Regulatory Effect And Mechanism Of CGAS On Macrophage Polarization

ObjectivesTo elucidate the regulatory effect and molecular mechanism of cGAS on macrophage polarization and its role in acute lung injury by using WT?cGAS-/-and TLR4-/-mice,so as to provide new ideas for the treatment of clinical related diseases.MethodsInterferon-?(IFN-?)in combination with lipopolysaccharide(LPS)to induce the polarization of M1 macrophages;Interleukin-4(IL-4)induces the polarization of M2 macrophages.Western blot and quantitative real-time PCR(qPCR)were used to detect the expression of M1 and M2 macrophage markers after macrophage polarization in WT and cGAS-/-mice macrophages.Macrophages of WT and cGAS-/-were stimulated with 100 ng/ml LPS respectively.MAPK and NF-?B were detected by western blot,the expression of inflammatory factor mRNA was detected by qPCR,and the level of IL-6 was detected by enzyme linked immunosorbent assay(ELISA).Mitochondrial DNA(mtDNA)was extracted from the cytoplasm of macrophages stimulated by LPS.The content of mtDNA in the cytoplasm was detected by qPCR.At the same time,mtDNA was transfected into mouse macrophages,western blot was used to detect the protein level of cGAS,and qPCR was used to test the relative mRNA level of inflammatory cytokines.Macrophages from WT and TLR4-/-mice were induced to polarize into M1 and M2 macrophages in vitro.The content of mtDNA in the cytoplasm,relative mRNA level of cGAS?M1 and M2 macrophage markers were detected by qPCR.mtDNA was transfected into macrophages from WT and TLR4-/-mice,and the content of cGAMP was detected by ELIS A,The macrophages from WT and cGAS-/-mice were treated with 5?M rapamycin,an inhibitor of mTORC1 pathway,western blot was used to detect the activation of mTORC1 pathway,and qPCR was used to detect the mRNA changes of M1 and M2 macrophages markers.WT and cGAS-/-mice were intraperitoneally injected with 50 mg/kg LPS to establish the acute lung injury model.The wet/dry ratio(W/D)of mice lung tissue was calculated.Hematoxylin and eosin staining(he)was used to detect the pathological damage of organs before and after administration.Immunohistochemical staining(IHC)and western blot was used to detect the expression of cGAS in lung tissue.The number of F4/80+macrophages and Ly6G+neutrophils in peripheral blood and spleen and the proportion of CD16/32+M1 macrophages and CD206+M2 macrophages in BALF were detected by flow cytometry.The proportion of CD16/32+M1 macrophages and CD206+M2 macrophages in lung tissue were detected by tissue immunofluorescence.The mRNA level of inflammatory factors and chemokines in lung was detected by qPCR.Results1.cGAS can promote the polarization of M1-like macrophages and mediate inflammatory response.In M1-like macrophages,the expression of cGAS was significantly up-regulated..Compared with WT mouse macrophages,cGAS-/-mouse macrophages expressed less iNOS,TNF?,IL-6,IL-12 and other M1 macrophage markers,and the synthesis of NO decreased;In M2-like macrophages,cGAS was significantly down regulated.Compared with WT mouse macrophages,cGAS-/-mouse macrophages highly expressed the markers of M2 macrophages such as Arg-1,YM-1,Fizz1 and IL-10.Compared with WT mouse macrophages,the activation of MAPK and NF-?B pathway in cGAS-/-mouse macrophages inhibited significantly after LPS stimulation in vitro,and the content of IL-6 in the medium was decreased.2.LPS can induce mtDNA releasing and activate cGAS through TLR4 receptor.Compared with the control group,the content of mtDNA released into the cytoplasm of macrophages was significantly increased after LPS stimulation.Transfection of mtDNA into WT mouse macrophages could significantly up regulate the expression of cGAS and promote the polarization of M1 macrophages.After TLR4 gene knockout,compared with WT mouse macrophages,the content of mtDNA and the production of cGAMP in the cytoplasm of TLR4-/-mouse macrophages were significantly decreased under LPS stimulation.Compared with WT mouse macrophages,the mRNA levels of cGAS and M1 macrophage markers such as iNOS,TNF-? and IL-12 were significantly decreased in TLR4-/-mouse macrophages induced by M1 polarization,while the mRNA level of M2 macrophage markers were almost unchanged.3.mtDNA can activate cGAS and promote the activation of downstream mTORC1 pathway.Compared with WT mouse macrophages,the activation of mTORC1 pathway in cGAS-/-mouse macrophages was inhibited.5?M rapamycin can significantly reverse cGAS mediated polarization of M1 macrophages.4.The level of systemic inflammation in cGAS-/-mice was significantly reduced.Compared with WT mice,the number of neutrophils and macrophages in peripheral blood and spleen of cGAS-/-mice was significantly decreased.In the model of acute lung injury,the expression of cGAS was significantly up-regulated.After cGAS knockout,the lung injury and pulmonary edema were significantly improved.At the same time,compared with WT mice,the total number of F4/80+macrophages and the proportion of CD16/32+macrophages in BALF of cGAS-/-mice decreased significantly,but the proportion of CD206+macrophages increased significantly.ConclusionLPS can induce the production of mtDNA through TLR4 receptor.mtDNA can activate cGAS and promote M1 macrophage polarization by positively regulating mTORC1 pathway.cGAS knockout can significantly reduce the systemic inflammatory response and promote the polarization of M1 macrophages to M2 macrophages in BALF.Innovation1.This project is the first to explain the regulatory effect of cGAS on macrophage polarization.2.cGAS can regulate the polarization of alveolar macrophages in acute lung injury.3.In this project,we studied the molecular mechanism of cGAS in regulating macrophage polarization,which laid a foundation for elucidating the mechanism of cGAS mediated inflammatory response,and provided new strategies and ideas for the treatment of related diseases in clinic.