Tangeretin Promotes Regulatory T Cell Differentiation By Inhibiting Notch1/Jagged1 Signaling In Allergic Rhinitis

Background: Allergic rhinitis(AR)comprises an immunoglobulin E(Ig E)-mediated type I hypersensitivity reaction that occurs when atopic individuals react to an inciting inhaled allergen,characterized by symptoms of sneezing,rhinorrhea,and nasal obstruction.Persistent or uncontrolled Th1/Th2/Th17/Treg cell responses are often associated with the pathological states of allergic rhinitis,in particular,excessive and/or abnormal Th2 responses are always involved.AR affects up to 40% of the population worldwide and is easy to relapse,it is also associated with a variety of comorbid conditions,such as rhinosinusitis,asthma and otitis media.Although not life threatening,the symptoms of AR as well as the comorbid diseases are frequently bothersome,adversely affect work,psychological health and quality of life,as well as imposing a significant social burden and costs.Despite rapid progress in the drug therapy,such as intranasal steroids,antihistamines,and leukotriene receptor antagonists,20% of AR patients do not show sufficient subjective and objective improvement.Allergen-specific immunotherapy is the only curative treatment option,however,it still faces multiple challenges,including efficacy,safety,compliance and comparative cost-effectiveness.Tangeretin is an O-polymethoxylated flavone that is abundant in the peel of citrus fruits,it selectively inhibits Notch1 signaling and demonstrates broad beneficial bioactivities,such as anti-oxidant,anti-inflammatory,and anti-cancer.However,the exact role of tangeretin on AR and the underlying mechanisms remain unclear.In this study,we will assess whether tangeretin functions in regulating T-regulatory cells(Tregs)and alleviating allergic rhinitis,aiming to identify effective therapies to manage airway inflammation in AR.This research was divided into three parts:The first part: The expression of Notch1/Jagged1 in AR patientsObjective:To investigate the expression of Notch related receptors and ligands in AR patients,and its relationship with allergy severity.Methods: 1.204 patients(103 cases of AR and 101 controls)were recruited from July to November 2018 in Department of Otolaryngology-Head and Neck Surgery,Renmin Hospital of Wuhan University,China.The expression of Notch1,Jagged1 and DLL1 in the serum was detected by ELISA kits.2.To investigate the relationship between Notch1/Jagged1 expression with allergy severity,AR patients with grades 1�3were recorded as AR-mild,and patients with grades 4-6 were recorded as AR-severe.The differences of Notch1/Jagged1 levels between these two group were verified.3.The expression of Notch1/Jagged1 were also analysed between house dust mites(H1)-mild or Dermatophagoides farinae(D1)-mild group and house dust mites(H1)-severe or Dermatophagoides farinae(D1)-severe group.4.The Pearson correlation coefficient(r)was calculated to assess the correlation between Notch1/Jagged1 expression and s Ig E levels.Results: 1.Compared with the control group,Notch1/Jagged1 expression were significantly up-regulated in AR patients(P<0.05),while DLL1 remained unaltered(P>0.05).2.Markedly increased Notch1/Jagged1 expression were observed in patients with severe AR than that with mild AR(P<0.05).3.The expression of Notch1/Jagged1 were also increased in H1(or D1)-severe group compared to H1(or D1)-mild group(P<0.05).4.Pearson correlation analysis showed that the expression of Notch1/Jagged1 were positively correlated with the levels of s Ig E(Notch1: r=0.7106,P<0.01;Jagged1: r=0.6361,P<0.01).Conclusion: Notch1/Jagged1 signaling was significantly up-regulated in allergic rhinitis and positively associated with the severity of AR.The second part: Tangeretin promotes regulatory T cell differentiation by inhibiting Notch1/Jagged1 signaling in AR model.Objective:To investigate the effect of tangeretin on allergic rhinitis and the underlying mechanisms.Methods: The specific pathogen free(SPF)C57BL/6 mice were grouped randomly(n= 10/group): Normal group,AR group,AR+Tan(tangeretin)group,and AR+DXM(dexamethasone)group.1.The allergic symptom scores,OVA-specific Ig E titers(ELISA),eosinophil infiltrates(HE stain),and goblet cells hyperplasia(PAS stain)were observed.2.The T-helper cell(Th1,Th2,and Th17)-related cytokines in splenic lymphocyte supernatant were detected by cytokine bead array kit.3.The expression of Notch1?Notch2?Jagged1 and Foxp3 were