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.

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Background The pathogenesis of inflammatory bowel diseases (IBD) is characterized by a dysregulated crosstalk between the host and the microbiome that leads to the development of inflammation and dysbiosis. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, particularly of butyrate-producing species such as Faecalibacterium prausnitzii. The epithelial NADPH oxidase dual oxidase 2 (DUOX2), which prevents bacterial colonization of the mucosa through the production of hydrogen peroxide (H2O2), is the only gene consistently altered in IBD patients before the onset of disease. However, the involvement of DUOX2 in IBD is not well understood. We aimed to define how inflammation and the microbiota regulate DUOX2 activity. Methods C57BI/6J males raised in specific-pathogen free (SPF) and germ-free (GF) conditions underwent a model of dextran sulfate sodium (DSS)-induced colitis for 6 days. We obtained colon specimens for histopathology and isolation of colon epithelial cells (CEC). We stimulated colonoids from wild-type (WT), toll-like receptor 4-KO (Tlr4-KO), and Duox2-KO mice with IFNγ, heat-killed adherent invasive Escherichia coli (AIEC) strain LF82, and heat-killed F. prausnitzii strain A2-165 and determined expression of Duox2, Duoxa2, and production of H2O2. We also treated colonoids with butyrate, a microbial metabolite with anti-inflammatory properties. Extracellular H2O2 production was analyzed by means of the Amplex Red assay, whereas gene expression was determined by qPCR. Results SPF mice undergoing DSS-induced colitis developed overt inflammation that was accompanied by upregulation of Duox2 and Duoxa2, as well as increased production of H2O2 in freshly isolated CECs. DSS-treated GF mice developed a mild inflammation that also caused increased H2O2 production and Duoxa2 upregulation. WT colonoid stimulation with IFNγ and the Crohn’s disease-associated pathobiont AIEC induced Duox2 and Duoxa2 expression, whereas heat-killed F. prausnitzii did not. Similarly, both IFNγ and AIEC promoted epithelial production of H2O2 in WT colonoids but not Duox2-KO colonoids, indicating that epithelial release of H2O2 in response to these stimuli is mediated by DUOX2. Response to AIEC additionally required functional TLR4. Although heat-killed F. prausnitzii did not alter H2O2 production, its metabolite butyrate caused a significant blockade in the release of H2O2 in response to both IFNγ and AIEC. Conclusions Our results show that both inflammation and pathobionts induce the expression and activity of DUOX2, which begets more inflammation. We posit that specific depletion of pathobionts or restitution with butyrate-producing bacteria such as F. prausnitzii may be beneficial in IBD.

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