Crosstalk Between Nasal Epithelial Cells And CD4+CD25+Foxp3+Treg Cells In Allergic Rhinitis

Allergic rhinitis(AR)is characterized by nasal mucosa hyperreactivity when exposured to allergens,such as house dust mites,animal dander,pollen,trigger allergen-specific B lymphocytes to produce Ig E.Although much is known about the imbalance of Th1/Th2 in AR,the role of nasal epithelial cells as the first line of mucosal defense to inhaled pathogens is concerned more and more but still undefined.We now realize that epithelial cells recognize allergens through expression of pattern recognition receptors and then mount the innate and adaptive immune responses.On allergen recognition,epithelial cells release cytokines,such as thymic stromal lymphopoietin and cells junction,which activate the dendritic cell network and other innate immune cells,such as basophils and type 2 innate lymphoid cells.Therefore epithelial cells are crucial in determining the outcome of allergen inhalation in allergic rhinitis and asthma.NECs produce chemokines and cytokines that recruit and activate local dendritic cells(DCs)after allergen exposure.Then they promote Th2 immune responses and are induced more immunologic activity,which result in cascade amplification of the downstream responses,following eosinophilis and basophils inflammation.Understanding about abnormalities in these functions in allergic patients will be helpful to consider novel therapeutic opportunities that target this key structure.Most recent studies focus on the NECs itself or the communication with DCs,although the true role of NECs in allergic disease is far from clear.It is now generally appreciated that peripheral regulatory T lymphocytes tolerance is essential for a normal immune response and successful immunotherapy of allergic disorders.A number of studies have demonstrated the role for Treg in mediating of allergic airways disease by suppression of DCs' role of inducement to genetate effector T cells,B lymphocytes which could generate specific Ig E,and the function of immunocytes including mast cell,esosinophils and basophils.They also have significant correlation with local tissue remodeling and inhibit the migration of effector T cells to tissues.The allergy-suppression effect leads Tregs to be a hotspot.There're numerous suppressive mechanisms,including the cell-to-cell contacting(eg.CD25,CTLA-4,ICOS)and releasing cytokines(eg.IL-10,TGF-beta,IL-4,PD-1).Understanding about Tregs characteristic will be helpful to consider novel therapeutic targets,however,there are still many questions confused us.Natural allergen specific Tregs was unfounded nor the effective activator.TGF-beta and IL-10 are not desirable,the former has disadvantages of carcinogenic and fibrosis-genic,the latter has very short half-life in-vivo.Therefore,we need more studies about Tregs.Ngugen's study suggested that TSLP directly impairs pulmonary Treg function which association with aberrant tolerogenic immunity in asthmatic airway in 2010.Based on these studies,it's not hard to find that TSLP is one of the most important epithelial-derived cytokines in AR,there might be a crosstalk between nasal epithelial cells and Foxp3+Treg cells.In this study,we hypothesize that NECs may communicate with Tregs directly by cytokines,and moderate the suppression fuction of Tregs in AR.In order to confirm the hypothesis,we designed the experiment plan including three parts and worked.In the first part,aimed to identify the communication of NECs and Tregs,we built a co-culture model of NECs and Tregs in vitro.We cultured the primary human nasal epithelial cells from healthy volunteers till 80% confluence,isolated CD4+CD25+Regulatory T cell form human PBMCs by using the magnetic isolated kit,built the co-culture model successfully.Examed surface markers by Flow Cytometry,then analysed the meaning of the high expression of CCR8 and CTLA-4(p=0.002,P<0.05;p=0.035,P<0.05)after co-culture compared to the single culture.In part two,aimed to determine the Treg-associated cytokines expressions from allergic NECs,AR patients with house dust mite specific Ig E and healthy controls were enrolled to collect the primary NECs.Examed the Treg-associated cytokines including TSLP,IL-10,TGF-?,IL-25,CCL1.Analysed the differences of TSLP?,CCL1?,IL-25?,TGF-?? m RNA in AR group(P<0.05,P<0.05,P<0.05,P<0.05)after incubating with Der p1 compared with healthy group before co-culture.In part three,aimed to elaborate the influence of NECs on Tregs suppress fuction in allergic disease.We examed the Tregs surface markers by Flow Cytometry,analysed the meaning of the low expression of CCR8 and CTLA-4(p=0.007,P<0.05;p=0.011,P<0.05)after co-culture compared to the healthy control.Detected there were nodifferences of concentrations TSLP?(p=0.016,P<0.05),CCL1?(p=0.047,P<0.05),IL-10(p=0.101,P>0.05),TGF-?(p=0.432,P>0.05)in the suspension compared to healthy control.Then conducted correlation analysis between NECs' cytokines and the surface markers of Tregs.The results of this study suggest that,primary human NECs could communicate with Tregs directly,the suppression function of Tregs was be maintained or mounted after co-culture with NECs.In AR,the abnormality of NECs immunologic activity moderate the suppression function by cytokines expression change including increased TSLP and decreased CCL1.The moderated suppression function including low expression of CTLA-4 and CCR8.CCL1/CCR8 might be the other pathway between NECs and Tregs in vitro besides TSLP/TSLPR,and may play a key role in immune network of AR.Our study confirmed the crucial role of nasal epithelial cells from a new view.The conclusions will be helpful to consider novel therapeutic targets in AR and airway allergic disease.