Single-cell Sequencing Combined With Public Databases To Mine The Mechanisms Of Systemic Sclerosis And Complications And Some Preliminary Verificatio

BACKGROUND:Systemic sclerosis(SSc)is an autoimmune disease characterized by autoimmunity,microangiopathy,skin and organs fibrosis,with heterogeneous clinical manifestations.The etiology and pathogenesis of SSc still remain unclear.Although lowdensity granulocytes(LDGs)have been shown to be involved in the pathogenesis of multiple autoimmune diseases,there is a lack of studies related to LDGs in the pathogenesis of SSc.METHODS:From October 2020 to November 2022,SSc patients and healthy controls(HCs)were recruited from the Department of Rheumatology and Immunology,Peking Union Medical College Hospital.First,it was confirmed whether peripheral blood LDGs were present in SSc patients using flow cytometry analysis and immunofluorescence.Then PBMCs from three primary dcSSc patients were collected for single-cell RNA sequencing analysis,and the transcriptome characteristics and possible pathogenic mechanisms of LDGs were explored in conjunction with the public database of HCs.RESULTS:A total of 20 SSc patients and 44 HCs were recruited in the first stage,with a male-to-female ratio of 3:17 and a mean age of 48±13 years old,and 44 healthy controls with a male-to-female ratio of 17:27 and a mean age of 41.3±17 years old,with no statistical difference between the two groups(p>0.05).Six patients(30%)were with family history of autoimmune diseases,including one with SSc;three patients(15%)had family history of tumors;11 patients had dcSSc,and 9 had lcSSc.In patients with skin involvement,the mRSS score was 2(2).In terms of clinical symptoms,the common clinical manifestations were raynaud’s phenomenon(1 5.75%),interstitial lung lesions(14.70%).acid reflux and heartburn(11.55%).capillary dilation(10.50%),arthritis/arthralgia(7,35%),and dysphagia and fingertip ulcers(5.25%each).Compared with healthy controls,the proportion of peripheral blood LDGs was significantly higher in SSc patients.LDGs from SSc patients had a higher capacity to generate neutrophil extracellular traps(NETs)than healthy controls and patients’ own normal density of neutrophils.Three patients with primary dcSSc were enrolled for single-cell sequencing of PBMCs,and single-cell sequencing data from public databases were combined for further analysis.Enrichment analysis suggested that the top 100 genes characteristically highly expressed in LDGs were mainly associated with NETs,phagocytic vesicles,superoxide anion production,respiratory burst,IL-17 signaling pathway,nuclear factor kappa-light-chainenhancer of activated B cells.The MCODE key subset of prompt genes is associated with the Glycation End product receptor(RAGE)binding,neutrophil chemotaxis,myeloid leukocyte activation,secretory granule membrane,respiratory burst,phagocytosis,external components of the plasma membrane,and enzyme activator activity.Conclusion:By using flow cytometry analysis and immunofluorescence,we found the presence of LDGs in the peripheral circulation of SSc patients and active production of NETs from LDGs.Based on single-cell sequencing of PBMCs from SSc patients and public database data,LDGs were found to be involved in the pathogenic mechanism of SSc possibly through RAGE/TLR4 binding to HMGB1,which promotes NETs generated by intracellular NADPH oxidase-dependent pathway.Our study enriches the cellular phenotype,transcriptome characteristics and possible pathogenesis of LDGs in SSc patients,and provides new ways for disease activity markers and therapy in SSc.Background:Systemic sclerosis(SSc)is an immune-mediated connective tissue disease with heterogeneous clinical manifestations.Although the pathophysiology of SSc is complicated and still unclear,previous studies indicated that monocyte/macrophage play an essential role in the pathogenesis of SSc.The purpose of this study is to explore the pathogenic effects of monocyte phenotype and pathways of programmed cell death(PCD).Method:Combined with the previous single-cell sequencing analysis results and public RNA databases,we analyzed the phenotype and transcriptome characteristics of monocytes in untreated patients with dcSSc from Peking Union Medical College Hospital,and investigated the type and role of the programmed death pathway in terms of gene expression and enrichment analysis,in combination with the results of single-cell