Comparative Analysis Of Peripheral Blood Immunoinflammatory Landscapes In Patients With Acute Biliary Tract Infection And Its Secondary Septic Shock Using Single-Cell RNA Sequencing

Objective:Acute biliary tract infection(ABTI)is an infectious disease of the biliary tract,which has potential lethality.Septic shock secondary to ABTI is characterized by acute onset,rapid progress and high rate of mortality.The pathogenesis of septic shock is intricate,and immune dysfunction is its important pathogenesis.A comprehensive immune evaluation of septic shock is a prerequisite to study its immune mechanism.In this study,the single-cell transcriptome sequencing technology(sc RNA-seq)was employed to compare the types and functions of peripheral blood immune cells of patients with ABTI and its secondary septic shock,thus providing a theoretical reference for the pathogenesis of ABTI with varying degrees of severity.Methods:3 peripheral blood samples were collected from acute biliary tract infection and its secondary septic shock,respectively.Peripheral blood mononuclear cells(PBMCs)suspension was prepared.We perform sc RNA-seq to obtain sequencing data,and carry out quality control,cell clustering analysis and labeling annotation successively on the data data.We compare and analyze differentially expressed genes(DEGs)of the two groups of immune cells and their subpopulations.In addition,gene ontology(GO)functional enrichment analysis was carried out on DEGs.Pseudo-time series analysis was carried out on neutrophils and T lymphocyte subpopulations in the two groups in the differentiation process.We analyze the expression differences of major signaling pathways between the two groups and explore the interaction relationship between major immune cells ultimately.Results:Through sc RNA-seq,this study has identified seven cell types and found that the functional enrichment results of up-regulated DEGs between most immune cells in septic shock were correlated with neutrophil activation and chemotaxis,indicating that neutrophils play a crucial role in the occurrence and development of septic shock.In this study,neutrophil progenitors(proNeus)were identified in the analysis of neutrophil subsets.The differentiation direction of proNeus to mature neutrophil-7 subset in peripheral blood was determined;Compared with patients suffering from ABTI,the gene phenotypes(ELANE,AZU1,and PRTN3)were significantly up-regulated in septic shock.In addition,the immunological characteristics of neutrophil subsets in these two kinds of patients were quite different.The number and function of each cell subset altered significantly.Moreover,the mutual effect between neutrophils and other types of immune cells was significantly different.Compared with patients with ABTI,the proNeus of patients with septic shock presented a state of "expansion",with upregulation of neutrophil degranulation,phagocytose,chromosome aggregation,and downregulation of the immune functions such as monocyte proliferation,T lymphocyte activation and proliferation.The neutrophil-7 subpopulation in septic shock is in a state of exhaustion,and its biological function shows the characteristics of weakening neutrophil migration,chemotaxis,antigen extraction and phagocytosis,etc.,and promotes the formation of septic shock immunosuppression,which makes infection difficult to control and aggravates the development of septic shock as a result.Analysis of monocytes and T lymphocyte subpopulations shows that the expression genes and biological functions of cell subpopulations in the two groups are closely related to pro-inflammatory response,anti-inflammatory response and immune regulation,and have significant differences.In addition,CXCL1-CXCR2 and other ligand-receptors are highly expressed in neutrophils and monocytes,enhancing interactions between immune cells.Conclusions:Through the screening and analysis of single-cell transcriptome sequencing data,this study found that there were significant differences in cell subtypes and molecular characteristics between ABTI and its secondary septic shock.Furthermore,this study also found that proNeus and other cell subtypes played a crucial role in the immune inflammation of septic shock,revealed the differentiation trajectory and biological function of neutrophils,and explored the mutual effect between main cell types.In this regard,it provided a reference for accurately evaluating the pathological severity of patients with septic shock and discovering the targets for therapy.