Investigation Of Pulmonary Immune Cell Response Profile And CD8~+T Cell Apoptosis Mechanism In Porcine Contagious Pleuropneumonia

Porcine contagious pleuropneumonia(PCP),caused by Actinobacillus pleuropneumoniae(APP),is a highly infectious respiratory disease commonly seen in pig farming facilities.It is characterized by hemorrhagic or necrotic fibrinous pneumonia and is associated with a high mortality rate.A comprehensive and systematic study on the immune response characteristics and immunopathological damage mechanisms of lung cells after infection with APP is still lacking.Therefore,analyzing the cellular immune response map of APP-infected pig lungs and investigating the molecular characteristics and mechanisms of pathological injury holds significant importance for the development of APP vaccines.In this study,we have established an APP infection model using Rongchang piglets,where lung tissues from the infected group(n=3)and control group(n=3)are collected.Single-cell suspensions are prepared for single-cell RNA-sequencing(scRNA-seq)analysis.A total of 59,062 cells were obtained and classified into 18 cell clusters.Among them,fibroblast-like cell clusters,epithelial-mesenchymal transition(EMT)-epithelial cell clusters,and T cell clusters emerged as the major components.Following APP infection,there was a significant abnormal increase in the number of fibroblast-like cells,accounting for over 50%of the total cell population in each sample.In contrast,the proportion of EMT-epithelial cells and T cells dropped significantly,indicating a marked disruption of the cellular homeostasis in lung tissues post-APP infection,leading to substantial damage to EMT-epithelial cells and T cells.Further analysis revealed that after APP infection,the lung experienced a significant enrichment of monocytes,and neutrophils cells.These cells played a crucial role in anti-infection by promoting the expression of interferon-inducible factors and inflammatory genes.The results of pseudotime trajectory analysis and GO enrichment analysis indicate that the infection of APP suppresses the differentiation of monocytes into alveolar macrophages(AM),and triggers endogenous apoptosis in AM,which are the primary reasons for the significant decrease in the number of AM after APP infection.These findings suggest that APP break the immune defense line of macrophages.In addition,heterogeneity analysis of T cell subpopulations identified 8 T cell subgroups,all of which showed a decrease in cell numbers post-APP infection.The CD8A~+γδT cells were activated following infection,initiating an immune response while upregulating the expression of genes associated with exhaustion and cell apoptosis.With the exception of na(?)ve CD4~+T and proliferative T cell clusters,the other subgroups exhibited enrichment in processes related to immune response and lymphocyte activation.This suggests that T cells likely play an important role in the early stages of the immune response to APP.In the pathogenesis of pulmonary fibrosis induced by APP,both epithelial cells undergoing EMT and fibroblast-like cells play crucial roles.EMT-epithelial cells not only exhibit a close relationship with fibroblast-like cells in Uniform manifold approximation and projection(UMAP)visualization plots but also show an upregulation in the expression of fibroblast markers SDC2 and COL15A1 post-infection,while downregulating the expression of epithelial markers EPCAM and FABP5.Furthermore,EMT-epithelial cells upregulate the fibrosis-promoting genes TGFB1 and HIF1A.Pseudotime trajectory analysis indicates that fibroblast-like cells differentiate from EMT-epithelial cells,suggesting that EMT-epithelial cells transform into fibroblast-like cells.Fibroblast-like cells also exhibit heterogeneity,with subgroup analysis identifying five subtypes of fibroblast cells.Proliferative fibroblast cells and COL1A1 fibroblast cells are commonly observed cell populations in healthy pigs.Despite the diverse functions of MT1A fibroblast,HBEGF fibroblast,and IFN fibroblast populations,all demonstrated roles in anti-infection immune responses and the promotion of fibroblast proliferation.Subsequently,cell communication analysis between fibroblast subgroups and other cell populations in pathological fibrosis revealed a significant enhancement of interactions with fibroblast subgroups post-APP infection.Notably,interaction scores increased the most with EMT-epithelial cells,vascular endothelial cells,and p DC.Further analysis revealed that,aside from COL1A1,the majority of fibroblast subgroups primarily enhance interactions with EMT-epithelial cells,vascular endothelial cells,and p DC through ligand-receptor pairs CD74/COPA.However,the COL1A1 fibroblast subgroup primarily strengthens interactions with other cells through the EGFR/MIF and EGFR/TGFB1 pathways.This suggests that post-APP infection,the fibroblast-like cells differ significantly from the common COL1A1 cell population,and indicates that CD74/COPA could serve as targets for disrupting fibroblast communication.To elucidate the pathological mechanisms mediated by APP,we conducted data analysis and flow cytometry,revealing a significant reduction in CD8~+T cells.Further experiments involving CD8~+T cell depletion and adoptive transfer in mice demonstrated that these cells mediate a decreased pulmonary APP burden and improved lung injury in mice by recruiting neutrophils and significantly upregulating the expression of GZMB,PRF1,and IL-8.Additionally,a negative correlation was observed between CD8~+T cells and the most abundant fibroblast-like cells,which are primarily enriched in functions related to regulating T cell apoptosis processes.Subsequently,co-culture of primary porcine lung fibroblasts with CD8~+T cells revealed that fibroblasts induce CD8~+T cell apoptosis by upregulating Gal-3 expression.Knocking down Gal-3 expression rescued CD8~+T cell apoptosis.RT-q PCR assays confirmed that fibroblasts induce extrinsic apoptosis in CD8~+T cells.Co-immunoprecipitation assays further revealed that fibroblasts induce extrinsic apoptosis in CD8~+T cells through the interaction of Gal-3/LAG3 ligand-receptor pairs.These findings suggest that rescuing the number of CD8~+T cells may represent a promising approach for ameliorating APP-mediated pathological damage.In summary,this study systematically reveals the cell atlas of the interaction between APP and the host at the single-cell level in porcine lungs.It provides a clear and comprehensive analysis of host immune cell response characteristics and pathological injury mechanisms,laying a theoretical foundation for the prevention and treatment of APP,while also providing rich data resources for the study of bacterial pneumonia.