| Tuberculosis(TB)is a chronic consumptive infectious disease for both human and animals caused by the infection of Mycobacterium tuberculosis complex(MTBC),which seriously affects the health of human and animals and is one of the major public health problems in the world.Although the BCG vaccine has saved tens of millions of lives,it has not significantly controlled the occurrence of TB,mainly because the pathogenesis of Mycobacterium tuberculosis(M.tb)infection has not been fully elucidated so far.This in turn has hampered the development of effective drugs and vaccines,so that TB remains a major threat to human and animal health.Studies have shown that alveolar macrophages(AM)are the first target cells to be infected when TB enters the lungs through the respiratory tract and the innate immune response of AM to infection is the first step in eliciting an acquired immune response and effective control of M.tb infection.The early confrontation result of M.tb and AM infection determines the further development of TB.Therefore,it will be of great help for in-depth reveal of the molecular mechanism of host AM clearance of M.tb to explore the early immune response events of AM to M.tb infection,that is,the occurrence of autophagy,the interaction between autophagy and inflammatory response,and the mutual regulation of lipid metabolism between bacteria and AM.In 2019,Chen Zhijian’s team reported for the first time in Nature the activation and "induced autophagy" function of cGAS-STING pathway,that is,binding with microbes or their own DNA in the cytoplasm to detect infection or tissue damage,and emphasized the importance of cGAMP-induced autophagy for DNA and pathogen clearance in the cytoplasm.This suggested that "whether cGAS-STING signal also interacts with the traditional TLRs signaling pathway in the early stage of M.tb infection of AM and combats M.tb infection by co-regulation of autophagy and inflammation response?".Chinese scholars have recently found that lncRNA MALAT1 is highly expressed in the peripheral blood of patients with active TB,which may be a biomarker for the diagnosis of active TB infection and it has also been found that macrophages infected with H37Rv could promote the up-regulation of MALAT1 expression,and silencing MALAT1 could enhance the clearance ability of macrophages to H37Rv.Bhatt Bharat(2020)found that MALAT1 is involved in regulating the lipid-rich environment of macrophages in M.tb infection,which is conducive to the growth of Mycobacteria.Accordingly,we speculate that MALAT1 may be involved in the regulation of M.tb infection by mediating the early autophagy of macrophages.Therefore,we combined the latest progress of cGAS-STING signaling pathway and lncRNA MALAT1 in autophagy to explore how cGAS-STING signaling pathway and lncRNA MALAT1 regulate autophagy of macrophages in the early stage of macrophage RAW264.7 infected with M.tb H37Ra and the "cross-talk" interaction between them.On this basis,we further isolated primary bovine macrophages and established early infection models of primary bovine alveolar macrophages of clinical strains of M.tb and M.bovis.Through whole transcriptomic and lipidomic sequencing and bioinformatics analysis,we deeply analyzed the differences in the early infection events of different clinical types of mycobacterial infection so as to provide theoretical reference for understanding the pathogenesis and prevention of M.tb.Through the research,we mainly obtained the following results:1.Study on the regulatory effects of cGAS signal on cGAS-STING signaling pathway,autophagy and TLR signaling pathway in M.tb-infected macrophages:Western-blot analysis after interference/overexpression of cGAS gene in RAW264.7 cells showed that the expressions of TBK1,p-TBK1,IRF3,p-IRF3,STING and p-STING in the cGAS-STING signaling pathway,as well as autophagy-related proteins ATG5,Beclin,LC3 Ⅱ and p62 were positively regulated with cGAS;cGAS activated the expression of p-STING,p-TBK1 and p-IRF3 in early H37Rainfected macrophages,downregulated the expression of STING,TBK1 and IRF3 and then mediated the activation of autophagy-related proteins ATG5,Beclin,LC3 II and p62.This phenomenon was further verified by the results of autophagic flow and autophagic flux analysis of the M.tb-infected RAW264.7 cell model by using the mRFP-GFP-LC3 autophagy double-labeled adenovirus.These results indicated that cGAS can promote H37Ra-induced autophagy in macrophages mediated by cGAS-STING