The Role Of ILC2 In The Pathogenesis Of Prior RSV Infection Protects Against The Subsequent Development Of Asthma

Objective:Bronchial asthma（asthma in short）is a kind of chronic respiratory disease jointly participated by multiple cells,particularly are mast cell,eosinophil and T lymphocyte;it features airway chronic inflammation,mucous secretion increase,enhancement of airway reactivity and airway remodeling.As a common chronic disease,the morbidity of asthma presents rising trend year by year,which has become serious global public health issue that threatens human health.Meanwhile,to research asthma pathogenesis,prevent and control the occurrence and development asthma allergic inflammation are attracting researchers’increasing attention.The pathogenesis of asthma is still unclear;more and more researches believe that immune imbalance is the foundation of asthma morbidity;both cellular immunity and humoral immunity participated in the morbidity and attack of asthma.Respiratory virus infection may affect Th1/Th2 balance so as to play important role in the formation as well as development of asthma,so it is the important factor inducing and aggravating asthma,among which respiratory syncytial virus（RSV）resulting in infants’lower respiratory tract infection is one of the major causes of asthma.Some researches indicate that,the children with trachitis due to RSV infection are more likely to get recurrent wheezing and asthma,whose reasons are related to Th2 cytokine secretion induced by RSV infection.On the contrary,some other reports verified that,RSV infection could induce the production of stronger Th1 cytokine IFN-γinside BALB/c mouse,lower Th2immune response,inhibit virus replication and alleviate airway inflammatory reaction.Some researches discovered that,RSV could lower OVA sensitized asthma mouse’s bronchial hyperresponsiveness through inducing the generation of Keratinocyte cytokine.Our previous researches also showed that,RSV infection could induce organism to synthesize IFN-γand IL-12,reduce the quantity ofγδT cells inside lung tissue,lower Th2 cytokine IL-4 and IL-10mRNA expressions inγδT cells while increasing Th1cytokine IFN-γmRNA expression.However,mechanisms that RSV infection affect allergen-induced allergic asthma are still largely unknown.Type 2 innate lymphoid cell（ILC2）is an innate immune cell discovered in recent years that belongs to innate lymphocyte family.It is the lymphocyte contains typical form of lymphocyte but lacks specific antigen recognition receptor.ILC2 originates from common lymphoid progenitor inside bone marrow,so it used to be called as Natural helper cells（NHCs）,nuocytes and innate type 2 helper cells（Ih2s）.ILC2 mainly produces Th2 cytokines,such as interleukin-5（IL-5）,IL-9 and IL-13;its function in allergic reaction response,parasitic infection and asthma is attracting more attention.As the important source of Th2 cytokine in organism,ILC2 plays significant role in the pathogenesis of allergic reactions such as asthma.Some research reported found that,influenza virus H3N1 infection could lead to bronchial hyperresponsiveness of asthma characteristic signs,while the generation of bronchial hyperresponsiveness relied on the IL-13 produced by ILC2 rather than adaptive immune response.Their researchdiscovered the influential role of ILC2 cells in respiratory inflammation and initially verified the function of innate immune reaction in asthma induced by virus and uncovered the new mechanism of virus infection induced asthma;that is the core function of IL-13 of innate immune cell source.By means of OVA inducing mouse acute asthma,Klein et al detected expressions of IL-5,IL-13 and IL-4 in its lung tissue.The results showed that,ILC2 in lung tissue was the important source of IL-5 and IL-13 in OVA induced acute asthma.As mentioned above,ILC2 couldplay an important role in OVA-induced asthma,and RSV infection could have certain effect of allergen induced airway inflammatory reaction.Then,whether innate immune cell ILC2 participated in this process and what its function as well as mechanism is are still unclear.Therefore,by utilizing RSV infected OVA sensitized mouse and detecting ILC2 biological activity variation inside mouse lung tissue,this research discussed about the function and mechanism of ILC2 in the occurrence as well as development of RSV infection induced asthma,which provided theoretical reference for the prevention and treatment of asthma.Methods:1.Virus Reproduction and TitrationHuman respiratory syncytial virus type A2（RSV A2）was propagated in monolayer of Hep-2 cells.Separated virus through 30%sucrose ultracentrifugation centrifugation and stored in-80℃refrigerator.Virus titerswere expressed