Characterization Of Host Immune Response After Pseudomonas Aeruginosa Inhalation Infection

Pseudomonas aeruginosa(PA)is the main pathogen of hospital acquired pneumonia(HAP)and ventilator associated pneumonia(VAP),which poses a serious threat to clinical medicine.As a multidrug-resistant opportunistic pathogen,PA has multiple pathogenic factors,its ability to adapt to the host microenvironment which directly affects efficiency of the host’s innate and adaptive immune response,and current therapies with antibiotics are limited in the treatment of PA infections.With the changing disease spectrum and increasing number of opportunistic infections,the role of the lung’s innate immune system in fighting infections has gained attention.Therefore,this study focuses on the characteristics of the host innate immune response after inhalational PA infection,we first constructed the mouse model infected with PA via aerosolized intratracheal(i.t.)inoculation,and resolves the characteristics at the protein,cellular,and gene expression level,aiming to identify targets for intervention in organismal inflammation,laying a foundation for in-depth understanding of the pathogenic mechanism of PA pulmonary infection,having important theoretical significance and potential application value for the prevention and control of PA infection.In this study,a C57BL/6J mouse model infected with PA strain PAO1 was successfully constructed via aerosolized intratracheal(i.t.)inoculation and evaluated by the survival curve,organ bacterial loads and histopathology.Next,host immune response characteristics were resolved at the protein,cellular,and gene expression level.At the protein level,large amounts of inflammatory proteins were detected in the bronchoalveolar lavage and mice serum after infection,indicating a dramatic inflammatory response in the lung and organism;At the cellular level,flow cytometry was used to detect dynamic changes in key cell populations and subpopulations in the lung.Neutrophils、monocytes、alveolar macrophages(AM)and natural killer(NK)cells responded to a high degree during infection,which may mediate host tissue injury.At the gene expression level,RNA-seq high-throughput sequencing technology was used to explore the changes in the time-series transcriptional profile of the host lung after infection,analyze the expression patterns and dynamic transcription characteristics of differentially expressed genes(DEGs),mine their functions,and systematically show the development process of the inflammation in detail.The results show that TNF,IL-17,MAPK and NF-k B signaling pathways played key roles in the immune pathogenic mechanism after infection.IFN-γsignaling promoted the activation and recruitment of NK cells and macrophages leading to inflammatory cell infiltration and an amplified inflammatory response in the lung at 24 hpi.Activation of MAPK pathway during infection induces apoptosis of AM and NK cells.The 48 hpi period may be a turning point in the inflammatory response,where the lung tissue parenchyma switches from a pro-inflammatory phenotype to an anti-inflammatory phenotype.The biological processes related to tissue repair are highly activated,suggesting the presence of lung injury and repair processes.Epithelial cells and fibroblasts play an important role in repair,tissue remodeling and regeneration after lung injury,with a subsequent focus on the role of non-immune cells in the progression of inflammation.The discovery of genes that may interfere with the development of inflammation: Saa3,Serpina3 n,Lcn2,Orm1 may serve as important targets for subsequent studies.Among them,Serpina3 n,which is associated with COPD and cystic fibrosis,and Orm1,which encodes an acute-phase response protein,have not been reported in relation to PAO1 inhalation infection and can be the key target genes for follow-up studies.To verify the role of key cells in PA inhalation infection,neutrophils,which are the most responsive in the inflammatory response,were first selected for verification to investigate their role in the process of acute lung injury caused by P.aeruginosa infection.A neutrophil-depleted mouse model was constructed based on the previous model.The results showed that neutrophil depletion increased mortality in mice,and neutrophil recruitment during infection effectively inhibited bacterial proliferation.We found that there was a negative feedback mechanism in the body after neutrophil depletion,which compensated for increased chemotaxis and activation of neutrophils;monocytes and macrophages more actively replaced depleted neutrophils in their function of clearing extracellular pathogens.Thus,neutrophils play an important role in the inflammatory homeostasis of acute lung injury caused by P.aeruginosa and are important regulators of inflammatory and immune responses.The heterogeneity of neutrophils during the host response can be further explored.In conclusion,this study first constructed a mouse model of PAO1 via aerosolized intratracheal inoculation and systematically revealed the characteristics of the host innate immune response after inhalational P.aeruginosa infection at the protein,cellular,and gene expression level,demonstrated the progression of inflammation,also demonstrated the important role of neutrophils in the regulation of immune response and inflammatory homeostasis,provided target genes and factors for subsequent studies.Our data therefore provide new insight into the pathogenesis of P.aeruginosa pulmonary infections and provide references for inhalational infection protection and immunotherapy.