| Part Ⅰ RSV NS1 induces airway inflammation and AHR in miceObjective Respiratory syncytial virus(RSV)is one of the main pathogens of lower respiratory tract infection in children.After infection,it can cause the sensitization state of the body and is closely related to the recurrent wheezing in the later stage.Its pathological mechanism has not been completely clarified.Our previous studies have shown that RSV NS1 can cause airway pathological changes as an independent pathogenic factor,but it is not clear whether it is related to AHR and sensitization.Based on previous studies,this study intends to further explore the effects of RSV NS1 on airway inflammation and lung resistance(RL)in mice,so as to provide ideas for the study of the pathogenic mechanism,prevention and treatment of RSV.Methods The animal models of RSV infection and plasmid transfection were constructed to verify the expression of RSV gene and plasmid protein in mouse lung tissue.1.Grouping of animals.BALB/C mice aged 4-6 weeks were randomly divided into RSV infection group,RSV + siNS1 intervention group,pNS1 transfection group and PBS(or empty vector transfection)as the control group.RSV virus solution(1.4×10^7 TCID50/ml,50μl),RSV virus solution + siNS1(100pmmol)and RSV NS1 recombinant expression plasmid(pNS1,5μg)were used to construct the mouse model of RSV infection / pns1 transfection.2.Construction of animal sensitization model and detection of airway resistance.Based on the above mouse infection(transfection)model,the sensitization/stimulation experiment was carried out with ovalbumin(OVA),and the RL was evaluated by whole-body plethysmography at the specified time after stimulation.3.Sample acquisition and detection.After measuring the airway resistance,the mice were euthanized at the specified time point,and the lung and alveolar lavage fluid(BALF)were taken.The lungs of mice were taken for histopathological examination.After centrifugation of alveolar lavage fluid,cell smears and staining microscopy were classified and counted.Result1.On the 5th day after RSV inoculation,the lungs of mice were taken for PCR amplification,and the gene expression of RSV glycoprotein(G)and phosphoprotein(P)was observed.NS1 protein expression was detected in the lower respiratory tract of mice 1,3,5,8,11 and 14 days after pNS1 treatment.2.RSV NS1 participates in airway inflammation pathology and AHR.Compared with the control group,RSV group and pNS1 group can cause obvious lung tissue injury in mice.Histopathological examination showed that pNS1 transfection can lead to edema,airway stenosis and a large number of inflammatory cell infiltration.BALF detection showed that the number of eosinophils,lymphocytes,macrophages and neutrophils in lung tissue increased(P < 0.05).Compared with the control group,the RL of mice in pNS1 transfection group and RSV infection group increased significantly(P < 0.05).The intervention of siNS1 could significantly reduce the lung injury,airway inflammatory cell infiltration and RL caused by RSV infection in mice(P < 0.05).3.pNS1 aggravates the pathological changes of lung tissue in sensitized mice.On the basis of OVA sensitization,pNS1 transfection further aggravates the production of mucus in mouse airway,the number of eosinophils,neutrophils,macrophages and lymphocytes in BALF(P < 0.05),and causes the increase of RL(P < 0.05).Conclusion RSV NS1,as an independent pathogenic factor,can cause airway inflammation and AHR in mice,and aggravate the pathological changes of respiratory tract in sensitized state.Part Ⅱ The immune regulation effect of RSV NS1 in miceObjective Our previous study have shown that RSV NS1 can cause airway pathological changes as an independent pathogenic factor,and can affect AHR and sensitization in mice.Based on previous studies,this study intends to further analyze the effects of NS1 on mouse immune cell differentiation,cytokine secretion and immune cell function,and then confirm the pathological mechanism of immune imbalance caused by NS1 by affecting immune tolerance.Methods1.Grouping of animals.BALB/c mice aged 4-6 weeks were randomly divided into RSV(6.3×10^6 TCID50/ml,50μl)infection group,RSV + siNS1(100pmmol)group,pNS1(5μg)group and PBS(or empty vector transfection)as the control group.2.Anti-OX40 L blocking experiment.Based on the above experiments,mice were pretreated with anti-OX40 L monoclonal antibody or control antibody(control Ig G)before and after plasmid transfection or RSV infection,and the above experiments were repeated.3.Sample acquisition and treatment.After euthanizing the mice at the specified time point,take the lung,spleen and peripheral blood.The proportion of Th1,Th2,Th17 and Foxp3 + regulatory T cells(Treg)in CD4 + T cells of mouse lung and spleen was detected by flow cytometry.The cytokines IL-4,IL-5,IL-6,IL-10,IL-17 and transforming growth factor β(TGF-β),tumor necrosis factor α(TNF-α)and interferon-γ(IFN-γ)in BALF or serum were detected by BCA protein assay.The concentrations of thymic stromal lymphopoietin(TSLP)and OX40 L in serum were detected by enzyme-linked immunosorbent assay.The total RNA was extracted from the lungs of mice,the expression of TSLP and OX40 L genes were detected by q RT-PCR,and the expression of RSV antigen was detected by immunofluorescence.The titer of RSV in mouse lung homogenate was determined by TCID50 method.4.The cell experiment.pNS1 was transfected into human bronchial epithelial cells(BEAS-2B),the