Notch2 Suppresses The Development Of Allergic Rhinitis By Promoting FOXP3 Expression And Treg Cell Differentiation

Background:Allergic rhinitis（AR）is an allergic disease characterized by nasal symptoms such as itching and congestion.Although there are many treatments,there are still some patients who cannot control symptoms.Poor compliance of immunotherapy based on desensitization treatment.Treg cells are CD4+T cells.FOXP3,a key transcription factor in the nucleus,mediates a variety of physiological functions such as cell proliferation,differentiation,maturation and apoptosis.Treg cells are one of the key cells to regulate immune response and maintain immune tolerance,but the causes and upstream regulatory targets of Treg cells’abnormal immune function in AR are not very clear.Notch signaling is composed of multiple Notch proteins,which have different roles.It has been found that Notch signaling is involved in allergic diseases by affecting the growth and development of T cells.In AR,Notch1 expression is elevated and may promote the occurrence and development of AR.However,the expression of Notch2 in AR is not clear,and the relationship between Notch2 and Treg cells and its specific role in AR are not clear.This study intends to explore how Notch2 affect Treg cells in AR,so as to provide a new direction for AR immunotherapy targeting Treg cells.This study is divided into the following three parts:The first part:The expression and significance of Notch2 in AR patientsObjective:To explore the expression of Notch2 and FOXP3 in AR patients and the relationship between Notch2 and allergen sIgE.Methods:1.Select patients（101 AR patients and 66 control patients）who received treatment in otolaryngology head and neck surgery,Renmin Hospital of Wuhan University from September 2019 to December 2020,collect their sera,and detect the expression of Notch2 in their sera by enzyme-linked immunosorbent assay（ELISA）.2.AR patients were divided into mild group（grade 1-3）and severe group（grade 4-6）according to the highest serum allergen grade,analyze and compare the difference of Notch2 expression between the two groups.3.Analyze and compare the H1（house dust mite）-mild or D1（Dermatophagoides farina）-mild group（grade 1-3）and H1-severe or D1-severe groups（grade 4-6）the difference of Notch2 expression.4.The relationship between Notch2expression and sIgE in serum of AR patients was analyzed by Pearson correlation coefficient.5.The patients who came to hospital for nasal endoscopic surgery due to deviation of nasal septum were collected at the same time.The lower turbinate mucosa of patients（19 AR patients and 17 control patients）was collected during operation,and hematoxylin eosin（HE）staining and periodic acid schiff（PAS）staining was used to detect the allergic inflammatory response of nasal mucosa.6.The expressions of Notch2 and FOXP3 and their relationship between the two groups were analyzed and compared by immunofluorescence and Western blot.Results:1.AR patients had a lower level of Notch2 in peripheral serum than that in the control group（P<0.01）.2.The expression of Notch2 in severe group decreased further than that in mild group（P<0.01）.3.The expression of Notch2 in H1-severe or D1-severe groups was also decreased compared with H1-mild or D1-mild group（P<0.01）.4.The expression of Notch2 in serum of AR patients was negatively correlated with the sIgE levels of house dust mite and dust mite（D1:r=-0.68,P<0.01;H1:r=-0.72,P<0.01）.5.Compared with control group,the number of eosinophils along with goblet cell proliferation increased in nasal mucosa of AR patients（P<0.01）.6.The expression of Notch2 abviously decreased in nasal mucosa of AR patients and so did the expression of NICD2 and FOXP3（P<0.05）.There was co localization of Notch2 and FOXP3 in nasal mucosa.Conclusion:The decrease of Notch2 expression is closely related to the occurrence of AR,and the expression of Notch2 may inhibit the occurrence and development of AR.The second part:Notch2 promotes the differentiation and immunosuppressive function of human and mouse Treg cellsObjective:To explore the effect of Notch2 on Treg cells and its molecular mechanism in vitro.Methods:1.CD4+CD25-T cells and Treg（CD4+CD25+）cells from human and mouse were separated by magnetic beads,and construct the over expressed Notch2intracellular lentivirus of human and mouse gene sequences respectively.2.CD4+CD25-T cells in human and mice were cultured in vitro and transfected with the corresponding over expressed lentivirus to culture,and flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+Treg cells.3.Treg cells in human and mice were cultured in vitro and transfected with the corresponding over expressed lentivirus,and co-cultured with the corresponding CD4+CD25-T cells,and the expression level of Th1/Th2/Th17 cytokines in the cell supernatant was