Molecular Mechanism Of Respiratory Tract Epithelial Cell Damage Caused By Glaesserella Parasuis And Its Synergistic Pathogenesis With Porcine Reproductive And Respiratory Syndrome Virus

Glaesserella parasuis（G.parasuis）is the pathogen that causes Gl（?）sser disease.As a common opportunistic pathogen in pig farms,it is one of the main pathogens causing porcine respiratory disease complexes（PRDC）,which can colonize the upper respiratory tract of pigs.G Parasuis can break through the physical barrier of respiratory epithelium,enter the deep tissues and cause systemic spread when the host is under stress or immune deficiency.In the acute phase of the disease,lesions include polyarthritis and polyserositis.Chronic infections can cause fibrosis of the surface of the lungs and heart cavity resulting in reduced growth rate.However,there are few studies on the strategies of different virulene strains of G Parasuis to cross the airway epithelial barrier,and the mechanism of G.parasuis colonization on epithelial cell damage is still unclear.In addition,the detection rate of co-infection between G.parasuis and other respiratory pathogens has increased in recent years,among which the co-infection of G.parasuis and PRRSV is one of the most common forms in epidemic investigations of various countries,causing serious economic losses to the pig industry.The co-pathogenic mechanism of G.parasuis and PRRSV in respiratory system is still unclear,which causes great trouble for disease prevention and control in pig farms.Due to the difference between in vivo and in vitro studies on the co-infection of G.parasuis and PRRSV,the establishment of an in vitro co-infection model that can simulate the respiratory microenvironment will provide an important strategy for research on the co-pathogenesis of G.parasuis and PRRSV in the respiratory system.To compare tissue invasive ability into the respiratory barrier and to explore the mechanism of epithelial cell damage induced by G.parasuis,this study used mice,precision-cut lung slices（PCLS）,and porcine well-differentiated respiratory epithelial cells to systematically evaluate the cytotoxic effects and injury mechanisms induced by serotype 3strain（G.parasuis 81-3）and serotype 5 strain（G.parasuis 80-5）.First,the pathogenicity test was executed on C57 mice to evaluate the virulence of different serotypes of G.parasuis;in addition,an infection model of G.parasuis on PCLS were established to analyze the infective ability and virulence of different strains in host lungs.And on this basis,the infection model of G.parasuis in well-differentiated airway epithelial cells was further established and the mechanism of G.parasuis impairing epithelial cell barrier was analyzed.At the same time,a co-culture of ALI system（Co-ALI）consisting of highly differentiated tracheal epithelial cells（wd PTEC）and porcine alveolar macrophages（PAMs）was established to analyze the synergistic pathogenic mechanism of G.parasuis and highly pathogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV）co-infection in respiratory system.The results obtained on basis of the above researches are as follows:（1）G.parasuis 81-3 and G.parasuis 80-5 showed significant virulence differences,and G.parasuis 80-5 led to more intense inflammatory response and pathological injury of respiratory system,caused more serious damage to ciliated epithelial cells,regulated more severe cytotoxicity and epithelial barrier destruction.（2）G.parasuis could adhere to and proliferate on polar epithelial cells in air-liquid culture system,and the upper respiratory tract was more conducive to the colonization of G.parasuis,while the lower respiratory tract was more sensitive to the cytotoxicity induced by G.parasuis.（3）The characteristics of G.parasuis infection in polarized epithelial cells showed that G.parasuis 80-5 could increase the spacing of adjacent epithelial cells in wd PTEC by regulating the transcription and protein levels of cell junctions（including ZO1,Claudin 3、Occludin and E-cadherin）and facilitate bacteria cross epithelial barrier,while G.parasuis81-3 promoted the invasion of bacteria into epithelial tissue mainly by disrupting tight junctions.（4）Transcriptome sequencing and related protein expression results showed that G.parasuis can regulate the expressions of genes and proteins related to intercellular junctions and apoptosis pathways.TNFαproduced by G.parasuis infected-epithelial cells is a key factor in the regulation of cell-cell junctions was confirmed by TNFα^-/-mice.In addition,the G.parasuis could regulate epithelial cell apoptosis independently through TNFα/TNFR1 and Fas/Fas L pathways was verified in TNFα^-/-mice and Fas^-/-mice.（5）The transcriptional and protein levels of PAMs polarization regulators were analyzed in the coinfection of G.parasuis and PRRSV in Co-ALI system,which indicated that PRRSV could promote the M2 polarization of PAMs and inhibited the G.Parasuis mediated M1polarization,thereby promoting the infection of HP-PRRSV in co-infection and inhibiting the proinflammatory process in Co-ALI system.（6）G.Parasuis colonization ability was significantly enhanced in the co-infection group,and G.Parasuis could promote the ability of PRRSV to cross the physical barrier of respiratory epithelial cells by regulating the functions of tight junction complex between epithelial cells and play a synergistic role in disease.In conclusion,G.parasuis 81-3 and G.parasuis 80-5 showed different virulence effects in mouse,PCLS and porcine respiratory epithelial cells.G.parasuis could disrupt the function of cellular junctional protein complexes between epithelial cells by stimulating TNFαsecretion.In addition,G.parasuis 80-5 has more destructive effects on adhesion junctions which could enlarge the space between adjacent epithelial cells and facilate G.parasuis to cross the respiratory epithelial barriers.In addition,G.parasuis could induce apoptosis of respiratory epithelial cells mainly through two extrinsic apoptosis pathways of TNFα/TNFR1 and Fas/Fas L independently.A co-infection model of G.parasuis 81-3 and HP-PRRSV in the Co-ALI system was established and we elucidated that G.parasuis 81-3promote HP-PRRSV replication and the ability to cross the respiratory barrier by regulating the polarization of PAMs and interfering with the junctional interaction between epithelial cells through chemokines and cytokines.The results of this study provide data support for the pathogenic mechanism of G.parasuis in the porcine respiratory system,provide a theoretical basis for the further study of the pathogenic mechanism of G.parasuis and PRRSV co-infection in the respiratory system,and provide new ideas for the optimization of co-infection model of porcine respiratory system.