total RNA was collected at the specified time,and the TSLP gene expression was detected by q RT-PCR.Result1.pNS1 regulates the distribution of immune cells in mice.Compared with the empty vector control group,the proportion of Th2 and Th17 cells in pNS1 transfected group increased(P < 0.05),while the proportion of Th1 and Treg cells decreased(P < 0.05).2.pNS1 regulates the expression of cytokines in mice.The serum concentration of IL-4,IL-5,IL-6,IL-17,TGF-β and TNF-α in pNS1 group increased,while IFN-γand IL-10 decreased(P < 0.05).3.RSV NS1 up-regulates the expression of TSLP.pNS1 transfection up-regulates the expression level of serum TSLP in mice.The expression level of TSLP m RNA in total RNA of mouse lung tissue increased within 2 hours after plasmid transfection,reached the peak at 12 hours,and returned to the physiological level at about 48 hours.RSV infection and pNS1 treatment of BEAS-2B cells also increased the expression of TSLP m RNA(P < 0.05).4.pNS1 up-regulated the expression of OX40 L.The level of OX40 L in serum increased significantly 24 hours after pNS1 transfection(P < 0.05);The expression level of OX40 L gene in mouse lung tissue began to increase 30 minutes after pNS1 transfection,reached the peak on the third day after transfection,and was still higher than the basic level one week after transfection.5.Blocking OX40 signal could inhibit RSV replication.The application of OX40 L neutralizing antibody to RSV infected mice can significantly reduce the RSV titer in mouse lung homogenate(P < 0.05).Immunofluorescence analysis of mouse lung tissue showed that the expression of RSV antigen decreased significantly after blocking OX40 signal compared with the control group(P < 0.05).6.Blocking OX40 L can reduce airway inflammation and AHR caused by pNS1.Anti OX40 L can reduce the inflammatory response of mouse lung tissue,reduce the number of goblet cells secreting mucus,and significantly reduce the number of eosinophils,lymphocytes and neutrophils in BALF(P < 0.05).The RL induced by methacholine in mice decreased significantly(P < 0.05).Conclusion RSV NS1 can regulate the changes of a variety of immune cells and cytokines including Treg cells.The inflammatory factor TSLP and its activated costimulatory factor OX40,which are up-regulated in respiratory epithelial cells in the early stage after infection,can inhibit RSV replication,but reduce immune tolerance and promote the formation of airway inflammation.Part Ⅲ RSV NS1 regulates Treg cells through OX40L/Akt/mTOR signalsObjective Our previous study have shown that RSV NS1 can regulate a variety of immune cells including Treg cells and cytokines including TSLP.The cytokine TSLP and its activated costimulatory OX40 play an important role in RSV replication and the formation of allergic airway inflammation caused by NS1.The interaction of OX40 / OX40 L has been proved to be involved in regulating the differentiation of CD4 + T cells and affecting the function of Th2 and Treg cells.Therefore,this study intends to further explore the role and molecular mechanism of OX40 signal in the immune regulation of NS1 in mice.Methods1.Grouping of animals.BALB/c mice aged 4-6 weeks were randomly divided into pNS1(5μg)transfection group and empty vector transfection control group.Mice were pretreated with rapamycin by intraperitoneal injection of anti-OX40 L monoclonal antibody(15mg/kg)or mTORC1 inhibitor rapamycin(3mg/kg)at the specified time before and after plasmid transfection.Control Ig G or vehicle normal saline were used as control factors respectively.2.Sample acquisition and treatment.After euthanizing the mice at the specified time point,the lungs and spleen were taken.The proportion of Treg cells in CD4 + T cells of mouse spleen was detected by flow cytometry.The expression of mTOR was detected by immunohistochemistry,the total protein was extracted,and the expression of mTOR pathway activation related protein(pmTOR,its upstream factor p Akt,downstream factor p S6K1)and Foxp3 protein were detected by Western blot.Result1.OX40 signal is involved in the regulation of pNS1 on Tregs.In pNS1 transfected mice,anti-OX40 L was used to block OX40 L signal,resulting in the increase of Foxp3 protein expression in mouse lung tissue(P < 0.05).The inhibitory effect of NS1 on Treg was significantly improved or even reversed(P < 0.05).2.pNS1 up-regulated the expression of mTOR in mouse lung tissue.mTOR positive antigen expression was observed in both pNS1 group and empty vector control group.The mean integrated optical density of mTOR positive area in pNS1 group was significantly higher than that in control group(P < 0.05).pNS1 increased the expression of p Akt,pmTOR and p S6K1 protein in mouse lung tissue(P < 0.05).3.OX40 L is involved in regulating of mTOR pathway.Blocking OX40 L can reduce the up regulation of p Akt,pmTOR and p S6K1 protein expression caused by pNS1(P< 0.05).4.mTOR pathway was involved in the regulation of pNS1 on Tregs.Rapamycin significantly inhibited the phosphorylation of S6K1,alleviated or even reversed the down-regulation of Foxp3 protein by pNS1(P < 0.05).Compared with the control group,the proportion of Treg cells in the spleen of rapamycin treated mice was significantly increased(P < 0.05).Conclusion RSV NS1 inhibits the differentiation of Treg cells by up regulating OX40 signal,which is partly mediated by regulating AKT-mTOR signal pathway. |
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