PAR2 promotes M1 macrophage polarization and inflammation via FOXO1 pathway

Abstract Macrophages polarization plays essential but different roles in most diseases such as atherosclerosis, adipose tissue inflammation, and insulin resistance. Our previous study revealed that protease‐activated receptor 2 (PAR2), a G‐protein coupled receptor influenced macrophage function, but little is known regarding the regulation of macrophage polarization process and its potential mechanisms. In the present study, bone marrow–derived macrophages (BMDM) isolated from C57/BL6 mice and cultured with L929‐conditional medium and murine macrophage cell line RAW264.7 were used to study the function of PAR2 activation in vitro. BMDM was stimulated by the small molecular PAR2 agonist, 2‐furoyl‐LIGRLO‐amide trifluoroacetate salt, followed by transcription factor microarray to screen the significantly activated signaling pathways under PAR2 activation. Western blot analysis, quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to evaluate the expression of targeted genes and transcription factors. Immunofluorescence was used to observe the subcellular distribution of transcription factors. Our results demonstrated that M1‐like polarization was presented by PAR2 agonist treatment with significant upregulation of interleukin‐1β, interleukin‐6, monocyte chemotactic protein‐1, and tumor necrosis factor‐α in BMDM and RAW264.7. Microarray identified forkhead box protein O1 (FOXO1) was significantly increased under PAR2 agonist stimulation, which was confirmed by qPCR and Western blot analysis. Immunofluorescence demonstrated that increased FOXO1 accumulated in the nucleus, which is necessary to promote transcription for targeted genes. We further knocked down FOXO1 expression using small interfering RNA, which alleviated PAR2‐induced proinflammatory gene expression. The PAR2/FOXO1 pathway mediated stimulation of proinflammatory genes was further confirmed by tryptase, an endogenous ligand of PAR2. In conclusion, this study demonstrated that PAR2 activation–induced M1 polarization and inflammation through the FOXO1‐dependent pathway.