analysed by western blotting.4.Flow cytometry was used to verify the proportion of splenic CD4+CD25+Foxp3+Treg cells.Results: 1.Compared with the normal group,the AR model demonstrated increased allergic symptom scores,OVA-specific serum Ig E titers,eosinophil infiltrates,and goblet cells hyperplasia(p<0.05).Mice treated with either tangeretin or DXM displayed markedly reduced allergic symptom scores and serum levels of OVA-specific Ig E production compared to the AR group,as well as significant reductions in nasal eosinophil infiltration and mucus hypersecretion(p<0.05).2.Compared with the normal group,The quantities of IL-6,IL-10,and IL-17 A were significantly increased following OVA challenge,whereas IFN-? and IL-2 decreased(p<0.05).Therapeutic administration of tangeretin during OVA challenges induced remarkable protection against Th1/Th2/Th17 imbalance with increased IFN-? and decreased IL-6,IL-10 and IL-17 A levels(p<0.05).3.Compared with the Normal group,the AR group showed an increase in Notch1/Jagged1 production,and decreased Notch2 and FOXP3 expression(p<0.05);compared with AR group,markedly decreased Notch1/ Jagged 1 production,as well as significantly increased FOXP3 expression were observed under tangeretin treatment(p<0.05),while Notch2 protein levels remained unaltered(p>0.05).4.Compared with those in the Normal group,the proportion of splenic CD4+CD25+FOXP3+Treg cells in the AR group were significantly decreased(p<0.05),whereas tangeretin-treated mice showed higher abundance of CD4+CD25+FOXP3+Treg than AR mice(p<0.05).5.No changes in Th1/Th2/Th17 cytokines,no changes in the expression of Notch1/Jaggd1 signal,and no changes in the differentiation of Treg cells and Foxp3 expression were found in the DXM treatment group(p>0.05).Conclusion: Tangeretin can alleviate AR development by inhibiting the expression of Notch1 and Jagged1,and promoting FOXP3/Treg cells differentiation.The third part: Tangeretin promotes regulatory T cell differentiation by inhibiting Notch1 expression on CD4+T lymphocytesObjective:To explore the molecular mechanism of tangeretin in promoting generation of FOXP3+i Tregs.Methods: 1.CD4+CD25�T cells were isolated from the spleen of 8-to12-week-old WT C57BL/6 mice using MACS columns.To achieve the efficient i Treg polarization in vitro,several stimulus concentrations(anti-CD3;anti-CD28;IL-2 and TGF-?)in culture medium were investigated.2.Naive CD4 + T cells were polarized into i Tregs in the presence of tangeretin(4,10,or 18?M)or DMSO control.The proportion of CD4+CD25+Foxp3+Treg cells were analysed by flow cytometry,and the expression of Foxp3 m RNA and Notch1 m RNA were detected by RT-PCR.Furthermore,the Pearson correlation coefficient(r)was calculated to assess the correlation between Foxp3 levels and Notch1 expression.Results: 1.CD4+CD25�T cells stimulated with 10?g/m L of plate-bound anti-CD3 antibodies,1?g/m L of anti-CD28 antibodies,15ng/m L of IL-2,and 10ng/m L of TGF-? provided best culture conditions for Treg-related in vitro experiments(78.5%),and CD4+CD25�T cells stimulated with 10?g/m L of plate-bound anti-CD3 antibodies,1?g/m L of anti-CD28 antibodies,10ng/m L of IL-2,and 8ng/m L of TGF-?showed lowest i Treg polarization(34.8%).2.Compared with 0?M,Notch1 m RNA expression was variably decreased by all concentrations of tangeretin in a dose-dependent manner,followed by a concomitant up-regulation of the proportion of CD4+CD25+FOXP3+Treg cells and its transcription factor Foxp3 m RNA levels.In addition,pearson correlation analysis showed that the expression of Foxp3 was negatively correlated with the levels of Notch1.Conclusion: Under certain concentration,tangeretin promotes the polarization of FOXP3+ i Tregs via control of Notch1 expression on CD4+T cells in a dose-dependent manner.In summary,this study confirmed that Notch1/Jagged1 signaling was significantly up-regulated in AR patients and positively associated with the severity of AR;In AR model,tangeretin can alleviate AR development by inhibiting the expression of Notch1/Jagged1 rather than Notch2,and promoting FOXP3+Treg cells differentiation;tangeretin promotes the polarization of Tregs via control of Notch1 expression on CD4+T cells in a dose-dependent manner in vitro.Thus,we concluded that tangeretin could promote regulatory T cell responses by inhibiting Notch1/Jagged1 expression,followed by promoting FOXP3/Treg cell differentiation and thus could serve as a novel curative therapeutic for AR.