Supplementary Figure 5: Pathogenic mechanisms of TRM cells in acute DSS-induced colitis.
(a) Immunohistochemistry staining for Foxp3 in colon tissue from WT, Hobit KO or DKO mice with acute DSS colitis. Left: Representative images, white arrows highlight positive cells. Right: Quantification of positive cells per high power field (HPF). n = 26 from three independent experiments; Bars: 50 µm (upper row), 25 µm (lower row). (b) Immunhohistochemistry staining for MPO in colon tissue from WT or DKO mice treated with DSS for three days. Left: Representative images, white arrows highlight positive cells. Right: Quantification of positive cells per HPF. n = 10 from two independent experiments. (c-f) Expression of pro-inflammatory cytokines in mRNA from LPMCs from the colon of WT, Hobit KO and DKO mice with acute DSS colitis as determined by qPCR (c, e) and in supernatants from anti-CD3/CD28-stimulated LPMCs as determined by multiplex assay (d, f). n = 17 (c, Il12a, e, Il13), n = 16 (e, Ifng, Il17a), n = 15 (c, Tnf) from two independent experiments, n = 8 from one representative out of two independent experiments (c, Il1b), n = 22 from three independent experiments (d, f). (g) mRNA expression of Gzmb in LPMCs from the colon of WT, Hobit KO and DKO mice with acute DSS colitis as determined by qPCR (left panel, n = 14 from two independent experiments). Granzyme B concentration in the supernatants of anti-CD3/CD28-stimulated LPMCs from such mice as determined by ELISA (right panel, n = 15 from two independent experiments). (h) Immunohistochemistry staining for EpCAM (red) and TUNEL (green) in the colon of WT, Hobit KO and DKO mice with acute DSS colitis Left: Representative images, white arrows highlight EpCAM+TUNEL+ apoptotic epithelial cells. Right: Quantification of EpCAM+TUNEL+ cells per high power field (HPF). n = 19 from two independent experiments. Bars: 50 µm (upper row), 25 µm (lower row). (a, b, h) Center values – mean; error bars – s.e.m.; (c-g) Boxplots – median with upper and lower quartile, whiskers – minimum and maximum; floating bars - median with minimum and maximum. (a-h) * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed student’s t-test (b, d IL-12, e Il17a, f IL-13), one-way ANOVA with Newman-Keuls post hoc test (all other panels).
