关键词: 结核分枝杆菌, 巨噬细胞, 极化, 活动性肺结核, 6KD早期分泌靶向蛋白

Abstract:

Objective To investigate the alteration of M1/M2 polarization of monocyte-macrophages from the peripheral blood of patients with active pulmonary tuberculosis， and the effect of Mycobacterium tuberculosis ESAT6 on the polarization of human THP-1 cells. Methods Whole blood and serum samples were collected from 14 patients with active pulmonary tuberculosis and 10 healthy controls. Peripheral blood mononuclear cells （PBMCs） were isolated from whole blood with heparin sodium using lymphocyte fluid. The mRNA levels of HLA-DR， CD11C， CD68， CD206 and Arg-1 in PBMCs from patients with active pulmonary tuberculosis were detected by real-time quantitative PCR. The secretion of cytokines （IL-2， IL-6， TNF-α， IFN-γ， IL-4， etc.） was detected by flow cytometry. Human THP-1 cells were induced by phorbol ester （PMA） to differentiate into macrophages-like cells， which were divided into M0 group， M1 group， M2 group， and M0+ESAT6 group. After 24 hours of stimulation， the mRNA levels of HLA-DR， CD11C， CD68， CD206 and Arg-1 were detected by real-time PCR. Following stimulation with ESAT6 for 6 h， 12 h and 24 h， the levels of cytokines （IL-2， IL-6， TNF-α， IL-4， etc.） in cell culture supernatant from THP-1 cells were detected by flow cytometry. Results Compared with the healthy control group， the mRNA expression levels of M1-polarized phenotypic molecules HLA-DR， CD11C and CD68 in PBMCs of the active pulmonary tuberculosis group were up-regulated （P < 0.05）. The mRNA expression level of M2-polarized phenotype molecule CD206 was decreased （P < 0.05）， while the expression level of Arg-1 mRNA was not statistically significant （P > 0.05）. Serum levels of M1-related proinflammatory cytokines IL-2， IL-6， IFN-γ and TNF-α were increased in patients with active pulmonary tuberculosis（all P < 0.05）， whereas decreased level of anti-inflammatory cytokine IL-4（P < 0.05）were found in serum samples from patients with active pulmonary tuberculosis. THP-1 macrophages were induced to differentiate into different phenotypes in vitro， and the HLA-DR mRNA expression level of cell M1 polarization phenotype molecule was statistically significant among all groups （F = 21.83， P = 0.000）. Pairwise comparison results showed that expressions of HLA-DR mRNA in M1 group and M0+ESAT6 group were significantly upregulated compared with M0 group （P < 0.05）， there was no significant difference between the other groups （P > 0.05）. However， there was no significant difference in the expression of CD68 mRNA among all groups （F = 2.480， P = 0.135）. There was no significant difference of mRNA expressions of CD206 and Arg-1 among all groups （F = 1.233， P = 0.3597； F = 6.059， P = 0.068）. There were no significant differences between the M1-related pro-inflammatory cytokine IL-2 and anti-inflammatory cytokine IL-4 at different time points of cell culture （P > 0.05）. Compared with the M0 and ESAT6 phenotypes， the levels of pro-inflammatory cytokines IL-6 and TNF-α in the M1 phenotype group were significantly increased at 12 h and 24 h （P < 0.05， P < 0.05， P < 0.001， P < 0.001； P < 0.05， P < 0.05， P < 0.01， P < 0.01）； but there was no significant difference between the other groups（P>0.05）. Conclusions The ability of peripheral blood monocyte-macrophages to polarize to M1 is enhanced， while the ability to polarize to M2 is weakened in patients with active pulmonary tuberculosis. Mycobacterium tuberculosis ESAT6 can promote the polarization of macrophages to M1， which affects the activity and progression of tuberculosis.

Key words: mycobacterium tuberculosis, macrophage, polarization, active pulmonary tuberculosis, early secretary antigenic target 6