Altered Circulating Immune Cell Subsets In Kawasaki Disease Revealed By ScRNA-seq

Part Ⅰ Altered monocyte subsets in Kawasaki disease revealed by scRNA-seqBackground:Kawasaki disease(KD)is a type of systemic immune vasculitis,which is characterized by the activation of immune system with unknown etiology.Monocyte is an important member of the innate immune system which play a key role in the occurrence of vasculitis,however its role in KD is still elusive because of its extremely heterogeneous and complex functions.We infer that there may be some other unknown monocyte subsets that regulate the occurrence of KD.With the development of scRNAseq,the overall transcriptome characteristics of monocytes can be revealed at the singlecell level and the subtle differences between different cell subsets could be identified.Therefore,the monocyte subsets that changed in KD patients could be identified,which may provide more effective therapeutic targets.Aim:We aim to fully reveal the single-cell transcriptome map of monocytes,comprehensively delineate monocyte heterogeneity in healthy and KD infants.The changed monocyte subsets in KD patients will be identified.Methods:Peripheral monocytes were enriched from peripheral blood mononuclear cells(PBMC)by negative magnetic sorting techniques from 2 healthy infants and 2 KD patients.scRNA-seq was performed after performing single cell labeling.Different cell subsets were obtained by unsupervised clustering and enriched functions were analyzed.Differentiation states were analyzed by Monocle.SELL+CD14+CD16-monocytes of PBMC from healthy and KD infants were validated using flow cytometry.Results:Three monocyte subsets were identified in healthy infants,including CD 14++CD16-monocytes,CD 14+CD16+monocytes and CD 14+CD16++monocytes.The three monocyte subsets represent a linear differentiation by cell trajectory analysis,and their different functions were revealed by functional enrichment analysis.CD14++CD16-monocytes were associated with response to bacterium and neutrophil activation.Function of antigen processing and presentation,and response to virus were activated CD 14+CD 16+monocytes.Functions of cytokine,cell proliferation and inflammation were enhanced in CD14+CD16++monocytes.The subset,SELL+CD14++CD16-monocytes,which expanded in KD patients,were poorly differentiated and related to neutrophil activation.Conclusion:Circulating monocytes of infants are divided into three subsets with different functions,and a liner differentiation relationship were revealed.SELL+CD 14++CD16-monocytes tend to increase in KD patients,which will provide potential biomarkers for KD diagnosis and treatment.Part Ⅱ Altered PBMC subsets in Kawasaki disease revealed by scRNA-seqBackground:Although the pathogenesis of KD vasculitis is still unclear,activation of the immune system plays a crucial role in KD vasculitis.It has been revealed that the number and phenotype of circulating immune cells have changed,but the key cells or molecules that caused KD is still unclear.It is possible that some unknown immune cells or molecules that play a key role in KD were missed,as the research methods used before were difficult to explore the extremely complex immune system of human.On the other hand,scRNA-seq could reveal the overall transcriptome of immune cells at the singlecell level,so we can analyze the interaction between different immune cells and find the immune cell subsets that play a key role in KD.Aim:We aim to reveal the map of transcriptome of PBMC in healthy and KD children using scRNA-seq and find the changed cell subsets in KD.The communication relationship between different cell subsets will be analyzed and the key cell subsets in KD will be identified.Methods:PBMC were separated from peripheral blood samples of 3 healthy infants and 3 KD infants.scRNA-seq was performed to acquire the transcriptomic atlas of PBMC.Bio-information analysis was used to identify different cell subsets in PBMC and reveal their functions,and cellphoneDB was used to explore communication relationship between different cell subsets.Results:32 PBMC subsets were identified after scRNA-seq of 72857 PBMCs,including 20 T cell subsets,3 B cell subsets,3 NK cell subsets,5 myeloid cell subsets and 1 endothelial progenitor cell subsets.Classical B cell subset and CD3E+B cell subsets increased significantly,while COTL1+NK cell subsets decreased significantly in KD patients.FCGR3A+CSF1R+myeloid cell subsets communicate well with COTL1+NK cell subsets in KD.And the ligand/receptor interactions,CD74-MIF and CD74-COPA,highly expressed and enriched significantly.Conclusion:The map of transcriptome of PBMC in healthy and KD children were revealed for the first time.The percentages of two B cell subsets and one NK cell subset were different between KD patients and healthy children.A strong communication relationship between FCGR3A+CSF1R+myeloid cell subsets and COTL1+NK cell subsets were revealed.And the ligand/receptor interactions,CD74-MIF and CD74-COPA,between the two cell subsets was revealed in KD.