Aire Regulates The Polarization Of M1 Macrophages Through MiR-155

Mutations in the Aire gene will lead to the Autoimmune Polyoendocrine diseases-Candidiasis-Ectodermal dystrophy（APECED）.APECED is mainly characterized by chronic mucocutaneous candidiasis,hypoparathyroidism and Addison’s disease.Studies confirm that Aire is a transcriptional regulator widely expressed in cells during the first stage of embryogenesis.Later,its expression was limited to a few cells involved in T cell tolerance,such as medullary thymic epithelial cells（mTEC）,B cells and bone marrow-derived cells（such as dendritic cells（DCs）and macrophages）,which play an important role in the elimination of negative selection of autoreactive T cells mainly by regulating the expression of a large number of tissue restricted antigens（TRAs）on these cells.So the Aire gene mutation can lead to multiple organs with autoimmune diseases.However,the cause of the Candida infection in patients with Aire mutations remains unclear.The diversity of the microenvironment can induce macrophages with different phenotypes and functions.IFN-γcombined with LPS can induce macrophage polarization to type 1 macrophage（M1）.M1 macrophages are mainly involved in inflammation and clearance of pathogens（such as Candida）;IL-4 induces its polarization to type 2 macrophage（M2）,and M2 macrophages mainly mediate tissue repair and angiogenesis.In addition,studies have shown that miR-155 is thought to be a closely related molecule to the inflammatory response and play a key role in inducing macrophage polarization to theM1.And studies have found that the expression of microRNAs（including miR-155）in thymic epithelial cells is regulated by the Aire gene.Therefore,based on the above background,we suppose whether the expression of Aire in the periphery could promote macrophage polarization to M1 by regulating the expression of miR-155 in macrophages,and thus providing possible explanations to the susceptibility of Candida in the patients with Aire mutation.Based on the above,we conducted the following studies:Ⅰ.The effect of Aire on polarization of M1 macrophageOur previous results showed that Aire can promote the polarization of mouse mononuclear macrophage cell line（RAW264.7）to M1 macrophages,but whether it also affects normal mouse peritoneal macrophages is unclear,so we first observed the effect of Aire on the mouse peritoneal macrophage polarization.1 The effect of Aire on M1 and M2 macrophages related molecules expressionAfter stimulated by IFN-γ+LPS or IL-4 for 24 hours,the expressions of M1-related molecules（IL-1α,IL-6 and iNOS）and M2-related molecules（Arg-1）mRNA expression levels were observed.The results showed that mRNA expression of M1-related molecules（IL-1α,IL-6 and iNOS）in unstimulated and stimulated groups in Aire^-/-mice peritoneal macrophages was significantly lower than that in WT mice.The mRNA expression of Arg-1 in Aire^-/-mice peritoneal macrophages after stimulation was higher than that in WT mice,but the expression of Arg-1 in untreated WT and Aire^-/-groups was not significantly different.The expression of M1-related molecules（IL-1α,IL-6 and iNOS）of macrophages in the stimulation of Aire group was significantly higher than that in the GFP group.The results in Aire group at unstimulation status showed the similar trends as the stimulation when it compare with GFP group,but there is not statistically significant.While M2 related molecules（Arg-1）mRNA expression levels were not lower than the GFP group.These resultsindicate that Aire can promote the polarization of M1 and inhibit the polarization of M2.2 The effect of Aire on the surface molecules expression of M1 and M2To further determine the effect of Aire on the macrophage polarization phenotype,the flow cytometry was used to detect M1 surface molecules（MHC-II and CD16/32）and M2 surface molecules（Dectin-1 and CD206）in the peritoneal macrophage of WT and Aire^-/-mice,transfected with GFP and with Aire lentivirus.The results showed that the expression levels of M1 surface molecules（MHC-II and CD16/32）in peritoneal macrophages of Aire^-/-mice were lower than those of WT mice,whereas the the expression of M2 surface molecules（Dectin-1 and CD206）were higher than those of WT mice.The expression levels of M1 surface molecules（MHC-II and CD16/32）in peritoneal macrophages of transfected Aire group were higher than those of GFP group,while M2 surface molecules（Dectin-1 and CD206）expression levels were lower than the GFP group.It is further shown that Aire can promote the polarization of M1 and inhibit the polarization of M2.II.Aire