asa 50%tissue culture infections dose（TCID50）.2.Establishment and Grouping of Animal ModelSpecific-pathogen-free grade,6-8 weeks old female BALB/c mice were randomly divided into 4 groups:normal control group（MOCK group）,RSV infection group（RSV group）,OVA asthma group and RSV infected asthma group（RSV/OVA group）.Applied intraperitoneal injection of OVA 20μg sensitization（containing 20μg OVA and 2.25mg aluminum hydroxide,dissolved in 100μL PBS）to OVA group at 0th and 12th day;placed mice in transparent and sealed vessel at 19-24th day to challenged with 1%OVA solution by aerosol inhalation;20 min each time and 1 time/d.Instilled 20μL virus suspension containing 1.0×10⁶ TCID50 RSV into nasal cavity 7 days before OVA firstsensitization to infect mice.Collected samples after 48 hours of OVA last aerosol inhalation.Collected samples of RSV group 7 days after nasal cavity infection by RSV suspension.3.Influence of priorRSV Infection on OVA asthma Mouse Airway Inflammation（1）Production and dyeing of lung tissue pathological specimensTook the inferior lobe of left lung of model mouse to prepare paraffin section and HE dyeing to observe lung tissue inflammatory infiltration and other pathological variation.PAS dyeing was used to obverse lung tissue goblet cell proliferation and mucus secretion.（2）Giemsa Staining to detect type and quantity of BALF inflammatory cellsLungs were lavaged through a tratracheal tube with 1 ml PBS;collected bronchoalveolar lavage fluid;took cell suspension as smear after centrifugation;applied Giemsa Staining;selected view randomly under microscope;counted 200 cells and the quantities as well as percentages of neutrophil,macrophage,eosinophil and lymphocyte.（3）ELISA method to detect the Th1/Th2 cytokine levels inBALFThe level of IL-4,IL-5,IL-13,IFN-γ,and IL-33 in BAL fluids was quantified using a mouse cytokine detection ELISA kit.（4）Measurement of protein gene expressionTotal RNA was extracted from lung homogenates using TRIzol reagent.The first-strand cDNA was synthesized from the RNA using SuperScript^TM RT reagent Kit.Quantitative real-time RT-PCR was performed to detect mRNA expression level of lung tissue local Th2 cytokine（IL-5,IL-13 and IL-4）and Th1 cytokine（IFN-γ）.4.Influence of prior RSV Infection on OVA-inducedasthma Mouse ILC2 Quantity and Biological activity（1）Preparation of lung tissue single cell suspensionAfter deeply anesthetize mouse with intraperitoneal injection of cholral hydrate,extracted model mouse lung tissue and cut up;cultured by shaking inside the nutrient solution containing 200μg/ml collagenase D and 40μg/ml DNaseⅠ;split red blood cell with erythrocyte lysis buffer and suspended in RPMI1640 medium solution after counting.（2）Identification of ILC2 in the lungsof mice with flow cytometryFor identification of ILC2s in the lungs of mice,each aliquot of lung parenchyma cells was incubated with anti-mouse CD16/32 and then incubated on ice for 20 min with either specific mAbs or isotype-matched control Igs.The following antibodies were used:APC-conjugated anti-CD45,PerCP-conjugated anti-ST2,FITC-conjugated anti-lineage antibodies.Lineage cocktail included:anti-CD3,-CD4,-CD5,-CD8,-CD11b,-Gr-1,-CD19,-B220,-DX5（or NK1.1）,and-TCRδ（eBioscience）.Stained lung cells（1×10⁶）were analyzed by flow cytometry.Pulmonary ILC2s were identified as CD45⁺Lin^-ST2⁺.（3）Flow cytometry detects the Th2cytokineproduction of ILC2Took the cell suspension prepared in above experiment and then fully blended it with PMA with irritant 50 ng/ml,500 ng/ml ionomycin and 10 ug/ml brefeldin A;placed in 37℃culture box for 5h incubation.Washed twice with 1ml 2%FBS-PBS,abandoned supernatant by centrifugation,added fluorescent antibody FITC-lineage cocktail,APC-anti-Mouse CD45 and Percp-anti-Mouse ST2.Added trans membrane solution to re-suspend cell,incubate in 4℃for 1h,added trans membrane washing liquor to wash for one time.Centrifuged for 6 min with 1500rpm under 4℃to remove supernatant.1ml PBS to re-suspend;added PE-anti-Mouse IL-5,PE-anti-Mouse IL-13 and PE-anti-Mouse IL-4 fluorescent antibodies and isotype controls;ice bath for 30 min away from light;re-suspended cell with 300μl 2%FBS-PBS;detected by flow cytometry.