| Background and Objective:Respiratory syncytial virus(RSV),a member of the family Paramyxoviridae,genus Pneumovirinae,is the most common cause of lower respiratory tract infections in infants and young children worldwide.The majority of children become infected with RSV during the first year of life,with almost all children exposed to RSV by the age of 2 ys.Each year an estimated 34-64 million annual RSV infections occur resulting in 3-4 million hospitalizations and 50,000-200,000 deaths worldwide in children less than 5 ys of age.It remains a major cause for infant death.Age at initial infection is critical in determining initial and subsequent immune responses to RSV.In addition to causing acute respiratory failure,RSV infection is associated with sequelae such as secondary bacterial infections and reactive airway disease.It has long been speculated that acute RSV infection during infancy correlates with a greater risk of allergic asthma later in life.Supportive care remains the cornerstone of current management and no other therapy has been shown to influence the course of the disease.There is currently no safe and effective RSV vaccine.The host immune response is thought to contribute to the severity of RSV-induced disease in both humans and animal models.Many research have been done.There are Thl/Th2 imbalances in children with capillary bronchitis caused by RSV,and various Th1 and Th2 cytokines,such as IFN-γ、TNF-α、IL-2、IL-4、IL-5、IL-6、IL-8 and IL-13,are involved in the airway inflammation.Thl cytokines,IFN-γ and TNF-α,play a role in the removal of the virus.Innate immunity in RSV infection plays an important role in airway inflammation too.In recent years,the role of natural helper cells and thymic stromal lymphopoietin(TSLP)have been concerned.IL-33,a newly identified member of the IL-1 cytokine family approximately 10 years ago,expressed in the nucleus of epithelial cells and released into the extracellular space following tissue damage.It was first described as a nuclear protein designated’nuclear factor from high endothelial venules’(NF-HEV).Constitutive and abundant expressing in normal human tissues,IL-33 is normally released by damaged or necrotic barrier cells(endothelial and epithelial cells),acting as an alarmin.IL-33 is a ligand for the orphan IL-1 family receptor ST2(growth stimulation expressed gene 2).As a cytokine,IL-33 signals through a heterodimer receptor complex comprising an IL-33－specific ST2(IL-1 receptor-like molecule),coupled with the commonly utilized subunit,IL-1R accessory protein(IL-1RAcP),which is shared by other members of IL-1 cytokine family.Together,ST2/IL-1RAcP forms a heterodimeric signaling complex to which multiple proteins enabling IL-33-induced signaling are recruited.These include TLR/IL-1R family pathway components,including the signaling adaptor MyD88,the ubiquitin ligase,TRAF6,and IRAKs.Activated TRAF6 and IRAK1 and-4,in turn,lead to the NF-κB,MAPKs p38,ERK and JNK activation,inducing IL-5 and IL-13 transcription.Soluble form of ST2(sST2),the decoy receptor,prevents productive interaction of IL-33 with ST2.The alarmin interleukin-33 is most typically associated with the initiation and enhancement of T helper 2(Th2)responses.In recent years,it was indicated that IL-33 can also drive Thl effector function.Thl cells and Th1-associated cytokines,such as INF-y,are crucial to control viral and intracellular bacterial infections.Now,evidence is accumulating that IL-33 also potently stimulates group 2 innate lymphoid cells(ILC2s),regulatory T(Treg)cells,CD8+ T cells and natural killer(NK)cells.IL-33 is now recognized to have a key role in innate and adaptive immunity.The role of IL-33 in airway inflammation caused by RSV infection is not clear.Mitogen-activated protein kinase(MAPK)signaling pathways have been identified in eukaryotic cells,and transmit different signal to the nucleus.MAPK pathways are responsible for regulating a variety of cellular activities,including growth,proliferation,differentiation,apoptosis and inflammation,in response to certain environmental stimuli.MAPK family includes three major distinct stress-activated protein kinases,namely c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),and p38 MAPK.There are 5 ERK subgroups,ERK1-ERK5,among them,ERK1 and ERK2 are more extensive and clearly researched,with the molecular weight of 44kDa and 42kDa respectively.Nuclear factor-kappa B(NF-κB)is a kind of important regulatory transcription factor in inflammatory response.There are 5 kinds of subunits of NF-κB,including p65,p50,p52,Rel and RelB.NF-κB exists in the form of inactive compounds with the inhibition protein,predominate in the cytoplasm.When the inhibition protein is degradated of by signal molecules,NF-κB falls off from its inhibition protein and activated,then transmit into the nucleus.By regulating the expression of target genes,NF-κB involved in