The Role Of IL-33/ST2 Signaling Pathway In OVA-induced Age-dependent Allergic Airway Inflammation

Background: Asthma is a chronic airway inflammatory disease,which is highly heterogeneous and can be divided into various subtypes in clinic.Allergic asthma is the most common type that most children and about 50% of adults with asthma belong to this type.Allergic asthma is mediated by type 2 immune response involving multiple innate and adaptive immune cells,typical features of which include eosinophil infiltration in lung tissue,increased levels of type 2 cytokines(IL-4/IL-5/IL-13),goblet cell metaplasia and increased mucus secretion in the airways,elevated IgE levels in serum and airway hyperresponsiveness.The role of age in the pathogenesis of allergic asthma is getting more and more attention.The sensitization phase of allergic asthma occurs mainly in infancy.The function of the immune system during this period is quite different from that of adults,which is manifested in the bias to Th2 response,and the related mechanism has not been fully elucidated.In recent years,an important progress in the field of respiratory allergic diseases is that people recognize the role of barrier epithelial cells in initiating,strengthening and maintaining type 2 immune response via their secretion of epithelial-derived cytokines(IL-33/TSLP/IL-25,etc.).Recent studies have found that mice can secrete large amounts of IL-33 during the alveolar formation period(before three weeks old),which may be an important mechanism that explains the type 2 response bias in the lung of young mice.Base on that,we used wild type(WT)mice,IL-33-deficient mice(IL-33KO)and IL-33 receptor-deficient mice(ST2KO)in C57BL/6 background,established OVA-induced allergic asthma model in different age groups,to explore the effects and mechanisms of IL-33/ST2 signaling pathway on airway allergic inflammatory response in neonatal and adult mice,with a view to providing a theoretical basis for clinical intervention strategies.Objective: To investigate the role of IL-33/ST2 signaling pathway in OVA-induced age-dependent allergic airway inflammation.Methods: I.According to the age of mice at the time of initial sensitization,we established the OVAinduced allergic asthma model of the neonatal group(1w at the initial sensitization)and the adult group(6-8w at the initial sensitization)respectively: WT,IL-33 KO and ST2 KO neonatal and adult mice in C57BL/6 background were used.During the sensitization period,OVA/Alum suspension was administered intraperitoneally on days 0 and 7(neonatal mice were given 50 μl of saline containing 10 μg OVA and 1 mg Alum;the dose of adult mice was doubled);During the challenge period,1% OVA solution was inhaled for 30 minutes each time for 5 consecutive days from the 14 th to the 18 th day.The control group was nebulized with saline.They were sacrificed and the required test samples were collected,24 hours after the last atomization.II.Experimental groups: The neonatal group and the adult group were further divided into 6 groups : wild type negative control group(WT Saline/Saline),wild type OVA group(WT OVA/OVA),IL-33 knockout negative control group(IL-33 KO Saline/Saline),IL-33 knockout OVA group(IL-33 KO OVA/OVA),ST2 knockout negative control group knockout mice(ST2KO Saline/Saline),ST2 knockout OVA group(ST2KO OVA/OVA).III.The effect of IL-33/ST2 signaling deficiency on allergic airway inflammatory response in neonatal and adult groups: the samples were collected at 24 hours after the last atomization: Lung were digested with collagenase D to prepare single cell suspension,and then Flow cytometry(FCM)was used to detect the proportion and number of eosinophils in lung;Lung tissue sections were stained with Periodic Acid-Schiff staining(PAS)and Hematoxylin-Eosin staining(H&E),and bronchi goblet cell metaplasia and inflammation infiltration in lung were observed under a microscope;The levels of IgE and IgG1 in serum,and the levels of IL-4,IL-5,IL-13,and IFN-γ in bronchoalveolar lavage fluid(BALF)were detected by ELISA.IV.The effect of IL-33/ST2 signaling deficiency on immune cells in lungs of neonatal and adult OVA/OVA groups: the proportion and number of main immune cells in the lungs,including DCs,ILC2 s,effector T cells,Th cells,and the expression level of OX40 L on the surface of DCs and ILC2 s cells were detected by FCM,and activated subsets of ILC2 and CD4~+ Th cell were measured by intracellular cytokine(IL-5/IL-13/IFN-γ)staining.V.The effect of IL-33/ST2 signaling deficiency on the expression of IL-33/TSLP/IL-25 and their receptors in neonatal and adult OVA/OVA groups: the levels of IL-33,TSLP and IL-25 in lung homogenate were detected by ELISA;the expression levels of IL-25 receptor(IL-17RB)and TSLP receptor(TSLPR)on the surface of DCs,ILC2 s and CD4~+ T cells were detected by FCM.VI.The role of TSLP and IL-25 in OVA-induced model of IL-33 KO adult mice: Anti-IL-25 and anti-TSLP blocking antibodies were given to OVA-induced model of IL-33 KO adult mice during the challenge period.Specifically,80 μl anti-IL-25(40μg)and 40μl anti-TSLP solution(40μg)were administered nasally 2 hours before each nebulization,mice were sacrificed 24 hours after the last atomization,and the detection items after collecting samples is the same as Ⅲ,Ⅳ.Results: I.In the neonatal group,after WT neonatal mice were sensitized and stimulated by OVA,they have a typical respiratory allergic inflammatory response,which