| Latent tuberculosis infections hinder the eradication of pathogens by the host's immune system,making them the primary reservoir of tuberculosis and a barrier to the fight against tuberculosis.Therefore,diagnosis and prevention of latent tuberculosis infection are the key to the prevention and treatment of tuberculosis.A group of antigens encoded by the dormant survival regulator(DosR)is crucial for the persistence of Mycobacterium tuberculosis in the host.In recent years,domestic and foreign research shows that DosR antigen has great potential in the diagnosis of latent infection and the development of new anti-TB vaccine.The antigen Rv1737c selected in this study is a kind of DosR regulator mainly expressed by Mycobacterium tuberculosis in incubation period.At present,there are few reports about this antigen and most of the functions are unknown.Objective:To explore the role and mechanism of DosR antigen Rv1737c in the regulation of innate immunity and inflammation in host and provide the experimental basis for the study of new anti-TB vaccine.Methods:Recombinant plasmid pET30a(+)-Rv1737c was constructed by genetic engineering and the recombinant protein Rv1737c(rRv1737c)was obtained by prokaryotic expression.Mice peritoneal macrophages and THP-1 cells were treated with rRv1737c.The expression of various effector molecules of macrophages stimulated by rRv1737c was detected by Western Blot and Real-Time PCR.The expression of costimulatory molecules on macrophages was detected by flow cytometry.The concentrations of cytokines in the supernatant of the cells were detected by ELISA.Subunit vaccine were made using rRv1737c and the adjuvant cholera toxin B subunit.C57BL/6mice were immunized with subunit vaccine at weeks 0,2,4.The immunogenicity of the vaccine was measured one week after the last immunization.Mice were infected by high-concentration BCG via the nasal route two weeks later.After three weeks,the effects and mechanisms of rRv1737c on immune protection and inflammation regulation were evaluated by detecting immune responses in mice as well as lung colony counts and histopathological lesions.Results:The protein rRv1737c was stably expressed in E.coil engineering bacteria,and the rRv1737c with high purity was obtained by nickel column affinity chromatography.For the first time,we have revealed a novel mechanism of Rv1737c in inducing tolerogenic phenotype of macrophage by modulating the expression of IDO1.Furthermore,Rv1737c interacted with macrophages through TLR2.It augmented NF-?B phosphorylation and co-stimulatory molecule(CD40/CD80)expression and promotes the expression of inflammatory cytokines(TNF-?,IL-1?,IL-6 and IL-8)secretion.Our findings showed that the mice immunized with recombinant Rv1737c(rRv1737c)adjuvanted with Cholera Toxin Subunit B(CTB)induced higher levels of antigen-specific antibodies(IgG,IgM,and IgA)than the sham-treated mice.Upon challenge infection with BCG,rRv1737c-CTB immunized mice showed inflammatory changes in the lungs but no difference in lungs bacterial load compared with the control mice.The immune effects due to rRv1737c immunization was associated with switching the macrophage phenotype from proinflammatory M1to activated M2 characterized by enhanced production of IL-10.The cell co-culture results showed that rRv1737c-treated macrophages can promote Th0 cell polarization to Th2.Our intracellular flow cytometric analysis further showed that the immunized mice after the BCG challenge had an increased frequency of the lung CD4~+T cells expressing IFN-?,IL-4 and IL-10 compared to the unimmunized mice.In line with this,culture of the lung cells with killed BCG resulted in the production of higher levels of multiple cytokines,such as IFN-?,IL-10 and IL-6,than those of control mice.Conclusions:DosR antigen Rv1737c induces macrophage activation by activating TLR2receptors.The mice immunized with rRv1737c-CTB were able to produce a strong antigen-specific immune response but showed more severe lung inflammatory lesions after BCG infection,which may be due to Th2-type immune responses induced by rRv1737c-activated M2 macrophages. |
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