sequencing and public RNA database.HCs and SSc patients were also recruited to verify the specific subtype of monocytes and their correlation with disease activity and clinical indicators.Result:Single-cell sequencing data from our center identified two key subpopulations of monocytes:CD 14+monocytes and CD 16+monocytes.There were significant gene expression differences between CD 14+monocytes and CD 16+monocytes compared to those from HCs.Enrichment analysis suggested that CD 14+monocyte transcriptome genes were closely associated with functions or pathways such as respiratory burst,cell differentiation,ficolin-1 granules,ROS production,NOD-like receptor signaling pathway and TLR pathway;CD 16+monocyte transcriptome genes were closely associated with T cell regulation,intercellular adhesion,antigen presentation,IL-17 signaling pathway,TLR pathways,NF-κB signaling pathway and ficolin-1 granules.In addition,the transcriptome enrichment analysis of the above cells was seen for programmed death pathways such as apoptosis,autophagy and necroptosis.Enrichment analysis of CD 14+monocytes in public database also showed apoptosis and necroptosis,and the expression levels of key genes of PCD-related pathways were different from HCs.Based on literature reports,we detected the proportion of CD14+CD16+monocytes and correlation with clinical indices in SSc patients and HCs,and found that the proportion of CD14+CD16+monocytes was significantly higher in SSc patients than in HCs,but there was no statistic correlation with disease activity and laboratory indices.Conclusion:The transcriptome enrichment analysis of key subpopulations of monocytes,CD 14+monocytes and CD 16+monocytes,in SSc patients showed pathways of PCD,like apoptosis,autophagy and necroptosis,and the analysis of public database data confirmed the existence of the above death pathways,and there were clear expression differences of key molecules related with PCD patheways,suggesting that in addition to apoptosis and autophagy,there may be other pathways of PCD in the peripheral monocytes of SSc patients,which play essential roles in the development of the disease.The proportion of peripheral CD 14+CD16+monocytes was higher in patients than in healthy controls,which might also play an important role in the pathogenesis of SSc,although there was no statistic correlation with disease activity,and inflammatory indexes.Background:Systemic sclerosis(SSc)is a rare autoimmune disorder characterized by autoimmunity,inflammation,vasculopathy,and fibrosis.Glycosylation of immunoglobulin G(IgG)has been shown to be linked to various autoimmune diseases.Nevertheless,the relationship between IgG glycosylation and SSc remains unknown.The purpose of this research is to get a better understanding of the features of IgG glycosylation in SSc as well as its connection with clinical and laboratory indices.Method:Patients with SSc and healthy controls(HCs)were recruited from the Rheumatology and Immunology Department,Peking Union Medical College Hospital,between April 2019 and January 2021.We gathered information regarding the demographics,clinical manifestations,laboratory test findings,and serum.In order to investigate the glycosylation of serum IgG,lectin arrays were utilized.Result:A total of 54 patients with SSc and 13 HCs were recruited.There were no differences in age,and gender between the two groups.Patients with SSc demonstrated statistically significant changes in specific lectins signal-to-noise(S/N)ratio of fucose,sialic acid,mannose,galactose,N-Acetylgalactosamine,N-acetyl-D-glucosamineand Galβ4GlcNAc,when compared with HCs.In SSc subgroups,26 lcSSc and 28 dcSSc were enrolled,there was no difference between the two groups in age or disease duration.In terms of clinical presentation,lcSSc patients had a higher proportion of arthralgia/arthritis(30.8%vs 3.6%,p=0.021)and for disease activity,compared to lcSSc patients,patients with dcSSc showed greater disease activity in terms of modified Rodnan skin score(mRSS,7.3 ± 5.8 vs 14.3±10.9,p=0.015),European Scleroderma Trials and Research Group disease activity index(6.2±3.7 vs 10.4±4.7,p<0.001)and Medsger disease severity(2.4±1.5 vs 3.9±1.7,p=0.002).For laboratory values,significant differences existed between the two groups in NLR,PLR,coefficient of variation of red cell volume Distributing Width,estimated glomerular filtration rate(eGFR),complement 4(C4)and fibrinogen.Lectin