Fig. 3: ROCK1/2-inhibition reduces aGVHD in mice and causes downregulation of genes related to IL6 JAK STAT3 signaling in CD11b⁺ cells.

a–k Mice underwent allo-HCT to induce aGVHD. Mice in the ROCK1/2-inhibitor group were treated intraperitoneal with 8 mg/kg ROCK1/2-inhibitor (dissolved in 100 µl PBS) from day 4 to day 13, mice in the vehicle group were treated intraperitoneal with an equal volume PBS. a Survival of mice was plotted by using the Kaplan–Meier method and compared by using a log-rank (Mantel Cox) test. Depicted are 10 mice in the vehicle group and 11 mice in the ROCK1/2-inhibitor group. The experiment was performed twice. b–d Histological aGVHD scoring of liver (b), colon (c) and small intestine (d) of mice on day 14 after allo-HCT that received either ROCK1/2-inhibitor or vehicle treatment. Depicted are 10 mice in each group. The plots show mean +/− SD and the P-value was calculated using a two-tailed unpaired student t-test. e The scatter plot shows the percentage of CD11b⁺ cells of all CD45⁺ cells in the lamina propria of small intestine on day 14 after allo-HCT, which equals 10 days of ROCK1/2-inhibitor or vehicle treatment. Depicted are 10 mice each group. The experiment was performed twice. The plot show mean +/− SD and the P-value was calculated using a two-tailed unpaired student t-test. f Principal Component (PC) Analysis based on gene expression, determined by mRNA-microarray of CD45⁺CD11b⁺ cells that were FACS-isolated from the spleen of mice on day 14 after allo-HCT, which equals 10 days of ROCK1/2-Inhibitor or vehicle treatment. Depicted are four mice in the ROCK1/2-inhibitor group (red) and three mice in the vehicle group (blue). g The heatmap shows downregulated genes involved in IL6_JAK_STAT3_signaling. Color scale represents the row-wise z-score intensity. * indicates significant changes in gene expression. h The scatter plot shows the MFI of Il-6 expression in CD11c⁺CD11b⁺ DCs isolated from the spleen on day 14 after allo-HCT. The experiment was performed three times, each data point represents one mouse and values were normalized to the mean of the control group. The plot show mean +/− SD and the P-value was calculated using a two-tailed unpaired student t-test. i Representative histograms of Il-6 expression in CD11c⁺CD11b⁺ DCs isolated from the spleen on day 14 after allo-HCT. j The scatter plot shows the MFI of Tnf expression in CD11c⁺CD11b⁺ DCs isolated from the spleen on day 14 after allo-HCT. The experiment was performed three times and each data point represents one mouse. The plot show mean +/− SD and the P-value was calculated using an two-tailed unpaired student t-test. k Representative histograms of Tnf expression in CD11c⁺CD11b⁺ DCs isolated from the spleen on day 14 after allo-HCT.