Figure 7.

IL-20 interferes with IFN/STAT2-dependent necroptotic cell-death pathway in IECs from patients with IBD. (A) Levels of STAT2 expression in Crohn’s disease, UC and non-IBD controls as detected by RNA-Seq. (B) Confocal imaging of pSTAT2 in biopsies from Crohn‘s disease, UC and non-IBD controls (n=5/group) with positive IECs indicated by arrows. (C) Three-dimensional organoids generated from colon biopsies of patients with IBD were either stimulated for 30 min with 25 ng/mL IFN-β or were left untreated and protein extracts were subjected to western blot analysis with anti-pSTAT2 and STAT2 antibodies. Results from two patients are shown. Beta-actin served as loading control. (D) Results of MLKL and pMLKL levels after the incubation of IBD organoids with IFN-β for 24 hours. (E) IBD organoids (n=3 different patients) were incubated with IFN-β in the presence or absence of necrosulfonamide for 36 hours and cell death was assessed by staining with propidium iodide to mark dead cells. Hoechst was used for normalisation purposes. (F) Western blot analysis of pSTAT2 and STAT2 in IBD organoids after stimulations with IL-20 and IFN-β. Scale bars, 75 µm in B. Statistics: Welch’s t-test in A, mean with 95% CI is displayed. (G) Schematic representation of possible interactions between IL-20 and type I IFN signals in homeostasis, during the active phase and the resolution phases of intestinal inflammation. IEC, intestinal epithelial cell; IFN, interferon; IL, interleukin; RNA-Seq, RNA-sequencing. *P<0.05; ****p<0.0001.