The Effect Of ERK/MAPK Signaling Pathway In The Differentiation Of Peripheral Blood Th17 Cells Of Patients With Pemphigus Vulgaris

BackgroundPemphigus vulgaris(PV)is an organ-specific autoimmune mucosal skin disease with a specific immunological basis.The main sign is producing the immunoglobulin G autoantibodies in the epidermal desmosome core.Anti-Desmoglein 3(Dsg3)antibodies cause the spinous layer to unravel and blister formation within the epidermis.The occurrence and development of this disease are closely related to the immune disorders of peripheral blood CD4+T cells.Previous studies had suggested that PV was an autoimmune disease dominated by T-helper 2 cells(Th2),but increased studies found that Th17 played an important role in the pathogenesis of PV.Retinoic acid orphan receptor yt(RORyt),as a specific transcription factor of Th17 cells,could be activated by the control of signal transducer and activator of the transcription(STAT)3.Thereby,Th17 cell differentiation is increased.The ERK/MAPK pathway could regulate Th cell differentiation,however,the role of regulating Th17 cells in different diseases is controversial.It has not been reported that ERK/MAPK pathway regulate the differentiation of Th17 cells in peripheral blood of PVPurposesDetermine the expression level of Th17-related factors and ERK/MAPK pathway in peripheral blood CD4+T cells of PV patients,and analyze its clinical significance.We further explore the role of Th17 in the pathogenesis of PV and the role of ERK/MAPK on the differentiation of Th17 cells in peripheral blood of PV.Method1.RT-qPCR was used to detect the expression of RORyt mRNA in peripheral blood CD4+T cells from PV patients and healthy controls.ELISA was performed to test the IL-17 serum level.The correlation between the expression levels of RORyt and IL-17 and the severity of disease in patients with PV were analyzed.Relevance and explore its clinical significance.2.Flow cytometry was used to detect the proportion of Th17 cells in peripheral blood mononuclear cells(PBMCs)in the patients with PV and healthy controls.3.We detected the expression levels of Th17-related factors and ERK/MAPK pathway molecules in peripheral blood CD4+T cells from PV patients and their healthy controls by WB.4.We used immunofluorescence to detected the protein expression and distribution of p-STAT3 and ERK/MAPK pathway molecules in CD4+T cells from peripheral blood of PV patients and healthy controls.5.Th17-cell differentiation in vitro.Flow cytometry was used to detect the proportion of Th17 cells without inhibitor and PD96059.RT-qPCR was used to detect the expression of RORyt mRNA.Result1.The increased expression of RORyt mRNA and IL-17 serum levels in PV patients and their correlation with disease severity.RT-qPCR analysis showed that the expression of RORyt/GAPDH relative mRNA in 14 PV patients was higher than that in 10 healthy controls(P=0.002).To determine whether the up-regulated RORyt mRNA expression levels were related to anti-Dsg3 antibody titers and ABSIS scores,we performed a spearman correlation analysis.The results showed that RORyt expression level was positively correlated with anti-Dsg3 antibody titer(r=0.561;P=0.0371)and ABSIS score(r=0.642;P=0.013).Subsequently,the serum IL-17 levels of 14 PV patients and 10 healthy controls were analyzed.The ELISA results showed that IL-17 serum levels were higher in PV patients than in healthy controls(P=0.028).There was also a significant positive correlation between IL-17 serum levels and anti-Dsg3 antibody titers(r=0.824;P<0.001),and ABSIS scores(r=0.814;P<0.001).2.Increased number of Th17 cells and the expression of Th17 lineage-related factors in the patients with P VFlow cytometry was used to measure the number of Th17 cells in PBMCs from 6 PV patients and 6 healthy controls.Compared with the healthy control group,the percentage of Th17 cells in PV patients was significantly higher(P<0.001).In addition,WB detected STAT3,p-STAT3,RORγt,and IL-17 protein levels in CD4+T cells.Compared with the healthy control group,the relative gray ratios of p-STAT3/GAPDH,RORγt/GAPDH,and IL-17/GAPDH in CD4+T cells of PV patients increased significantly(P<0.001;P=0.016;P=0.002).Further immunofluorescence staining showed that the accumulation of p-STAT3 protein in the cytoplasm of CD4+T cells was higher than that in control group..3.The expression of MAPK pathway protein in CD4+T cells in peripheral blood of PV patients increased.The relative gray ratio of p-MEK/GAPDH and p-ERK/GAPDH in peripheral blood CD4+T cells of PV patients increased significantly(P<0.001;P<0.001).Immunofluorescence staining futher confirmed that p-MEK1/2 and p-ERK1/2 protein accumulation in CD4+T cell cytoplasm of PV patients’ peripheral blood increased.The results of this study indicate that the activity of the ERK/MAPK signaling pathway in PV is enhanced.4.Inhibition of ERK/MAPK pathway could block the Th17 cell differentiation in vitro.In the presence of IL-6,TGF-β,IL-23,IL-1β and anti-IFN-γ/anti-IL-4 antibodies,CD4+T cells were induced to differentiate into Th17 cells in vitro and added to the cultured cells PD98059 or an equal volume of DMSO were used to determine the ratio of Th17 cell differentiation and RORyt mRNA expression in two groups of cultured cells.The results showed that when ERK activation was blocked,the number of Th17 cells decreased(P=0.004).In addition,the expression level of RORγt mRNA and IL-17 titers in the control group was also significantly higher than that in the experimental group.ConclusionIn summary,the present study indicated the important role of Th17 cells in the pathogenesis of PV.Aberrantly increased expression of ERK/MAPK proteins may lead to a deterioration of pathogenic Th17-type immunity and result in the pathogenesis of PV.MEK inhibitor PD98059-treated CD4+T cells from patients with PV could prevent Th17 cells differentiation in vitro.Further investigation into the mechanism may offer a novel idea to understand the pathogenesis of PV and could provide a novel potential molecular target for treatment of this disease.