To Explore The Relationship Between CD4~+T Cells And The Patients With Pemphigus Vulgaris Of Different Severity And The Mechanism Of SOCS3 Affecting Its Differentiation

Pemphigus vulgaris(PV)is an autoimmune bullous disease that occurs in the skin and mucous membranes,with erythema,intraepidermal blisters,and erosions as the main clinical manifestations.The disorder of immune function is one of the reasons for the pathogenesis of PV.Besides autoantibodies,the number of CD4+T cells and the proportion of their subsets were abnormal in PV patients.Therefore,in recent years,the research on PV has gradually shifted from humoral immunity to cellular immunity based on CD4+ T cells.However,it is still unclear whether changes in CD4+T cells can be used as a clinical indicator to evaluate the progression of PV disease severity;The possible mechanisms of the imbalance in the proportion of CD4+T cell subsets in PV and the relationship between their and acantholysis associated with disease activity is also unclear.Therefore,this study explores these questions by reviewing clinical cohorts and experiments in vitro.Part Ⅰ Assessment of clinical laboratory indexes as objective comprehensive indicators of patients with pemphigus vulgaris of different severity:A single-center retrospective studyObjective:Current evaluation systems for disease activity in patients with PV pay less attention to clinical laboratory indicators,and the changes of these indicators may be earlier than the appearance of skin lesions.However,the relationship between changes in these indicators and the degree of disease activity is unclear.Therefore,this study evaluated the utility of clinical laboratory indicators,mainly lymphocyte subpopulations,as objective indicators of disease activity in PV.Methods:Retrospective study including PV patients with active phase who had not received systematic use of glucocorticoids or had received minimal treatment(≤10mg/d prednisone acetate)at least two months,and had not received rituximab before admission to the Department of Dermatology of Affiliated Hospital between January 2013 and May 2022.PV patients were divided into three groups by disease activity according to the PDAI score:mild(0-8),moderate(9-24)and severe(≥ 25).The level of anti-Dsg antibodies,albumin to globulin ratio(A/G),blood lipids,and the percentage of lymphocyte subpopulations were collected.We assessed the correlations of quantitative variables by Spearman correlation(rs).Multivariable linear regression adjusted the variables associated with the disease activity of PV(PDAI score).Results:(1)A total of 211 PV patients’ medical records between January 2013 and May 2022 were included in this study.According to the PDAI score,the 211 medical records were divided into mild(21 cases,9.95%),moderate(124 cases,58.77%)and severe(66 cases,31.28%).(2)The level of anti-Dsgl(rs=0.356,P<0.001)and anti-Dsg3(rs=0.222,P=0.007),the count of neutrophil(rs=0.161,P=0.019)and platelet(rs=0.199,P=0.004),neutrophil-to-lymphocyte ratio(NLR,rs=0.155,P=0.024),and platelet-to-lymphocyte ratio(PLR,rs=0.210,P=0.002)were positively correlated with PDAI score.However,A/G(rs=-0.269,P<0.001),low density lipoprotein(rs=-0.142,P=0.047),the percentage of CD4+T cells(rs=-0.337,P=0.007)and CD4/CD8(rs=-0.277,P=0.030)were negatively correlated with PDAI score.(3)Multiple linear regression showed that the factors associated with PDAI score were higher the level of anti-Dsgl antibody(P=0.001),higher NLR(P=0.006),and lower A/G(P<0.001)and the percentage of CD4+T cells(P=0.042).Conclusions:(1)The decrease in the proportion of CD4+T cells may be related to the progression of disease activity during the onset of PV.(2)High the level of anti-Dsg1 antibody and NLR combined with low A/G,and the percentage of CD4+T cells could to explain the PDAI score.These findings might provide more a comprehensive and objective evaluation system to reflect the disease activity of PV by laboratory indicators.Part Ⅱ Low SOCS3 expression in CD4+T cells from pemphigus vulgaris patients enhanced Th1-and Th17-cell differentiation and exacerbated acantholysis via STAT activationObjective:In the first part of this study,we found that the decrease in the proportion of CD4+ T cell in PV patients was associated with an increase in disease severity.And previous studies have shown that the proportions of CD4+T subset cells and secreted cytokines are unbalanced during the progression of the disease.However,the mechanism causing this phenomenon and the relationship between the imbalanced proportion of CD4+T subset cells and acantholysis are not clear.This study looked for differential genes between CD4+T cells in PV patients and healthy controls,and explored the effect of differential genes on CD4+ T cell subsets and the relationship between unbalanced CD4+ T cell subsets and acantholysis.Methods:(1)In PV patients,the proportions of CD4+T cells(Th1,Th2,Th17 and Treg cells)in peripheral blood and the levels of secreted cytokines were measured.The correlation between the above contents and disease severity or anti-Dsg antibodies level was analyzed.