signaling pathway.Western-blot and ELISA analysis of TLR signaling pathway in H37Ra-infected macrophages with cGAS interference/overexpression showed that cGAS positively regulated the expression of TLR signaling pathway related proteins TLR-6,TRAF6,NF-κB p65,p-NF-κB p65 and MYD88,as well as the expression of downstream inflammatory factors IL-1β,IL-6 and TNFα.This indicates that cGAS positively mediates the activation of TLR signaling pathway and the secretion of inflammatory cytokines in H37Ra-infected macrophages.2.Study on the regulatory effect of STING signal on cGAS-STING signaling pathway,autophagy and TLR signaling pathway in M.tb-infected macrophages:Western-blot analysis after interference/overexpression of STING gene in RAW264.7 cells showed that the expressions of cGAS,TBK1,p-TBK1,IRF3,p-IRF3 and p-STING in the cGAS-STING signaling pathway,as well as autophagy-related proteins ATG5,Beclin,LC3 Ⅱ and p62 were positively correlated with STING;STING activated the expression of p-STING,p-TBK1 and p-IRF3 in early H37Ra-infected macrophages,downregulated the expression of TBK1 and IRF3 and then mediated the activation of autophagy-related proteins ATG5,Beclin,LC3 Ⅱ and p62.Furthermore,mRFP-GFP-LC3 autophagy double-labeled adenovirus was verified to analyze autophagic flow and autophagic flux.These results demonstrated that STING promoted autophagy regulation of H37Ra-infected macrophages mediated by cGAS-STING signaling pathway.Further Western-blot and ELISA analysis of TLR signaling pathway in infected cell model showed that STING signal positively regulated the expression of TLR-6,TRAF6,NF-κB,p-NF-κB P65 and MYD88 in TLR signaling pathway and also mediated the secretion of inflammatory cytokines IL-1β,IL6 and TNFα.STING promoted the activation of TLR signaling pathway and secretion of inflammatory cytokines in H37Ra-infected macrophages.3.Studies on LncRNA MALAT1’s interaction with CGAS-STING signaling pathway and regulation of autophagy and TLR signaling pathway in M.tb-infected macrophages:After H37Ra infected RAW264.7 macrophage,the expression of MALAT1 was found to be upregulated by Realtime-qPCR analysis.Realtime-qPCR and Western-blot analysis of RAW264.7 infected cell models before and after MALAT1 interference showed that the expression of MALAT1 was positively regulated the expression of cGAS,p-STING,p-TBKl and P-IRF3 in the cGAS-STING signaling pathway,downregulated the expression of STING,TBK1 and IRF3,and then mediated autophagy-related proteins ATG,Beclin,p62 and LC3II activation.Furthermore,mRFP-GFP-LC3 autophagy double-labeled adenovirus was utilized to analyze and verify the autophagic flow and flux.After MALAT1 interference,Western-blot and ELISA analysis before and after RAW264.7 infection showed that the protein expression levels of TLR6,TRAF6,MYD88,NF-κB p65 and p-NF-κB P65 in TLR signaling pathway were significantly down-regulated and the secretion of inflammatory cytokines IL-1β,IL-6,TNF-α and IFN-β decreased;H37Ra infection further activated the TLR signaling pathway and the secretion of inflammatory factors.Further analysis of the expression levels of MALAT1 in cGAS and STING interference/overexpressing RAW264.7 cells before and after H37Ra infection showed that the expressions of cGAS,STING and MALAT1 were positively correlated.4.Whole transcriptomic analysis of primary bovine alveolar macrophages(BAM)infected with clinical strains of M.tb and M.bovis:We constructed cDNA libraries of 3 strains of Mycobacterium tuberculosis(M.tb)and 3 strains of Mycobacterium bovis(M.bovis)clinical strains infecting primary BAM for 4 h and 6 h respectively,and performed whole-transcriptome sequencing and bioinformatics analysis.The findings are as follows:(1)M.bovis infection caused up-regulation of 235 genes and down-regulation of 135 genes,upregulation of 119 lncrnas and down-regulation of 99 lncrnas in BAM;of the 370 differential genes obtained in M.bovis vs.CK,compared with M.tb infection,M.bovis triggered more expression activation of the 21 genes related to immune signaling pathways,chemokines and inflammatory cytokines in BAM.But M.bovis did not induce significant changes in nutrient metabolism-related pathways.(2)M.tb infection resulted in up-regulation of 194 genes and down-regulation of 138 genes,upregulation of 103 LncRNAs and down-regulation of 111 LncRNAs in BAM;GO enrichment analysis of 332 differentially expressed genes in the comparison of M.tb and CK showed that 25 genes were associated with immune system and signal transduction functions,and were involved in lipid metabolism,immune response,inflammatory response and signal transduction.