Abstract
Interferons (IFN) are key immunoregulatory cytokines in Inflammatory Bowel Disease (IBD) pathogenesis. Type I IFNs (IFN-I) prime the host epithelium to respond to the gut microbiota and-derived metabolites as part of innate-immune signalling to preserve mucosal homeosta sis. Anti-TNF antibodies treated IBD patients exhibit increased IFN-I-induced genes. Whether an enhancement of the IFN-I pathway leads to altered innate epithelial repair pathways in IBD is unknown. Human IBD single-cell RNA-Sequencing datasets (N=5) were analyzed for IFN-I targets of interest via Ingenuity Pathway Analysis and Hegemon. Sensitivity of colonic organoids derived from Crohn’s Disease (CD) responders (R) or non-responders (NR) to butyrate (0.1mM≈depleted patient butyrate levels; N=6) and inflammation (50ng/ml TNF+IL1β+IL6), was determined by measuring proliferation, permeability via FITC-dextran translocation and IFN-I levels using an Interferon PCR-Profiler. Inflamed epithelia of IBD patients exhibit an enrichment of IFN-I genes associated with innate-immune signalling pathways, microbial recognition, wound healing, and antiviral pathways. Inflamed NR-CD organoids exhibit a 3.1-fold increase in permeability (p<0.0001) and a 55%-loss in prolifera tion (p=0.015) in response to 0.1mM butyrate compared to inflamed R-CD and control colonoids. Comparatively, in controls, butyrate induces a 45.5%-increase in proliferation (p=0.0018) and a 2.3-fold decrease in permeability (p=0.0003), suggesting that epithelial repair and barrier integrity is impaired in NR-CD patient epithelia in a butyrate-depleted inflamed environment. Consistently, transcript levels of specific IFN-I targets*- Il7r, Ifih1, Ifi6, Ifi44, Sp110, Irf1, Cxcl10, Ifit3 are significantly decreased (p<0.02-0.045) in inflamed NR-CD organoids compared to R-CD organoids treated with butyrate, suggesting that IFN Is may be associated with epithelial repair in inflammatory, butyrate-depleted epithelia of anti-TNF treated R-CD patients and its lack thereof in NR-CD patients. In the native intestinal mucosa, however, CD patients non-responsive to Infliximab (anti-TNF), shows highly ele vated levels of the IFN-I targets* whereas responsive patients show restoration of IFN-Is to near control levels. Hence, suggesting that an immune trigger drives the increased expression of IFN-I targets* in CD patients non-responsive to anti-TNFs leading to a persistent hyper activation of innate signalling, microbial recognition, and pathways that initiate repair. In conclusion, we show for the first time that a unique panel of IFN-I targets* robustly predicts clinical non-response to anti-TNFs in NR-CD patients. Through its unique role in driving epithelial repair and barrier integrity in anti-TNF treated R-CD epithelium, this study demon strates for the first time a novel andnon-canonical rolefor IFN-Isin intestinal mucosal healing.