| Background:Primary Sj?gren’s syndrome(pSS)is a chronic systemic autoimmune disease with the main clinical manifestations of dry mouth and dry eyes,and some patients may develop extra-glandular multi-organ involvement.The histologic hallmark of pSS is T and B cell infiltration into salivary and lachrymal glands.Among the infiltrated lymphocytes,CD4+T cells are the dominant cells,including Th1,Th17,follicular helper T(Tfh)effector cells.Th1 cells infiltrate in pSS salivary glands and primarily produce interferon-y and tumor necrosis factor-α,which induces salivary gland epithelial cell dysfunction by promoting fas-mediated apoptosis,altering tight junction function and increasing permeability.Th17 cells are the main subset that secretes interleukin-17 which have been used to establish a kind of animal model of experimental Sj?gren’s syndrome.Tfh cells are a specialized CD4+T pedigree,which is the most essential B cell helper cells.Tfh cells express the surface molecules such as C-X-C motif chemokine receptor 5(CXCR5),programmed cell death protein-1(PD-1),inducible T cell co-stimulator(ICOS),CD40 ligand(CD40L),and secrete IL-21,IL-4,CXCL13 et al.Tfh cells interact with B cells in the germinal center through direct connection,ligand-receptor interactions,or cytokines,promoting B cell activation,proliferation and differentiation,and autoantibody production.Tfh cell differentiation is mainly modulated by the nuclear transcription factor BCL6.The number and percentage of Tfh cells is increased in both the peripheral blood and salivary glands of patients with pSS and correlate with abnormal B cell differentiation and disease activity of pSS,suggesting that Tfh cells play an important role in the pathogenesis of pSS.Objective:This study aims to explore the molecular mechanisms that regulate the abnormal CD4+T cell activation and Tfh cell differentiation in patients with pSS from the perspective of naive CD4+T cells.Methods:We first examined the activation,proliferation,apoptosis and differentiation of pSS and healthy control(HC)naive CD4+T cells by flow cytometry.Then differentially expressed genes(DEGs)were identified using RNA sequencing of naive CD4+T cells from three pSS patients and three age and gender-matched HCs,which was confirmed by quantitative PCR in another independent pSS cohort.The pathway enrichment analysis of Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Gene Set Enrichment Analysis(GSEA),and protein-protein interaction network analysis were performed to help find the pivotal signaling pathways.DEGs were overexpressed in HC naive CD4+T cells and were silenced in pSS naive CD4+T cells using nucleofection to determine the genes regulating Tfh cells.ChIP-seq combined with ATAC-seq was performed to elucidate the underlying epigenetic mechanism whereby the gene regulated Tfh cells.Finally,we treated naive CD4+T cells with pSS-related cytokines to explore the regulatory factors of the target gene.JAK-STAT signaling pathway inhibitors were used to investigate the key molecules in this activated pathway.Results:pSS naive CD4+T cells had higher potentials of activation,proliferation,apoptosis,and differentiation towards Tfh cells.Thymocyte selection-associated high mobility group box(TOX)expression was upregulated in pSS naive CD4+T cells and promoted CD4+T cell activation and Tfh cell polarization.TOX silencing in pSS naive CD4+T cells downregulated expression of B cell lymphoma 6(BCL6)and altered levels of multiple Tfh-associated signature genes.ChIP-seq analysis implied that TOX bound to the BCL6 locus,where there was accessible region found by ATAC-seq.Interferon-αinduced TOX overexpression,which was attenuated by JAK and STAT1 inhibitors.Conclusions:Our data suggest that TOX in pSS naive CD4+T cells is upregulated which facilitates Tfh cell differentiation.Mechanistically,interferon-α induces TOX overexpression in naive CD4+T cells through JAK-STAT1 signaling pathway and TOX regulates BCL6 expression.Therefore,this study implies that the interferon-α-JAK-STAT1-TOX axis participates in the pathogenesis of pSS and might be new therapeutic target for patients with pSS.Background:Eosinophilic granulomatosis with polyangiitis(EGPA),formerly called Churg-Strauss syndrome,is the rarest type of antineutrophil cytoplasmic antibody(ANCA)-associated vasculitis.EGPA causes systemic,necrotizing,granulomatous inflammation of small and medium-sized vessels.Hallmarks of EGPA include asthma,eosinophilia in peripheral and tissues.EGPA affects various systems such as skin,respiratory system,kidneys,digestive system,heart,and nervous system.EGPA-associated cardiac and neurological lesions do great harm to patients and had high incidence and poor prognosis.However,systematic cohort study about cardiac and neurological involvement in Chinese EGPA population is currently lacking.Objectives:This study was aimed to analyze and summarize the clinical features,independent associated factors,treatment and outcomes of cardiac and neurological involvement in EGPA from a single-center Chinese