The Effect And Mechanism Of TPH Cells In Dermatomyositis

Objective:Dermatomyositis(DM),which belongs to idiopathic inflammatory myopathies(IIM)is a heterogeneous disease characterized by symmetrical muscle weakness,elevated serum level of muscle enzymes,chronic inflammatory cell infiltration,muscle fiber degeneration and necrosis.Compared with healthy people,the capillaries in the muscle area of DM patients are usually less and abnormally thickened,presenting with hyperplasia and necrosis.Perhaps,these aberrant morphologic changes could lead to local ischemia that,in turn,could cause muscle fiber destruction resembling microinfarcts and perifascicular atrophy.Muscle biopsy shows that macrophages,CD4~+T cells,and a small number of B cells are the main components of infiltrative cells.A recent study on rheumatoid arthritis has revealed a unique subset of CD4~+T cells named T peripheral helper(T peripheral helper,Tph)cells that play a role as a Bcell helper in extranodal sites of rheumatoid joints.Numerous previous studies reported that abnormal numbers of circulating Tph cells in multiple autoimmune diseases.Moreover,it has been demonstrated that CX3CR1~+Tph-like cells infiltrate the inflammatory foci of Ig G4-related disease.These results suggest that Tph cells may be involved in the pathogenesis of autoimmune diseases.In this study,we aimed to investigate Tph cells in DM patients,and explore the role of such cells in the muscle involvement and its possible mechanism.Methods:1.Altered peripheral helper T cells in peripheral blood and muscule tissue of the patients with dermatomyositisFresh peripheral blood,muscle tissue and clinical data of patients with DM were enrolled in this study.Healthy persons matched with sex and age,and patients with non-IIM were collected as controls.The frequency of Tph cells were evaluated by flow cytometry.Expression of Tph cells and B cells was examined in muscle tissue by immunohistochemistry(IHC).The correlation of Tph cells in DM patients with clinical characteristics was determined.2.The effect and mechanism of CX3CR1~+peripheral helper T cells in dermatomyositisFresh peripheral blood,muscle tissue and clinical data of patients with DM were enrolled in this study.Healthy persons matched with sex and age,and patients with non-IIM were collected as controls.The frequency of CX3CR1~+Tph cells were evaluated by flow cytometry.Expression of CX3CR1~+Tph cells was examined in muscle tissue by IHC.The correlation of CX3CR1~+Tph cells in DM patients with clinical characteristics was determined.CD4~+T cells were separated from PBMC of DM patients and cultured in vitro,stimulated by recombinant human CX3CL1 protein of different concentrations(50 n M,100 n M)and JAK2 inhibitor baricitinib.The cell viability was detected by CCK8 assay.CD4~+T cells were stimulated by recombinant human CX3CL1 protein of different concentrations(50 n M,100 n M).The frequency of CX3CR1~+Tph cells were evaluated by flow cytometry.Furthermore,CX3CR1~+CD4~+T cells were separated from DM patients and cultured in vitro,stimulated by recombinant human CX3CL1(100 n M)and baricitinib.The frequency of IFN?~+CX3CR1~+Tph cells,the expression of phos-JAK2 and phos-STAT1 were detected by flow cytometry and Western Blot,respectively.CX3CR1~+CD4~+T cells were stimulated by recombinant human CX3CL1 protein of different concentrations(50 n M,100 n M).The expression of chemokines CX3CL1 was detected by ELISA.Co-culturing CX3CR1~+CD4~+T cells and B cells from DM patients,the chemotaxis of B cells was observed by migration transwell assay and flow cytometry.Results:1.Altered peripheral helper T cells in peripheral blood and muscule tissue of the patients with dermatomyositisFlow cytometry revealed that circulating Tph cells were decreased in peripheral blood of DM patients compared with healthy controls(HCs).However,muscular expression of Tph and B cells was upregulated in patients with DM compared to that in the controls by IHC.Interestingly,the increased B cells accumulated around Tph cells in infiltrated lesions.The frequency of circulating Tph cells was positively correlated with CD3~+T cells,CD4~+T cells,and CD8~+T cells,whereas negatively correlated with erythrocyte sedimentation rate(ESR),interleukin(IL)-6,and IL-10 levels.Furthermore,the abnormal circulating Tph cells in peripheral blood were recovered after glucocorticoid treatment.2.The effect and mechanism of CX3CR1~+peripheral helper T cells in dermatomyositisFlow cytometry revealed that circulating CX3CR1~+Tph cells were decreased in peripheral blood of DM patients compared with healthy controls.he frequency of circulating Tph cells was negatively correlated with lactic dehydrogenase(LDH)and IL-10 levels.CX3CR1~+Tph cells are the major infiltrating cells in muscle tissue of DM patients.Recombinant human CX3CL1 protein significantly increased the frequency of CX3CR1~+Tph cells,and the effect became more pronounced with increasing doses.In CX3CR1~+CD4~+T cells,recombinant human CX3CL1 protein significantly increased the frequency of IFN?~+CX3CR1~+Tph cells;the frequency of IFN?~+CX3CR1~+Tph cells decreased after the addition of JAK2 inhibitor baricitinib.Compared with the control group,the phosphorylation of JAK2 and STAT1 in CX3CL1 group upregulated significantly,and recovered after the addition of baricitinib.Co-culturing CX3CR1~+CD4~+T cells and B cells from DM patients,the chemotaxis of B cells was observed in CX3CL1 group by migration transwell assay.The recombinant human CX3CL1 protein increased the level of chemokines CXCL13 in CX3CR1~+CD4~+T cells culture supernatant.Conclusion:1.Altered peripheral helper T cells in peripheral blood and muscule tissue of the patients with dermatomyositis,the increased B cells accumulated around Tph cells in infiltrated lesions.These results indicate that Tph cells might be involved in the immunopathogenesis of DM and play a crucial role in the pathologic T–B cell interactions that exist in ELS.2.CX3CR1~+Tph cells,as the major Tph cells existing in the inflammatory muscle tissues of dermatomositis patients,on the one hand,promote the expression of IFN-?through the JAK2-STAT1 pathway;on the other hand,promote the chemotaxis of B cells by producing the chemokine CXCL13,which may provide new insights for the treatment and development of dermatomositis.