| Background:Allergic rhinitis is one of the most common respiratory diseases in the world,manifested by continuous sneezing,clear water-like runny nose,nasal obstruction and nasal itching.About 500 million people in the world are suffering from allergic rhinitis.The prevalence rate of allergic rhinitis in central cities of China is 8.7%-24.1%,and it is increasing year by year.Although allergic rhinitis does not endanger life,it will seriously affect the quality of life and work efficiency of patients,and have a negative impact on academic,employment,emotional and social functions.The huge social and economic burden caused by allergic rhinitis can not be ignored.Allergic rhinitis is characterized by the involvement of CD4~+T cell subsets and related immune mediators,including specific cytokines and chemokines.Allergic rhinitis has long been considered to be the result of up-regulation of T helper(Th)2 cells and relative absence of Th1 cells.The initiation of Th2 reaction involves enormous mechanisms,including allergic inflammation mediated by eosinophil infiltration,including many cytokines and chemokines,which leads to pathological changes such as increased vascular permeability,increased exudation,inflammatory cell infiltration,tissue edema and increased excitability of nerve endings.Although Th2 cell mediated immune response mechanism can explain many characteristics of allergic rhinitis,this simple inflammatory model is not enough to fully explain its immune mechanism.In recent years,the successive discoveries of Th17 and Regulatory T(Treg)cells complicate the existing Th1/Th2 balance pattern and expand our understanding of the pathogenesis of allergic rhinitis.Recently,a new independent Th cell subset,Th9,has been identified,which is characterized by high expression of IL-9.IL-9 can affect inflammation and normal tissue cells,increase the number of lymphocytes,eosinophils and mast cells,enhance mast cell response to allergens,and promote the expression of mucin.Various T cell subsets regulate inflammation mainly by secreting specific cytokines.For example,Th1 cells secrete interferon(IFN)-?,Th2 cells secrete interleukin(IL)-4,IL-5,IL-13,Th9 cells secrete IL-9 and Th17 cells secrete IL-17.Th9 cells have been reported to be involved in the development of allergic asthma,but their effects on allergic rhinitis remain unclear.Objective:In view of the important role of IL-9 in other inflammatory diseases and the protective effect of neutralizing antibodies against IL-9 under these conditions,we infer that IL-9 or Th9 cells may be an important factor in allergic rhinitis and that neutralizing IL-9 antibody therapy may be effective in allergic rhinitis.Therefore,we first detect whether there is Th9 cell subset in allergic rhinitis mouse model,and then study the changes of CD4~+T cell subset and the improvement of symptoms in allergic rhinitis mouse model after the intervention of IL-9 neutralizing antibody.Methods:Forty female mice aged 6-8 weeks were divided into four groups with an average of 10 mice in each group:normal control group,allergic rhinitis group,IL-9neutralizing antibody group and homologous control group.On days 0,2,4,6,8,10,12,14,mice were sensitized by intraperitoneal injection of 1 mg/ml ovalbumin(OVA)and 20 mg/ml aluminium hydroxide 100?l.From the 15th day to the 25th day,100?g OVA was dissolved in 20?l saline every day and dripped into the nasal cavity of mice to stimulate.The mice of IL-9 neutralizing antibody and control group were given 10?g IL-9 neutralizing antibody and anti-IL-9 homologous control antibody 30 minutes before OVA stimulation.The mice of control group were sensitized and stimulated by saline instead of OVA at all stages.On the 25th day,the number of scratches and sneezes was recorded within 10 minutes of the last intranasal administration.Two hours after the last administration on the 25th day,the mice were executed.Five mice in each group were immersed in 4%polyformaldehyde and decalcified in 10%ethylenediaminetetraacetic acid for 28 days.The specimens were paraffin-embedded and coronal sections with a thickness of 4 microns.Then eosinophils were detected by hematoxylin and eosin staining.The nasal mucosa of the remaining five mice in each group was divided into two parts:one was prepared into single cell suspension,centrifuged and the supernatant was taken.The protein levels of IFN-?,IL-4,IL-9 and IL-17 in nasal mucosa were detected by cytometric bead array;the number of Th1,Th2,Th9,Th17 and Treg cells in nasal mucosa were detected by flow cytometry.In the second part,tissue was stored at-80?for RNA extraction.The expression of Th cell-related cytokines(IFN-?,IL-4,IL-9 and IL-17),Th cell-related specific transcription factors(T-bet,Gata3,PU.1,Irf4 and Ror?t)and Treg cell-specific transcription factors Foxp3 were detected by real-time polymerase chain reaction.Result:The allergic rhinitis mouse model was successfully constructed.Compared with the control group,the nasal symptoms of the mice were significantly aggravated,and the number of eosinophils in nasal mucosa was significantly increased.After intervention with IL-9 neutralizing antibody,the corresponding nasal symptoms were improved and the number of eosinophils decreased.Th9 reaction in mucosa of allergic rhinitis mouse model increased.The levels of Th cell-related cytokines IFN-?,IL-4,IL-9 and IL-17 in the nasal mucosa of allergic rhinitis mice were significantly higher than those in the control group.After neutralizing IL-9,the levels of these four cytokines were effectively controlled.The expression of IL-4,IL-9 and IL-17 in allergic rhinitis was significantly higher than that in control group.After intervention with IL-9 neutralizing antibody,the expression of these three cytokines was effectively controlled,while the expression of IFN-?in allergic rhinitis was slightly higher than control group,but no statistical difference.However,after neutralizing IL-9,the expression of IFN-?decreased significantly.The expression levels of Gata3,PU.1,Irf4 and Ror?t mRNA in allergic rhinitis were significantly higher than those in control group.After the intervention of IL-9 neutralizing antibody,the expression levels of Gata3,PU.1,Irf4 and Ror?t mRNA showed a downward trend,while the level of T-bet gene was not significantly different among the four groups.Compared with control group,the expression level of Foxp3 gene was significantly lower in allergic rhinitis,and showed an upward trend after neutralizing IL-9 antibody.Th2,Th9 and Th17 cell percentages in allergic rhinitis were significantly higher than those in control group.After intervention with IL-9 neutralizing antibody,their percentages decreased,but there was no significant difference in Th1 cell percentage among the four groups.Treg cell percentage in allergic rhinitis was significantly lower than those in control group.After using IL-9 neutralizing antibody,the percentage of Treg cell percentage increased.Conclusion:For the first time,we confirmed that Th9 cells were involved in the development of allergic rhinitis,and IL-9 neutralizing antibody had a broad anti-inflammatory effect.It can not only improve the symptoms of allergic rhinitis,but also inhibit Th2,Th9 and Th17 responses and enhance regulatory T response.We hope that the results of this study will improve people's understanding of the pathogenesis of allergic rhinitis and help to develop new antibodies to treat the disease. |
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