Characteristics Of Pulmonary Cytokine Response And Immunologic Mechanism Of IL-21 As Actinobacillus Pleuropneumoniae Infection

Actinobacillus pleuropneumoniae(APP)can cause infectious pleuropneumoniae in pigs,which is one of the pathogenic bacteria seriously harmful to pig industry.APP mainly invades the tonsils and upper respiratory tract of pigs,colonizes,reproduces in the lungs,and induces acute infection death and chronic infection with hemorrhagic,fibrinous,necrotizing pleurisy and pneumonia as the main characteristics.Cytokines play an important role in immune response and homeostasis balance during infection.The lung related to the outside has active immune responses and obvious pathogenic feathers.However,the comprehensive and in-depth researches on the characteristics of cytokines induced by APP and their roles in pathogenesis are currently lacking.Therefore,in this study,piglets and mice were used as infection models to dissect the feathers of pulmonary cytokine response and the role and mechanism of cytokines in APP-induced pneumonia.The main researches are as follows:1.Characteristics of pulmonary immune response in porcine pleuropneumonia and its comparison with mouse APP infection modelIn this study,piglets were experimentally infected intranasally with APP,and the lung tissue,sera and BALF were collected at different time points after infection.Transcriptome sequencing and immunohistochemical staining were performed on lung tissue,and 29 cytokines in BALF and sera were detected by ELISA.The results showed that the cytokine response in sera was different from and earlier than that in BALF.At the early stage of APP infection,the lung immune responses were mainly negatively regulated,and forming local excessive inflammation at the later stage.To further explore the immune response characteristics,mice APP infection model were established,and the characteristics of immune cell response in lung,spleen and peripheral blood were analyzed by mass cytometry,while the changes of cytokines were detected by ELISA.The results showed that each tissue had strong tissue-specificity,and several tissue-or APP-specific subsets were identified.The changed cytokines in mice were not completely consistent with piglets,but the overall change trend was similar.Therefore,based on the literature,the course of APP infection and the degree of lung injury,IL-1?and IL-18 significantly increasing in piglet sera and BALF at the early stage of infection(6 h),and IL-21 increasing only in BALF at the middle and late stage of infection(24-48 h)were selected as the key cytokines.2.Analyze the relationship between lung cytokines produced post APP infectionIn order to understand the cascade relationship of cytokines during APP infection,precision-cut lung slices(PCLS)of healthy piglets were successfully prepared.The relationship among 7 major cytokines that significantly increased after APP infection was studied.APP infected IFN-?,IL-5,IL-21 or IL-6 deficiency mice models were established,and 31 cytokines were dynamically detected by Luminex.The results showed that there was a cascade trigger relationship between major cytokines after APP infection,and the combination of IL-18 and IL-21 can promote IL-17A,GM-CSF,IFN-?and IL-21 expression.In the APP infection model of cytokine deficient mice,IL-21deficiency led to an obvious increase of proinflammatory cytokines such as GM-CSF,IL-18 and IL-12,and a decrease in anti-inflammatory cytokine IL-10,aggravating lung injury.However,in IFN-?deficient mice,TNF-?,IL-6,IL-4,IL-17A and IL-18 were rapidly up-regulated in the early stage of infection(6 h)and then followed by rapid inflammatory inhibition lately,which was the key to the recovery of pneumonia.3.NLRP3 mediates APP-induced lung macrophage death in an inflammasome-independent manner to promote the infection.IL-1?and IL-18 were significantly increased in BALF and sera after APP infection in piglets.To understand the role of NLRP3 in APP infection,NLRP3^-/-,ASC^-/-and caspase1/11^-/-mice were used.The study found that NLRP3 deficiency did not significantly improve the defense against APP infection by inhibiting IL-1?and IL-18secretion.NLRP3 significantly reduced the basal levels of NK,NKT,and CD4⁺T cells in the lung,and considerably reduced the proportion of NK and NKT cells after infection,inhibiting the clearance of APP.In addition,NLRP3 did not affect the proportion of lung neutrophils,macrophages,and monocytes,but promoted the death of lung macrophages.Transcriptome sequencing analysis of APP-infected bone marrow-derived macrophages from WT and NLRP3^-/-mice showed that NLRP3promoted macrophage death,increasing the ability of macrophages to express CXCL5and IL-1?and polarize toward M2 macrophages.4.Elucidating the role and mechanism of IL-21/IL-21R signaling pathway against APP infectionIL-21/IL-21R signaling plays an important role in various immune diseases and immune cell development,however,there is no systematic study about it on pulmonary immune response in bacterial pneumonia.Here,IL-21R^-/-mice were used to explore the effect of IL-21/IL-21R on the composition and function of lung immune cells after APP infection by mass cytometry and flow cytometry.The role and mechanism of key immune clusters were also clarified.The results showed that IL-21R^-/-mice were more susceptible to APP than WT mice,and secretion of pro-inflammatory cytokines(IFN-?,TNF-?,and IL-6),neutrophil activation,inflammatory monocytes recruitment,and the numbers of M1 macrophages in the lungs were significantly reduced in IL-21R^-/-mice,weakening the natural immune response in lung.Compared with IL-21R^-/-mice,unrecognized Ly6C and Ly6G double positive CD4⁺T cells appeared in the lungs of WT mice after APP infection,which had strong secretion capacity of IL-10,IL-21,granzyme B and perforin,and directly promoted the proliferation and phagocytic bactericidal function of macrophages.Thus,this study elucidated that IL-21induced Ly6C⁺Ly6G⁺CD4⁺T cell could cooperate with macrophages against APP infection.In conclusion,the current study clarified the dynamic response characteristics of cytokines and the relationships among the key cytokines in piglets post APP infection,and revealed the function and mechanism of NLRP3 and IL-21 during APP infection.It enriched the cognition of lung immune response and provided a theoretical basis for the prevention and treatment of porcine infectious pleuropneumonia.