Study Of Thymic Stromal Lymphopoietin's Role In The Mechanisms Of RSV-induced Asthma Exacerbation

BACKGROUND AND AIM:Bronchial asthma(asthma) is a chronic respiratory disease characterized by chronic airway inflammation,mucus hypersecretion,airway hyperresponsiveness and airway remolding,which has a great harmful impact on health.The initiation and development of asthma are regulated by diverse factors,which determine the differentiation of lymphocytes,the type of immune responses,the synthesis of cytokines and the process of tissue's repair.In spite of the great progresses in fundamental researches of asthma,the current treatments to control the acute exacerbation of asthma and severe asthma are still unsatisfied.The development of molecular virology techniques,in particular of reverse transcription-polymerase chain reaction(RT-PCR) assays for the identification of respiratory viruses in biological samples,has provided a more sensitive tool to evaluate the role of respiratory viruses in the natural history of asthma.Clinical studies performed using RT-PCR suggest that the proportion of virus-induced asthma exacerbations is likely be around 80-85% in school-aged children and 60-75%in adults of the total number of exacerbations. respiratory syncytial virus(RSV) is a single strand negative-sense RNA virus,which is the most common pathogen causing the infection of children's airway.it's infection has been verified as a main cause of children's asthma,and was regarded as an important dangerous factor in development and exacerbation of asthma.But the human's immune response to RSV is incomplete and transient,the adults had been infected by RSV may be infected again by the same strain of RSV.The destructive effects of microorganism invasion cause the evolution of host defense.In mammals,the immune system is composed of two branches,the innate immune system and the adaptive immune system,The innate immune response is the first line of host defense and is responsible for immediate recognition and control of microbial invasion.The innate immune response relies on evolutionarily ancient germline-encoded receptors,the pattern-recognition receptors(PRRs),which can recognize the conservative and invariant pathogen-associated molecular patterns (PAMPs) of microorganisms.Toll like receptors are the most distinctive receptors in the recognition of virus.The airway epithelial cell was regarded as a line of physical barrier,which defense harmful inhalants and microorganisms.current researches have shown the airway epithelial cells play a key role in the initiation and acceleration of host airway defense.It can sense stimulation and initiate the innate immune response to induce cytokines production,which regulate the adaptive immune response by interacting with local dendritic cells(DCs).The airway epithelial cells can affect DCs through secreting thymic stromal lymphopoietin(TSLP),which can promote development of DCs to regulate naive CD4~+T cells' differentiation to Th2 type.The TSLP-DCs induced Th2 CD4~+T cells produce a very unique cytokine profile,including IL-4, IL-13,IL-5 and TNF-a.one study demonstrated that in all TLRs' ligands only TLR3 ligand(dsRNA) induced great TSLP mRNA expression in normal human bronchial epithelial cells.This suggest the formation of dsRNA during viral replication possibly induce TSLP expression to exacerbate the Th2 inflammation reaction,and aggravate the mucosal inflammation in airways of asthmatics.Although asthma is characterized by Th2-type inflammation,the normal CD4~+T cell response to viral infection is thought to be predominantly of the Th1 type.The abnormal reactive inflammation in respiratory viral infection may be skewed toward inappropriate and potentially harmful Th2 responses.The purposes of this research are to investigate whether RSV has effects on the expression of innate immune response receptor TLR3 and Th2 cytokine TSLP during the virus' infection in human respiratory airway epithelial cells,to investigate whether anti-TLR3 antibody can inhibit the expression of TSLP in RSV-infected airway epithelial cells,to investigate whether local Th1/Th2 microenvironment has effects on the expression of TSLP by adding recombinant human IFN-γand recombinant human IL-4 into culture medium,and to investigate whether ribavirin and Dexamethasone have effects on the expression of TSLP in RSV-infected airway epithelial cells.subsequently to research whether the change of TSLP is concerned with asthma we investigate the expression of TSLP in RSV-induced asthma exacerbation and it's effects on airway inflammation in mice.finally we investigate different drugs' effects on expression of TSLP and airway inflammation in mice.METHODSIn vitro tests1.