| Bacterial pneumonia is a common and frequently occurring disease,which causes sustained serious economic losses to the breeding industry.Due to the increase of bacterial resistance,the lack of commercial broad-spectrum and cross-protective vaccines,the change of bacterial pneumonia pathogen spectrum and the widespread multi-pathogen mixed infection,the prevention and control effect of antibiotics and vaccines are not ideal,so the new prevention and control strategies are urgently needed.Recent studies have found structural cells can act as regulators for immune cells to maintain a stable environment within the tissue.In the region microenvironment of the lung,there are many studies on the anti-infection immunity of alveolar macrophages,but the studies on the immune defense of alveolar macrophages with neighboring structural cells are still lack.Actinobacillus pleuropneumoniae(APP)can cause infectious pleuropneumonia in pigs,presenting typical pathological changes of pulmonary infection such as hemorrhagic,cellulosic,necrotizing pleurisy and pneumonia,and is a typical pathogen of bacterial pneumonia in pigs.Therefore,in this study,APP was used as the model bacteria strain to analyze the effect and mechanism of porcine trachea epithelial cell(PTEC)in regulating porcine alveolar macrophage(PAM)against APP to explore the candidate preparations for the prevention and treatment of bacterial pneumonia.The main researches are as follows:1.Clarifing the role of PTEC-exosome in APP-induced PAM deathAPP can induce PAM death.Studies found that APP-infected PTEC supernatant(PTEC-CS)can significantly inhibit APP-induced PAM death,reduce intracellular bacterial load and the overexpression of pro-inflammatory cytokines IL-18,IFN-γ,and TNF-α.Further,it was found that exosome was the effective component in PTEC-CS to play the protective role.Moreover,infection experiments in mouse confirmed that exosomes derived from PTEC could significantly reduce clinical symptoms,lung tissue load,lung index,lung tissue pathological changes and injury degree,and improve the survival rate of mouse.These results suggest that PETC-derived exosomes could protect PAM from damage caused by APP.2.Identifing the effective components of PETC exosomes that play the role for resisiting PAM damage induced by APPIn order to identify the effective components in exosomes that play anti-infective roles,Small RNA sequencing of PETC exosomes was performed in this study.KEEG and GO analysis of the top 25 miRNAs showed that miRNA was significantly enriched in the aspects of resistance to pathogen infection,autophagy,TNF-αinflammatory response,phagocytosis and PI3K/AKT/m TOR signaling pathway.Among them,the content of miRNA-21-5p was the highest in exosomes,and its GO and KEEG analysis mainly focused on the regulation of antimicrobial,NADPH-induced injury,inflammatory response,autophagy,PI3K/AKT/m TOR,pyroptosis,TNF-α and telomere lifespan,which suggested that miRNA-21-5p was involved in the process of APP infection.Further in vitro experiments showed that miRNA-21-5p could significantly reduce PAM damage and cytotoxicity caused by APP,and improve the bactericidal ability of PAM.APP infection experiments in mouse showed that the administration of miRNA-21-5p significantly reduced the clinical symptoms,lung tissue bacterial load and lung tissue pathological damage of mouse,and improved the survival rate of mouse,indicating that miRNA-21-5p is an effective component of exosome in anti-infection.3.The molecular mechanism of miRNA-21-5p for PAM in resisting to pulmonary bacteriaThe effects of miRNA-21-5p on cell death types during APP infection were first detected by RT-qPCR and Western blot.The results showed that miRNA-21-5p could significantly promote PAM autophagy,inhibit excessive secretion of ROS,inhibit pyroptosis,and improve the bactericidal ability of PAM.miRNA-21-5p target genes were predicted by miRanda and Target Scan databases,then the dual luciferase reporting test verified that miRNA-21-5p exerts its protective effect for PAM by conbining the 3’UTR of PIK3 CD and inhibiting its gene expression.Moreover,in vitro and vivo experiments,it was found that the protective effect and mechanism of miRNA-21-5p were also applicable to bacterial pneumonia caused by methicillin-resistant Staphylococcus aureus(MRSA)and Klebsiella pneumoniae(K.pneumoniae).4.Elucidating the mechanism by which Carbon dots targeting PIK3 CD can combine with miRNA-21-5p to resist pulmonary bacterial infectionCarbon dots(CDots)nanomaterials have a wide range of biological functions and have good prospects in antimicrobial applications.In this study,CDots were synthesized by hydrothermal method with polyethylenimide(PEI)and ascorbic acid(VC),which has good biocompatibility and biosafety.In vitro studies showed that CDots could significantly reduce the death of macrophages infected with APP,MRSA and K.pneumoniae by promoting the M1 polarization,and improve the phagocytosis and bactericidal ability of macrophages.Molecular dynamics simulation found that both CDots and miRNA-21-5p could target PIK3 CD,si RNA interference technique was used,it was verified that the CDots could bind in PIK3 CD,inhibit the activation of PIK3/AKT/m TOR signaling pathway,and further promote the M1 polarization of macrophage.This protective effect of CDots was confirmed in mouse models infected with APP,MRSA,and K.pneumoniae,moreover,miRNA-21-5p combined with CDots had more significant effect for resisting pulmonary bacterial infection.In conclusion,this study clarified the role and main components of structural cell-PTEC exosomes in regulating alveolar macrophages resistance to pulmonary bacterial infection,revealed the molecular mechanism of miRNA-21-5p and CDots nanoparticles resistance to pulmonary bacterial infection,and proved that the combination of the two had stronger anti-infection effect.This study not only enriched the cognition of structural cells in regulating immune cells,but also provided new targets and new ideas for the common prevention and treatment of bacterial pneumonia such as APP,MRSA and K.pneumoniae. |
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