The Allergic Asthma Mechanism Of Derf31,which Is A New Allergen Of Dermatophagoides Farina

Blackground:Published data indcate that the pathogenesis of asthma is associated with genetics,lifestyle and environment.Among numerous allergens,inhalant allergens such as house dust mites are the most important factors in the induction of airway allergy.Dust mites have regional differences.In southern area of China,the secretions,and corpses of dermatophagoides farina is the main allergens of asthma,rhinitis and atopic dermatitis.Almost 60% asthma patients are sensitive to dust mites.When stimulated by allergens,lung epithelial cells release multiple cytokines,including thymic stromal lyphopoietin(TSLP),IL-33 which up-regulate the number of type 2 innate lymphocytes(ILC2s).Previous studies found that ILC2 s has close relationship with allergic disease.Objective: In our previous studies,Derf31 was identifed as a new allergen.In the present study,we prepared Derf31,and sought to elucidate the role of Derf31 in the development of airway allergy from both cell and animal experiments.Methods:(1)Using prokaryotic gene expression systems to produce the proteins of Derf31 and Derf 1,the target proteins were purified by affinity chromatography.(2)Cell experiments: DC2.4 cells were stimulated with Derf31;the co-stmulatory molecules of the cells were assessed by FACS;BMDCs were prepared and co-cultured with splenocytes,the levels of Il-4 and IFN-? were determined by ELISA;splenocytes were stained with CFSE;the cell proliferation was assessed by FACS.A549 cells were stimulated with Derf31;the levels of TSLP,IL-33 were determined by ELISA and RT-PCR.The signal transduction pathways of TSLP were determined:Derf31 were used to stimulate lung cells of normal mice or A549 cells,respectively;the cells were treated with anti-mouse TLR2 Ab or TLR4 signaling inhibitor.The supernatants or cells were collected to be analyzed the expression of IL-3 and TSLP.(3)Animal experiments: To develop airway inflammation in mice by Derf31 or Derf1.The mice were sacrificed.The serum specific antibody levels,cytokines,the histology of lung tissues were analyzed.BALF and lung homogenates were prepared to be analyzed cytokine levels,counts of eosinophil by flow cytometry and optical microscopy.The lung cells were stained with PE-CD90 1?PerCP-IL-33 R ? FITC-Lineage and APC-CD45 to detected the number of ILC2 s.meanwhil,the total RNA of lung tissues was prepared to be analyzed by RT-qPCR to determine the expression of IL-13,TSLP and IL-33.Results: We expressed and purified the protein of Derf31 and Derf1.The co-stimulated molecules CD40?CD80 and CD83 were up-regulated when DC2.4 cells were stimulated with Derf31.Meanwhile,Derf31 could drive Th0 cells to differentiate into Th2 cells and promote the proliferation of CD4+ T cells.The co-culture system of lung cells and Derf31 showed that in the presence of antibody rather than TLR4 inhibitors,the expression of TSLP were significantly down-regulated.Derf31 could induce airway allergy in mice.The levels of eosinophil,ILC2 s,TSLP and IL-33 were higher in airway tissue than that in control groups.Conclusion: Derf31 can enhance the co-stimulated molecules of DCs and driveTh0 cells to Th2 cells;TSLP and IL-33 leves from epithelial cells are up regulated by Derf31 via activating TLR2.Derf31 can induce eosinpohilic airway allergy,and enhance lung-resident ILC2 s.