Exploring The Heterogeneity And Bactericidal Activity Of Macrophages By Single-cell RNA Sequencing

Macrophages are composed of a variety of specific subtypes,which play an important role in resisting pathogen invasion and regulating inflammation.Functional heterogeneity is the key to their response to various environments.However,the current phenotypic classification system does not reflect the complexity of macrophages in terms of immune activity and gene expression.In addition,when macrophages pretreated with specific components of bacteria are infected by bacteria,their ability to kill bacteria will be enhanced,but the mechanism of the enhancement is unclear.We used mouse bone marrow to induce differentiation to obtain macrophages,and set up an experimental group and a control group.The experimental group was pretreated with BLP(bacterial lipoprotein)for 24 hours,and the control group had no pretreatment.Then the two groups of macrophages were simultaneously infected with Staphylococcus aureus,the macrophages were harvested at 0 h(no bacterial infection),1 h,3 h and 6 h after bacterial infection,and the single cell transcriptome sequencing(scRNA-seq)was performed.By analyzing the scRNA-seq data of approximately 80,000 macrophages,we obtained 13 macrophage subtypes and revealed a series of macrophage activation states,the complexity of which is higher than the traditional M1 and M2 polarization states.Some subtypes are consistent with previous findings,while others were first identified in our study.After that,we compared the gene expression patterns of the experimental group and the control group based on the defined macrophage subtypes,detailed their functional differences in each subtype,and identified some genes(Marco,Slpi,Acp5,Fpr2,Lcn2,Saa3)that may be related to the macrophages capacity to clear bacteria.Finally,we compared the two groups of macrophages on the horizontal and vertical axes of time,and found that the experimental group had two specific macrophage subtypes,which were directly caused by the pretreatment of BLP.During the entire stage of S.aureus infection,the immune response of macrophages was increasing,the number of subtypes would decrease,and other subtypes would be transformed into several major functional subtypes.In summary,this study provided a single-cell map of mouse macrophages with S.aureus infection and we discovered new macrophage subtypes.The mechanism of enhanced bactericidal ability of macrophages after BLP pretreatment was explored from the single cell level.