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Objective The main objective of the present study was to investigate themacrophage polarization in the formation and development of tuberculous granulomaand to evaluate antigen-presenting function of macrophgae in tuberculous granuloma.Methods Refer to the relevant literature and pre-experimental methods,peripheral blood mononuclear cells (PBMC) were obtained from healthy PPD testnegative volunteers who had no family members with tuberculosis(TB) and theninfected with mycobacterium tuberculosis H37Rv for forming in vitro model ofhuman tuberculosis granuloma,which mimics in vivo human granuloma. UninfectedPBMC were set as control group.To detect the secretion of cytokines in thetuberculous granuloma microenvironment,the cell culture supernatant (IL-6, IL-10,IL-12, TNF-α, IFN-γ, and CCL18) was collected at different time points (24h,3d,6d,9d and12d) and detected by ELISA.Then the expression of CXCL10, CXCL11,CCR7, CCL17and CCL18were analyzed by Real-time PCR at different timepoints.After in vitro granuloma model was formed,non-adherent cells were removedand the granuloma were decentralized by trypsin digestion.Then the surface markersHLA-DR,CD86,CD163(scavenger receptor),CD206(mannose receptor) of granulomamacrophages which were collected by adhesive culture for4h were detected by flowcytometry (FCM).Results The human tuberculous granuloma in vitro model formed at6-daypost-infection.In the early stage of granuloma fomation,cell surface expression ofmacrophages marker CD163on the infected group was decreased compared to theuninfected group (P<0.05). Furthermore the level of cytokines and chemokines TNF-α,IFN-γ,IL-10, IL-12,CXCL10, CXCL11, CXCL17,CCL18were upward indifferent degrees in the cell culture supernatant of in vitro mode.In later developmentof granuloma,the expression of CD163was higher than the uninfected group(P<0.05),while HLA-DR, CD86expression were significantly reduced (P<0.01).Thesecretion of CCL18, IL-6, IL-10were significantly increased in the cell culturesupernatant of in vitro mode while TNF-α,IFN-γ,IL-12were declined.Furthermore The expres-sion of CXCL10, CXCL11, CCR7were significantly reduced followingCCL17, CCL18upregulated.Conclusion Both M1and M2macrophages existed in human tuberculousgranuloma.In the early stage of granuloma formation,macrophages was mainlymarked by M1polarization,By contrast,the later development of granuloma mainlymarked by M2polarization,and the antigen-presenting function of macrophagesmaybe significantly weakened。
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