The Mechanism Of Influenza Virus NS1 And Endothelial Cell S1PR1 In Regulation The Inflammatory Factors ICAM-1 And IFN-? For ARDS

Acute respiratory distress syndrome(ARDS),the most common form of severe acute respiratory failure,is easily missed and has a high case fatality rate.So far,there is a lack of treatment.Acute inflammation and injury of alveolar-capillary membrane resulted in a highpermeability of epithelial and endothelial,neutrophilsand acute inflammation recruited into alveolar cavity.A large number of inflammatory mediators can reduce alveolar active substance then cause collapse of distal alveolar and consolidation,lead to hypoxemia and pulmonary edema,trigger ARDS.Influenza A virus is one of the most common pathogens causing viral pneumonia and ARDS,when influenza virus infects the alveolar epithelium,it replicates and induces apoptosis releasing new virions that could infect the endothelium.Infected epithelial cells also release inflammatory mediators,which could induce epithelial leak,neutrophils recruited into the lungs,epithelial-endothelial barrier dysfunction,alveolar edema and respiratory failure.Human alveolar epithelial cell infected with influenza virus A H1N1 produced antiviral IFN?,which were critical for the clearance of viruses.IFN-P through autocrine way to promote their own production,but influenza virus can affect the production of IFN-? through antigen drift or microenvironment change,which evade immune surveillance.The non-structural protein 1(NS1)of influenza A viruses inhibits the production of IFN-? by various mechanisms,inhibiting the virus detector retinoic acid-inducible gene(RIG-1);NS1 inhibits the activation of NF-kB and IRF3.NS1 of the influenza A H5N1 was demonstrated to inhibit the activation of signal transducers and activators of transcription 1/2(STAT1/2)to decrease the expression of IFN-?.However,whether NS1 of the influenza A(H1N1)inhibits the production of IFN-? in human alveolar epithelial cells by affecting the phosphorylation of STAT1/2 and the loop of IFN-?-mediated autocrine process of IFN-P production remains elusive.This study explores whether influenza A(H1N1)virus NS1 down-regulated interferon receptor 1/2(IFNAR1/2)by inhibiting the activation of STAT1/2 and reducing the production of IFN-?.It was thought that airway epithelial cells play a major role in inflammatory storms of influenza infection,because it is the main place of influenza virus replication,and can secrete a variety of inflammatory and chemotactic factors,recruit a large number of immune cells to lung,destroy the lung microvascular endothelial barrier,which mediated lung injury.Some recent research show that endothelial cells play a major role in the inflammatory storms of influenza virus infection,S1PR1 agnosim suppresses early proinflammatory cytokines and innate immune cell recruitment during influenza virus infection.When influenza virus infects the endothelial cells,activation endothelial cells can upregulate the expression of ICAM-1,thereby enabling the the recruitment of leucocytes to the alveolus.The large number of neutrophils and macrophages could damage the alveolar-capillary membrane and contrubite to pulmonary edema through releasing inflammatory factors,eventualLy lead to ARDS.This study explores whether stimulating S1PR1 inhibits the production of inflammatory storms by inhibiting the expression of ICAM-1 when influenza virus infects HPMEC.What are the specific mechanisms that inhibit the expression of ICAM-1.Chapter 1 The mechanism of endothelial cell S1PR1 regulate the inflammatory factor ICAM-1 in ARDSInfluenza virus infection involves a multitude of people worldwide,and endothelial cells are an important target of lung inflammation caused by influenza A virus infection.The roles of endothelial sphingosine 1-phophate receptor1(S1PR1)in the regulation of molecules involved in leukocyte recruitment during influenza A virus infection still remain unknown.To investigate whether SlPR1-?