influences the polarization of M1 macrophages through miR-155The above studies have confirmed that Aire can affect the polarization of mouse peritoneal macrophages to M1,but the mechanism is not yet clear.Studies have shown that miR-155 plays an important role in the polarization of M1,and the expression of miR-155 in thymic epithelial cells is regulated by Aire.Therefore,we hypothesized whether Aire could also affect the polarization of M1 macrophages by regulating miR-155.For this reason,we performed the following experiments.1 Aire regulates the expression of miR-155 in macrophagesFirst,in order to determine whether Aire can regulate the expression of miR-155in macrophages,we detected the expression levels of miR-155 in Aire^-/-mice and Aire-overexpressed peritoneal macrophages by RT-qPCR.The results showed that miR-155 expression levels in peritoneal macrophages from Aire^-/-mice were significantly lower than those from WT mice.The expression levels of miR-155 in peritoneal macrophages transfected with Aire were significantly higher than those transfection with GFP.These results suggest that Aire could up-regulate the expression of miR-155 in mouse peritoneal macrophages.2 Aire influences macrophage polarization to M1 through miR-155To further investigate whether Aire influences macrophage polarization to M1through modulating miR-155,we interfered the expression or function of miR-155 on the basis of the above two cell models,thereby establishing Aire^-/-mice miR-155overexpression（miR-155 mimics）and Aire overexpressed miR-155 inhibition（miR-155 inhibitor）macrophage model,and the expression levels of IL-1α,IL-6,and iNOS in these cells were detected by RT-qPCR.The Griess method was used to detect the contents of NO in the supernatant of the above cells.FACS was used to detect the expression of M1 surface molecules（MHC-II and CD16/32）in the above cells.The results showed that the expression levels of M1 related molecules（IL-1α,IL-6 and iNOS）in Aire^-/-mice miR-155 mimics stimulation group were significantly higher than Aire^-/-mice miR-155 mimics NC stimulation group and lower than WT miR-155mimics stimulation group,and M1 related molecules（IL-1α,IL-6 and iNOS）expression levels in the Aire^-/-mice were lower than those in the WT group.The expression levels of M1 related molecules（IL-1α,IL-6,and iNOS）in Aire overexpressing miR-155 inhibitor group were significantly lower than Aire overexpressed miR-155 inhibitor NC stimulation group and significantly higher than GFP miR-155 inhibitor stimulation group.The expression of M1 related molecules（IL-1α,IL-6 and iNOS）in the Aire group was higher than that in the GFP group,but the expression of IL-1αwas not significantly different between the two groups.ELISA detection of IL-1αand IL-6 protein content consistent with its mRNA results.The result of NO content was consistent with the change of iNOS.FACS results showed that the expression of MHC-II and CD16/32 in Aire^-/-mice miR-155 mimics stimulation group was higher than Aire^-/-mice miR-155 mimics NC stimulation group and lower than WT miR-155 mimics stimulation group.The expression of MHC-II and CD16/32 in Aire over-expressed miR-155 inhibitor group was lower than Aire over-expressed miR-155 inhibitor NC stimulation group and higher than GFP miR-155 inhibitor-stimulated group,and the expression of CD16/32in Aire inhibitor group was overall higher.There was no significant difference in the expression of GFP inhibitor.The above results indicate that Aire may promote the polarization of M1 macrophages by regulating the expression of mi R-155.3 The effect of Aire on the expression of SOCS1 in macrophagesTo further explore the molecular mechanism of Aire’s effect on the polarization of M1 macrophages through miR-155,we detected the SOCS1 level in the Aire^-/-mice and Aire overexpression peritoneal macrophages by RT-qPCR and immunofluorescence at mRNA and protein levels,respectively.mi R-155 regulates the expression of SOCS1,a downstream target molecule in the process of M1 polarization.The results showed that the mRNA expression of SOCS1 in Aire^-/-mice was higher than that in the WT mice.The expression of SOCS1 mRNA in Aire group was lower than that in the GFP group.The results of immunofluorescence observation were consistent with the results of RT-qPCR,and the expression of SOCS1 in the over-expression Aire unstimulated group was significantly decreased.The above results indicate that Aire affects the polarization of M1 macrophages through miR-155 expression,and one of the molecular mechanisms may be through the influence of SOCS1.