analysis revealed statistical differences in galactose Nacetylglucosamine and mannose-specific lectin S/N ratio.For correlation analysis,NLR,PLR,glomerular filtration rate,complement 4,and mRSS score were correlated with multiple sets of lectin signal-to-noise ratios.Based on the lectin S/N ratio,a binary logistic regression model was developed to predict SSc occurrence.Conclusion:IgG glycosylation was discovered to be associated to the illness status as well as the disease activity of SSc after conducting a comparative examination of serum IgG glycosylation levels in HCs and several subgroups of SSc patients at our center.Also,a binary logistic regression model was established based on S/N Ratio.The level of galactose-specific binding lectin GSLI-B4 S/N ratio was correlated with the risk of SSc development.Background:SSc is a disease characterized by extensive vasculopathy,autoimmunity and inflammation,and progressive fibrosis of the skin and internal organs.Genetic susceptibility,epigenetic effects,and environmental factors may play an important role in the development of the disease.SSc is one of the most prevalent autoimmune diseases associated with tumors,lung cancer being one of the most prevalent forms.However,the correlation between systemic sclerosis and lung cancer is still unclear,and this study seeks to investigate the potential molecular mechanism underlying this association.Method:Inpatients of SSc with tumors were enrolled from Peking Union Medical College Hospital between January 1983 and February 2023.Demographic and clinical data were collected and summarized.Simultaneously,gene expression profiles of lung tissue,GSE48149 pertaining to SSc with interstitial lung diseases and GSE101929 related to nonsmall cell lung cancer,were obtained from the Gene Expression Omnibus database,and common differentially expressed genes(DEGs)were identified.Then,protein-protein interaction(PPI)network was constructed using the STRING database,and DEGs were screened by Cytoscape software plug-ins MCODE and CytoHubba.Enrichment analysis of hub genes was conducted.The TRUUST database analyzed the transcription factors(TFs)controlling hub genes.Finally,hub genes and TFs were verified by gene expression profiles of array.Results:A total of 894 SSc inpatients were diagnosed in the center,including 63 patients with secondary tumors,the incidence rate was 7.0%.the average age of tumor occurrence was 53.3 ±13.1 years old.and the course of disease from the initial symptom of systemic sclerosis to the diagnosis of tumors was 13.2±9.6 years.Non-small cell lung cancer(NSCLC)is the most prevalent form of tumor(33.3%),with with interstitial lung diseases(ILD)complicating 67.0%of NSCLC patients.By analyzing data from gene expression profiles of array,we screened 134 DEGs,including 49 up-regulated and 85 down-regulated genes.PPI Network were constructed,and using MCODE and CytoHubba,5 clusters were identified,and 15 genes with the highest MCC score were deemed hub genes for subsequent analysis.Enrichment analysis of hub genes demonstrated that phosphatidylinositol 3-kinases(PI3Ks)-serine/threonine protein kinase(AKT)signaling pathway plays a critical role in the pathogenesis of both diseases.To verify hub genes,additional gene expression profiles of peripheral blood mononuclear cells(PBMCs)of SSc and NSCLC,GSE117928 and GSE39345,as well as lung tissue of SSc,GSE81292 and GSE39345,were used,and binary logistic regression models for the two diseases were constructed based on the hub genes,with good model prediction and classification.TRUUST database was used and identify 10 transcription factors(TFs),including transcription factor CP2,E2F transcription factor 1(E2F1),POU class 5 homeobox 1,CCAAT/enhancer binding protein β(CEBPB),and Y box binding protein 1,and validated by the original datasets,and found that the expression levels of CEBPB differed from the control group in both disease groups.Conclusion:Using public database,we determined that PI3K-AKT and CEBPB axes may represent a potential mechanism of association between SSc-ILD and NSCLC.S100A12,TYMS,and SFTPD expression levels in PBMCs play an important role in identifying NSCLC and SSc.The conclusions,which are based solely on variations in RNA levels and bioinformatics analysis,still require experimental confirmation.In addition,future large,well-designed epidemiological studies are required to confirm this conclusion.