Fig. 3. RT-MPs reprogram macrophages through Jak-STAT and MAPK pathways.
(A) Quantification of the accumulation of PKH26-labeled RT-MPs in various cell types in MPE mice (means ± SEM, n = 5 mice). (B) Representative images of macrophages (green) that were recruited and phagocytosed red RT-MPs over time in the CX3CR1+/GFP window chamber. RT-MPs were stained with red fluorescence dye PKH26 and subcutaneously injected into the window. Scale bar, 50 μm. (C) Flow cytometric analysis of RT-MPs internalization by macrophages at multiple time points (n = 3). (D) Heat map of RNA-seq on RT-MP–treated and untreated BMDM-M2 cells. (E) RT-qPCR analysis of the expression levels of M1- and M2-associated mRNAs in RT-MP–treated BMDM-M2 cells. The results indicate up-regulation of M1-associated mRNAs and down-regulation of M2-associated mRNAs. (F) The top 20 up-regulated functional pathways in RT-MP–treated and untreated groups, as determined by KEGG analysis. (G) Protein expression levels of p-STAT1, p-p38, p-ERK, p-JNK, and β-actin in BMDM-M2 cells treated with RT-MPs at the indicated time points were analyzed by Western blotting. (H) Flow cytometric analysis of CD86, major histocompatibility complex II (MHC II), and CD206 expression in BMDM-M2 cells treated with RT-MPs and GSH-incubated RT-MPs. MFI, mean fluorescence intensity. (I) Flow cytometry gating strategy for measurement of macrophages from MPE mice. (J) Quantification of CD206 expression in MPE mice after treatment with RT-MPs. (K) RT-MP–treated BMDM-M2 cells showed increased phagocytosis of LLC cells compared to that of control BMDM-M2 cells. Flow cytometric analysis was performed to evaluate the phagocytosis rate. FITC, fluorescein isothiocyanate; PE, phycoerythrin. (L) In the CX3CR1+/GFP mouse window chamber, RT-MP–treated macrophages were recruited and phagocytosed a greater number of LLC-RFP cells. Representative images were shown here. Scale bars, 50 μm. **P < 0.01 and ***P < 0.001.