Effects Of Mycobacterium Tuberculosis HBHA And ESAT-6 On Macrophage Polarization

Tuberculosis is one of the most deadly infectious diseases caused by Mycobacterium tuberculosis,which seriously endangers human health and public safety.Mycobacterium tuberculosis can invade multiple host systems and organs and cause tuberculosis in different parts.Macrophages are at the forefront of immune defense of the host and play an important role in resisting the invasion of exogenous microorganisms.Activated macrophages express different functional phenotypes,stimulated by different micro-environmental signals.Proinflammatory and microbicidal macrophages(M1)dorminate at the early stage of infection,and shift to M2 with infection into late stage,which weaken the capability of inflammatory damage and the bacteria control,resulting in the replication and persistence of the bacteria.Previous reports have shown that Mycobacterium tuberculosis HBHA and ESAT-6 can modulate the activation of macrophages and affect the infection process and outcome of Mycobacterium tuberculosis.Our research focuses on the effect of HBHA and ESAT-6 on macrophage polarization.Part 1 Methods: First,the dual luciferase reporter vector of IL-10 promoter was constructed.The activity ratio of firefly luciferase and Renilla luciferase was detected in the recombinant vector transfected Raw264.7 cells in order to indentify the expression activity of the recombinant vector.SDS-PAGE and Western blot was applied to analysize and identify the ESAT-6 protein expressed by the prokaryotic expression system and purified through the His GraviTrap column.After transfected with pGL3-IL10 promoter recombinant vector and,as the internal control,pRLSV40 vector simultaneously,Raw264.7 cells were treated with HBHA and ESAT-6,then the luciferase activity of each treatment group was tested.Results: The dual luciferase reporter vetor of IL-10 promoter was successfully constructed,and the luciferase activity of recombinant vector was lower than that of pGL3-promoter(positive control)group(P < 0.01),but significantly higher than that of pGL3-basic(negative control)group(P < 0.01).The ESAT-6 protein was expressed in a soluble form and the specific ESAT-6 antibody detected a protein band whose molecular weight was consistent with the theoretical value.The ratio of luciferase activity in HBHA and ESAT-6 treatment group was not statistically significant compared with the control group,indicating HBHA and esat-6 could not regulate the activity of IL10 promoter.Part 2 Methods: Mouse BMDM,Raw264.7 cells and THP-1 cells were treated with LPS+IFN-?,IL-4,HBHA and ESAT-6,respectively.IL-6,IL-12 and TNF-? in the supernatants of cell culture were detected by ELISA.Quantitative Real-time PCR was performed to measure the expression of molecular markers of macrophage polarization [inducible nitric oxide synthase(iNOS),TNF-?,CXCL-10,Arg-1,CCL-22 and CD206] at mRNA level.Western blot was employed to detect the expression of iNOS and Arg-1 at protein level.Finally,the Mycobacterium tuberculosis H37Rv-?Rv0475 strains knocked out the gene Rv0475 coding HBHA were constructed through plasmid and were transformed pMV261-Rv0475 vector to construct HBHA complemental strains,Mycobacterium tuberculosis H37Rv-c-Rv0475.Western blot assay was employed to test the expression of iNOS and Bcl-2 in the Raw264.7 cells infected by both strains and Mycobacterium tuberculosis wild strains.Results: Increased secretion of IL-6,IL-12 and TNF-? in the supernatants of cell culture and enhanced expression of iNOS and TNF-? at mRNA level were observed after treating BMDM with Mycobacterium tuberculosis HBHA.Similarly,after HBHA treatment,the secretion of IL-6 and the expression of TNF-? were also up-regulated in Raw264.7 cells;the the expression of CXCL-10 at mRNA level was augmented in treated THP-1 cells.Mycobacterium tuberculosis HBHA and ESAT-6 had similar effects on murine and human macrophages.Neither of them could increase the expression of Arg-1,a molecular marker of M2 macrophages,in BMDM at protein level,but both enhanced the expression of iNOS,a molecular marker of M1 macrophages,in BMDM and Raw264.7 cells.In Raw264.7 cells infected with Mycobacterium tuberculosis H37Rv-?Rv0475,the expression of iNOS protein decreased,compared with the wild type infected group.This demonstrated the effect of HBHA and ESAT-6 on M1 polarization of macrophages.Part 3 Methods: HBHA protein after dialysis was labelled with fluorescence and then the fluorescence labeling efficiency was qualitatively evaluated by Dot blot.HuProt ? human proteome chip was applied to screen target protein interacting with HBHA.After that,pGEX-5X-1/HBHA expression plasmid was constructed to produce the HBHA protein,and GST pull down further verified the ECSIT,Oncostatin M and PLEKHA8 of Raw264.7 macrophages interacting with HBHA.Results: 41 positive signal points were filtered out through the human proteome chip.GST pull down assay confirmed that HBHA can interact with ECSIT,Oncostatin M and PLEKHA8,respectively.Conclusion: Mycobacterium tuberculosis HBHA and ESAT-6 can induce the polarization of macrophages to M1 phenotype;the proteins of macrophages that mediate the function of HBHA may be ECSIT,Oncostatin M and PLEKHA8.