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration and time of PolyⅠ:C stimulationUsing 10%DMEM culture medium containing different concentration of PolyⅠ:C(0μg/ml,1μg/ml,5μg/ml,10μg/ml,25μg/ml,50μg/ml) to cultivate 16HBE for 3 hours,real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.Using 10%DMEM culture medium containing 10μg/ml PolyⅠ:C to cultivate 16HBE for different hours(1 h,2 h,3 h,6 h,12 h,24 h),real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.2.The expression of TLR3 in 16HBE after RSV infectionHep-2 cells were inoculated to amplify RSV,indirect immunofluorescence assay was used to verify the amplification of RSV in 16HBE,Reed-Muench method was used to calculate RSV's virulence.indirect immunofluorescence assay was used to detect the expression of TLR3 in 16HBE after 24 hours of RSV infection.3.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE and production of TSLP protein after RSV infection and interventionsThe experiment was designed to divide 6 groups:control group,RSV group, RSV/Anti-TLR3 group,RSV/IFN-γgroup,RSV/L-4 group,RSV/Dexamethasone group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.4.The effect of ribavirin on the comparative expression of TSLP mRNA and production of TSLP protein in RSV-infected 16HBEThe experiment was designed to divide 4 groups:control group,RSV group; RSV/ribavirin group;ribavirin group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.Animal experiments1.The effects of RSV infection on production of TSLP and airway inflammation in asthmatic mice32 BALB/c mice were divided into 4 groups:control group,OVA group,RSV group,OVA/RSV group. Airway hyperresponsiveness to MeCh(Sigma;0,3.125,6.25,12.5,25 and 50 mg/ml in isotonic saline) was assessed by whole body plethysmography(Buxco), Penh value of different groups were recorded and compared.Cytokines(IL-4,IFN-γ,IL-5,IL-13) in mice serum were examined by using ELISA assay.To evaluate the pulmonary immune response,BAL fluid was isolated,total cells recovered were counted,and the cellular composition of BALF was determined using Giemsa's staining.TSLP in BALF was examined by using ELISA assay.Pulmonary histopathology:On protocol day 26,mice lungs were excised and fixed.These tissues were then embedded in paraffin,cut sections and stained with hematoxylin and eosin(H&E);periodic acid-Schiff(PAS) to show mucus production in airway goblet cells.TSLP Immunohistochemical staining of lung specimens was observed in different groups.The production of TSLP was examined by using western blotting,Gel-Pro ANALYZER 4.5 software was used to analyze the comparative production of TSLP protein.2.The effects of different drugs on secretion of TSLP and airway inflammation in RSV-induced exacerbation of asthma32 BALB/c mice were divided into 4 groups:OVA/RSV group,PolyⅠ:C group, ribavirin group,Dexamethasone group.Methods of examination in Airway hyperresponsiveness to MeCh,Cytokines (IL-4,IFN-γ,IL-5,IL-13,TSLP) assay,BALF's cell classification,pulmonary histopathology and TSLP's western blotting were described above.Statistical analysisStatistical analysis was performed using SPSS 13.0 software.All data were expressed as mean±SEM.One-way analysis of variance(one-way ANOVA) and LSD,SNK or Dunnett's T3 methods were used to determine differences between experimental groups according to test of homogeneity of variance.Significance was accepted when p＜0.05.RESULTS:Results of in vitro tests1.The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration of PolyⅠ:C stimulation were upregulated along with PolyⅠ:C concentration.Peak expression of TSLP mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(29.84±5.32-fold compared to control,P＜0.01). Peak expression of TLR3 mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(4.46±0.8-fold compared to control,P=0.001).The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different time of PolyⅠ:C stimulation were induced to peak levels at 3h,corresponding copies of TSLP mRNA was increased to 4.48±0.35-fold compared to 1h stimulation(P＜0.001),TLR3 mRNA was increased to 1.66±0.12-fold compared to 1h stimulation(P＜0.001).2.Hep-2 cells inoculated by RSV show great typical cytopathic effect,the supernatant of culture medium was collected to get viruses,the virulence of virus was adjusted to MOI=2.indirect immunofluorescence assay show RSV amplication in Hep-2 cells.16HBE cells inoculated by RSV were cultured subsequently for 24 hours, indirect immunofluorescence assay show higher production of TLR3 when compared to control.3.The expression of TSLP mRNA and TLR3 mRNA in 16HBE after 6 hours of RSV infection were increased(1.63±0.08-fold and 1.54±0.34-fold compared to control respectively).The expression of TSLP mRNA(0.83±0.18-fold compared to control) was decreased when recombinant human IFN-γwas added,lower than RSV group(P＜0.001).The expression of TSLP mRNA(2.61±0.45-fold compared to control) was enhanced synergistically when recombinant human IL-4 was added, higher than RSV group(P=0.025).The expression of TSLP mRNA(1.23±0.2-fold compared to control) was decreased when anti-TLR3 antibody was added,lower than RSV group(P=0.034).Dexamethasone inhibited the increased level of TSLP mRNA expression in RSV-infected cells(P＜0.001).different interventions have no evident effects in expression