cohort.Methods:A total of 110 EGPA patients were longitudinally consecutively enrolled and followed up who were admitted to our hospital from 2007 to 2019.We reviewed their clinical records and determined the independent factors associated with cardiac and neurological involvement using univariate and multivariate logistic regression.We calculate the optimal cut-off value for predicting the cardiac lesions by receiver operating characteristic curves and analyzed the cumulative survival rates by Kaplan-Meier curves.Results:1.Cardiac manifestations:Cardiac involvement was present in 36.4%(40/110)of EGPA patients,which mainly manifested as pericardial effusion(16.4%,18/110),myocardial involvement(13.6%,15/110),and heart failure(8.2%,9/110).The mean age of patients with cardiac manifestations was 42.1±14.23 years with no female/male predominance.Compared with the cardiac-unaffected group,the cardiac-affected group showed a lower rate of biopsy-proved vasculitis(0%versus 20%,p=0.002).The eosinophil count[odds ratio(OR)=1.142,95%confidence interval(CI)1.029-1.267]was independently associated with cardiac manifestations in EGPA,with a cut-off value of 3.66 X 109/L[area under the curve=0.692,p=0.001].Regarding treatment,the cardiac-affected group displayed a higher ratio of glucocorticoid pulse combined with intravenous cyclophosphamide(40%versus 21.4%,p=0.037),and intravenous immunoglobulin combined with glucocorticoid plus intravenous cyclophosphamide(17.5%versus 4.3%,p=0.035)than the control group.Outcomes(p=0.131)and survival(p=0.1972)were not significantly different between the two groups.2.Peripheral neuropathy(PN):PN occurred in 46.4%of EGPA patients.Among the patients with PN,paresthesia and muscle weakness were observed in 82%and 33%of patients,respectively.Both the upper and lower limbs were involved in 51%of patients.30%of EGPA patients had symmetrical multiple peripheral neuropathy,whereas only 16.4%presented with mononeuritis multiplex.Compared to patients without PN,patients with PN had a higher erythrocyte sedimentation rate,C-reactive protein,rheumatoid factor,Birmingham Vasculitis Activity Score(BVAS),and positivity of myeloperoxidase-antineutrophil cytoplasmic antibodies.Regarding manifestations,patients with PN tended to develop weight loss and arthritis or joint pain.Notably,ANCA positivity,arthritis or joint pain,and higher BVAS were found to be independently associated factors for PN in EGPA.Patients with PN more frequently need glucocorticoid pulse and intravenous cyclophosphamide.With the longest follow-up of 11.0 years,we found that age and cardiac involvement were risk factors for survival,and female was the protective factor.3.Central nervous system(CNS)involvement:CNS lesions were observed in 17.3%(19/110)of EGPA patients.Ischemic lesions were the most common manifestations,accounting for 63.2%of the 19 cases,followed by posterior reversible encephalopathy syndrome(36.8%),spinal cord involvement(15.8%),medulla oblongata involvement(15.8%),and intracranial hemorrhages(15.8%).Compared to the control group,patients with CNS involvement were of older age(51.7±11.56 vs.43.7±13.78 years,p=0.019)and had a higher ratio in the digestive tract involvement(52.6%vs.28.6%,p=0.042).Multivariate analysis revealed that age,disease duration,and fever were the potential independent risk factors for CNS involvement of EGPA.Glucocorticoids combined with cyclophosphamide were the basic strategic therapy(94.7%).Intrathecal injections of dexamethasone and methotrexate were administered to 21.1%of the patients.Although seven patients relapsed during glucocorticoid reduction,seventeen patients finally achieved clinical remission.One patient died of acute intracerebral hemorrhage within one month,and another patient died of gastrointestinal perforation.Outcomes and cumulative survival show no significant differences between the two groups.Conclusions:1.Cardiac involvement:In this single-center Chinese EGPA cohort,cardiac manifestations are observed in 36.4%of patients,which primarily presents as myocardial involvement,pericardial effusion,and heart failure,independently associated with eosinophil count.Glucocorticoid combined with cyclophosphamide is the treatment cornerstone for EGPA patients with cardiac manifestations.2.Peripheral neuropathy:PN in this EGPA cohort frequently displays with symmetrical multiple peripheral neuropathy.Positive ANCA,arthritis or joint pain,and higher BVAS are the independently associated factors of PN in EGPA.Glucocorticoids with immunosuppressants are vital therapeutic strategy.3.CNS involvement:CNS lesions are uncommon in EGPA with various manifestations,and ischemic lesions are the most frequent.Age,disease duration,and fever are independent factors associated with CNS involvement in EGPA.The therapy of glucocorticoids combined with cyclophosphamide and intrathecal injections yields favorable responses.Acute intracranial hemorrhage and gastrointestinal perforation may be the principal causes of death in EGPA. |
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