PD-L1对细菌脓毒症免疫抑制的影响及其机制

Effects of PD-L1 on immunosuppression of bacterial sepsis and its relevant mechanism

目的:探讨在脂多糖(LPS)所致细菌脓毒症免疫抑制中树突状细胞(DCs)程序性细胞死亡配体-1(PD-L1)的表达情况及其相关信号通路。方法:细菌脂多糖刺激骨髓来源树突状细胞诱导淋巴细胞免疫抑制模型,实验分为5组:对照组(Con)、脂多糖组(LPS)、2-(4-吗啉基)-8-苯基-4H-1-苯并吡喃-4-酮+脂多糖组(LY294002+LPS)、吡咯烷二硫代甲酸铵盐+脂多糖组(PDTC+LPS)和脂多糖+封闭PD-L1组(LPS+αPD-L1)。小鼠骨髓来源单核细胞用含粒细胞巨噬细胞集落刺激因子(rmGM-CSF 10 ng/ml)和白介素4(rmIL-41 ng/ml)的10%胎牛血清1640培养基于CO_(2)培养箱37℃静置培养4 d后,LPS(10 ng/ml)处理DCs静置12 h获得PD-L1高表达的DCs作为免疫抑制刺激细胞。通路抑制剂LY294002(10μmol/L)、PDTC(20μmol/L)作用1 h阻断PI3K和NF-κB信号。采用流式细胞分析、激光共聚焦显微成像检测LPS诱导树突状细胞PD-L1表达及磷脂酰肌醇3激酶/丝氨酸苏氨酸激酶B(PI3K/AKT)信号通路活化情况;BrdU细胞增殖实验和γ-干扰素酶联免疫斑点实验检测LPS诱导树突状细胞PD-L1表达上调对抗原特异性T细胞增殖反应及细胞毒性T细胞杀伤作用的影响。结果:与对照组比较,LPS组DCs表面PD-L1阳性细胞百分比升高(P<0.01),PD-L1荧光信号强度增强,且主要分布于细胞表面和细胞质,DCs介导的T细胞增殖水平降低(P<0.01),γ-干扰素斑点形成细胞数下降(P<0.01)。与LPS组比较,LY294002+LPS组、PDTC+LPS组和LPS+αPD-L1组PD-L1荧光信号强度降低,T细胞增殖水平升高(P<0.01),γ-干扰素斑点形成细胞数上升(P<0.01),改善树突状细胞介导的T细胞免疫抑制现象。结论:PD-L1是介导脂多糖所致细菌脓毒症免疫抑制的关键分子,PI3K信号、NF-κB信号也参与此免疫抑制过程。

Objective:To investigate the expression of programmed death ligand-1(PD-L1)in dendritic cells(DCS)and its related signaling pathway in lipopolysaccharide(LPS)-induced immunosuppression of bacterial sepsis.Methods:Stimulating with bacterial LPS,bone marrow-derived dendritic cells could induce T lymphocyte immunosuppression imitating bacterial sepsis model.The experiments were divided into 5 groups:control group,LPS group,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one(LY294002)+LPS group,pyrrolidinedithiocarbamate(PDTC)+LPS group and LPS+anti-PD-L1 group with 6 multiple wells in each group.After mice bone marrow source monocytes were cultured with rmGM-CSF(10 ng/ml)and rmIL-4(1 ng/ml)in 10%fetal bovine serum 1640 for 4 days,DCs cells were treated with with 10 ng/ml LPS for 12 h to obtain immunosuppressive cells with high expression of PD-L1.Pathway-inhibitors LY294002(10μmol/L)and PDTC(20μmol/L)were used to block PI3K and NF-κB signals.Flow cytometry and confocal laser scanning microscopy were used to detect the PD-L1 expression and phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signal activation on DCs.BrdU cell proliferation assay andγ-interferon enzyme-linked immunospot assay were used to detect ovalbumin specific T lymphocyte proliferation response and cytotoxic T cell response,respectively.Results:Compared with the control group,the percentage of PD-L1 positive cells and PD-L1 red fluorescence intensity of DCs were all increased(P<0.01),while DCs-mediated T cell proliferation andγ-interferon spot-forming cell number were decreased(P<0.01).PI3K inhibitor LY294002,NF-κB inhibitor PDTC and PD-L1 blocking antibody could significantly reverse the inhibition of DCs mediated T lymphocytes immunosuppression above(P<0.01).Conclusion:PD-L1 was a key molecule that mediates immunosuppression in lipopolysaccharide induced bacterial sepsis.PI3K Signal and NF-κB signal were also involved in this immunosuppressive process.