The Role Of IL-35 In The Pathogenesis Of Sarcoidosis

Background and Objective Our previous study found that imbalance of Treg/Th17 cell and Treg dysfunction may be one of the important causes of sarcoidosis.IL-35 is a novel anti-inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells,suppression of Th17 cell development and the induction of suppressive iTr35 cells(IL-35-producing Tregs).Therefore,we infer that Treg dysfunction and imbalance of Treg/Th17 cell is associated with the reduction of IL-35 secretion or abnormality of IL-35 introducing intracellular signaling.So,this study will investigate which cells of lymphocyte secret IL-35 and its expression.We will also investigate biological functions of IL-35 cell in sarcoidosis.The aim of the study is to search for the pathogenesis and a potential therapeutic target in sarcoidosis.Methods ELISA was adopted to measure plasma IL-35 levels in sarcoidosis patients(n=98)and normal controls(n = 98).The mRNA expression levels of p35,EBI3 and Foxp3 in peripheral blood mononuclear cells(PBMCs)were studied based on real-time quantitative PCR.The correlation between plasma IL-35 levels in patients with sarcoidosis and clinical parameters was analyzed by Pearson’s correlation analysis.The percentage of Treg cells in peripheral blood of patients with sarcoidosis and healthy controls was detected by flow cytometry,Meanwhile,the expression of IL-35 on CD4 + T cells,CD8 + T cells and Treg cells was detected by flow cytometry.Results The plasma IL-35 levels in sarcoidosis patients(43.56±11.77 pg/mL)was significantly lower than normal controls(55.18± 11.53 pg/mL),the difference was statistically significant(P<0.001).The plasma IL-35 levels in treated patients were significantly higher than untreated patients(47.38 ± 12.02 pg/mL vs 40.82 ± 10.89 pg/mL;P = 0.0059).The plasma IL-35 levels in sarcoidosis patients was positively correlated with the diffusing capacity of the lungs for carbon monoxide(DLCO%)predicted values(r = 0.76,P<0.001),but it showed no significantly correlation with other clinical parameters.The expression levels of EBI3 and Foxp3 mRNA in the sarcoidosis group were significantly lower than in the normal control group(P= 0.0016,P= 0.0391,respectively),while the expression level of p35 gene mRNA was not significantly different between the two groups(P=0.5816).There was a significant positive correlation between the relative expression of EBI3 and Foxp3 gene in sarcoidosis group and normal control group(r=0.786,P<0.001;r=0.730,P<0.001,respectively).There was a low positive correlation between p35 and Foxp3 gene expression in patients with sarcoidosis(r = 0.383,P= 0.037),However,there was no significant correlation between the two genes expression in the normal control group(r=0.208,P=0.271).Flow cytometry showed that the average proportion of Treg/CD4+ T cells in peripheral blood of 10 patients with sarcoidosis(5.82 ± 0.51%)was not significantly different from 10 healthy controls(5.71 ± 0.90%),(P＝0.7413).No IL-35-expressing cells were found in CD4 + T cells,CD8 + T cells and Treg cells.Conclusion The plasma IL-35 levels in patients with sarcoidosis patients was significantly reduced,and the plasma IL-35 levels were elevated by glucocorticoid and/or immunosuppressive agents therapy,suggesting that IL-35 may be involved in the inflammatory process of sarcoidosis and play an important role in the pathogenesis of sarcoidosis.The plasma IL-35 levels in sarcoidosis patients is positively correlated with the DLCO%predicted values,suggesting that it can be used as a biomarker for predicting the progression of pulmonary disease in sarcoidosis.The expression levels of EBI3 and Foxp3 mRNA in sarcoidosis group were significantly lower than normal control group,and the relative expression of the EBI3 and p35 gene was positively correlated with Foxp3.This suggests that a decrease in the number of Treg cells in peripheral blood of patients with sarcoidosis leads to a reduction in EBI3 and p35 gene production,resulting in a decrease in heterodimer formed by EBI3 and p35 and a final reduction in IL-35 synthesis.To confirm the above deduction,this study used flow cytometry to detect human T lymphocyte subsets,but did not find IL-35 positive cells,and this discrepancies may be associated with a small number of cells secreting IL-35 in the human T lymphocyte subsets,or that IL-35 may be secreted primarily by other cell subsets.