A Study On The Cell Clusters Differentiation Of PBMCs Derived Early Septic Patients Basing On Single-Cell Sequencing Technology

Background:Sepsis is a serious lifer-threatening organ dysfunction caused by infection,with a mortality rate of 22%-55%,which is mainly caused by immune dysfunction,including excessive inflammation and immunosuppression.Peripheral blood mononuclear cells(PBMCs)are the cells with a single nucleus in the peripheral blood,mainly including lymphocytes and monocytes.PBMCs are widely involved in immune function and inflammatory response,which are relatively stable in normal state and play an important role in maintaining the homeostasis of body.It is generally believed that the infection can alter significantly the components,gene expression and cell function of PBMCs,causes immune disorders and eventually lead to the occurrence and development of sepsis.A more in-depth and comprehensive understanding of the alterations of PBMCs under the condition of sepsis is conducive to the diagnosis and treatment of sepsis.Purpose:To characterize panoramically the alteration of PBMCs in sepsis at single-cell transcriptional level.Materials and Methods:We isolated PBMCs from 7 septic patients and 4 donors who met the inclusion criteria.Based on BD Rhapsody,PBMCs were generated by single cell RNA sequencing,cell types were clustered and identified by unsupervised clustering and annotation analysis,gene expression profiles and signaling pathways for each cell clusters are described and compared in both sepsis and normal groups.Results:In sepsis,PBMCs were profiled for 6 kinds of cell clusters:T-lymphocytes,B-lymphocytes,dendritic cells,natural killer cells and natural killer T cells.In the early stage of sepsis,the proportion of monocytes increased and other 5 cell clusters decreased or did not change significantly.Compared with the normal group,T lymphocytes,dendritic cells and monocytes significantly enhanced the activity of inflammatory response related pathway,while B lymphocytes obviously weakened the above pathway,and the related pathways about apoptosis were activated while the activity of proliferation was inhibited in both dendritic cells and monocytes,the alterations activity of bile acid metabolism,myogenesis were opposite in NK cells and NKT cells.The alterations of these pathways activity were directly reflected in the alterations of gene expression profiles.These cell clusters can be further clustered into 19 subsets by tSNE cluster,early sepsis caused alterations in the composition,gene expression profile and signal pathway of each cell subset,which were mainly related to the enhancement of inflammatory response and the inhibition of immune function,and there was significant heterogeneity between different subsets.Among them,in the 6 subsets of monocytes,the cell proportion of subset 7 and 11 was positively correlated with the clinical indexes PaO2/FiO2 and NEWS SCORE,In addition,the expression of some representative genes of each subset was closely related with the clinical indexes of sepsis,for example,S110a12 expression in subsets 6 and 7 was positively correlated with age and APACHE Ⅱ score,but negatively correlated with PaO2/FiO2 and SOFA score;NEAT1 expression of subset 7 was positively correlated with age,serum creatinine and APACHE Ⅱ score,but negatively correlated with SOFA score.Conclusion:The characteristic alterations of PBMCs composition,gene expression profile and signal pathway caused by early stage of sepsis were comprehensively described at the single-cell transcriptional level,revealing the distribution characteristics of differentiated subsets of monocytes in sepsis,which provides a unique perspective for understanding the progress of sepsis and clinical treatment.However,the differentiation pathways and their regulatory mechanisms of monocytes in sepsis had not been involved in this study,more in-depth researches are needed.