（4）Real-time quantitative RT-PCR to detect IL-5,IL-13 and IL-4 mRNA expressions in ILC2 cellsSorted model mouse lung tissue ILC2 cells,extracted total RNA by using Triozl reagent.Adopted real-time quantitative PCR method to detect IL-5,IL-13 and IL-4mRNA expressions in ILC2 cells.5.Influence of Adoptive Reaction of ILC2 on RSV/OVA Model mouse Lung InflammationWe adoptively transferred ILC2s to RSV/OVA mice to examine the role of ILC2s on RSV-affected,OVA-induced allergic airway inflammation.Na?ve BALB/C mice were intraperionteally（i.p.）injected with IL-33（400 ng/dose each）,once daily for three days.24 hours after the last injection,lung tissue was harvested and CD45⁺Lin^-ST2⁺ILC2s were FACS-sorted.Purified cells were resuspended to 1×10⁶ cells/ml in PBS and 2×10⁴cells in 200μl of PBS was injected into each mouse via the tail vein one hour prior to RSV infection.6.Influence of RSV infection on IL-33/ST2 mRNA Expression of Asthma Mouse Lung TissueAdopted Real-time RT-PCR technology to detect expression level of lung tissue local cytokine IL-33 and ST2LmRNA.7.IL-33 affect the number and function of ILC2 via IL-33/ST2 pathwayThe number and Th2 cytokine production of ILC2 were determined by flowcytometry after stimulated with IL-33.We also performed qRT-PCR to detect the expression of IL-4,IL-5 and IL-13 mRNA in the ILC2 cells after treated with anti-ST2mAbs to block IL-33/ST2 signaling.8.IL-33 activates MAP Kinases in ILC2 cellsWe performed qRT-PCR to determine expression of ERK-1、ERK-2、IRAK-1、IRAK-4、TRAF-6、JNK-1、JNK-2、NF-kB、P38 and MyD88 mRNA in ILC2 cells after stimulated with IL-339.Statistics ProcessApplied SPSSl6.0 software to statistical analysis,expressed experimental result with±S.Quantitative data were analyzed with a one-way ANOVA to compare results among groups.Bonferroni and Games-Howell post-hoc tests were applied if significant differences were found among groups.P<0.05 indicated statistical significance of determination.Results:1.PriorRSV infection alleviated asthma mouse airway inflammatory reaction and lowered quantity of inflammatory cells inside asthma mouse lung tissue.OVA sensitized and challengedBALB/c mouse airway epithelium thickened,lumen narrowed,and the quantity of inflammatory cells,eosnophils,neutrophils and macrophages increased obviously.Prior RSV infection inhibited OVA induced lung inflammatory reaction and alleviated inflammatory cell infiltration inside model mouse lung tissue.2.Prior RSV Infection affect the levels ofThl/Th2 cytokine in the BALFofAsthma Mouse.PriorRSV infection obviously decreased the levels of IL-5 andIL-13in the BALF ofasthma mouse,but increasedIFN-γlevel.3.Prior RSV Infection Influenced Asthma Mouse Lung Thl/Th2 Cytokine mRNA ExpressionPriorRSV infection obviously inhibited the expressions of asthma mouse lung tissue cell IL-4,IL-5 andIL-13 mRNA,but it reinforced local IFN-γmRNA expression level.4.Prior RSV Infection Reduced the number and Th2 cytokine productionof ILC2 Cells inAsthma Mouse Lung TissuePrior RSV infection obviously lowered the number and Th2 cytokine IL-5,IL-13productionof ILC2 cells in the asthma mouse lung tissue.5.Prior RSV Infection Influenced Th2 Cytokine mRNA Expression in ILC2 cells.Prior RSV infection obviously inhibited the IL-5 andIL-13 mRNA expressions of asthma mouse lung ILC2 cells.6.Influence of ILC2 Cells Participating in RSV infection on Asthma Mouse Airway Allergic InflammationAfter RSV/OVA mouse were adoptive transfer of the ILC2 cells,the quantity of lung inflammatory cells increased obviously,among which the percentages of eosnophils and neutrophils increased obviously.In bronchoalveolar lavage fluid,the content of Th2cytokines（IL-5,IL-13 and IL-4）increased;in lung tissue,Th2 cytokines（IL-5,IL-13and IL-4）expression reinforced;however,the content of Th1 cytokine IFN-γin bronchoalveolar lavage fluid decreased and mRNA expression in lung tissue weakened,which verified the influential effect of ILC2 cells’participation into prior RSV infection on asthma mouse airway allergic inflammation.7.Prior RSV Infection Lowered IL-33 and ST2L mRNA Expression of Asthma Mouse Lung TissueIn this research,we analyzed mouse lung tissue IL-33 and ST2LmRNA expression situation based on real-time quantitative PCR technology.When comparing the results with that in OVA group,mouse lung tissue IL-33 and ST2LmRNA expression in RSV/OVA group decreased obviously,so the difference was statistically significant（P<0.05）.8.ILC2 are induced in the lung by IL-33/ST2 signal pathwayAdministration of IL-33 induced increased ILC2 numbers in lung.Treatment of mice with IL-33 also inducedproductionofIL-4,IL-5 and IL-13 in lung ILC2.Incubation of the lung ILC2 cells with anti-ST2 antibody diminished the mRNA expression of IL-4,IL-5 and IL-13.9.IL-33 induces ILC2 cells via MAPK signaling pathway.The relative expression of MyD88,IRAK-4 and P38 in ILC2 cells were enhanced afterstimulated with IL-33.Conclusion:1.Prior RSV infection lowers allergen induced airway inflammatory reaction.2.Prior RSV infection controls process of airway allergic inflammatory reaction through changing quantity of ILC2 cells and cytokine secretion activity via lowering IL-33/ST2pathway.3.IL-33 induces ILC2 cells via MAPK signaling pathway.