Immunoblot: 1:1,000 Total ERK Cell Signaling 137F5 Immunoblot: 1:1,000 BrdU Accurate OBT0030 Histochemical staining: 1:200 Immunofluorescence and bromodeoxyuridine staining. Immunofluorescence was performed on 5-µm-thick FFPE intestinal tissue sections using a goat polyclonal anti-H1R antibody (Santa Cruz Biotechnology, Santa Cruz, Dallas, TX). Slides were deparaffinized in xylene and hydrated with a series of washes using graded alcohols, followed by antigen retrieval with Tris-EDTA buffer (pH 8.0, 1 mM EDTA, 10 mM Tris) in a steamer for 30 min. After a wash with TBS-T (50 mM Tris·Cl), slides were blocked with 10% goat serum for 30 min and then incubated with primary antibodies in TBS-T buffer at 4°C overnight. Dilutions for antibodies were listed in Table 1. After being washed with TBS-T, slides were incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) for 30 min, followed by DAPI counterstaining to visualize nuclei. For immunofluorescence staining of cultured cells, HCT116 cells were fixed with 4% paraformaldehyde for 10 min followed by 0.5% Triton X-100 incubation for 5 min and then blocked as described above. Immunofluorescence images were generated by fluorescence microscopy (Nikon Eclipse 90 Ni-E). For bromodeoxyuridine (BrdU) labeling, mice were injected with 200 µl of BrdU 2 h before death (Invitrogen) and staining was performed on FFPE sections of colon tumors using a rat anti-BrdU antibody (Accurate, Westbury, NY) and then visualized with the ABC-Elite Kit using DAB (Vector Laboratories, Burlingame, CA). Immunoblots. Lysates from HCT116 cells or tissues were obtained with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Supernatants containing proteins were resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked with 5% dry milk and incubated with primary antibodies overnight at 4°C. After washes with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and developed with ECL substrate (GE Health Care, Buckinghamshire, UK). Densitometric analyses of immunoblots were performed using Band Leader software. RNA extraction and quantitative RT-PCR analysis. Total RNA was extracted using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Reverse transcription was prepared from 1 μg of total RNA using SuperScript II reverse transcription kit (Roche). Quantitative PCR analyses were then performed using Fast SYBR Green (Life Technologies) and amplified on the Applied Biosystems 7900HT instrument. Ribosomal RNA (18S) was used as a control. Primers were listed in Table 2. Relative levels of gene expression were analyzed using the comparative C T method (2−∆∆C T method). Table 2. List of primer sequences and their corresponding applications Gene Name Forward (5′-3′) Reverse (5′-3′) Applications HRH1 CACACTGAACCCCCTCATCT ATTTTGTTGCATCCCCTCAG qPCR HRH2 ACCAGCAAGGGCAATCATAC CATGATCAGTAGCGGGAGGT qPCR Egr1 GAGCGAACAACCCTATGAGC TGGGATAACTCGTCTCCACC qPCR CCL20 CCAAGAGTTTGCTCCTGGCT TGCTTGCTGCTTCTGATTCG qPCR IL8 CACCGGAAGGAACCATCTCA GGAAGGCTGCCAAGAGAGC qPCR IL6 GTATGAACAACGATGATGCACTTG ATGGTACTCCAGAAGACCAGAGGA qPCR 18S GGGTCGCGTAACTAGTTAGCATG CTTAGTTGGTGGAGCGATTTGTC qPCR TOPO-Hrh1 CTAGGATCCGCCACCATGAGCCTTCCCAACACCTC CTAGAATTCTTAGGAACGAATGTGCAGAATTTTTTTG Cloning TOPO-Hrh2 CTAGGATCCGCCACCATGGAGCCCAATGGCACGGTTC CTAGAATTCTTAAGCACTGATATGTAGTGATGG Cloning Cell viability assay. HCT116 cells (1 × 103/well) were plated and cultured in a 96-well plate overnight. Cells were treated with dimaprit dihydrochloride (25 μM), 2-pyridylethylamine dihydrochloride (25 μM), or both for 4 days. Cell viability was determined using a Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, MD), and the absorbance was read at 450 nm using a spectrophotometer. For transfected HCT116 cells, cell viability was determined after treatment with 100 nM histamine for 4 days. Statistical analyses. Statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software, San Diego, CA). All data were normally distributed and examined using parametric tests. Data for tumor counts were analyzed by one-way ANOVA with a Bonferroni post hoc test. The band densities of immunoblots at different time points were analyzed by two-way repeated measures ANOVA with a Bonferroni post hoc test. The results of quantitative PCR (see Figs. 4 and 5) and cell viability assay were analyzed by one-way ANOVA with a Bonferroni post hoc test. The results of quantitative PCR studies (see Fig. 2) and BrdU-positive cell counts were analyzed using unpaired, two-tailed Student's t-test. Statistical test results are presented as means ± SD. Significance was set at P < 0.05, P < 0.01, and P < 0.001, as indicated in figures.

