白细胞介素-37在糖尿病肾脏疾病患者中的表达及其对CD8^(+)T细胞杀伤功能的调控作用

Expression of interleukin-37 in patients with diabetic kidney disease and its regulatory activity to the cytotoxic function of CD8^(+)T lymphocytes

目的观察白细胞介素(interleukin,IL)-37在糖尿病肾脏疾病(diabetic kidney disease,DKD)患者中的表达,并评估外源性IL-37对DKD患者CD8^(+)T细胞功能的调控作用。方法采用横断面研究方法,纳入20例健康对照者、36例2型糖尿病(diabetes mellitus type 2,T2DM)患者及47例DKD患者。采集外周血,分离血浆和外周血单个核细胞。酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测血浆IL-37、可溶型IL-1受体8(IL-1 receptor 8,IL-1R8)水平。流式细胞术检测CD8^(+)T细胞中IL-18受体α链(IL-18 receptorαchain,IL-18Rα)、IL-1R8水平和免疫检查点分子水平。纯化CD8^(+)T细胞,使用重组IL-37刺激培养,与人胚胎肾293(human embryonic kidney 293,HEK293)细胞直接接触或间接接触共培养。ELISA法检测穿孔素(perforin)、颗粒酶B(granzyme B)、干扰素-γ(interferon-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。通过检测乳酸脱氢酶水平计算靶细胞死亡比例。结果DKD患者血浆IL-37水平[(63.42±23.30)ng/L]低于健康对照者[(143.02±50.67)ng/L]和TD2M患者[(87.88±40.62)ng/L](t=8.848,P<0.001;t=3.456,P<0.001)。血浆IL-37对个体发生T2DM、T2DM患者发生DKD的预测效能较好[曲线下面积分别为0.797(95%CI 0.676~0.917,P<0.001)和0.691(95%CI 0.576~0.807,P=0.003)]。DKD患者血浆IL-37水平与血尿素氮(r=-0.313,P=0.032)和血肌酐水平(r=-0.477,P<0.001)呈负相关,与估算肾小球滤过率呈正相关(r_(s)=0.478,P<0.001)。DKD患者IL-1R8^(+)CD8^(+)细胞比例显著高于健康对照者和T2DM患者(33.60%±9.47%比16.29%±5.97%、17.13%±4.85%,t=7.545、9.516,均P<0.001),且与空腹血糖、血尿素氮、血肌酐、估算肾小球滤过率无相关性(均P>0.05)。IL-18Rα^(+)CD8^(+)细胞比例、可溶型IL-1R8水平、免疫检查点分子占CD8^(+)T细胞比例在健康对照者、T2DM患者、DKD患者之间的差异无统计学意义(均P>0.05)。DKD患者CD8^(+)T细胞分泌穿孔素和颗粒酶B水平均显著高于健康对照者[(108.78±12.42)ng/L比(94.60±10.07)ng/L,t=3.096,P=0.005;(261.34±48.79)ng/L比(166.28±30.80)ng/L,t=3.387,P=0.002]和T2DM患者[(108.78±12.42)ng/L比(92.58±14.71)ng/L,t=3.263,P=0.003;(261.34±48.79)ng/L比(170.66±39.24)ng/L,t=2.627,P=0.014],但CD8^(+)T细胞分泌IFN-γ和TNF-α水平在健康对照者、T2DM患者、DKD患者间的差异无统计学意义(均P>0.05)。在直接接触共培养中,IL-37刺激后CD8^(+)T细胞诱导HEK293细胞死亡比例降低(13.03%±4.97%比17.88%±5.19%,t=2.235,P=0.037),上清中穿孔素[(222.02±25.79)ng/L比(294.30±25.58)ng/L,t=6.603,P<0.001]、颗粒酶B[(416.27±90.24)ng/L比(524.71±115.53)ng/L,t=2.454,P=0.023]、IFN-γ[(23.66±4.20)ng/L比(35.18±8.51)ng/L,t=4.026,P<0.001]和TNF-α[(1.62±0.29)μg/L比(2.09±0.57)μg/L,t=2.302,P=0.034]水平均降低。在间接接触共培养中,CD8^(+)T细胞诱导HEK293细胞死亡比例、穿孔素、颗粒酶B水平在无刺激和IL-37刺激之间的差异均无统计学意义(均P>0.05),但IL-37刺激后上清中IFN-γ[(23.56±6.24)ng/L比(32.56±9.90)ng/L,t=2.550,P=0.019]和TNF-α[(1.41±0.31)μg/L比(2.10±0.44)μg/L,t=4.011,P<0.001]水平降低。结论DKD患者外周血IL-37水平降低,外源性IL-37可抑制DKD患者CD8^(+)T细胞的杀伤活性。

