Figure 1.

MSP-RON signaling activates an immunomodulatory gene expression signature in macrophages. A. RNA sequencing was performed on F4/80 positive-sorted peritoneal macrophages treated with MSP for 7 hours (n = 3). Euclidian distance matrix shows distinct clustering of MSP-treated wild-type (WT) macrophages. MSP-treated (+) and untreated (-) RON TK-/- macrophages exhibited similar gene expression profiles. In this plot, darker color indicates higher similarity between samples. Color key and overlaid histogram indicates the frequency of observations at a given Euclidian distance value. B. Numbers of differentially-expressed genes as determined by different fold-change thresholds. Gene sets were obtained by comparing MSP-treated WT macrophages with MSP-treated RON TK-/- macrophages (FDR q-value <0.01). C. Changes in mRNA expression levels of selected immune costimulatory and coinhibitory ligands. Linear fold change was calculated in MSP-treated WT macrophages compared to MSP-treated RON TK-/- macrophages. p-values were obtained with Benjamini-Hochberg multiple testing correction. ND: not detected. D. Representative flow cytometry histograms of WT and RON TK-/- peritoneal macrophages after 24 hours of treatment with (+) MSP or with vehicle control, (-)MSP. Histograms were pre-gated on CD11b(hi) F4/80(hi) RON(+) resident macrophages (see Fig S5 for representative flow cytometry data). Dashed lines indicate the fluorescence values at the population peak in MSP-stimulated WT samples. E. Quantification of mean fluorescence intensity (MFI) of indicated markers in WT or RON TK-/- macrophages (n = 5 for both) with and without MSP treatment by flow cytometry. F. Flow cytometric analysis of expression levels of PD-L1 and CD80 in MSP-treated macrophages with or without RON-selective kinase inhibitors BMS777607 and merestinib (LY2801653). Two-tailed t-test was used to compare staining intensity. (ns) p > 0.05, (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001, (****) p ≤ 0.0001.