白细胞介素-35调控CD4+CD25+CD127 dim/-调节性T细胞在呼吸机相关性肺炎发病中的作用

摘要

目的 观察呼吸机相关性肺炎(ventilator associated pneumonia,VAP)患者外周血和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中CD4+CD25+CD127dim/-调节性T细胞(regulato-ry T cells,Tregs)和白细胞介素(interleukin,IL)-35的变化,并分析IL-35对Tregs的调控在VAP发病中的作用.方法 收集23例VAP患者和11例健康志愿者(normal controls,NCs),分离外周血单个核细胞、血清和BALF,通过酶联免疫吸附试验检测IL-35水平,通过流式细胞术检测CD4+CD25+CD127dim/-Tregs比例.纯化BALF中CD4+CD25+CD127dim/-Tregs,利用重组人IL-35对纯化的Tregs刺激培养后与自体CD4+CD25-T细胞共培养,检测细胞增殖和分泌细胞因子水平.结果 外周血中Tregs的比例和血清IL-35水平在VAP患者和NCs之间差异无统计学意义.但在VAP患者中,感染部位BALF中Tregs的比例和IL-35水平较非感染部位显著升高,且BALF中Tregs的比例与IL-35水平呈显著正相关(r=0.441,P=0.035).经抗感染等综合治疗后,病情好转的患者BALF中Tregs的比例和IL-35水平较治疗前显著下降,Tregs分泌IL-35的水平亦显著下降,但病情未好转或恶化的患者BALF中Tregs的比例和IL-35水平与治疗前比较则无明显变化,Tregs分泌IL-35的水平亦无明显变化.同时,病情好转患者Tregs抑制细胞增殖能力和分泌IL-10水平均较病情未好转或恶化患者减弱.利用重组人IL-35刺激治疗前患者BALF中纯化的Tregs可增强其免疫抑制功能,主要表现为细胞增殖减弱、抑制性细胞因子(IL-35和IL-10)分泌增多以及促炎因子(IFN-γ和TNF-α)分泌减少,但是,IL-35对治疗好转患者BALF中纯化Tregs的功能无显著影响.结论 IL-35和Tregs在VAP患者BALF中显著升高,且IL-35可增强Tregs的免疫抑制功能.IL-35调控Tregs功能可能参与了VAP的发病.%Objective To investigate the changes in CD4+CD25+CD127 dim/- regulatory T cells ( Tregs) and interleukin ( IL)-35 in peripheral blood and bronchoalveolar lavage fluid ( BALF) samples from patients with ventilator-associated pneumonia ( VAP) . Methods A total of 23 patients with VAP and 11 normal controls ( NCs) were enrolled in this study. Peripheral blood mononuclear cells, serum and BALF samples were isolated and collected. Levels of IL-35 were measured by enzyme linked-immunosorbent assay. Percentages of CD4+CD25+CD127dim/-Tregs were measured by flow cytometry. CD4+CD25+CD127dim/-Tregs in BALF samples were isolated and purified, which were then stimulated with recombinant human IL-35 and co-cultured with autologous CD4+CD25-T cells. Cells and supernatants were harvested for analysis of cell proliferation and cytokine secretion. Results No significant difference in peripheral Tregs and serum IL-35 was found between patients with VAP and NCs. The percentages of Tregs and the levels of IL-35 in BALF samples collected from infectious sites were remarkably higher than those collected from non-infectious sites in patients with VAP. Moreover, there was a positive correlation between Tregs and IL-35 in BALF ( r=0. 441, P=0. 035). Tregs and IL-35 in BALF samples as well as Treg-secreted IL-35 were significantly re-duced in patients who had good response to therapy. However, no significant change in these parameters was observed in patients who had poor response to therapy. Besides, suppression of cell proliferation and IL-10 secretion that were related to Tregs were inhibited in patients whose condition was improved as compared with those in patients who had no response to therapy. Stimulation with recombinant human IL-35 enhanced the immunosuppressive function of purified Tregs that were separated from BALF of treatment-na?ve patients with VAP, which was mainly marked by suppressed cell proliferation, increased secretion of inhibitory cytokines (IL-35 and IL-10), and decreased secretion of proinflammatory cytokines (IFN-γ and TNF-α). However, IL-35 had little effect on the activities of Tregs that were separated from patients with VAP who responded to therapy. Conclusion Both IL-35 and Tregs are increased in BALF of patients with VAP and IL-35 en-hances the immunosuppressive function of Tregs, which indicates that IL-35-mediated modulation of Tregs might take part in the pathogenesis of VAP.