重症肌无力患者白细胞介素-36表达及其对调节性T细胞和Th17细胞平衡的调控

Expression of interleukin-36 and its modulation on the balance between regulatory T cells and Th17 cells in patients with myasthenia gravis

目的观察重症肌无力(MG)患者白细胞介素(IL)-36的表达,研究IL-36对MG患者调节性T细胞(Tregs)和Th17细胞的调控作用。方法入组2016年7月至2021年8月新乡市中心医院就诊的MG患者51例(MG组)和健康对照者25名(对照组),采集外周血,分离血浆和外周血单个核细胞(PBMCs),采用酶联免疫吸附试验检测血浆IL-36α、IL-36β、IL-36γ、IL-36RA、IL-35、IL-17水平,流式细胞术检测Tregs和Th17细胞比例,实时定量聚合酶链反应法检测叉头盒蛋白3(FoxP3)和维甲酸相关孤独核受体γt(RORγt)mRNA表达。用重组IL-36β(5 ng/ml)刺激MG患者的PBMCs或纯化的Tregs,分析Tregs和Th17细胞比例、IL-35和IL-17分泌、FoxP3和RORγt mRNA表达、Tregs抑制活性的变化。结果IL-36α、IL-36γ、IL-36RA水平在对照组和MG组之间的差异均无统计学意义(均P>0.05)。MG组IL-36β水平高于对照组[(73.43±13.91)pg/ml比(60.91±12.65)pg/ml,t=3.79,P<0.001)。MG组Tregs比例[(4.67±1.33)%比(6.32±1.81)%,t=4.48,P<0.001]、IL-35水平[(50.06±7.93)pg/ml比(65.37±8.90)pg/ml,t=7.59,P<0.001]、FoxP3 mRNA(1.03±0.14比1.57±0.46,t=7.78,P<0.001)低于对照组,而Th17细胞比例[(1.05±0.15)%比(0.94±0.21)%,t=2.61,P=0.011]、IL-17水平[(40.61±13.13)pg/ml比(33.09±11.48)pg/ml,t=2.44,P=0.017]、RORγt mRNA(1.26±0.16比1.03±0.13,t=6.08,P<0.001)高于对照组(均P<0.05)。上述指标在不同性别之间、早发型和晚发型之间、非胸腺瘤组和胸腺瘤组之间、不同Osserman分型之间的差异均无统计学意义(均P>0.05),与定量重症肌无力(QMG)量表评分均无明显相关性(均P>0.05)。重组IL-36β对MG患者PBMCs增殖无影响(P=0.248),可降低Tregs比例[(3.05±0.66)%比(4.18±1.07)%,t=4.23,P<0.001]、IL-35分泌[(48.12±10.93)pg/ml比(56.96±13.73)pg/ml,t=2.36,P=0.023]和FoxP3 mRNA表达(0.99±0.17比1.18±0.13,t=4.01,P<0.001),但对Th17细胞比例、IL-17分泌和RORγt mRNA表达无影响(均P>0.05)。重组IL-36β刺激的Tregs免疫抑制活性减弱,表现为细胞增殖升高[(0.83±0.12)×10^(5)个比(0.69±0.15)×10^(5)个,t=3.02,P=0.005]和IL-35分泌降低[(28.71±10.08)pg/ml比(37.12±10.47)pg/ml,t=2.39,P=0.023]。结论MG患者中升高表达的IL-36β可能主要通过抑制Tregs功能调控Tregs/Th17细胞平衡。

Objective To investigate interleukin(IL)-36 expression in patients with myasthenia gravis(MG),and to study the modulatory function of IL-36 on regulatory T cells(Tregs)and Th17 cells in MG patients.Methods Fifty-one MG patients(MG group)and 25 healthy controls(control group)were enrolled in this study in Xinxiang Central Hospital between July 2016 and August 2021.Peripheral blood was collected.Plasma and peripheral blood mononuclear cells(PBMCs)were isolated.Plasma IL-36α,IL-36β,IL-36γ,IL-36RA,IL-35,and IL-17 levels were measured by enzyme-linked immunosorbent assay.The percentages of Tregs and Th17 cells were measured by flow cytometry.Forkhead box protein P3(FoxP3)and retinoid-related orphan receptor gamma t(RORγt)mRNA expressions were measured by real-time polymerase chain reaction.PBMCs or purified Tregs from MG patients were stimulated with recombinant IL-36β(5 ng/ml).Changes of Tregs and Th17 cell percentages,IL-35 and IL-17 secretions,FoxP3 and RORγt mRNA expressions,as well as immunosuppressive activity of Tregs were analyzed.Results There were no statistically significant differences of IL-36α,IL-36γ,or IL-36RA between the control group and the MG group(all P>0.05).IL-36βlevel was notably higher in the MG group compared with the control group[(73.43±13.91)pg/mlvs(60.91±12.65)pg/ml,t=3.79,P<0.001].Treg percentage[(4.67±1.33)%vs(6.32±1.81)%,t=4.48,P<0.001],IL-35[(50.06±7.93)pg/mlvs(65.37±8.90)pg/ml,t=7.59,P<0.001]and FoxP3 mRNA expression(1.03±0.14vs 1.57±0.46,t=7.78,P<0.001)was lower,while Th17 cell percentage[(1.05±0.15)%vs(0.94±0.21)%,t=2.61,P=0.011],IL-17[(40.61±13.13)pg/mlvs(33.09±11.48)pg/ml,t=2.44,P=0.017]and RORγt mRNA expression(1.26±0.16vs 1.03±0.13,t=6.08,P<0.001)was higher in the MG group(P<0.05).There were no statistically significant differences of above indices between different genders,onset ages,afflicting with thymoma,or different Osserman types(allP>0.05).There were no statistically significant correlations between above indices and quantitative myasthenia gravis(QMG)score(allP>0.05).Recombinant IL-36βstimulation did not affect PBMCs proliferation in MG patients(P=0.248),and reduced Tregs percentage[(3.05±0.66)%vs(4.18±1.07)%,t=4.23,P<0.001],IL-35 secretion[(48.12±10.93)pg/mlvs(56.96±13.73)pg/ml,t=2.36,P=0.023]and FoxP3 mRNA expression(0.99±0.17vs 1.18±0.13,t=4.01,P<0.001),but did not affect Th17 cell percentage,IL-17 secretion or RORγt mRNA expression(allP>0.05).Recombinant IL-36βstimulation inhibited immunosuppressive activity of Tregs,which presented as enhanced cellular proliferation[(0.83±0.12)×10^(5) vs(0.69±0.15)×10^(5),t=3.02,P=0.005]and reduced IL-35 secretion[(28.71±10.08)pg/mlvs(37.12±10.47)pg/ml,t=2.39,P=0.023].Conclusion Increased IL-36βcontributed to the regulation of Tregs/Th17 cell balance probably through inhibition of Tregs function in MG patients.