The Role And Mechanism Of OVA-induced Autophagy In DCs To Promote The Occurrence And Development Of AR

Background:Allergic rhinitis(AR)refers to a type I hypersensitivity reaction mainly mediated by IgE after exposure to allergens.Many cells,such as mast cells,eosinophils,lymphocytes and inflammatory mediators involve in the pathegenesis of AR,which is a chronic non-infectious inflammatory diseases of the nasal mucosa.The pathogenesis of AR is mainly a Th1/Th2/Th17-mediated immune imbalance.Currently the treatment of AR is mainly glucocorticoids,antihistamines and immunotherapy,but AR is still difficult to cure.Finding new mechanisms of AR pathogenesis can provide new ideas for AR treatment.Autophagy is a normal physiological process responsible for maintaining the homeostasis and survival mechanism of normal cells.The process involves the degradation of damaged proteins and organelles by lysosomes,and is also involved in the phagocytosis of foreign bodies(viruses,bacteria,fungi)and other processes.Under normal physiological conditions,intracellular autophagy is usually at a low level,but when cells are under stress,autophagy levels are rapidly up-regulated.Dendritic cells assumes the role of source and switch in the pathogenesis of AR.This study intends to explore the changes of autophagy,the main cells undergoing autophagy,and the relationship between autophagy and CD4+T cells during the pathogenesis of AR,so as to provide a new method for the treatment of AR.This study is divided into the following three parts:The first part:Expression and significance of autophagy markers in DCs in nasal mucosa of AR patientsObjective:To investigate the expression level of LC3 Ⅱ and relationship with DCs in nasal mucosa of AR patients.Methods:45 patients(23 AR patients,22 controls)recruitment were from December 2018 to December 2019 in Department of Otolaryngology Head and Neck Surgery,Renmin Hospital of Wuhan University,who underwent nasal endoscopic surgery.Western Blot was used to analyze the expression levels of LC3 Ⅱ in the specimens of two groups.Double mmunofluorescent staining was used to analyze the expression levels of LC3 Ⅱ in the DCs of the specimens of two groups.Results:Compared with the control group,LC3 Ⅱ was highly expressed in the inferior turbinate mucosa of AR patients,and the expression level of autophagy significantly increased.Compared with the control group,the CD11c+cells,LC3 Ⅱ+cells,LC3 Ⅱ+and CD11c+double positive cells in the inferior turbinate mucosa of AR patients significantly increased.Statistical analysis showed that autophagy cells and dendritic cells in the nasal mucosa of AR patients significantly increased and dendritic cells undergoing autophagy significantly increased(P<0.05).Conclusion:The expression level of LC3 Ⅱ and DCs in the nasal mucosa of AR patients increased and autophagy DCs increased.Autophagy in DCs may be closely related to the pathogenesis of AR.The second part:Therapeutic effects of autophagy inhibitors on AR animal models and their effects on autophagy in DCsObjective:To investigate the level of autophagy markers expression in AR animal models and the possible mechanisms that affect the genesis and development of AR.Methods:The specific pathogen free(SPF)C57BL/6J mice were divided into 3 groups at random and received corresponding modeling and treatment(8 mice/group):normal control group,AR group,and 3-MA group(autophagy inhibitor).1.Compare the allergic symptom scores of mice in each group;enzyme-linked immunosorbent assay(ELISA)was used to-eosin(HE)staining was used to detect eosinophilic infiltration in nasal mucosdetect the expression level of serum OVA-IgE titers in each group of mice;hematoxylina;goblet cells hyperplasia(PAS stain)was used to detect the proliferation of goblet cells.2.Western blot was used to detect the expression levels of proteins closely bound up with autophagy(LC3 Ⅱ,ATG5,P62)in the nasal mucosa tissue in each group.3.The expression of LC3 Ⅱ in the nasal mucosa of each experimental group was analyzed by double immunofluorescent staining.Results:1.Compared with the normal control group,the nasal symptom score and the expression level of OVA-IgE titer in the serum of AR mice increased,and the eosinophils and goblet cells in the nasal mucosa significantly increased(P<0.05).Compared with the mice in the AR group,the nasal symptom score and serum OVA-IgE titer decreased and the infiltration of eosinophils and goblet cells in the nasal mucosa was alleviated(P<0.05)in 3-MA group.2.Compared with the normal control group,LC3 Ⅱ,ATG5 were significantly up-regulated(P<0.05)and P62 was significanly