Mechanism Of M~6A Methyltransferase METTL3 In Polarization Of Macrophages Infected With Tuberculosis

Background:Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB)infection.In recent years,with the emergence of multi-drug resistant MTB,the prevention and control of TB has brought great challenges.Macrophage,as the frontier line of immune defence,and is the important part of MTB infection.Research has shown that M1/M2 polarization of macrophages is closely related to the progression and pathogenesis of MTB infection.However,our mechanistic understanding of macrophages polarization in MTB infection,remains vague.N6-methyladenosine(m~6A)modification,as one of the most vital chemical modififications,is a dynamic process and is reversible to some extent.The m~6A modification is catalyzed by methyltransferase and can be removed by demethylase.It has been extensively studied and implicated that m~6A can be recognized by m~6A reading protein or binding proteinin and related to regulating stability,splicing,localization,and translation of mRNA,as well as regulating gene expression at the post-transcriptional level.RNA N6-Methyladenosine Methyltransferase-Like3(METTL3)is the most pivotal catalytic subunit of methyltransferase complex,which is involved in the translation and nucleation of mRNA.Recent studies have shown that,METTL3 mediated m~6A plays an important regulatory role in the process of immune response.However,the role and mechanism of METTL3 in tuberculosis is not yet known.Objective:To investigate the expression of METTL3 in macrophages with MTB infection,its role in macrophage polarization and related mechanisms.Methods:1.RT-qPCR was used to compare the expression of m~6A-related genes in PBMC of TB patients and healthy donors.THP-1-derived macrophages were infected by H37Ra in MOI=10,RT-qPCR and Western blot were used to compare the expression of METTL3 after infection(0h,4h,6h,12h,24h,48h).2.THP-1 from human peripheral blood were induced to M1 and M2 macrophage by LPS+IFN-γand IL-4 respectively in vitor.We further investigated the METTL3expression in macrophages under different condition by RT-qPCR and Western Blot.3.Constructing overexpressed、silenced METTL3 lentivirus vectors and packaging in lentivirus to get recombinant lentiviru.THP-1 were transfected by recombinant lentivirus and stable strains were screened out,the expression of METTL3 and m~6A level were identified.The OE-METTL3 and sh-METTL3 THP-1 were infected by H37Ra,RT-qPCR was used to detect the expression of polarization surface markers in macrophages at different time points,and Arginase activity and Nitrite level were detected by colorimetric method.IL-10、IL-12p40、TNF-α、CCL17 in cell supernatant were detected by ELISA.Bacterial load was detected by solid culture method.4.The expression of SOCS2 in sh-METTL3 THP-1 cells was detected by RT-qPCR.Me RIP-qPCR was used to detect the level of m~6A modification of SOCS2 mRNA by METTL3.The effect of m~6A on SOCS2 expression was detected by the Double luciferase gene report assay.The effect of METTL3 on SOCS2 mRNA stability was detected by RNA stability assay.SOCS2 small interfering RNA(si-SOCS2)was transfected in sh-METTL3 macrophages,and the polarization phenotype,cytokine secretion level and bacterial load of macrophages were detected.5.Constructing silenced m~6A binding protein YTHDF2 lentivirus vectors and packaging in lentivirus to get recombinant lentiviru.The THP-1 were infected by H37Ra after recombinant Lentivirus transduction to establish MTB infection model.The expression of SOCS2 was detected by RT-qPCR.RIP-qPCR was used to detect the interaction between YTHDF2 and SOCS2 mRNA.The effect of YTHDF2on SOCS2 mRNA stability was detected by RNA stability assay.Results:1.METTL3 mRNA and protein expression in pulmonary tuberculosis patients’PBMCs were significantly down-regulated compared with healthy donors.In MTB infection model,the results were consistent in the infection model.2.Both RT-qPCR and Western blot showed that METTL3 was down-regulated in M1 macrophages and up-regulated in M2 macrophages.3.Overexpression of METTL3 increased m~6A level of macrophages,while METTL3 silencing down regulated m~6A level.In OE-METTL3 THP-1 after MTB infection,the intracellular Arginase activity of macrophages was significantly upregulated and Nitrite level was decreased,and M2 markers including Arg1,CCL17,CCL18,CD163 and IL-10 were up-regulated and M1 markers including HLA-DR,IL-1β,TNF-α,i NOS and IL-12p40 were down-regulated.In sh-METTL3 THP-1 cell after MTB infection,the changes of Arginase activity and Nitrite level were opposite,and the expression of M1 markers TNF-αand IL-12p40was up-regulated and M2 markers CCL17,IL-10 was down-regulated.4.Colony counting results showed that the overexpression of METTL3 promoted the intracellular survival of MTB,while the silencing of METTL3 enhanced the bactericidal ability of macrophages.5.SOCS2 expression was up-regulated after METTL3 silencing in macrophages.Me RIP-qPCR showed that m~6A antibody was enriched in SOCS2 with m~6A modification.METTL3 silencing down-regulated the m~6A level of SOCS2 mRNA and increased SOCS2 mRNA stability.6.In rescue experiment,si-SOCS2 transfected into sh-METTL3 macrophages induced the M2 polarization phenotype and reduce the bactericidal ability of macrophages.7.RT-qPCR showed that SOCS2 mRNA expression was up-regulated after YTHDF2 silencing and the stability of SOCS2 mRNA increased.Conclusion:MTB infection can down-regulate the expression of METTL3 in macrophages,and then down-regulate the m~6A level of its target gene SOCS2 mRNA,enhancing the stability of SOCS2 mRNA,and induce the polarization of macrophages towards M1.This study is the first to reveal the role of m~6A modification in the anti-tuberculosis immunity of macrophages,contributing to a further understanding of the immune response mechanism of tuberculosis.