Characteristics Of Lung Immune Cell Response Induced By Actinobacillus Pleuropneumoniae And The Immune Function Of CD14^+/- Neutrophils

Porcine contagious pleuropneumonia（PCP）is a highly contagious contact respiratory disease caused by Actinobacillus pleuropneumoniae（APP）and causes huge economic losses to the pig industry.At present,due to a large number of APP serotypes,the lack of cross-protection of vaccines,the serious secondary infection of interbacterial or mixed infection between bacteria and viruses,antibiotic resistance and the promulgation of the"prohibition of resistance and limited antibody"orders,the prevention and treatment of PCP is still very difficult.As a target organ for APP infection,the immune response characteristics of the lungs determine the development of the disease.Therefore,from the perspective of host immunity,the system reveals the characteristics of APP-induced lung immune response,confirms the subpopulation and function of key immune cells in the process of disease development,and not only provides new targets for APP prevention and treatment,but also provides new ideas for other infectious diseases of the lungs.Therefore,in this study,a mouse model of APP infection was first constructed,and the composition and changes of mouse lung immune cells at different time points after APP infection were analyzed by high-throughput mass spectrometry flow cytometry combined with bioinformatics analysis.The results showed that in the process of APP infection,the immune cells of the lungs had strong heterogeneity,and a total of 27 myeloid cell subpopulations and 21lymphoid cell subpopulations with different phenotypes were identified.At the same time,this study plotted the map of APP-infected immune cells,and identified the time-specific immune cell subsets of infection,even including Ly-6G⁺CD4⁺T cells that have not yet been identified.Surprisingly,this study identified 17 neutrophil subsets in the lungs,with phenotypic clusters of 5heterogeneous neutrophil subsets,of which CD14⁺neutrophils were mainly concentrated in the lung tissue repair phase（24 h）,suggesting that the neutrophil population with differential expression of CD14 may be associated with the process of APP infection.Subsequently,we used flow cytometry,single-cell sequencing techniques,and bioinformatics analysis to further study the basic characteristics of neutrophil subsets with differential expression of CD14 in mice and piglets.Flow cytometry mice CD14^+/-neutrophils IL-17A,TNF-α,IFN-γ,IL-10 and IL-21 secretion capacity and MHC-II,CD86 and APP expression capacity,compared with CD14^-neutrophils,CD14⁺neutrophils have a strong cytokine secretion capacity,can secrete pro-inflammatory factors and anti-inflammatory factors.Compared with CD14⁺neutrophils,CD14^-neutrophils exhibit a stronger antigen retrieval capacity.Single-cell sequencing analysis of piglet lung cells determined the presence of CD14^+/-neutrophils in piglets,and cytokine secretion and antigen formulation related gene expression and GO and KEEG analysis of Top genes in 2groups of cells yielded similar results to those in mouse lungs.In addition,flow cytometry found that CD14⁺and CD14^-neutrophils were prevalent in lung tissue infected with Gram-negative bacteria such as Klebsiella pnenmoniae,Escherichia coli,and Pseudomonas aeruginosa.In order to further study the function of CD14⁺neutrophils,we combined with LPS in vitro induced CD14⁺neutrophil test and adoptive transfer method to transfer CD14⁺neutrophils to normal mice lungs and then infected with APP.Detection of mouse survival rate,clinical symptoms,and bacterial load,we found that CD14⁺neutrophils will aggravate mouse death in the early stage of infection,inhibits the lungs’ability to sterilize bacteria,but accelerate the recovery of clinical symptoms of mice in the later stage of infection,and infer that they may play an anti-inflammatory role in vivo.Finally,the function of immune cells is regulated by various immune molecules such as cytokines,due to the expression of IL-5 receptors on the surface of neutrophils,so this study constructed an IL-5 knockout(IL-5^-/-)mouse APP infection model,using flow cytometry,immunofluorescence,RT-q PCR,plate coating to detect the bactericidal ability of IL-5^-/-and wild（wild type,WT）mouse neutrophils,reactive oxygen species（ROS）expression,neutrophil extracellular traps（NETs）release and sterilize granular protein m RNA.The number of CD14^+/-neutrophil subsets in lung tissue before and after infection in IL-5^-/-and WT mice was also detected.The results showed that the clinical symptoms of IL-5 deletion mice were aggravated,the mortality rate was increased,and the resistance to APP infection was weakened.Further studies found that IL-5 deletion led to CD14⁺neutrophilia in mice in an uninfected condition,and reduced neutrophilic bactericidal ability by inhibiting the formation of neutrophil NET rather than ROS production and killing granule proteins.In summary,this study systematically analyzes the characteristics of APP-induced pulmonary immune response,constructs a time-specific map of APP infection,identifies key immune cell subsets during infection,and reveals the immune function of CD14⁺and CD14^-neutrophil subsets and their impact on IL-5,which not only enriches people’s understanding of the immune response to APP infection,but also provides new ideas and targets for the prevention and treatment of APP and its related infectious pneumonia.