detected by cytokine bead array kit（CBA）.4.Mouse CD4+CD25-T cells were stained with CFSE antibody,and co-cultured with transfected mouse Treg cells.The proliferation of CD4+CD25-T cells was detected by flow cytometry.Results:1.Notch2 can promote the differentiation of human and mouse CD4+CD25-T cells into Treg cells in vitro.At the concentration of MOI=25,50,100,Notch2 can promote the differentiation of human Treg cells in a dose-dependent manner（P<0.05）.2.Compared with the oe Vec group,human Treg cells after transfection can inhibit the expression of human IL-2,TNF-α,IL-4,IL-6 and IL-10（P<0.05）.Transfected mouse Treg cells could inhibit the expression of IL-2,TNF-α,IL-4,IL-6 and IL-10（P<0.05）,and also inhibit the proliferation of CD4+CD25-T cells（P<0.05）.Notch2 could enhance the immunosuppressive ability of Treg cells.Conclusion:Notch2 can promote the differentiation of human and mouse CD4+T cells into Treg cells and enhance the immunosuppressive function of Treg cells.The third part:Notch2 promotes the differentiation of Treg cells and reduce the allergic inflammatory response in miceObjective:To explore the effect of up regulating Notch2 on AR model mice and its mechanism.Methods:1.Forty SPF C57 female mice were randomly divided into five groups according to different intervention factors:Control group,Basic group,AR group,AR^{oe Vec}group and AR^{oe Notch2}group.2.The allergic symptoms of mice in each group was evaluated,and the expression of OVA-sIgE in serum was detected by enzyme-linked immunosorbent assay（ELISA）,and the allergic inflammatory reaction of nasal mucosa was detected by hematoxylin eosin（HE）staining and periodic acid schiff（PAS）staining.3.The expression of Notch2 and FOXP3 in nasal mucosa of mouse in each group was analyzed and compared by western blot.4.Chromatin immunoprecipitation and polymerase chain reaction（Ch IP-PCR）were used to detect the interaction mechanism between Notch2 and FOXP3.5.Flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+Treg cells in spleen of mice in each group.6.Spleen lymphocytes were cultured in vitro,so as to detect the expression of Th1/Th2/Th17 cytokines in cell supernatant by cytokine bead array kit（CBA）.Results:1.Compared with the Control group,allergic symptom score,serum OVA-sIgE,nasal mucosal eosinophil infiltration and goblet cell metaplasia had no significant difference in the basic group and in AR group increased significantly（P<0.05）.Compared with AR group,there was no significant change in AR^{oe Vec}group,while in AR^{oe Notch2}group,allergic symptom score and serum OVA-sIgE expression decreased（P<0.05）,nasal mucosal eosinophil infiltration and goblet cell metaplasia decreased significantly（P<0.01）.2.The expression of Notch2 and FOXP3 was higher in the basic group and lower in AR group than that in the control group（P<0.05）.Compared with AR group,expression of Notch2 and FOXP3 had no significant difference in AR^{oe Vec}group,but increased in AR^{oe Notch2}group（P<0.05）.3.The study of their interaction confirmed that Notch2 could directly regulate FOXP3 promoter transcription,and at least two binding sites have been identified（P<0.01）.4.Compared with the control group,the Basal group had a higher proportion of CD4+CD25+FOXP3+Treg cells and the AR group lower（P<0.05）.Compared with the AR group,the proportion of CD4+CD25+FOXP3+Treg cells had no obvious difference in AR^{oe Vec}group,and increased in AR^{oe Notch2}group（P<0.05）.5.Compared with Control group,there was no significant change in Basal group,the expression of IL-2 and IFN-γdecreased,IL-6,IL-10 and IL-17A increased in AR group（P<0.05）.Compared with AR group,the expression of Th1/Th2/Th17 cytokines had no significant difference in AR^{oe Vec}group,and the expression of IL-2 and IFN-γincreased,IL-6,IL-10 and IL-17A decreased in AR^{oe Notch2}group（P<0.05）,suggesting that up-regulation of Notch2 can also reverse the imbalance of Th1/Th2/Th17 immune response in AR mice.Conclusion:Up-regulation of Notch2 expression in AR mice can alleviate allergic inflammatory response through promoting FOXP3 expression and Treg cell differentiation.In summary,this study confirmed that the expression of Notch2 in serum and nasal mucosa decreased in AR patients,the expression of serum Notch2 was negatively correlated with allergen sIgE,and there was co-localization of Notch2 and FOXP3 in nasal mucosa;Notch2 can promote the differentiation of human and mouse CD4+T cells into Treg cells and enhance the immunosuppressive function of Treg cells;In AR mouse model,Up-regulation of Notch2 expression in AR mice can alleviate allergic inflammatory response through promoting FOXP3 expression and Treg cell differentiation.In conclusion,this study concluded that Notch2 suppresses the development of allergic rhinitis by promoting FOXP3 expression and Treg cell differentiation.