Objective To investigate interleukin-37(IL-37)expression in patients with diabetic kidney disease(DKD),and to assess the regulation of exogenous IL-37 on CD8^(+)T cell function in DKD patients.Methods A cross-section study was carried out.Twenty healthy controls,thirty-six patients with diabetes mellitus type 2(T2DM),and forty-seven DKD patients were enrolled in the study.Peripheral blood was collected.Plasma and peripheral blood mononuclear cells were isolated.IL-37 and soluble IL-1 receptor 8(IL-1R8)levels in the plasma were measured by enzyme-linked immunosorbent assay(ELISA).IL-18 receptorαchain(IL-18Rα),IL-1R8 and immune checkpoint molecules levels in CD8^(+)T cells were measured by flow cytometry.CD8^(+)T cells were purified,and were stimulated with recombinant IL-37.CD8^(+)T cells were co-cultured with HEK293 cells in either direct contact or indirect contact manner.Levels of perforin,granzyme B,interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)were measured by ELISA.The proportion of target cell death was assessed by measuring lactate dehydrogenase level.Results Plasma IL-37 levels in DKD patients[(63.42±23.30)ng/L]were significant lower than those in healthy controls[(143.02±50.67)ng/L]and T2DM patients[(87.88±40.62)ng/L](t=8.848,P<0.001;t=3.456,P<0.001).Plasma IL-37 level had good predictive values for T2DM in health individuals and for DKD in T2DM patients[the area under the curve was 0.797(95%CI 0.676-0.917,P<0.001)and 0.691(95%CI 0.576-0.807,P=0.003),respectively].Plasma IL-37 level was negatively correlated with urea nitrogen(r=-0.313,P=0.032)and creatinine(r=-0.477,P<0.001),and positively correlated with estimated glomerular filtration rate(eGFR)(r_(s)=0.478,P<0.001)in DKD patients.IL-1R8^(+)CD8^(+)cell proportion in DKD patients(33.60%±9.47%)was significantly higher compared to healthy controls(16.29%±5.97%)and T2DM patients(17.13%±4.85%)(t=7.545,9.516,both P<0.001),but did not correlate with fast blood glucose,urea nitrogen,creatinine,or eGFR(all P>0.05).There were no statistical differences of IL-18Rα^(+)CD8^(+)cell proportion,soluble IL-1R8 level,or immune checkpoint molecule proportion in CD8^(+)T cells among healthy controls,T2DM patients,and DKD patients(all P>0.05).Perforin and granzyme B secretions by CD8^(+)T cells were significantly elevated in DKD patients compared with healthy controls[(108.78±12.42)ng/L vs.(94.60±10.07)ng/L,t=3.096,P=0.005;(261.34±48.79)ng/L vs.(166.28±30.80)ng/L,t=3.387,P=0.002]and T2DM patients[(108.78±12.42)ng/L vs.(92.58±14.71)ng/L,t=3.263,P=0.003;(261.34±48.79)ng/L vs.(170.66±39.24)ng/L,t=2.627,P=0.014].There were no significant differences of either IFN-γor TNF-αsecretions by CD8^(+)T cells among healthy controls,T2DM patients,and DKD patients(all P>0.05).In direct contact co-culture manner,CD8^(+)T cell-induced HEK293 cell death was down-regulated(13.03%±4.97%vs.17.88%±5.19%,t=2.235,P=0.037).The levels of perforin[(222.02±25.79)ng/L vs.(294.30±25.58)ng/L,t=6.603,P<0.001],granzyme B[(416.27±90.24)ng/L vs.(524.71±115.53)ng/L,t=2.454,P=0.023],IFN-γ[(23.66±4.20)ng/L vs.(35.18±8.51)ng/L,t=4.026,P<0.001]and TNF-α[(1.62±0.29)μg/L vs.(2.09±0.57)μg/L,t=2.302,P=0.034]were also reduced as well.In indirect contact co-culture manner,there were no significant differences of CD8^(+)T cell-induced HEK293 cell death,perforin,or granzyme B levels between no stimulation and IL-37 stimulation(all P>0.05).IFN-γand TNF-αlevels in the supernatants were reduced in response to IL-37 stimulation[(23.56±6.24)ng/L vs.(32.56±9.90)ng/L,t=2.550,P=0.019;(1.41±0.31)μg/L vs.(2.10±0.44)μg/L,t=4.011,P<0.001].Conclusion IL-37 level is reduced in DKD patients.Exogenous IL-37 suppresses the cytotoxicity of CD8^(+)T cells in DKD patients.