Figure 3.

MMP13 is produced by intestinal fibroblasts upon IL36R activation. (A) RNA was isolated from colons of wildtype animals that were treated with an anti-IL36R antibody or an isotype control antibody (each 250µg, twice a week) during chronic DSS-induced colitis. Mmp13 expression was detected by qPCR (n= 7-8 per group). (B) Wildtype animals were injected with 2µg IL36R ligand mix and the expression of Mmp13 was analyzed by qPCR in colon lysates at the next day compared to the PBS injected controls. (n= 6 per group) (C) Representative pictures from co-stainings of MMP13 with vimentin (VIM), Pdpn or αSMA of CD patients with stenosis from the IF cohort are shown. Arrows indicate double positive cells. (D) Colon fibroblasts were enriched from wildtype animals and the cells were used for stimulation with IL36R ligands (100ng/ml) or PBS as control over 9 days. Fresh stimulants were given every third day. Quadruplicates were used for bulk RNA sequencing. The adjusted p value of the 20 highest regulated genes (threshold p<0.05, log2fc>2, mean count>50) is depicted. (E) Murine colon fibroblasts were used for chronic IL36R stimulation (100ng/ml) over 7 days. New cytokines were added every 2-3 day. Supernatants were used for detection of MMP13 protein by ELISA (n= 6). (F) Colon fibroblasts were enriched from untreated wildtype and Myd88-/- mice. The cells were stimulated for 4h with IL36R ligands (100ng/ml) or PBS (n= 6-7 per group) and gene expression was detected by qPCR. Quantitative data were analyzed by unpaired t test (*p>0.05, **p>0.01, one-tailed) and mean values are shown with standard deviation in (A, B, E, F) Scale bar represents 50µm.