Expression of interleukin-37 in patients with diabetic kidney disease and its regulatory activity to the cytotoxic function of CD8 +T lymphocytes
布海霞 1徐可 1韩晓静 1王焕 1周艳红 2杨克魁
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作者信息
1. 新乡市中心医院/新乡医学院第四临床学院肾脏内科，新乡 453000
2. 新乡市中心医院/新乡医学院第四临床学院内分泌科，新乡 453000
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摘要
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 receptor α chain，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 receptor α chain (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 (allP>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-γ or TNF-α secretions 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/Lvs. (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/Lvs. (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-γ and TNF-α levels in the supernatants were reduced in response to IL-37 stimulation [(23.56±6.24) ng/Lvs. (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.
Abstract
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 receptor α chain (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 (allP>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-γ or TNF-α secretions 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/Lvs. (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/Lvs. (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-γ and TNF-α levels in the supernatants were reduced in response to IL-37 stimulation [(23.56±6.24) ng/Lvs. (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.
关键词
糖尿病,2型/白细胞介素类/糖尿病肾病/CD8阳性T淋巴细胞/白细胞介素⁃37
Key words
Diabetes mellitus, type 2/Interleukins/Diabetic nephropathies/CD8-positive T-lymphocytes/Interleukin-37
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