| Tuberculosis (TB) is a chronic infectious disease and a kind of immune disorder disease, caused by the Mycobacterium tuberculosis (Mtb) infection. Current studies suggest that tuberculous granuloma plays a dual role in TB occurrence, development and prognosis. As the initiating and main effector cells of the TB immune, macrophages involve in Mtb infection and the formation and development of granulomas. Macrophages are highly plastic and complex cells, in different microenvironments, macrophages might diaplay different phenotypes and show different functions. In order to further explore the phenotype and the the role of macrophages during Mtb infection, clarify the formation and development of tuberculous granulomas and the possible regulatory mechanism, we had performed four parts of researches as following:1 The introduction and phenotype identification of the polarization of macrophages and its bactericidal effects during Mtb infectionMonocyte-derived macrophages (MDM) from human peripheral blood were induced to polarize by IFN-γ+LPS and IL-4 respectively in vitro. Using unstimulated MDM as control, macrophages stimulated with IFN-y+LPS or IL-4 were detected for the gene expression of CXCL10, CXCL11, CCR7, CCL17 and CCL18 by Real-time PCR, the levels of memberane proteins HLA-DR, CD86, CD 163 (scavenger receptor) and CD206 (mannose receptor) by flow cytometry (FC), the levels of cytokine IL-6, IL-10, IL-12, TNF alpha, IFN -γand CCL18 in cell culture supernatant by ELISA.Results showed the M1 macrophage and M2 macrophage were successfully induced by stimulation with IFN-γ+LPS and IL-4 respectively. We further detected the phenotyphe and cytokines/chemokines profilts of Mtb infected macrophage, and found that Mtb infection could induce mixed phenotype of M1 and M2 macrophage. Bactericidal effect research demonstrated that different polarization induction could significantly affect the bactericidal effect of macrophage. To our knowledge, this is the first research which clarified the polarization change of macrophage in the course of Mtb infection and the roles of M1 and M2 macrophages in Mtb infection.2 Identification the phenotype of human tuberculous granulomas macrophageIn this study, biopoy spcimen and paraffin section preparation from the lung tissue of TB patients were asssyed by immunohistochemical staining and Real-Time PCR. Results showed that there are a large number of activated macrophages infiltration. The expression of moleallar makers of both M1 (iNOS) and M2 macrophages (CD206) were detected in tuberculous granulomas involving macrophages. Real-time PCR detection showed that the expression of M1 macrophages related genes CXCL10 and CXCL11 as well as M2 macrophages related gene CCL17 and CCL18 were increased in tuberculous granulomas. These results demonstrated that macrophages in tuberculous granulomas display a mixed phenotype of Ml and M2 macrophage.3 The polarization phenotypic and functional shift of macrophage in the course of formation and development of tuberculous granulomasIn this study, in vitro model of tuberculous granulomas was constructed by infection of peripheral blood mononuclear cells (PBMC) with Mtb H37Rv strain. The phenotype and function shift of macrophage in the tuberculous granulomas were monitored in the course of Mtb infction and development of granulomas. Results showed that the Mtb infection can activate the granulomas macrophages and enhance respiratory burst function. We investigated the type-shift of macrophages in the process of granulomas development by FC, Real-time PCR and ELISA. It was shown that most of macrophages were Ml macrophages at the early stage of the granulomas, while most of the macrophages were M2 macrophages at the later stage of the granulomas, which can lead to Mtb escape. The part of the study results suggest that the type and function of macrophages in formation and evolution of tuberculous granulomas.4 The influence of macrophage polarization and redox state on the formation and development of tuberculous granulomaIn this study, macrophages were induced to M1 macrophages and M2 macrophages by IFN-y+LPS or IL-4, respectively, then used to construct the in vitro model of tuberculous granulomas. Results showed that IFN-γ +LPS stimulation could promote the formation of granulomas and the clearance of Mtb. Oil red-O stain showed that Mtb triggered the differentiation of human granulomas macrophages into foamy macrophages, M2 macrophages can significantly promote the formation of foamy macrophages. In addition, we also foud that granulomas macrophages’ glutathione (GSH) levels gradually decreased after Mtb infection, which promoted the granuloma in the oxidative stress state. The antioxidant N-acetyl-L-cysteine (NAC) can correct the intracellular oxidative stress significantly, enhancing bactericidal activity of macrophages to Mtb and is conducive to controlling the infection of Mtb. The results of the research provided a possibility of application in the clinical treatment of TB in the future, just as the new treatment strategies by inducing M1 macrophages to control Mtb latent infections, provide new targets and research approach for tuberculous granuloma tissue reducing the bactericidal activity because of macrophages induced by the oxidative stress. |
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