Discordant effects of ex-vivo JAK inhibition on inflammatory responses in colonic compared to ileal mucosa

Background & aims: Janus kinase (JAK) inhibitors modulating JAK-STAT (signal transducers and activators of transcription) signaling pathway, are used for the treatment of patients with inflammatory bowel diseases (IBD). We aimed to identify the molecular effects of JAK inhibition in the human intestinal mucosa, considering the IBD location and phenotype. Methods: Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), or non-IBD controls (NC) were treated ex-vivo with the JAK inhibitor, tofacitinib. Phosphorylated STAT (p-STAT) levels were assessed by Western blot and Immunofluorescence. Inflammatory genes expression was assessed with Nanostring nCounter system. Human intestinal organoids were used to assess JAK inhibitors' effects on p-STATs and iNOS expression. Results: Explants were collected from 68 patients (NC=28; UC=20; CD=20). JAK inhibition reduced p-STAT1/3/5 expression in all explants. While p-STAT inhibition rates varied among patients (10%-88%), higher inhibition rates were observed in colonic compared to ileal explants. Significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, while only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including STAT1 and NOS2. Various JAK inhibitors reduced IFN-γ-induced increase in p-STAT1 and iNOS expression in organoids. Conclusions: A site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying drug impact and evaluating personalized treatment effects. Keywords: Tofacitinib; JAK inhibitors; Human intestinal mucosa; Organoids ### Competing Interest Statement KK, HAT, JA, ESB, NW, LHM, AF, ALB, ZL, HBE, IAB, SCK and KMR declare no conflict of interests. IG reports: research support from Gilead, Boehringer Ingelheim, Pfizer, and AbbVie. HY reports: institutional research grants from Pfizer and the ISF; consulting fees from AbbVie, Janssen, Pfizer, Takeda, Bristol Myers Squibb, and Elly Lilli; honoraria for lectures from AbbVie, Janssen, Pfizer, and Takeda; participation in a Data Safety Monitoring Board or Advisory Board for AbbVie, Pfizer, Takeda, Bristol Myers Squibb, and Elly Lilli. JEO reports: speaker fees from Takeda, Pfizer, Jansen, AbbVie, and Novartis; grant support from Pfizer; participation in Advisory Board of Takeda. ID has served as a speaker, consultant, and advisory board member AbbVie, Altman Research, Athos, Celltrion, Celgene/BMS, Ferring, Eli-Lilly, Gilead, Galapagos, Gutreat, Harp Diagnostics, Iterative Scopes, Janssen, Pfizer, Roche/Genentech, Sangamo, Sublimity, Sandoz, Takeda, Prometheus