DISCUSSION Gut microbe-derived histamine can suppress colon cancer in a mammalian host, and the impact of microbial histamine on the development of colonic neoplasms depends on the relative balance between H1R and H2R signaling pathways. In our mouse model studies, H2R signaling suppressed inflammation-associated tumorigenesis, whereas H1R signaling appears to promote gut inflammation and colonic carcinogenesis. In human colon cancer-derived cells and immune cells, H2R signaling counteracted H1R-mediated MAPK activation, and MAPK activation contributes to the development of chronic intestinal inflammation and cancer. These findings in human cell culture and mouse models are consistent with patient outcomes based on gene expression profiles in human CRC, whereby elevated HRH1 and reduced HRH2 gene expression were correlated with worse patient outcomes. Together, the data suggest that the ratio of HRH2/HRH1 expression in human tissue could represent a new prognostic biomarker for IBD and CRC and such insight could point the way to new disease preventive and therapeutic strategies that rely on histamine-receptor signaling. Intestinal epithelial cells secrete proinflammatory chemokines, including IL-8 and CCL20, to recruit immune cells to local sites of antigen presentation or injury and elicit inflammation. We demonstrate that histamine elevates expression of both IL8 and CCL20 through H1R/MAPKs in intestinal epithelial cells, such as ERK and JNK and that these effects were reversed by H2R activation. Prior studies showed that histamine promotes IL-8 expression through H1R signaling in human bronchial epithelial cells (3). We have extended this observation to intestinal epithelial cells, which may include both H1R and H2R signaling pathways to maintain intestinal homeostasis. Tilting toward increased H1R (relative to H2R) signaling may promote chemokine production and induce inflammation. Consistent with our results, R2 database analysis showed elevated HRH1 gene expression in the IBD mucosa (data not shown). In addition, several previous studies reported increased proinflammatory cytokines, such as IL-8 and CCL20, in the IBD mucosa (36, 47). In the inflamed gut mucosa, infiltrating immune cells are a major source of cytokines, in which IL-6 is a key contributor to the development of IBD and inflammation-associated CRC (6). We show that LPS-induced IL6 expression was elevated via H1R signaling and suppressed via H2R signaling by histamine in human monocytes and mouse macrophages. Our findings agree with those of other studies, where it has been shown that histamine suppresses LPS-driven proinflammatory cytokine secretion (TNF, IL-12, and CXCL10) in dendritic cells (18) and in peritoneal macrophages via H2R signaling in mice (44). Our results suggest that long-term use of H2R blockers might promote colonic tumor initiation in IBD. In contrast, H1R antagonists (conventional antihistamines) and H2R agonists may suppress inflammation by potently suppressing LPS-induced p38 MAPK activation in macrophages. The p38 MAPK pathway is a key signaling pathway in chronic inflammation and cancer development (73). In IBD, activation of p38 signaling results in elevated TNF expression (69) and in mouse models of colon cancer; inactivation of p38 MAPK signaling significantly suppressed colon tumor development (9). Therefore, a combination of a H2R agonist and a H1R antagonist may effectively suppress p38 MAPK and represent a promising new preventative or therapeutic approach for IBD and CRC. In clinical trials, CNI-1493, a nonselective p38 and JNK inhibitor, alleviated severe Crohn's disease in a small cohort study, suggesting that p38 could be a potential target in IBD treatment (28). However, a larger cohort study using a specific p38 inhibitor, BIRB 796, failed to treat active Crohn’s disease, due to serious adverse events (59). The p38 signaling pathway may be targeted with microbiome-based treatment strategies, possibly reducing