(3)Through the enrichment analysis of the mRMA differential expression of genes related to cGASSTING,autophagy and TLR signaling pathways in the transcriptome,it was found that the expressions of cGAS,ATG5,TLR6 and TRAF6 were up-regulated,and the expressions of IRF3 and TBK1 were down-regulated after M TB and M.Bovis were infected with BAM.(4)By constructing the central gene WGCNA,11927 LncRNAs and miRNAs were selected for further analysis,and 19 differential genes were identified by hierarchical clustering.PCK1,PTGS2 and CNTNAP1 related to M.tb infection,as well as CXCL3 and RAG2 genes related to M.bovis and M.tb were used as candidate genes to construct gene co-expression interaction networks with M.tb or M.bovisBAM respectively.PTGS2 was identified as a key gene involved in M.tb-BAM interaction and was negatively correlated with BTA-Mir-574,BTA-Mir-29b,BTA-Mir-15a,BTA-Mir-9-5p,BTA-Mir-30a-5p and 66 LncRNAs and positively correlated with 38 LncRNAs.M.tb can up-regulate the expression of btamiR-574 and bta-miR-9-5p.The results of KEGG pathway enrichment analysis showed that the genes involved in the M.tb-BAM interaction network were mainly involved in fat digestion and absorption related to nutrient metabolism,as well as various amino acid metabolism pathways.CCL20,a key gene involved in the regulatory network of M.bovis-BAM interaction,was negatively correlated with bta-miR7857-5p and 16 LncRNAs and positively correlated with 4 LncRNAs.Among the LncRNAs associated with key genes,the expression of various LncRNAs was significantly affected by M.bovis or M.tb infection at the early interaction stage.The results of KEGG pathway enrichment analysis showed that the genes involved in M.bovis-BAM interaction mainly played roles in immune-related pathways including MAPK signaling pathway,TNF signaling pathway,sphingolipid signaling pathway,and p53 signaling pathway.The above results suggest that M.tb can activate the immune system of BAM and regulate host nutrient metabolism for colonization and development,while M.bovis is recognized by the host,leading to immune system activation to defend against its invasion.5.Bilipidomic analysis of primary BAM infected with clinical strains of M.tb and M.bovis for 4 h and 6 h:Bilipidomics analysis was performed in both bacterial and host cells after BAM was infected with 3 strains of Mycobacterium tuberculosis(M.tb)and 3 strains of Mycobacterium bovis(M.bovis)clinical strains.(1)Bacterial lipidome analysis:17 lipids were found to contain high levels of M.tb,and five lipids were identified as key lipids in M.tb lipid metabolism.45 lipids were high in M.bovis,and five lipids were identified as key compounds of M.bovis.In contrast M.bovis contains polyketides,sphingolipids and lipids and lipid-like molecules not significantly identified in M.tb.From this,we concluded that the lipid compositions of M.tb and M.bovis were significantly different.(2)Host cell lipidome analysis:37 lipids were increased in M.tb-infected BAM,and polyketide and glycerophospholipid were found to be the most increased(P<0.0001)by enrichment analysis.49 lipids were highly accumulated in M.bovis-infected BAM and 18 glycerophospholipid-related lipids were found to be highly increased in BAM by enrichment analysis.Therefore,infection of M.tb and M.bovis resulted in marked differences in lipid metabolism of BAM.The lipid network was further constructed using 62 lipids of M.tb and M.bovis,and 86 lipids in M.tb and M.bovis-infected macrophages.Through analysis,it was found that one M.tb and four M.bovis lipids caused the differences in lipid metabolism in macrophages in response to mycobacterial infection.In conclusion,in the early immune response of M.tb infected macrophages,the cGAS-STING signaling pathway mediates the activation of autophagy and TLR signaling pathways,as well as the secretion of inflammatory factors and the expression of MALAT1 and has a positive regulatory relationship with MALAT1.Whole transcriptome and lipidomics revealed that M.tb and M.bovis activated immune and metabolism-related signaling pathways in the early stage of macrophage infection with significant differences,which provided a theoretical reference for in-depth study of the pathogenesis of tuberculosis. |
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