-arrestin2-NF-kB pathway is involved in the expression of ICAM-1 in human pulmonary microvascular endothelial cells induced by H1N1 influenza virus and further affect the production of inflammatory cytokines.The expression of IL-8,MCP-1,IFN-?,IFN-? and ICAM-1 mRNA in human pulmonary microvascular endothelial cells was infected by influenza A(H1N1)virus.Human pulmonary microvascular endothelial cells were transfected with S1PR1 plasmid and small interfering RNA,and then were infected with influenza virus and stimulated by S1PR1-specific agonist CYM5442,the mRNA expression levels of IL-8,IL-1?,IFN-a,IFN-P,IL-6,TFN-a,Rantes and MCP-1 were detected by real-time quantitative PCR,the expression of IL-6,Rantes and MCP-1 were detected by ELISA.The S1PR1 and ?-arrestin2 plasmids were packaged by lentivirus and transfected into human pulmonary microvascular endothelial cells,then were stimulated with influenza virus and S1PR1 agonist CYM5442,the protein expression levels of S1PR1,?-arrestin 2,p-P65,P65 and ICAM-1 were detected by Western blot.HPMEC could be infected with H1N1 and responded to produce pro-inflammatory cytokines,chemokines,type ? interferons,and cellular adhesion molecules.After influenza virus infection and S1PR1 agonist intervention,compared with the control group,the expression levels of IL-8,IL-1?,IFN-a,MCP-1,IFN-?,IL-6,TFN-a and Rantes mRNA in the S1PR1 silencing group were increased by 3.1,2.1,2.5,1.5,2.3,2,1.6 and 2.1 times,respectively;In the control group,the expression levels of IL-6,IL-1?,TFN-a,Rantes,IFN-a,IFN-?,MCP-1 and IL-8 mRNA were increased by 3.1,1.7,2.1,2.6,3.9,4.3,4.2 and 3 times higher than those in the S1PR1 overexpression group,respectively;Western blotting showed that activation of S1PR 1 reduce the production of ICAM-1,inhibition of NF-kB signaling pathway via a dosage and time-dependent manner.Activated S1PR1 signaling regulates the production of cellular adhesion molecules by inhibiting NF-kB activation via a ?-arrestin2-dependent manner.H1N1 influenza virus infects of human pulmonary microvascular endothelial cells produce a large number of inflammatory cytokines.Agonism S1PR1 reduces the production of pro-inflammatory cytokines during influenza virus infection.Activated SIPR1 signaling regulates the production of cellular adhesion molecules by inhibiting NF-kB activation via a ?-arrestin2-dependent manner.Chapter 2 The mechanism of non-structural protein 1 regulate the inflammatory factor IFN-?Influenza virus evades immune responses via various mechanisms including modifying the immune microenvironments.Influenza virus non-structural protein 1(NS1)encoded by the virus genome inhibits type I interferon(IFN)signaling pathways,which is essential for the clearance of virus.However,the precise mechanisms of NS 1-mediated immune suppression remain poorly understood.To investigate whether influenza A(H1N1)virus NS 1 inhibits the expression of IFN-?through the IFNAR1/2-STAT1/2 pathway.The human alveolar epithelial cell line A549 cells were cultured and infected with H1N1 influenza virus.The anti-IFN-?neutralizing antibody and human recombinant IFN-P were respectively added to detect the expression of IFN-P mRNA.A549 cells were transfected with NS1 plasmid and infected with influenza virus.The expression of IFN-?,IFNAR1/2 mRNA and IFNAR1/2 protein level were detected.A549 cells were transfected with NS1 plasmid and stimulated with IFN-?,the expression of STAT1/2 was detected.Methods used in this experiment were flow cytometry,western blot,and real-time quantitative PCR.The expression of IFN-? mRNA in A549 cells was significantly increased after influenza A(H1N1)virus infection.The anti-IFN-? neutralizing antibody and human recombinant IFN-? inhibited and up-regulated the IFN-? mRNA in A549 cells respectively.A549 cells transfected with H1N1 NS1 lost the ability to produce IFN-P upon H1N1 infection or IFN-? stimulation.NSI inhibited the expression of interferon receptors.Meanwhile,NS1 prohibited the activation of signal transducers and activators of transcription STAT1/2,as well as the consequent IFN-P production.IFN-? production is an IFN-? dependent autocrine process after influenza virus infection in A549 cells.In the course of H1N1 influenza virus infection,NS1 reduces the expression of IFN-? by reducing the expression of IFNAR1/2,inhibiting the activation of STAT1/2.