the early immune response,inflammatory reaction,cell growth and apoptosis.The role of IL-33 in viral disease is disputable.Although IL-33/ST2 is strongly associated with asthma,it is currently unclear whether IL-33 acts directly on airway inflammation caused by respiratory syncytial virus(RSV).Elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV in acute phase.It also be seen that the amount of IL-33 protein in nasal washes was decreased in RSV-infected children.RSV infects primary airway epithelial cells and triggers the generation of IL-33,leading us to hypothesize that IL-33 receptor(ST2)signalling,MAPKs and NF-κB,may be involved in RSV induced airway inflammation.In this study,rat alveolar epithelial cell Ⅱ(AEC Ⅱ)were cultured in vitro to explore the rule of IL-33 express of AECⅡ infected by RSV.Then we established airway inflammation model caused by respiratory syncytial virus in immature SD rats by intranasal inoculation with RSV.We observed the pathological changes of lung tissue,the immunopathology state and the protein expression levels of ST2,P-ERK1/2,P-JNK,P-p38 and P-p65-NF-κB,to demonstrate that IL-33 signalling is required for regulation of airway inflammation and type2 responses in a rat model of acute respiratory syncytial virus infection.By using blockers,we explored the role of IL-33 neutralizing antibody in airway inflammation caused by respiratory syncytial virus in immature SD rats.Methods:1.Experiment of in vitro cell.Human respiratory syncytial viras type A(RSV A)was propagated in Hela cells.Recovery rat AEC Ⅱ,the cell culture supernatant were collected at 3 h,12 h,24 h after the RSV infection to measuring the concentration of IL-33 by ELISA.The expression levels of IL-33 mRNA of AECⅡ were examined by real-time fluorescence quantitative polymerase chain reaction between control group and RSV group among different time points.2.Experiments on animals.Specific pathogen-free 3-4-wk-old male SD rats were kept in pathogen-free conditions.The rats were randomly divided into normal control group(NC,n=10),RSVgroup(RSV,n=10)、RSV+a-IL-33 group(RSV+a-IL-33,n=10)and RSV+IgG group(RSV+IgG,n=10).Rats of model group were challenged intranasally with human RSV A on days 0 and 1.Normal control rats received intranasal liquid supernatant without virus.For blocking IL-33 signalling,rats of RSV+a-IL-33 group were treated intranasally twice with IL-33 neutralizing antibody at 3 hours before the first dose RSV infection and 12 hours after the second dose RSV infection.Rats of RSV+IgG group were treated intranasally twice with IgG at 3 hoturs before the first dose RSV infection and 12 hours after the second dose RSV infection.As a control group,normal control rats received intranasal liquid supernatant without virus.All rats were killed on day 4.Measure the levels of IL-33 of BALF and IL-33 mRNA of lung.The pathological changes of lung tissues were detected by H&E staining.The cells of BALF is counted and sorted by Wright-Giemsa staining.Cytokines of plasma,including IFN-γ,IL-5 and IL-13,were quantified by ELISA.The level of IL-33 in bronchial alveolar lavage fluid was also quantified by ELISA between control group and RSV group.The expression levels of IL-33 mRNA and ST2 mRNA of lung were examined by real-time fluorescence quantitative polymerase chain reaction between control group and RSV group.The expression levels of and ST2,P-ERK1/2,P-JNK,P-p38 and P-p65-NF-κB protein of lung were examined by Western blot analysis.Results:1.The relative expression of ST2 mRNA of AEC Ⅱ and the concentration of IL-33 in the rat AEC II cell culture supernatant.Compared with normal control group,the expression of IL-33 mRNA of AEC II in the RSV group were higher among different time points(P<0.05).Compared with normal control group,the concentration of IL-33 in the rat AEC II cell culture supernatant in the RSV group were significantly higher among different time points(P<0.05).2.The general form of lung tissue of SD rats.Compared with the NC group,the general form of lung tissue of the RSV group and RSV+IgG group presented more obvious congestion and hemorrhagic spots on the surface of lung.While the lung lesions of RSV+a-IL-33 group were lighter with few congested areas.3.Lung sections of SD rats by H&E staining.Compared with the NC group,the RSV group and RSV+IgG group exhibited pulmonary inflammatory cell infiltration,wide alveolar septa,bronchial epithelial cell necrosis,bronchial collapse and stenosis.While the lung pathological changes of RSV+a-IL-33 group were improved obviously,with slighter inflammatory cell infiltration and epithelial cell necrosis.4.Semiquantitative analysis of the severity of peribronchial inflammation.The semiquantitative analysis score of the severity of peribronchial inflammation was lower in the RSV+a-IL-33 group than in RSV group and RSV+IgG group(P<0.05).There is no significant difference in