活动性肺结核相关巨噬细胞M1/M2极化的改变及ESAT6对巨噬细胞极化的影响

Changes of M1/M2 macrophages polarization associated with active pulmonary tuberculosis and the effect of ESAT6 on macrophage polarization

目的探讨活动性肺结核患者外周血中单核-巨噬细胞M1/M2极化的变化及结核分枝杆菌ESAT6对人源THP-1细胞极化的影响。方法收集14例活动性肺结核(活动性肺结核组)和10例健康人(健康对照组)肝素钠抗凝全血和血清,采用淋巴细胞液分离肝素钠全血中单个核细胞(PBMCs),通过实时-定量PCR仪检测确诊的活动性肺结核患者PBMCs中HLA-DR、CD11C、CD68、CD206、Arg-1的mRNA水平,应用流式细胞仪检测细胞因子(IL-2、IL-6、TNF-α、IFN-γ、IL-4等)的分泌情况。将人源THP-1细胞经佛波酯(PMA)诱导分化,将其变成巨噬细胞样细胞后,分为M0组、M1组、M2组、M0+ESAT6组,刺激24 h后,荧光定量PCR检测HLA-DR、CD11C、CD68、CD206、Arg-1的mRNA水平。ESAT6分别刺激THP-16 h、12 h、24 h后,流式技术检测细胞培养上清中相关细胞因子的水平(IL-2、IL-6、TNF-α、IL-4等)。结果与健康对照组相比,活动性肺结核组外周血PBMCs中M1极化表型分子HLA-DR、CD11C、CD68的mRNA表达水平均上调(P<0.05);M2极化表型分子CD206 mRNA的表达水平下降(P<0.05),Arg-1 mRNA的表达水平差异无统计学意义(P>0.05)。活动性肺结核组患者血清M1极化相关的促炎性细胞因子IL-2、IL-6、TNF-α、IFN-γ水平均上升(P<0.05),抗炎性细胞因子IL-4水平降低(P<0.05)。体外诱导THP-1巨噬细胞分化为不同表型,细胞M1极化表型分子HLA-DR mRNA表达水平在各组之间有统计学意义(F=21.83,P=0.000),两两比较结果显示,M1表型组、M0+ESAT6表型组均较M0组明显上调(P<0.05),其余组间差异无统计学意义(P>0.05);但CD68 mRNA表达水平在各组之间差异无统计学意义(F=2.480,P=0.135)。细胞M2极化表型分子CD206、Arg-1 mRNA水平在各组之间差异无统计学意义(F=1.233,P=0.3597;F=6.059,P=0.068)。M1极化相关的促炎性细胞因子IL-2及抗炎性细胞因子IL-4在各组细胞培养不同时间节点均差异无统计学意义(P>0.05)。与M0和ESAT6表型组相比,M1表型组促炎性细胞因子IL-6和TNF-α水平在培养12 h、24 h均显著升高(P<0.05,P<0.05,P<0.001,P<0.001;P<0.05,P<0.05,P<0.01,P<0.01);其余组间差异无统计学意义(均P>0.05)。结论活动性肺结核外周血单核-巨噬细胞向M1极化的能力增强,向M2极化的能力减弱。结核分枝杆菌ESAT6可以促进巨噬细胞向M1极化,影响肺结核病情活动及进展。

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-γand TNF-αwere 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-αin 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.