白细胞介素-36在糖尿病肾病患者中的表达及其对单核细胞功能的调控作用

Expression of interleukin-36 and its regulatory effect on monocyte function in patients with diabetic kidney disease

目的分析白细胞介素-36(IL-36)家族成员在糖尿病肾病(diabetic kidney disease,DKD)外周血中的水平,探讨重组人IL-36α对DKD患者单核细胞功能的调控作用。方法连续入组2型糖尿病患者41例、DKD患者36例、对照者20名,分离血浆和外周血单个核细胞,酶联免疫吸附法检测血浆IL-36α、IL-36β、IL-36γ、IL-36受体拮抗剂(IL-36Ra)水平,分选单核细胞,实时定量PCR检测单核细胞中IL-36受体亚基mRNA表达。使用重组IL-36α刺激单核细胞,检测培养上清中毒性分子和细胞因子水平,流式细胞术检测程序性死亡受体-1(PD-1)和细胞毒性T淋巴细胞相关蛋白4(CTLA-4)表达。单核细胞与Vero细胞共培养,评估单核细胞的杀伤功能。结果 2型糖尿病组和DKD组血浆IL-36α、IL-36β水平显著高于对照组,DKD组血浆IL-36α水平亦高于2型糖尿病组(P<0.05)。IL-36γ和IL-36Ra水平在3组之间差异无统计学意义(P>0.05)。单核细胞中IL-36受体亚基mRNA表达在3组之间差异无统计学意义(P>0.05)。DKD组肿瘤坏死因子α(TNF-α)水平高于对照组和2型糖尿病组(P<0.05),PD-1和CTLA-4水平低于对照组和2型糖尿病组(P<0.05)。DKD组单核细胞诱导Vero细胞死亡的比例显著高于对照组和2型糖尿病组(P<0.05)。重组人IL-36α刺激后,DKD患者单核细胞分泌颗粒酶B和TNF-α水平显著升高(P<0.05),单核细胞诱导Vero细胞死亡的比例显著升高(P=0.024)。结论 DKD组单核细胞分泌颗粒酶B和IL-36α水平升高诱导单核细胞功能增强。

Objective To analyze the level of interleukin-36(IL-36)family cytokines in peripheral blood,and explore the regulatory role of recombinant human IL-36αin monocyte function in patients with diabetic kidney disease(DKD).Methods A total of 41 type 2 diabetes mellitus(T2DM)patients,36 DKD patients,and 20 controls were consecutively enrolled.Plasma and peripheral blood mononuclear cells(PBMCs)were isolated.Enzyme-linked immunosorbent assay(ELISA)was used to measure plasma levels of IL-36α,IL-36β,IL-36γ,and IL-36 receptor antagonist(IL-36Ra).PBMCs were sorted,and real-time quantitative PCR was performed to detect the mRNA expression of IL-36 receptor subunits in monocytes.Monocytes were stimulated with recombinant IL-36α,and levels of cytotoxic molecules and cytokines in the culture supernatant were measured.Flow cytometry was used to assess the expressions of programmed death receptor-1(PD-1)and cytotoxic T-lymphocyte-associated protein 4(CTLA-4).Co-culture of monocytes with Vero cells was performed to evaluate monocyte cytotoxicity.Results Plasma levels of IL-36αand IL-36βin the T2DM and DKD groups were significantly higher than those in the control group.The DKD group also showed higher plasma levels of IL-36αcompared to the T2DM group(P<0.05).There were no significant differences in IL-36γand IL-36Ra levels among the three groups(P>0.05).The mRNA expression of IL-36 receptor subunits in monocytes was comparable among the three groups(P>0.05).The DKD group had higher level of tumor necrosis factor-alpha(TNF-α)compared to the control and T2DM groups(P<0.05).The levels of PD-1 and CTLA-4 were lower in the DKD group than those in the control and T2DM groups(P<0.05).The proportion of monocyte-induced Vero cell death was significantly higher in the DKD group compared to the control and T2DM groups(P<0.05).After stimulation with recombinant human IL-36α,monocytes from DKD patients showed a significant increase in the secretion of granzyme B and TNF-α(P<0.05),as well as an increased proportion of monocyte-induced Vero cell death(P=0.024).Conclusion In DKD patients,elevated IL-36αand granzyme B levels in monocytes enhance monocyte function.