The Establishment Of Nasal-Only Delivery Allergic Rhinitis Model Challenged With Different Ovalbumin Concentration And Its Related Mechanism

Part I: Establish the model of allergic rhinitis in mice with different dose of allergenObjective: Predominantly,two animal models are used for allergic rhinitis(AR),which are established by intraperitoneal(IP)injection plus local challenge and nasal-only delivery.The differences between these two models are not fully understood.Moreover,dose-response relationship to allergens remains unclear.Methods: In this study,mice were sensitized by nasal drops(without adjuvant,once daily for 9 weeks)to set up a nasal-only delivery AR model.Five different doses of OVA nasal drops were served to explore the dose-response to allergens.Allergic symptoms,serum antibodies(IgE,Ig G2 a,Ig G1),spleen supernatant and NALF(Nasal Lavage Fluid)cytokines(IL-4,IL-5,IFN-r)and infiltrated eosinophils of the nasal mucosa were observed.Results: The allergic symptoms,serum antibodies,cytokines and infiltrated eosinophils were significantly higher in the high OVA concentration compared with those of the control group.Different OVA concentrations associated with the severity of allergy.Within a certain concentration range,OVA concentration positively related to the severity of symptoms,IgE antibody level and Th2 bias.Meanwhile,serum antibodies(IgE,Ig G1)and cytokines(IL-4,IL-5 in spleen and IL-4 in NALF)were significantly higher in the classical IP injection group than in the nasal drip groups.Conclusion: The IP injection model and the nasal-only delivery model are two typical models for AR that causes different immune response.A positive dose-response relationship in the nasal-only delivery model is observed from 25 mg/ml to 0.025 mg/ml.Part II: The mechanism of action of different doses of allergens on immature dendritic cells in vitroObjective:According to the animal model of natural allergic rhinitis,different doses of allergen stimulated mice to produce different degrees of allergic reactions.In allergic reactions,allergens may only be triggered by DC antigen presentation before triggering subsequent reaction.Therefore,the different functional states of DC can have a great impact on the subsequent differentiation direction of na?ve T cells.Studies have reported that the effects of different doses of allergens on T cells play a role by altering the amount of antigen presented,and little is known about how antigen dose affects dendritic cells.Therefore,we speculate that the allergen dose is first induced by different DCs of different functional states and presented to different levels of na?ve T cells to induce differentiation in different directions.Methods:Mature dendritic cells were isolated and cultured in vitro.After stimulation with different doses of allergen for 24 hours,the expression of cell surface antigen(CD40?CD11c?CD86?MHC-??CD80)was detected by flow cytometry.The difference factors of different doses of stimulation were analyzed by RNA-seq technique.DCs stimulated with different doses of antigen were co-cultured with sorted na?ve T cells for 72 h.Flow cytometry was used to detect Th1(CD4+IL-4+)?Th2(CD4+IFN-r+)?Th17(CD4+IL-17+)?Treg(CD4+CD25+Foxp3+).Results:This study found that the positive rates of dendritic cell maturation markers CD40,CD11 c,CD86,MHC-II,and CD80 increased with increasing OVA dose,and RNA was stimulated with different doses of allergens.RNA-seq shows a number of key metrics that differ significantly.After co-culture of dendritic cells induced by different doses of allergens with na?ve T cells,the flow detection results showed that the ratio of CD4+IL-4+ cells was higher in the 10?g group and the ratio of CD4+IFN-r+ cells in the 100?g and 1000?g groups.Higher;in the 10000?g group,the proportion of CD4+CD25+Foxp3+ cells increased,and the proportion of CD4+IL-4+ and CD4+IFN-r+ cells decreased.Conclusion:Different doses of allergens can stimulate dendritic cells with different mature states and determine the direction of initial T cell differentiation.The weak allergen-stimulated DC can induce na?ve T cells to differentiate into Th2 direction.When the antigen dose is increased,the na?ve T cells can be induced to differentiate into Th1.When the antigen exposed by the DC exceeds a certain dose,the na?ve T cells are induced to differentiate into Treg cells.