BACKGROUND Indole-3-carbinol (I3C) and other aryl hydrocarbon receptor agonists are known to modulate the immune system and ameliorate various inflammatory and autoimmune diseases in animal models, including colitis induced by dextran sulfate sodium (DSS). MicroRNAs (miRNAs) are also gaining traction as potential therapeutic agents or diagnostic elements. Enterohepatic Helicobacter (EHH) species are associated with an increased risk of inflammatory bowel disease, but little is known about how these species affect the immune system or response to treatment. AIM To determine whether infection with an EHH species alters the response to I3C and how the immune and miRNA responses of an EHH species compare with responses to DSS and inflammatory bowel disease. METHODS We infected C57BL/6 mice with Helicobacter muridarum ( H. muridarum ), with and without DSS and I3C treatment. Pathological responses were evaluated by histological examination, symptom scores, and cytokine responses. MiRNAs analysis was performed on mesenteric lymph nodes to further evaluate the regional immune response. RESULTS H. muridarum infection alone caused colonic inflammation and upregulated proinflammatory, macrophage-associated cytokines in the colon similar to changes seen in DSS-treated mice. Further upregulation occurred upon treatment with DSS. H. muridarum infection caused broad changes in mesenteric lymph node miRNA expression, but colitis-associated miRNAs were regulated similarly in H. muridarum- infected and uninfected, DSS-treated mice. In spite of causing colitis exacerbation, H. muridarum infection did not prevent disease amelioration by I3C. I3C normalized both macrophage- and T cell-associated cytokines. CONCLUSION Thus, I3C may be useful for inflammatory bowel disease patients regardless of EHH infection. The miRNA changes associated with I3C treatment are likely the result of, rather than the cause of immune response changes.

Enterohepatic Helicobacter species are epidemiologically linked to increased inflammatory bowel disease; however, little research has been done to elucidate potential contributions of individual species. We hypothesized that Helicobacter muridarum (Hm) would alter the course of DSS-induced colitis and the response to indole-3-carbinol (I3C), an anti-inflammatory phytochemical. We treated Hm-infected C57BL/6 mice with1% DSS +/− 40 mg/kg I3C and measured inflammatory biomarkers. We found that H. m exacerbated DSS-induced colitis and increased the percentage of Th17 cells in the mesenteric lymph nodes and spleen and increased IL-17 in colonic tissue compared to the DSS group. Also, we found that Hm bacteria itself produced inflammation and pathology. I3C, on the other hand, ameliorated colitis and shifted the Treg/Th17 balance in DSS+H.m-infected mice. We found that I3C treatment of DSS + H.m-infected mice decreased the expression of pro-inflammatory IL17 and RORC as well as increased anti-inflammatory Foxp3 when compared to the untreated group. The decreased expression of RORC correlated with increased miR-let7a-2 and miR-29a-3p expression and increased FoxP3 correlated with decreased miR-874 expression following I3C treatment. Moreover, I3C reduced the abundance of certain taxa, such as Clostridiales, Actinobacteria, and Erysipelotrichales, and increased the abundance of Ruminococcus. In summary, H. muridarum causes baseline inflammation and exacerbates colitis via microRNA-mediated increases in Th17 cells, while I3C ameliorates colitis via increased Treg populations.

Enterohepatic Helicobacter (EHH) species are bacteria that colonize the colons and biliary ducts of humans and other mammals. Virtually nothing is known about the mechanisms of EHH-induced colitis in wild-type mice or humans. Indole-3-carbinol (I3C), a chemical extracted from cruciferous vegetables, ameliorates Dextran sulfate sodium (DSS)-induced colitis. We hypothesized that specific EHH species would alter the course of DSS-induced colitis and possibly the responses to I3C treatment. We infected C57BL/6 mice with human- and rodent-associated EHH species and measured the effects on DSS-induced colitis and response to I3C treatment. We found that H. muridarum(H.m) and H. cinaedi exacerbate DSS-induced colitis and delay recovery. Furthermore, I3C ameliorated colitis and shifted the Treg/Th17 balance in H.m-infected mice. Moreover, the microRNAs expression pattern was altered in H.m-infected mice when compared to that of uninfected mice. Interestingly, enhanced colitis, as well as increased Th17 cells in spleens and mesenteric lymph nodes, were observed in H.m-infected DSS-treated mice(DHM). I3C treatment of DSS + H.m-infected mice (DMI) decreased the expression of pro-inflammatory IL17 and RORC as well as increased anti-inflammatory Foxp3 when compared to DHM group. These immunological changes correlated with the microRNAs expression. This demonstrates that the presence of EHH species alters susceptibility to DSS-induced colitis. Importantly, mice with exacerbated colitis respond to I3C. In the future, we plan to study the mechanisms underlying the effects of EHH on colon cancer development and the efficacy of I3C in both the colitis and colon cancer models.

Enterohepatic Helicobacter (EHH) species are gram-negative bacteria that colonize the colons and sometimes the biliary tracts of humans, primates, rodents, and other mammals. Though ~20% of the human population may be infected, EHH species are poorly studied in humans. H. hepaticus is known for causing colitis in mice with severe immunologic abnormalities; however, virtually nothing is known about the influence of EHH species on colitis in wild-type mice, which are more relevant to humans. Dextran sulfate sodium (DSS) treatment and DSS plus azoxymethane (AOM ) are widely used as mouse models of ulcerative colitis and colitis-associated colon cancer. We hypothesized that specific EHH species would alter the course of DSS-induced colitis and colitis-associated colon tumorigenesis in mice. We infected C57BL/6 mice with human- and rodent-associated EHH species +/− DSS, or +/− DSS/AOM and measured the effects on pathology and inflammation markers. We found that H. cinaedi, H. pullorum, H. fennelliae exacerbate DSS-induced colitis and delay recovery, whereas H. hepaticus reduced the effect of DSS-induced colitis severity. H. hepaticus also prevented tumor development in the AOM/DSS colon cancer model. We measured the proinflammatory and the anti-inflammatory cytokines in serum and colon homogenates and found that cytokine induction varied among the EHH species. Therefore, EHH species alter susceptibility to DSS-induced colitis and DSS-AOM colon cancer. These results suggest that EHH species could increase or decrease the risk of inflammatory bowel disease or colorectal cancer in humans, making it important to identify the infecting species.