The Effect And Mechanism Of Mycobacterium Tuberculosis Transmembrane Protein Rv3737 On Macrophage Polarization

Objective: To understand the effect of Mycobacterium tuberculosis transmembrane protein Rv3737 on macrophage polarization,and to investigate whether Rv3737 affects the survival of Mycobacterium tuberculosis in macrophages by regulating macrophage polarization and the regulatory mechanism.Methods: Our group constructed Mycobacterium smegmatis overexpressing Rv3737(MS＿Rv3737)and Mycobacterium tuberculosis knocking out Rv3737(H37Rv△Rv3737)in the previous stage,and the controls were Mycobacterium smegmatis containing empty vector(MS＿Vec)and Mycobacterium tuberculosis standard strain(H37Rv-WT),respectively.The recombinant strains were validated by Western-blot and PCR and cultured in 7H9 liquid medium for growth curve assay.Using MS＿Rv3737 and MS＿Vec or H37Rv△Rv3737 and H37Rv-WT strains to infect RAW264.7,the intracellular viability of Mycobacterium in macrophage was detected by counting colony forming units;Real-time PCR was performed to detect the m RNA level of M1 polarization-associated cytokines i NOS,IL-6,TNF-α,IL-1β,MCP-1,IL-12 b and M2 polarization-associated cytokines Arg-1,TGF-β,IL-10.ELISA was performed to detect the protein level of i NOS,IL-6,TNF-α,Arg-1 in cell culture supernatants;the surface markers associated with macrophage polarization CD86 and CD206 were detected by flow cytometry,and the functional markers i NOS and Arg-1 were detected by Western-blot.The expression of key signaling molecules in macrophage polarization-related signaling pathway was detected by Real-time PCR to screen the target pathway,and the phosphorylation level of P65 and IκB in NF-κB signaling pathway and P38,JNK and ERK in MAPK pathway were detected by Western-blot.Finally,inhibitors of NF-κB and MAPK signaling pathway were added,and the regulation of these two pathways on the above macrophage polarization cytokines was examined by Real-time PCR and ELISA.Graphpad prism 6.0 software was used for data collation and differential analysis to understand whether Mycobacterium tuberculosis transmembrane protein Rv3737 regulates the survival of Mycobacterium tuberculosis in macrophages by affecting macrophage polarization.Results: Rv3737 overexpression strain MS＿Rv3737 and empty vector strain MS＿Vec were successfully validated by Western-blot and PCR,and Rv3737 knocked out strain H37Rv△Rv3737 was validated by PCR.Both overexpression and knockout of Rv3737 did not affect the in vitro growth of Mycobacterium tuberculosis and Mycobacterium smegmatis,however,after the recombinant strains infected macrophage,the colony forming unit results suggested that overexpression of Mycobacterium smegmatis Rv3737 promoted the survival of Mycobacterium smegmatis in macrophage,while knockout of Mycobacterium tuberculosis Rv3737 inhibited the survival of Mycobacterium tuberculosis in macrophage;overexpression of Rv3737 significantly promoted elevated m RNA level of the M2 polarization-related cytokines Arg-1,TGF-β,and IL-10 and elevated protein level of Arg-1,in contrast,knockout of Mycobacterium tuberculosis Rv3737 significantly inhibited the expression of M2 polarization-related cytokines in macrophages,while M1 polarization cytokines i NOS,IL-6,and TNF-α,IL-1β,MCP-1,IL-12 b m RNA level and i NOS protein level were elevated;meanwhile,flow cytometry results showed that Mycobacterium smegmatis overexpressing Rv3737-infected macrophage promoted CD206 level.The transcript level of molecules regulating macrophage polarization were detected by Real-time PCR,and the results showed that Rv3737 mainly regulated the transcript level of AP-1 and NF-κB,the phosphorylation level of p65 and IκB in NF-κB signaling pathway and P38,JNK and ERK in MAPK signaling pathway were decreased after overexpression of Rv3737 on Mycobacterium smegmatis by Western-blot assay,and in contrast,the phosphorylation level of these related molecules were increased after infection of macrophage by the Rv3737 knockout strain.Finally,the expression of related cytokines were detected by Real-time PCR and ELISA after inhibition of NF-κB and MAPK-related pathways,and the results showed that the cytokines were not significantly altered after inhibition of MAPK pathway,indicating that Rv3737 mainly regulated the polarization of macrophage through NF-κB pathway.Conclusion: Overexpression of Mycobacterium smegmatis Rv3737 and knockout of Mycobacterium tuberculosis Rv3737 did not affect the in vitro growth of mycobacteria,but Rv3737 significantly promoted the survival of Mycobacterium tuberculosis in macrophage,and Rv3737 promoted the polarization of macrophage to M2 type and inhibited the production of pro-inflammatory cytokines,and this effect was mainly achieved through the regulation of NF-κB signaling pathway.