Single-cell RNA And ATAC Sequencing Revealed The Molecular Characteristics And Differentiation Tracks Of B Cells From Peripheral Blood In Systemic Lupus Erythematosus And Primary Sjogren’s Syndrome

BackgroundSystemic lupus erythematosus(SLE)and primary Sjogren’s syndrome(p SS)are common chronic and refractory autoimmune diseases in women.The simultaneous occurrence of these ADs in individual patients(multiple autoimmunity)and in members of a core family(family autoimmunity)suggests that they share a common etiology,including genetic,epigenetic factors,and sex hormones.SLE and p SS share many clinical manifestations,serological features,and immunological features,and the loss of tolerance of B cells,leading to the continuous production of autoantibodies and immune complexes,is critical to their pathogenesis.However,the pathogenesis of SLE and p SS is different.Traditional methods have neglected to investigate the mechanism at the single-cell level.The study of B lymphocytes in SLE and p SS using Single-cell sequencing analysis may help to reveal the important pathogenic mechanisms of SLE and p SS,identify new diagnostic markers and therapeutic targets,and provide evidence for precision medicine.ObjectiveIntegrated single-cell transcriptomic sequencing(sc RNA-seq)and single-cell assay for transposase-accessible chromatin sequencing(sc ATAC-seq)were used to define B lymphocyte subsets and to study their molecular characteristics and differentiation tracks in SLE and p SS.MethodsA case of SLE patient admitted to our hospital from October 2020 to August 2022 was selected,the clinical data of the patient was recorded,and the SLE Disease Activity Index was evaluated.Meanwhile,one patient with primary Sjogren’s syndrome was selected as the case control and one healthy volunteer was selected as the healthy control.30ml of peripheral venous blood was extracted from all subjects,peripheral blood mononuclear cells were extracted,and CD45~+CD19~+B lymphocytes were screened by flow sorting technique,single-cell suspension was prepared,and c DNA library was established.The original data were obtained using sc RNA-seq and sc ATAC-Seq.Arch R and SPSS23.0 software packages were used for bioinformatics analysis and statistical data processing,respectively.Results1.Our sc RNA-seq data identified eight distinct cell subsets.Naive B1 and Naive B2cell subsets are specifically amplified in the CD45~+CD19~+B cell composition of SLE and p SS.Naive B cells in p SS and SLE share down-regulated differential genes IGHA1,IGLC3,and IL4R.The most up-regulated differential genes in Naive B cells of SLE are interferon-related genes IFI44L,PLCG2 and IFI44.In Naive B cells of p SS,the most up-regulated differential genes were FOS,FOSB and CD69,which are related to the JUN family.2.Differentially expressed genes of Naive B cells in SLE and p SS were enriched to similar signaling pathways,both significantly enriched to TNF signaling pathway,Th17cell differentiation,Th1 and Th2 cell differentiation,Osteoclast differentiation,Leishmaniasis,IL-17 signaling pathway,and Cocaine addiction.3.As B cells differentiate,their functions become more refined and distinct.Our high-resolution data revealed the functional heterogeneity of different B-cell subsets.4.Integrated sc RNA-seq and sc ATAC-seq data revealed four distinct B-cell subsets in SLE and p SS,with a differentiation path of Naive B cells(NB),DN B cells(DNB),Activated memory B cells(AB),and Classical memory B cells(CMB).The characteristic of Naive B cell expansion was still present.5.Through analysis of transcription factors(TFs)motif enrichment and B-cell differentiation trajectories,we identified a group of TFs profiles that contribute to B-cell fate determination.Selective gain and loss of these TFs form a complex,iterative,and interacting process that may influence the differentiation and maturation of B-cell subsets.Conclusion1.In this study,we identified different B cell subsets of p SS and SLE,described the differences and similarities in the transcription profiles of Naive B cells amplified in the peripheral blood of p SS and SLE,and described the functional heterogeneity of different B cell subsets in SLE and p SS,providing a certain reference for the identification of p SS and SLE.2.We established an epigenetic map of B cell differentiation in p SS and SLE.In p SS and SLE,Naive B cells gradually differentiated into DNB cells under the regulation of a group of TFs profiles that contribute to B-cell fate determination.The results of our research improved understanding of B lymphocyte differentiation in SLE and p SS.