The Role Of Interleukin-35 In The Pathogenesis Of Systemic Sclerosis

Objective: To study the expression of interleukin(IL)-35 in serum and skin tissues of patients with systemic sclerosis(SSc),and to describe its potential association with SSc-related characteristics.To explore the role of IL-35 in the pathogenesis of SSc.Methods: The clinical data and specimens of 41 newly diagnosed patients with systemic sclerosis and 50 normal controls were collected.The levels of IL-35 in serum of SSc patients before and after treatment and normal control group were detected by enzyme-linked immunosorbent assay(ELISA).The levels of INF-gamma,IL-4,IL-17 A and IL-10 in serum of SSc patients before treatment and normal control group were detected by ELISA.Flow cytometry(FACS)was used to detect the proportion of Th1,Th2,Th17 and Treg cells in peripheral blood of SSc patients and normal controls.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the mRNA expression of IL-17 A,Epstein-Barr virus inducible gene 3(EBI3),IL-12 subunit alpha(P35),transforming growth factor(TGF)-beta 1,type I collagen,FOXP3,interleukin-12 receptor beta 2(IL-12 R beta 2)and gp130 in skin tissues of 6 SSc patients and 6 normal controls.The expression levels of IL-35 subunitEBI3 protein and P35 protein in skin tissues of 6 SSc patients and 6 normal persons were detected by immunohistochemistry.Western blot was used to detect the phosphorylation activation of IL-35 signal transduction and transcription activator(STATs)in the skin of 6 SSc patients and 6 normal persons.Finally,to analyze the correlation of serum IL-35 with Th1/Th2/Th17/Treg lymphocyte,their related cytokines(interferon-?,IL-4,IL-17 A,IL-10),skin sclerosis score(MRSS)and chest high resolution CT(HRCT)of SSc patients.To ananlyze the correlation of the IL-35 with TGF-beita1,Type I collagen and the skin MRSS score in the skin tissues of SSc patients.Result:1.ELISA results showed that compared with the normal control group,the serum IL-35 level in SSc patients increased significantly(48.86 ±30.87 vs.83.48 ±55.76,pg/ml)(P=0.006),and decreased significantly(83.48 ±55.76 vs.54.68 ±32.22,pg/ml)after 3 months of routine treatment(P < 0.001).Before treatment,there was no significant difference in serum IL-35 levels between lcSSc and dcSSc patients(93.26 ± 56.83 vs 76.55±55.14,pg / ml)(P = 0.696).2.ELISA results showed that compared with the normal control group,the serum level of IL-17 in SSc patients was significantly increased(17.72±5.28 vs.23.99 ±14.37,pg/ml)(P=0.002),the serum level of IFN-gamma was significantly decreased(20.46 ±7.56 vs.12.28 ±5.14,pg/ml)(P=0.032),and the serum level of IL-4 was significantly increased(18.95 ±8.75 vs.48.24 ±21.74,pg/ml)(P<0.001).There was no significant difference in serum IL-10 level(1.86 ± 1.10 vs.2.77±1.53,pg / ml)(P =0.152)between SSc patients and normal controls.Besides,Serum IL-35 level was positively correlated with serum IL-4level(r= 0.320,P= 0.041),but not with other cytokines detected in SSc patients.3.Serum IL-35 level was positively correlated with thoracic HRCT score(rho = 0.316,P= 0.044)in SSc patients before treatment,but not with skin MRSS score and disease duration.Serum IL-4 of SSc patients level was positively correlated with thoracic HRCT(rho = 0.424,P= 0.006).4.The results of flow cytometry showed that compared with the normal control group,the proportion of CD4+INF-gamma+Th1 cells in SSc patients was significantly decreased(12.23±4.64 vs.7.24±2.11,P=0.007),the proportion of CD4+IL-4+Th2 cells in SSc patients was significantly increased(2.47±0.89 vs.5.38±1.82,P=0.001),and the proportion of CD4+IL-17A+Th17 cells in SSc patients was significantly increased(0.93±0.28 vs.1.69±0.56,P=0.013).There was no statistical difference in the proportion of CD4+CD25+Foxp3+Tregs cells(4.01±1.07 vs.3.56±0.97,P=0.294)between SSc patients and normal controls.As a result,the ratio of Th1 cells to Th2 cells in SSc patients was significantly lower than that in normal control group,and the ratio of Th17 cells to Tregs cells had a trend to be higher than that in normal group.The serum IL-35 level of SSc patients was positively correlated with the proportion of Treg cells(r = 0.434,P= 0.005)and negatively correlated with the proportion of Th1 cells(r =-0.330,P= 0.035).5.RTFQ-PCR results showed that compared with the normal control group,the mRNA expression of IL-17 A in skin tissue of SSc patients was increased(1.02 ±0.24 vs.5.12±1.25,P=0.001),the mRNA expression of EBI3 was increased(1.04±0.33 vs.5.29±2.32,P=0.008),the mRNA expression of P35 was increased(1.01±0.17 vs.4.32±2.53,P=0.025),and the mRNA expression of FOXP3 was increased(1.04±0.32 vs.3.42±1.70,P=0.026),the mRNA expression of TGF-beta 1 was increased(1.13±0.29 vs.3.55±1.20,P=0.002),and the mRNA expression of type I collagen was increased(1.11±0.30 vs.4.21±1.78,P=0.007),while the mRNA expression of IL-12 R beta 2 and gp130 had no statistical difference between the patients with SSc and the normal control group.The mRNA expression of EBI3 was positively correlated with that of P35.The mRNA expression of EBI3 and P35 were positively correlated with the mRNA expression of type I collagen and had no correlation with the skin MRSS score.The mRNA expression of TGF-beta 1 was positively correlated with the skin MRSS score and the mRNA expression of P35 and type I collagen,and had no correlation with the mRNA expression of EBI3.6.Immunohistochemical results showed that compared with the normal control group,the IOD/Area value of EBI3 protein in skin tissue of SSc patients was significantly higher(0.378±0.038 vs.0.615±0.136,P < 0.001),and the IOD/Area value of P35 protein was significantly higher(0.343±0.034 vs.0.551±0.143,P=0.002).7.Western Blot results showed that compared with the normal control group,the phosphorylation level of STAT1 pathway in skin tissue of SSc patients was significantly increased(P-STAT1/GAPDH: 0.2221±0.0146 vs.0.4609±0.0665,P<0.05),and the phosphorylation level of STAT4 pathway was also significantly increased(P-STAT4/GAPDH: 0.1986±0.0276 vs.0.4749±0.0609,P< 0.05).Conclusion:1.The serum level of IL-35 in SSc patients increased significantly.The increased serum level of IL-35 was positively correlated with Th2-like cytokine IL-4 and the proportion of Treg cells,but negatively correlated with the proportion of Th1 cells.The serum levels of IL-35 and IL-4 were both positively correlated with the severity of pulmonary fibrosis in SSc patients.IL-35 mayplay a synergistic role with Th2-like cytokines in the pathogenesis of SSc pulmonary fibrosis.2.IL-35 may participate in the pathogenesis of SSc skin fibrosis through the synergistic effect with TGF-beta 1 by activating channel proteins STAT1 and STAT4.