Dynamic Immune Ecosystem Of Dengue Infection Revealed By Single-cell Sequencing

Dengue infection disease is one of the most common human arboviral diseases worldwide,which can result in severe complications.A dysfunctional immune response in dengue infective patients is a recurrent theme impacting symptoms and mortality,but the heterogeneity and dynamics of immune infiltrates during dengue infection remain poorly characterized.Traditional bulk transcriptomics provides valuable tools in revealing the mechanisms of dengue infection,but the heterogeneity of single cell is obscured during the averaging process,and the emerging single-cell sequencing technology provides a valuable tool for investigating the underlying mechanism of viral infections.Objectives : In this study,we analyzed the dynamic changes of the immune micro-environment in different stages of dengue infection based on single-cell RNA sequencing technology.In this study,we identified the key regulators of dengue infection,and clarified the potential role of these regulators in different stages.The interaction analysis of immune cells identifies signaling pathways that play important roles in different stages of dengue infection and further addresses the dynamic alteration of the immune micro-environment during dengue infection.Methods: We first downloaded single cell RNA sequencing data of different stages of dengue infection from the Gene Expression Omnibus(GEO)database,including 4 normal control samples,2 fever samples and 4 severe samples,and all single cells were from peripheral blood.In all 13,127 single cells,immune cells were included in downstream analysis.Quality control,cell clustering,visualization,and identification of differential genes were completed using the R package Seurat,and functional enrichment analysis of cell-specific expressed genes was completed by GO enrichment analysis.The spearman correlation analysis between specifically expressed genes was used to identify co-expression modules in immune cells during dengue infection.We next used Single-Cell r Egulatory Network Inference and Clustering(SCENIC)to identify transcription factors that play important roles in different stages of dengue infection and performed functional enrichment analysis based on TF target genes shared across different infection stages.As cell-cell interactions play important roles in normal human organismal activities as well as complex diseases,we used the R package Cell Chat to resolve the dynamic changes of cell communication during dengue infection,and identified key signaling pathways associated with dengue infection.Results: We obtained single-cell RNA sequencing data for 8,796 immune cells,and after quality control,7,923 high-quality single-cell were retained.Analysis of immune cell components at different stages of dengue infection suggested that the patient’s immune microenvironment has been significantly changed during dengue infection.In particular,we found that c DC is mainly derived from Control and that B cells,monocytes and T cells were more enriched in Fever stage.By analyzing the expression program of celltype-specific genes during dengue infection,we found that these genes are significantly associated with immune functions and respond to viral invasion in a celltype-specific co-expression module manner,and these modules could be regulated by TFs such as PAX 5,IRF 7,KLF 4 and IRF 8.Hence,We next systematically identified the TFs playing key roles during dengue infection,and found that most of these TFs showed celltype-specific and stage-specific transcriptional activity.Although five TFs are transcriptionally active at all stages of dengue infection,they target different target genes at different dengue infective stages and thus suggest different biological functions.These results suggested that the transcriptional regulatory networks are rewired during dengue infection.Furthermore,we used Cell Chat to construct a complex immune cell interaction network during dengue infection and observed a closer interaction between immune cells during the phase of viral infection,especially in Severe.Furthermore,we compared altered interactions in signaling pathways at different stages of dengue infection,identifying that the IFN-II and CXCL signaling pathways play important roles during dengue infection.Conclusions:Through the comprehensive analysis of single-cell sequencing data of 10 patients associated with dengue infection,we comprehensively characterized the immune cell atlas during dengue infection and analyzed the dynamics of immune microenvironment.We identified the key regulators during dengue infection and the signal pathways associated with the interaction between immune cells through bioinformatics methods.Together,our comprehensive analysis of dynamic immune ecosystem of dengue infection provided novel insights for understanding the pathogenesis of and developing effective therapeutic strategies for dengue infection.