the score between the RSVgroup and the RSV+ IgG group(P>0.05).5.The levels of IL-5,IL-13 and IFN-y of plasma.The concentrations of Th2-associated cytokines IL-5,IL-13 and Thl-associated cytokine IFN-γ in plasma were,significantly higher in the rats infected by RSV,including the RSV group,RSV+ a-IL-33 group and RSV+ IgG group,than in the NC group(P<0.05).The level of Th2-associated cytokine IL-5 and IL-13 in plasma was lower in the RSV+a-IL-33 group than in the RSV group and RSV+ IgG group(P<0.05).There is no significant difference in the concentration of IL-5 and IL-13 of plasma between the RSV group and RSV+IgG group(P>0.05).The concentrations of Thl-associated cytokine IFN-y of plasma was higher in the RSV group,RSV+ a-IL-33 group and RSV+IgG group;than in the NC group(P<0.05).While,there is no significant difference in the concentration of IFN-y of plasma among the RSV group,RSV+a-IL-33 group and RSV+IgG group(P>0.05).The ratios of Th1-associated cytokine and Th2-associated cytokine,IFN-γ/IL-5,in the RSV group,RSV+a-IL-33 group and RSV+IgG groupwere lower than in the NC group(P<0.05).The ratio of IFN-y/IL-5 was higher in the RSV+a-IL-33 group than in the RSV group and RSV+ IgG group(P<0.05).There is no significant difference in the ratio of IFN-y/IL-5 between the RSVgroup and the RSV+ IgG group(P>0.05).6.The expression of IL-33 protein of BALF and IL-33 mRNA in lung.Compared with the NC group,the level of IL-33 of bronchial alveolar lavage fluid(BALF)and IL-33mRNA of lung were both higher in the RSV group than in the NC group(P<0.05).7.The cell counting and sorting in BALF.The cell counting of BALF were significantly higher in the rats infected by RSV,including the RSV group,RSV+ a-IL-33 group and RSV+ IgG group,than in the NC group(P<0.05).The number of cells were lower in the RSV+a-IL-33 group than in the RSV group and RSV+ IgG group(P<0.05).There is no significant difference in the cell counting between the RSV group and RSV+IgG group(P>0.05).The percentage of lymphocyte,neutrophils and eosinophils of BALF were higher in the RSV group,RSV+ a-IL-33 group and RSV+IgG group,than in the NC group(P<0.05).There is no significant difference in the percentage of lymphocyte,neutrophils and eosinophils in BALF between the RSV group and RSV+IgG group(P>0.05).The macrophages percentage of BALF in the RSV group,RSV+a-IL-33 group and RSV+IgG groupwere lower than in the NC group(P<0.05).The macrophages percentage of BALF was higher in the RSV+a-IL-33 group than in the RSV group and RSV+IgG group(P<0.05).There is no significant difference in macrophages percentage of BALF between the RSV group and the RSV+ IgG group(P>0.05).8.The relative expression of ST2 mRNA and ST2 protein in lung.Compared with normal control group,the expression of ST2 mRNA and ST2 protein in lung tissue were higher in the RSV group,RSV-a-IL-33 group and RSV+ IgG group(P<0.05).The expression of ST2 mRNA and ST2 protein in lung tissue were lower in the RSV+a-IL-33 group than in RSV group and RSV+IgG group(P<0.05).There is no significant difference in the expression of ST2 mRNA and ST2 protein between the RSVgroup and the RSV+ IgG group(P>0.05).9.The relative expression of MAPK protein in lung.There is no significant difference in the expression of P-ERK1/2 protein in lung tissue among the four groups(P>0.05).Compared with normal control group,the expression of P-JNK and P-p38 protein in lung tissue were higher in the RSV group,RSV+ a-IL-33 group and RSV+ IgG group(P<0.05).The expression of P-JNK and P-p38 protein expression in lung tissue were lower in the RSV+a-IL-33 group than in RSV group and RSV+ IgG group(P<0.05).There is no significant difference in the expression of P-JNK and P-p38 protein between the RSVgroup and the RSV+ IgG group(P>0.05).10.The relative expression of P-p65-NF-KB protein in lung.Compared with normal control group,the expression of P-p65-NF-KB protein in lung tissue was higher in the RSV group,RSV+ a-IL-33 group and RSV+ IgG group(P<0.05).The expression of P-p65-NF-κB protein expression in lung tissue was lower in the RSV+a-IL-33 group than in RSV group and RSV+ IgG group(P<0.05).There is no significant difference in the expression of P-p65-NF-κB protein between the RSVgroup and the RSV+ IgG group(P>0.05).Conclusions:1.The expression of IL-33 of rat ACE Ⅱ increases after infected by RSV.2.The expression of IL-33 is increased in lung of immature SD rats infected by RSV.3.IL-33/ST2 pathway is involved in the Th2 bias airway inflammation of RSV-infected immature SD rats during the early phase of RSV infection by activating the JNK,P38 signaling and NF-κB.4.Treatment with IL-33 neutralizing antibody diminishes airway inflammation with Th2 bias caused by RSV in immature SD rats by closing the IL-33 and down-regulating the expression of ST2,which blocks the IL-33/ST2 signaling pathway. |
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