(2)Detection and analysis of differential mRNA in CD4+T cells from PV patients and healthy controls(HCs)by next-generation sequencing.Suppressors Of Cytokine Signaling 3(SOCS3)were found to be underexpressed in CD4+T cells of PV patients,and this mRNA was further investigated.(3)The mRNA expressions of SOCS3,STAT1 and STAT3 in CD4+T cells of PV and HCs were measured by PCR.(4)Three shRNA interference sequences targeting SOCS3 gene were designed and synthesized,and transfected into primary CD4+T cells from HCs in vitro.The silencing efficiency of CD4+T cells in each group was verified by PCR at 72h after transfection,and the optimal shRNA interference sequences were screened.(5)SOCS3-knockdown primary CD4+T cells were prepared.The proportions of CD4+T cells(Th1,Th2,Th17 and Treg cells)and the levels of cytokines secreted by CD4+ T cells were detected by flow cytometry and ELISA at 72h,96h and 120h after transfection,respectively.PCR was used to detect the mRNA expression of STAT1 and STAT3 in CD4+T cells at 72h after transfection.The proteins levels of p-STAT1,STAT1,p-STAT3 and STAT3 in CD4+T cells at 72h after transfection were detected by WB.(6)The pathogenic IgG from the serum of PV patients was isolated and purified and coincubated with HaCaT cells to construct a cell model of acantholysis.The pathological effects of IgG fractions from PV patients(PV-IgG)on cultured HaCaT cells was evaluated by a dispase-based dissociation assay and immunofluorescence staining of Dsg3.(7)A coculture system of SOCS3-knockdown primary CD4+T cells with HaCaT cells was constructed in vitro.After two days of coculture,the CD4+T cells were collected for flow cytometry and WB analysis,and the supernatants of the CD4+T cells were collected for ELISA analysis.HaCaT cells were collected,inflammatory factors mRNA expression and acantholysis were assessed.Results:(1)In PV patients,the proportions of Th1(P=0.016),Th17(P=0.045)cells,Th17/Treg(P=0.042)and the levels of IFN-y(P=0.013)were significantly increased.(2)SOCS3 mRNA in CD4+T cells of PV patients was significantly decreased(P=0.008),whereas STAT1(P=0.043)and STAT3(P=0.004)mRNA were significantly increased.(3)72h after transfection,the mRNA expression of SOCS3 in shSOCS3-1,shSOCS3-2,and shSOCS3-3 groups were significantly lower than in shControl group.Among them,CD4+T cells transfected with shSOCS3-1 exhibited a>85%decrease in SOCS3 expression at the transcriptional level(P<0.001)compared with control transfected cells or cells transfected with a nontargeting shRNA control(shControl).Therefore,we chose shSOCS3-1-transfected CD4+T cells for further experimentation.(4)72h after transfection,compared with CD4+T-shcontrol group,the proportions of Th1(P<0.001),Th17(P=0.006)and Treg(P<0.001)cells were significantly increased in the CD4+T-shSOCS3-1 group.And after 120h transfection,the proportion of Th1 and Th17 cells increased significantly,and the increase ratios were 50.33%and 38.94%,respectively.(5)72h after transfection,compared with CD4+T-shcontrol group,the levels of IFN-y(P<0.001),IL-17(P=0.001)and TGF-β(P=0.004)were significantly increased in the CD4+T-shSOCS3-1 group.And the results after 120h transfection were consistent with the results after 72h transfection.(6)72h after transfection,compared with CD4+T-shcontrol group,the protein levels of p-STAT1(P=0.001)and p-STAT3(P=0.003),and the mRNA expression levels of STAT1(P=0.004)and STAT3(P=0.033)were significantly increased in the CD4+T-shSOCS3-1 group.(7)After coincubation of PV-IgG with HaCaT cells,compared with the HaCaT-PBS group and HaCaT-HC-IgG group,the numbers of cell fragments in the HaCaT-PV1-IgG group(P=0.001 and P=0.007)and HaCaT-PV2-IgG group(P<0.001 and P=0.001)were significantly increased.And the continuous intercellular fluorescence of Dsg3 was disrupted in the HaCaT-PV1-IgG group and HaCaT-PV2-IgG group.In addition,HaCaT-PV-IgG significantly increased IFN-γ(P<0.001,P=0.001)and IL-6 mRNA(P=0.001,P=0.036)than HaCaT-PBS and HaCaT-HC-IgG group.(8)In the coculture system,the changes of indexes related to Th1 and Th17 cells were mainly detected.The proportions of Th1(P<0.001)and Th17 cells(P<0.001),and the levels of IFN-y(P<0.001)and IL-17(P<0.001)were significantly higher in the CD4+T-shSOCS3-1+HaCaT-PV-IgG group than those in the other five groups.(9)In the coculture system,the number of cell fragments(P<0.001)in the HaCaT-PV-IgG+CD4+T-shSOCS3-1 group was significantly higher than those in the other five groups.However,the WB results showed that the Dsg3 proteins levels in the HaCaT-PV-IgG+CD4+T-shSOCS3-1 group were significantly lower than those in the other five groups(P<0.001).Conclusions:Low SOCS3 expression in CD4+T cells from PV patients leads to overactivation of STAT,which causes CD4+T cells to overdifferentiate into Th1 and Th17 cells.Additionally,PV-IgG-induced local inflammation in skin lesions,which is mediated by IFN-γ and IL-6,can aggravate this phenomenon.Furthermore,low SOCS3 expression in CD4+ T cells further exacerbates PV-IgG-induced acantholysis.Therefore,upregulating the expression of SOCS3 in CD4+T cells of PV patients and maintaining the balance of the IFN-γ/STAT1/SOCS3 and IL-6/STAT3/SOCS3 pathways can alleviate acantholysis in patients with PV.Based on the present results,we shifted the understanding of pemphigus from a classical Th2-mediated disease to a Th1/Th17 synergistic disease.