The Role Of Interleukin-35 In Patients With Myasthenia Gravis

Objectives Myasthenia gravis(MG)is a chronic inflammatory autoimmune disease characterized by the presence of antibodies that target different protein components of the neuromuscular junction.Antibodies against the acetylcholine receptor(ACh R)are present in 85% of patients with MG.The interaction between T cells,B cells and cytokines plays an important role in the immunoinflammatory response in MG.Interleukin(IL)-35,a newly discovered member of the IL-12 family,downregulates the inflammatory response by inhibiting the differentiation of T helper cells and secretion of proinflammatory cytokines.The role of IL-35 in MG has not been reported yet.This study aimed to explore the role of IL-35 in MG through serum,cellular,and protein levels.Methods Part ? Forty-one patients with MG in acute phase were enrolled in the study.35 ageand sex-matched healthy controls(HCs)were enrolled.The clinical data,Myasthenia Gravis Foundation of America(MGFA)postintervention status were collected.The levels of IL-35 in peripheral blood of MG patients were detected by enzyme linked immunosorbent assay(ELISA).Flow cytometry was used to detect the proportion of T and B cell subsets in MG patients.Among the 24 MG patients with Minimal Manifestations(MM)-3,the levels of IL-35 and the proportion of T,B cell subsets in peripheral blood of MG patients before and after treatment were evaluated.Part ?I Forty-one patients with MG and 35 age-and sex-matched HC were enrolled in the study.The proportion of IL-35 secreting T and B cells was analyzed by flow cytometry,and then the proportion of IL-35 producing T and B cells between different subtypes of MG was compared.Eight patients with MG before treatment and eight ageand gender-matched HC were randomly selected.The CD4+ T cells were sorted out by flow cytometry.Then IL-35 interventions were given to MG group.Whether IL-35 can be positively feedbacked to IL-35 secreting T cell could be detected by PCR and flow cytometry.Part ? Six patients with MG before treatment were randomly selected.CD4+ T cells were sorted out by flow cytometry,then divided into 3 groups including control group Th17 differentiation group and IL-35 intervention group.The effect of IL-35 on Th17 cell differentiation was detected by PCR and flow cytometry.In this section,we aimed to clarify the effect of IL-35 on Th17 in MG and its possible mechanism.Results Part ? 1.The level of IL-35 in peripheral blood of MG patients before treatment was lower than that of HC(1.94 ± 1.78ng/m L,4.53 ± 3.35ng/m L)(p = 0.040);The level of IL-35 in generalized MG(GMG)patients(1.04 ± 1.15ng/m L)was lower than that of ocular MG(OMG)(2.75 ± 2.38ng/m L)(p = 0.002).The level of serum IL-35 in MG patients with thymoma(0.61 ± 0.41ng/m L)was lower than that without(2.11 ± 2.01ng/m L)(p = 0.023).2.After treatment,the level of IL-35 in MG patients with MM-3(5.17 ± 2.71ng/m L)was significantly higher than that before treatment(1.19 ± 1.09ng/m L)(p < 0.001).3.The percentage of CD19+IL-10+B cell in patients with MG before treatment was lower than that in HC(p = 0.008).The proportion of CD19+IL-10+B in 24 MG patients after treatment(MM-3)was significantly higher than that before treatment(p <0.001).4.The proportion of CD4+CD25+ T in patients with MG before treatment was lower than that in HC(p < 0.001).The proportion of Th1 and Th17 cells in patients with MG before treatment was significantly higher than that in HC(p = 0.039,p <0.001).The proportion of CD4+CD25+ T cells increased in 24 MG after treatment(p = 0.013).While,the proportion of Th17 cells after treatment decreased(p = 0.006).5.Correlation analysis between serum IL-35 levels and clinical data in MG showed that: the serum level of IL-35 in MG patients was negatively correlated with QMG(r = 0.002,p =-0.465)and MG-ADL score(r =-0.361,p = 0.020);IL-35 level was negatively correlated with Th17 cells(r =-0.335,p = 0.031)and positively correlated with CD19+IL-10+ B cells(r = 0.372,p = 0.017).6.The serum level of IL-17 in MG patients was significantly higher than that in HC(77.28 ± 27.1 pg/m L)(42.57 ± 13.49 pg/m L)(p < 0.001);GMG patients(90.51 ± 25.7 pg/m L)was significantly higher than that in OMG patients(58.61 ± 16.10 pg/m L)(p< 0.001);Patients with thymoma(100.48 ± 22.54 pg/m L)was higher than that without(71.66 ± 25.33 pg/m L)(p = 0.005).The level of serum IL-17 in MG patients after treatment(MM-3)was lower than that before treatment(p = 0.002).There was a significant negative correlation between serum IL-35 and IL-17 in patients with MG(r =-0.514,p = 0.001).Part ?I 1.The proportion of CD4+CD25+IL-35+ cells in MG patients before treatment was lower than that in HC(p < 0.001),and GMG lower than OMG(p = 0.003);Patients with thymoma was lower than that without(p = 0.001).After treatment,the proportion of CD4+CD25+IL-35+ cells increased compared with before treatment(p <0.001).2.The proportion of CD19+IL-35+ in MG patients before treatment was lower than that of HC(p < 0.001);GMG patients was lower than that in OMG patients(p = 0.001);Patients with thymoma was lower than that without(p = 0.008);and the proportion of CD19+IL-35+ cells in MG patients after treatment(MM-3)increased compared with before treatment(p = 0.001).3.The level of EBI3 and P35 m RNA in MG after IL-35 intervention was significantly higher than that in MG without IL-35 intervention and HC group(p < 0.001,p < 0.001,p <0.001,p <0.001),and the percentage of CD4+CD25+IL-35+ T cells was higher than that of the non-intervention group and the HC group(p < 0.001,p =0.004).Part ? 1.The level of IL-17 A and ROR?t m RNA in the differentiated group was higher than that in the blank group and IL-35 intervention group(p < 0.001,p = 0.004,p < 0.001,p < 0.001).There was no significant difference in IL-17 A or ROR?t m RNA between the IL-35 intervention group and the blank group(p = 0.096,p = 0.392).2.The percentage of CD4+IL-17A+ T cells in the differentiated group was significantly higher than that in the blank group and the IL-35 intervention group(p < 0.001,p = 0.001).There was no significant difference in the percentage of CD4+IL-17A+ T cells between the IL-35 intervention group and the blank group(p = 0.284).3.IL-35 may play an immunosuppressive role in MG by activating the signaling pathway of STAT1 to inhibit the differentiation of Th17 cells.Conclusions1.The imbalance of T,B cell subsets,especially the T cell subsets play an important role in the progression and prognosis of MG.IL-35 may play an important role in the prognosis of MG.The reduction of IL-35 in acute phase of MG might be related to the decrease of Treg and Breg.IL-35 may play an immunosuppressive role through the regulation of Th17 cell.2.The proportion of IL-35 secreting T and B cells in acute phase of MG was consistent with the change of IL-35 cytokine,suggesting that the imbalance of IL-35 producing T and B cells was involved in the mechanism of MG.IL-35 could play an immunosuppressive role through the positive feedback on CD4+ T cells to further promote the release of IL-35.3.IL-35 in MG could inhibit the differentiation of Th17 cells,which may be through activation of STAT1 signaling pathway.