adverse effects via a luminal treatment strategy. The role(s) of histamine on the prevention or development of human cancers remains controversial. For example, histamine increased cell proliferation in human hepatocellular carcinoma and melanoma by acting as an autocrine or paracrine growth factor (7, 39), while a different study showed that histamine suppressed cell proliferation in pancreatic cancer (13). In considering histamine’s specific effects, allergic diseases may affect cancer risk. Several studies showed that a history of allergies yielded an inverse correlation with disease risk including particular human cancers (2, 27, 54). In a large-scale prospective study in women, those with a history of allergic disease including allergic rhinitis and asthma had a significantly lower incidence of colorectal cancer (54). A history of allergies or other atopic conditions and glioma susceptibility were found to be inversely correlated (2). In addition, a large-scale population-based case-control study revealed a significantly lower incidence of pancreatic cancer among those who had a prior history of allergies (27). The concomitant use of antihistamines, which suppress H1R signaling, may be useful for treatment of allergic disease and prevention of specific human cancers based on our findings. Meanwhile, allergy may be a risk factor for lymphatic-hematopoietic malignancies, and prostate and breast cancers (48, 49). In different types of allergic diseases, histamine may induce or exacerbate allergic reactions, mainly through H1R and H4R. Locally manipulating the balance of H1R and H2R signaling could be an ideal strategy to leverage the ability to modulate cancer treatment. Furthermore, the net effect of H2R signaling on human colon tumors may depend on the microenvironment of the tumors. A recent study demonstrated that cimetidine treatment promoted tumor growth in orthotopically implanted human colon tumors in nude mice, while cimetidine treatment suppressed tumor growth when implanted subcutaneously (1, 58). This finding sheds light on the role of the tumor microenvironment affecting tumor growth. Therefore, treatment with H1R or H2R antagonists may differentially influence primary and metastatic tumors in cancer patients. Mammalian microbiome science is providing opportunities for scientists to interrogate known and new signaling pathways for possible contributions to cancer outcomes. Our studies with microbial histamine extend the literature on a metabolite known for more than a century, but a new “microbiome lens” enables investigators to examine histamine-receptor signaling as a communication link between gut microbes and mammals. As a result of these investigations, we can target specific human genes and signaling pathways using thousands of data points from databases linking cancer gene expression profiles and patient survival data (e.g., R2 and PROGgene). By exploring H1R and H2R signaling pathways using this approach, we detected an association between the greater ratio of H2R/H1R gene expression and longer survival in colorectal cancer. Such findings establish a direction for future studies in patients and may contribute to the development of molecular diagnostics using tissue-based gene expression. New therapeutics could be developed by combining strategies for blocking H1R signaling and promoting H2R signaling, in combination with compounds targeting other signaling pathways. Finally, such combination strategies could be used to prevent colorectal cancer in at-risk patient populations.

GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant P30-DK-56338 (to Texas Medical Center Digestive Disease Center), National Cancer Institute Grant U01-CA-170930 (to J. Versalovic), and unrestricted research support from BioGaia AB (to J. Versalovic).

DISCLOSURES J. Versalovic serves on the scientific advisory boards of Biomica and Seed Health and also receives unrestricted research support from Biogaia, AB.