Tim-3对早期梅毒患者外周血CD56dimNK细胞的影响及初步机制

Expression of Tim-3 on peripheral CD56dimNK cells of patients with early syphilis and its preliminary mechanisms

目的:研究Tim-3在早期梅毒患者外周血CD56 dim NK细胞中的表达情况及与细胞因子的相关性,并初步探讨其潜在的分子机制。方法:流式细胞术检测梅毒患者及正常对照外周血中NK细胞比值及NK细胞中Tim-3的表达水平,分析Tim-3表达与血清中IFN-γ水平的相关性。流式细胞仪分选CD56 dim NK细胞,培养并用IL-2刺激其增殖,干扰Tim-3后,流式细胞术及ELISA法检测IFN-γ水平。Western blot检测梅毒患者CD56 dim NK细胞干扰Tim-3前后JAK-STAT1通路蛋白变化。同时干扰Tim-3及STAT1蛋白表达,进一步分析Tim-3调控CD56 dim NK细胞分泌IFN-γ的确切分子机制。结果:梅毒患者及正常对照外周血中NK细胞比值无统计学差异(P>0.05)。梅毒患者NK细胞中Tim-3的表达水平明显上调(P<0.05),特别是CD56 dim NK细胞(P<0.01),且Tim-3的表达与血清中IFN-γ水平呈负相关性(P<0.01,r=-0.450 6)。梅毒患者CD56 dim NK细胞JAK-STAT1通路蛋白在IL-12和IL-18的刺激下较正常对照明显下调,干扰CD56 dim NK细胞中Tim-3的表达后则JAK-STAT1通路蛋白表达上调,同时上清中IFN-γ分泌水平增高(P<0.01)。同时干扰Tim-3及STAT1蛋白表达后IFN-γ升高现象消失(P>0.05)。结论:Tim-3通过JAK-STAT1通路调控CD56 dim NK细胞IFN-γ分泌,高表达的Tim-3水平可能抑制了梅毒螺旋体的清除进程,Tim-3可作为梅毒疾病进展的潜在分子标志物。

Objective: To investigate the expression of Tim-3 on peripheral CD56 dim NK cells in patients with syphilis,and to explore its potential molecular mechanisms. Methods: Flow cytometry was used to detect the ratio of circulating NK cells and the Tim-3 expression level on NK cells in patients with syphilis.The correlation between Tim-3 level and the serum IFN-γ was analyzed by using the Spearman′s rank test.Then,the CD56 dim NK cells were sorted by flow cytometry,and stimulated with IL-2.The Tim-3 and IFN-γ levels were measured by flow cytometry and ELISA methods after interference with Tim-3.Western blot was used to detect the changes of the JAK-STAT1 pathway in CD56 dim NK cells before and after interference with Tim-3.To analyze the molecular mechanism for Tim-3 regulating CD56 dim NK cells via controlling of IFN-γ secretion,co-transfection with Tim-3 and STAT1 interference plasmids was performed. Results: The ratio of NK cells in peripheral blood between patients with syphilis and controls was no significant difference ( P >0.05).The expression levels of Tim-3 were significantly up-regulated on NK cells,especially on CD56 dim NK cells in patients with syphilis ( P <0.05, P <0.01,respectively).Furthermore,the expression of Tim-3 was negatively correlated with serum IFN-γ levels ( P <0.01, r =-0.450 6).The JAK-STAT1 pathway in CD56 dim NK cells in patients with syphilis was down-regulated when stimulation with IL-12 and IL-18,by contrast with that the JAK-STAT1 pathway was up-regulated after interference with Tim-3 in CD56 dim NK cells.The levels of IFN-γ were consistently increased ( P <0.01).Meanwhile,the elevated IFN-γ levels were reduced after interfering with both Tim-3 and STAT1( P >0.05). Conclusion: Tim-3 regulates IFN-γ secretion on CD56 dim NK cells via the JAK/STAT1 pathway,and the higher expression of Tim-3 may inhibit the clearance of Treponema pallidum in patients with syphilis.Tim-3 may serve as a potential molecular marker for diagnosis of syphilis.