The Effects Of Acid-sensitive Ion Channel 1A(ASIC1a) On The Polarization Of M2 Macrophage

Objective:Acid-sensitive ion channel（ASIC）is an H⁺-gated and Na⁺selective cation channel,which is the member of the epithelial sodium channel（ENaC）superfamily and could be activated by acidic environment.ASIC1s are expressed in the central nervous system,peripheral nervous system and nonneuronal cells such as dendritic cells（DCs）,macrophages,chondrocytes,osteoclasts,synoviocytes,pulmonary artery smooth muscle cells and so on.ASIC1 involves in multi-physiological and pathological processes,such as pain,learning,fear and ischemic stroke.Macrophages are widely considered important effector cells of the innate immune system.Depending on the phenotypes,macrophages are classified into the classically activated macrophages（M1）and alternatively activated macrophages（M2）.They play a vital role in the primary immune response related to pathogens,inflammation and tissue repair.Our previous study found that macrophage expresses functional ASIC1,M1 macrophages express higher membrane molecules CD80 and CD86 after ASIC1a knockout.It was suggested that ASIC1a may inhibit the antigen presentation of M1macrophages.The role of ASIC1a in the polarization process of M2 macrophages has not been studied.The aim of the present study are to investigate whether ASIC1a plays a role in the polarization of IL-4 induced mouse bone marrow-derived macrophage（BMM）and peritoneal macrophage（PM）into M2 phenotype,and discuss the mechanism of ASIC1a in M2 type polarization process.Material and Methods:BMM and PM were isolated from WT and ASIC1a^-/-mouse,the percentage of F4-80 positive macrophages were analyzed by flow cytometry assay.Then M2 macrophages were induced by IL-4.The M2 macrophage markers,Arg1,FIZZ1,YM1,Mgl1 and Mgl2 mRNA were detected by RT-PCR,and the specific protein Arg1 and ASIC1 were detected by Western blotting（WB）.The expression of M2 surface molecule CD206 and intracellular FIZZ1 were evaluated by flow cytometry.BMM and PM were induced by IL-4 after different time（30min/60min/180min）,the protein expression of STAT6 phosphorylation was detected by WB.All experiment data was reported as mean±standard deviation.GraphPad Prism 6 was used for statistical ananlysis.ANOVA,Student t test were used as indicated.Differences were considered at P﹤0.05.Results:The percentage of F4-80 positive macrophages in BMM/PM were 96.6%and91.1%.I.The BMM derived from WT and ASIC1a^-/-mouse:After induced by IL-4for 24 h,the gene expression of Arg1,FIZZ1,YM1,Mgl1 and Mgl2 in ASIC1a^-/-group were significantly lower than that in the WT group.And the protein expression of Arg1 were also down-regulated.The expression of surface CD206 and intracellular FIZZ1 of BMM in ASIC1a^-/-group were significantly lower than that in the WT group.After induced by IL-4 for different time（0min/30min/60min/180min）,the protein expression of p-STAT6/STAT6 in WT and ASIC1a^-/-group had no significant difference.Ⅱ.The PM derived from WT and ASIC1a^-/-mouse:After induced by IL-4for 24h,the gene expression of Arg1,FIZZ1,YM1,Mgl1 and Mgl2 and the protein expression of Arg1 in ASIC1a^-/-group were significantly lower than that in the WT group.The expression of surface CD206 and intracellular FIZZ1 of PM in ASIC1a^-/-group were significantly lower than that in the WT group.After induced by IL-4 for different time（30min/60min/180min）,the the protein expression of p-STAT6/STAT6in WT and ASIC1a^-/-group had no significant difference.Conclusions:ASIC1a knockout causes a significant down-regulation of BMM and PM polarization to M2 phenotype,and this effect was not caused by STAT6phosphorylation in the IL-4 signaling pathway.