of TLR3 mRNA except for Dexamethasone(inhibited the increased level of TLR3 mRNA expression in RSV-infected cells).The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection were increased(1.55±0.36)(1.9-fold compared to control,P＜0.001).The comparative production of TSLP protein(1.13±0.31) was decreased when recombinant human IFN-γwas added,lower than RSV group(P=0.020).The comparative production of TSLP protein(1.99±0.37) was enhanced synergistically when recombinant human IL-4 was added,higher than RSV group(P=0.014).The comparative production of TSLP protein(1.28±0.28) was decreased when anti-TLR3 antibody was added,but no significance was found when compared to RSV group(P=0.114).Dexamethasone significantly inhibited the increased production of TSLP protein in RSV-infected cells(0.53±0.11)(P＜0.001).4.The expression of TSLP mRNA in 16HBE after 6 hours of RSV infection was increased.Ribavirin inhibited the increased level of expression.TSLP mRNA expression in ribavirin group(1.41±0.18-fold compared to control) was lower than RSV group(1.99±0.25-fold compared to control)(P=0.006).The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection was increased.Ribavirin significantly inhibited the increased level of TSLP protein.The comparative production of TSLP protein in ribavirin group(1.16±0.17) was lower than RSV group(1.97±0.19)(P＜0.001).Results of animal experiments1.Airway responsiveness to MeCh increased along with its concentration.Mice in the OVA/RSV group showed reduced airway function with the most high Penh compared to mice in other groups.a significantly increased Penh compared to OVA mice from 6.25 mg/ml MeCh(318.66±50.87 vs 187.95±38.73,P＜0.001),different drug treatments somewhat inhibited the enhancement of Penh(P＜0.01).a significantly decreased Penh in mice of different treatment groups compared to OVA/RSV mice at 50 mg/ml MeCh(P＜0.05),Dexamethasone is the most effective drug in decreasing Penh compared to other treatment groups(P＜0.001).2.Cytokine levels in mice serum were examined to study the Th1/Th2 polarization in different groups.levels of Th1/Th2 cytokine(IL-4,IL-5,IL-13,IFN-γ, TSLP) were elevated in OVA/RSV mice,higher than OVA mice(P=0.001, P=0.003,P=0.02,P＜0.001 respectively).All treatments inhibited the production of IL-4 and IL-5(P＜0.05),PolyⅠ:C and dexamethasone inhibited the production of IL-13(P=0.008,P=0.001 respectively),ribavirin and dexamethasone inhibited the production of IFN-γ(P＜0.01).3.numbers of total cells,eosinophils,neutrophils and lymphocytes in OVA/RSV group were the highest compared to all other groups(P＜0.01).Treatment groups all inhibited the elevation of total cells and eosinophils(P＜0.01).PolyⅠ:C and dexamethasone decreased elevation of lymphocytes,but ribavirin group showed no significant difference with OVA/RSV group(P=0.051).PolyⅠ:C and dexamethasone inhibited elevation of neutrophils when compared to OVA/RSV group(P=0.01,P=0.011 respectively). 4.H&E staining and PAS staining showed the most severe Airway inflammation in mice lung of OVA/RSV group,epithelial cells hypertrophy /hyperplasia,mucus hypersecretion,tremendous lymphocytes infiltration were observed.All treatment groups inhibited the airway inflammation to some degree, dexamethasone is the most effective drug in inhibiting inflammation. Immunohistochemical staining showed the most significant production of TSLP in OVA/RSV mice,treatment groups showed decreased production of TSLP.5.TSLP protein in mice lung was examined,the comparative production of TSLP protein in OVA/RSV group(0.93±0.18) was the highest compared to other groups.Ribavirin inhibited production of TSLP protein(P=0.031),dexamethosone was the most effective drug in inhibiting production of TSLP protein(P＜0.001).CONCLUSIONS:1.dsRNA promotes expression of TSLP mRNA by stimulating innate immune receptor TLR3 in human bronchial epithelial cells 16HBE..2.RSV replication after infection promotes the production of TLR3 in 16HBE.3.RSV infection stimulates 16HBE to product TSLP;IFN-γinhibit production of TSLP induced by RSV infection;but IL-4 synergistically enhance its production, these results demonstrate different Th1/Th2 microenvironments have different effects on RSV-induced TSLP production;TLR3 blockade can inhibit RSV-induced TSLP production moderately,this result demonstrates indirectly TLR3 is an important factor in RSV-induced TSLP production;dexamethosone significantly inhibit RSV-induced TSLP production,this result supports the useful effect of corticosteroids on treating RSV-induced TSLP production.4.Anti-virus drug ribavirin can inhibit RSV-induced TSLP production in 16HBE,this result indicates ribavirin may has therapeutical effect on treating RSV-induced asthma exacerbation.5.RSV infection promotes the production of TSLP in OVA sensitized asthmatic mice and aggravates inflammation in mice lung.6.PolyⅠ:C,ribavirin and dexamethasone can inhibit the production of TSLP to some degree in OVA sensitized asthmatic mice,and alleviate inflammation in mice lung.