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Background Patients with inflammatory bowel disease (IBD) have intestinal inflammation and are treated with immune-modulating medications. In the face of the coronavirus disease-19 pandemic, we do not know whether patients with IBD will be more susceptible to infection or disease. We hypothesized that the viral entry molecules angiotensin I converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are expressed in the intestine. We further hypothesized that their expression could be affected by inflammation or medication usage. Methods We examined the expression of Ace2 and Tmprss2 by quantitative polymerase chain reacion in animal models of IBD. Publicly available data from organoids and mucosal biopsies from patients with IBD were examined for expression of ACE2 and TMPRSS2. We conducted RNA sequencing for CD11b-enriched cells and peripheral and lamina propria T-cells from well-annotated patient samples. Results ACE2 and TMPRSS2 were abundantly expressed in the ileum and colon and had high expression in intestinal epithelial cells. In animal models, inflammation led to downregulation of epithelial Ace2. Expression of ACE2 and TMPRSS2 was not increased in samples from patients with compared with those of control patients. In CD11b-enriched cells but not T-cells, the level of expression of ACE2 and TMPRSS2 in the mucosa was comparable to other functional mucosal genes and was not affected by inflammation. Anti-tumor necrosis factor drugs, vedolizumab, ustekinumab, and steroids were linked to significantly lower expression of ACE2 in CD11b-enriched cells. Conclusions The viral entry molecules ACE2 and TMPRSS2 are expressed in the ileum and colon. Patients with IBD do not have higher expression during inflammation; medical therapy is associated with lower levels of ACE2. These data provide reassurance for patients with IBD.

BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitisassociated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H 2 O 2 ) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H 2 O 2 . Epithelial H 2 O 2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H 2 O 2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H 2 O 2 . The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The interrelationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2, a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2, chronic inflammation caused IECs to release higher levels of H2O2. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively, these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa.

This pilot study shows the feasibility of using lamina propria phagocytes to characterize the microbiota in IBD patients. The approach used in this study can narrow the spectrum of potentially dysbiotic bacterial populations and clinically relevant gene expression signatures in IBD patients.