were manifested as: the proportion and number of eosinophils in lung increased significantly,and the number of goblet cells in the respiratory tract increased with inflammatory cell infiltration.Type 2 cytokines such as IL-4,IL-5 and IL-13 in BALF,as well as IgE and IgG1 in serum were significantly increased.Neonatal mice with IL-33/ST2 signaling pathway loss have a certain degree of increase in the proportion and number of eosinophils in lung after OVA challenge,but significantly lower than the WT OVA/OVA group,and the other indicators are not different from the negative control group.II.In the adult group,OVA-induced asthma model was also successfully established in WT adult mice.After the adult mice with IL-33/ST2 signaling pathway deletion treated by OVA,certain indicators were similar to the WT positive group,except for the number of eosinophils in the lung and the IL-5 level in BALF,which were lower than those of the wild-type positive control group.III.In the neonatal group,by treating with OVA,the number and proportion of c DC2 s,mo DCs, ILC2s and effector Th2 cells(IL-5~+ Th cells and IL-13~+ Th cells)in the lung,proportion of IL-5~+ ILC2 s and IL-13~+ ILC2 s,and the expression of OX40 L on surface of c DC2 s and mo DCs,and ILC2 s were significantly higher in WT mice than that of the negative control group.After IL-33/ST2 signaling pathway-deficient group was treated with OVA,the proportion of c DC2 s in the lung tissue,the number and proportion of ILC2 s,and the proportion of IL-13~+ ILC2 s increased to some extent,but it was much lower than that of the WT OVA/OVA group,other indicators are no different from the negative control group.IV.In the adult group,by treating with OVA,the number and function of DCs,ILC2 s,and Th2 cells in the lung in WT mice showed the same trend as the WT neonatal group.After the IL-33/ST2 signaling pathway-deficient mice were treated with OVA,the proportion and number of DCs subsets of ST2 KO group were significantly lower than that of WT OVA/OVA group,while only mo DCs in IL-33 KO group showed the same trend,and c DC of it was not significantly different from the WT OVA/OVA group.There was no significant difference in the expression of OX40 L on the surface of DCs in all positive groups of three types of mouse.The number and effect of ILC2 of IL-33/ST2 signaling pathway-deficient mice in the adult group after OVA challenge was significantly lower than that in the WT positive group.The number of CD4~+ effector T cells and the proportion and number of IL-5~+ Th2 cells were still lower than those in the WT positive group.There was no significant difference in the proportion and number of IL-13~+ Th cells between positive groups with three types mice.V.IL-33,TSLP and IL-25 were expressed in the lung of the WT neonatal group at steady state,while only TSLP and IL-25 were expressed in adult group.After OVA modeling,the levels of IL-33 and TSLP in the WT neonatal group increased further,while in the adult group,IL-33/TSLP/IL-25 increased.Among them,the level of IL-33 in the neonatal group under steady state and after OVA modeling was significantly higher than that in the adult group.The absence of IL-33/ST2 signaling pathway in the neonatal group had little effect on the expression patterns of TSLP and IL-25.The absence of IL-33/ST2 signaling pathway in the adult group caused the level of TSLP and IL-25 significantly increased after OVA sensitization and stimulation,and higher than the WT positive control group.VI.In the neonatal group,the mice deficient in IL-33/ST2 signaling pathway were stimulated by OVA,and then TSLP receptor on the surface of c DC2 s and mo DCs,the TSLP receptor and IL-25 receptor on the surface of ILC2 and CD4~+ T cells were significantly lower than those of WT Positive control group.In the adult group,the IL-33/ST2 signaling pathwaydeficient mice were stimulated by OVA,and then TSLP receptor on the surface of DC subgroups were significantly increased,and were higher than those in the WT positive control group.The TSLP receptor on the surface of ILC2 and CD4~+ T cells was no difference with WT positive control group.There was no IL-25 receptor expression on the surface of DCs in the lung of mice in each group.VII.IL-33 KO adult mice had allergic respiratory inflammation after being stimulated by OVA.Anti-IL-25/anti-TSLP treatment during the challenge period could significantly alleviate the OVA-induced allergic inflammatory response of the respiratory tract,which is manifested by a marked decrease in the infiltration of eosinophils in the lung,a significant reduction in the infiltration of goblet cell metaplasia and inflammatory cells,and it was accompanied by significant down-regulation of the number of DCs subpopulations,Th2 cells and ILC2 s.Conclusion: I.The deficiency of IL-33/ST2 signaling pathway in neonatal group could cause a reduction in the number and function of DCs,ILC2 s and Th2 cells in lung,and significantly alleviate the OVA-induced allergic airway inflammation.II.The deficiency of IL-33/ST2 signaling pathway in adult group could increase the expression of TSLP,IL-25 and its receptor,and could still maintain the number and function of downstream DCs,ILC2 s and Th2 cells,and could still have allergic inflammation of the respiratory tract after treating with OVA.III.IL-33 KO adult mice can significantly alleviate OVA-induced allergic airway inflammation after treatment with anti-IL-25/anti-TSLP.