down-regulated(P<0.05)in AR group;compared with the AR group,LC3Ⅱ,ATG5 expression decreased(P<0.05)and P62 was significanly up-regulated(P<0.05)in 3-MA group.3.Compared with the normal control group,the counts of CD 11c+cells,LC3 Ⅱ+cells,and CD 11c+LC3 Ⅱ+cells in the nasal mucosa of AR mice significantly increased;compared with the AR group,the 3-MA treatment could reduce the numbers of CD 11c+cells,LC3 Ⅱ+cells,and CD 11c+LC3 Ⅱ+cells in AR group.Conclusion:Autophagy increased in AR mouse animal models and gathers in DCs.The therapeutic effect of autophagy inhibitors on AR may be related to the inhibition of autophagy in DCs.The third part:Effects of OVA-induced autophagy of DCson differentiation of CD4+T cells and allergic inflammatory responseObjective:To explore the effect of OVA-induced autophagy in DCs;to explore its effect on CD4+T cell differentiation and allergic inflammatory response.Methods:1.BMDCs were obtained by in vitro induction of bone marrow washings from The specific pathogen free(SPF)C57BL/6J mice of 6 weeks old.2.BMDCs were divided into control group(BMDCs,no OVA treatment),low concentration group(OVA,100μg/ml),high concentration group(OVA,500μg/ml),and autophagy inhibitior group(OVA 500μg/ml+5mM 3-MA,3-MA group).3.The expression of CD11C,CD80,CD86 and MHC Ⅱ in DCs of each group was detected by flow cytometry.4.The expression levels of autophagy-related proteins(LC3 Ⅱ,ATG5,P62)in BMDCs were detected by Western blot.5.The expression level of autophagosomes in BMDCs was observed by electron microscopy.6.The spleens of 8-week-old specific pathogen free(SPF)C57BL/6J mice was taken to make single cell suspension to be co-cultured with the BMDCs for 48 hours.7.Flow cytometry was used to detecte the proportion of Th1/Th2/Th17 cells in each group and the cytokine bead array kit(CBA)were used to detected each group’s Th1/Th2/Th17-related cytokines in the co-culture supernatant.Results:1.The expression of CD11C,CD80,CD86 and MHC Ⅱ in DC cells in each group exceeded 90%(P<0.05).2.Level of CD80 and CD86 expression on the surface of DCs showed no significant discrepancy among the control group,low concentration group,high concentration group and 3-MA group(P>0.05).The expression of MHC Ⅱ increased in high concentration group(P<0.05),while there showed no significant difference in expression of MHC Ⅱ among the control group,the low concentration group and the 3-MA group(P>0.05).3.Compared with the control group,the level of autophagy marker proteins LC3 Ⅱ and ATG5 expression increased along with the increase of the concentration of OVA and was the highest in the high concentration group(P<0.05).Compared to the control group,the expression of P62 gradually reduced with the increase of the concentration of OVA and was the lowest in the high concentration group(P<0.05).After 3-MA treatment,the expression of LC3 Ⅱ and ATG5 significantly decreased and P62 increased,similar to the control group.4.After OVA stimulation,a large number of autophagosomes appeared in DCs,which increased along with the increase of OVA concentration(P<0.05).However,after treatment by 3-MA,autophagosomes in DCs significantly reduced in 500 μg/ml OVA-stimulated DCs(P<0.01).5.Compared with the control group,the numbers of Th1 cells(CD4+T-bet+)and Treg cells(CD4+CD25+FOXP3+)in the lymphocytes of the low concentration group and high concentration group decreased as the concentration of OVA increased(P<0.05),while the numbers of Th2 cells(CD4+GATA3+),Th9 cells(CD4+PU-1+),Th17 cells(CD4+RORγt+)and Tfh cells(CD4+CXCR5+)increased following the concentration of OVA(P<0.05).Compared with the low concentration and high concentration groups,the number of Th1 cells(CD4+T-bet+)and Treg cells(CD4+CD25+FOXP3+)in the 3-MA group significantly increased(P<0.05),while the numbers of Th2 cells(CD4+GATA3+),Th9 cells(CD4+PU-1+),Th17 cells(CD4+RORγt+)and Tfh cells(CD4+CXCR5+)obviously decreased(P<0.05).6.Compared with the control group,the levels of IL-2,TNF-α and IFN-γ expression decreased in the co-culture supernatant of the OVA group,while the levels of IL-4 and IL-6 expression increased(P<0.05)following the concentration of OVA;compared to the control group,the levels of IL-10 and IL-17 expression increased in the co-culture supernatant of the high concentration group.Compared to the OVA group,the levels of IL-2,TNF-α and IFN-γ expression increased and the levels of IL-4,IL-6,IL-17 and IL-10 expression decreased(P<0.05)in the 3-MA group.Conclusion:OVA-induced autophagy of DCs promotes the development of AR inflammatory response by increasing the antigen presentation of DCs and leading to the Th1/Th2/Th17 imbalance.