ABSTRACT
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-36β level was notably higher in the MG group compared with the control group [(73.43±13.91) pg/ml vs (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/ml vs (65.37±8.90) pg/ml, t=7.59, P<0.001] and FoxP3 mRNA expression (1.03±0.14 vs 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/ml vs (33.09±11.48) pg/ml, t=2.44, P=0.017] and RORγt mRNA expression (1.26±0.16 vs 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 (all P>0.05). There were no statistically significant correlations between above indices and quantitative myasthenia gravis (QMG) score (all P>0.05). Recombinant IL-36β stimulation 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/ml vs (56.96±13.73) pg/ml, t=2.36, P=0.023] and FoxP3 mRNA expression (0.99±0.17 vs 1.18±0.13, t=4.01, P<0.001), but did not affect Th17 cell percentage, IL-17 secretion or RORγt mRNA expression (all P>0.05). Recombinant IL-36β stimulation inhibited immunosuppressive activity of Tregs, which presented as enhanced cellular proliferation [(0.83±0.12)×10 5vs (0.69±0.15)×10 5, t=3.02, P=0.005] and reduced IL-35 secretion [(28.71±10.08) pg/ml vs (37.12±10.47) pg/ml, t=2.39, P=0.023]. Conclusion:Increased IL-36β contributed to the regulation of Tregs/Th17 cell balance probably through inhibition of Tregs function in MG patients.