Study On Phenotypic Change Of Human Monocyte During Infection Of Mycobacterium Tuberculosis And Its Potential Mechanism

Object:To investigate the peripheral blood mononuclear cells phenotypic changes in tuberculosis patients and its possible mechanisms;To study the effect of Mycobacterium tuberculosis 6-Kilodalton early secreted antigenic target(ESAT-6) on Interleukin 12 (IL-12) production from human THP-1 monocytes.Methods:The expression of CD13,CD23,CD209,CD205 and CD206 on CD14 positive cells of the peripheral blood mononuclear cells in tuberculosis patients were analyzed by flow cytometry;The 1:5 diluted peripheral blood from patients or normal persons were cultured for 96h with or without ESTA-6,the supernatant were collected and the release of cytokines were detected by ELISA method.In vitro experiment,THP-1 monocytes were treated with PPD and H₃₇Rv with or without IL-4 for 48h,then cells were collected for detection of CD13,CD23 and CD209 by FACS analysis.In the study of effect of ESAT-6 on monocyte,recombinant ESAT-6 was purified by nickel-nitrilotriacetic acid metal affinity chromatography under native conditions.Human THP-1 monocytes were treated with ESAT-6,in present of LPS or not at different culture periods or doses. Supernatants were collected for IL-12 detection by ELISA.Signaling pathway inhibitors were used to find the signaling pathway of ESAT-6.Results:CD14⁺CD13⁺ were significantly higer in tuberculosis patients compared to healthy donors;PPD and heated-treated H₃₇Rv can synergisticly promote expression of CD13,CD23 and CD209 in THP-1 cells;Mycobacterium tuberculosis ESAT-6 antigen dose-dependently induced IL-12 release from THP-1 monocytes;Lipopolysaccharide (LPS) had synergic effect on ESAT-6 induced IL-12 production from THP-1 monocytes. The enhancement effect of ESAT-6 on IL-12 production was promoted by selective inhibitor of JNK MAPK,SP600125,while it was inhibited by PKR selective inhibitor, 2-AP. Conclusions:There is phenotypic change in peripheral monocyte of tuberculosis patients.The secretary protein and the bacterial structure protein may induce the expression of CD13 on monocyte,especially in presence of Th2 cytokine,IL-4.ESAT-6 may induce monocytes activation and enhance IL-12 release.This may have important significance of protection in the early period of infection.