The Co-blockade Of IL-13 And IL-25 Suppresses Airway Inflammation And Remodeling In A Mouse Model Of Asthma

Asthma is a chronic inflammatory disease of the airways involving many cells and cellular components.Repeated inflammation of the airway and unremitting remodeling provoke the irreversibility of pulmonary dysfunction and the resistance to current drugs in chronic bronchial asthmatics.Traditionally,asthma has been viewed as the hallmark Th2(adaptive immune response cell)disorder of the lungs and is characterized by increased levels of IL-4,IL-5 and IL-13.Importantly,a number of investigators have reported that IL-13 plays a key role in leading to the airway inflammation and remodeling.But anti-IL-13 therapies in clinical trials did not consistently demonstrate significant benefits.On one hand,the function of the Th2 cytokines complements one another.On the other hand,there are complex interactions between adaptive immune response and innate immune response.Recent studies report that ILC2s(innate immune response cells)are critical for the occurrence and development of asthma in mice.These cells have been discovered in mice that reside in the mucosa and response to IL-25,IL-33 and TSLP released by a damaged epithelium.The discovery of innate immune response provides a clue for the occurrence of Th2 inflammation in the lung and partly explains the reason why target block Th2 immune response cannot get consistently clinical benefit.Although asthma can be induced by IL-25,IL-33 and TSLP,IL-33 may be higher in virus-associated disease,TSLP may be higher in toxin-associated disease(such as cigarette smoke),and IL-25 may be higher in patients with allergen-induced asthma.Furthermore,IL-25 also drives IL-33 and TSLP production in the lung.These data demonstrate that IL-25 role in allergic asthma is more outstanding.Consequently,the most effective approaches for asthma patients who are resistant to current treatments may be combination therapies that suppress multiple cytokines and a range of disconnected pathways that separately contribute to asthma pathogenesis.The objective of this study was to test the hypothesis that the co-inhibition of adaptive immune responses and innate immune responses by a combined blockade of IL-13 and IL-25 with soluble IL-13 receptor ?2(s IL-13R)protein and soluble IL-25 receptor(s IL-25R)protein can down-regulate allergen-induced airway inflammation and remodeling effectively in mice.Methods:1.OVA was used to induce asthma model in mice,cell counts in BALF,HE staining and Masson staining to observe the preparation of model.2.Real-Time PCR was used to detect IL-13 and IL-25 m RNA in lung of asthma model.ELISA was used to detect the protein level of IL-13 and IL-25 in BALF and lung supernatant.IL-13 and IL-25 was located by immunohistochemistry.3.To observe the change of airway inflammation by cell counts in BALF and HE staining after the co-blockade of IL-13 and IL-25.Ah R was induced by Methacholine and assessed by lung resistance.4.The level of IL-4,IL-5,IL-13,IL-25,IL-33 and TSLP in BALF or lung supernatant was detected by ELISA.5.PAS staining and MUC5 AC was analyzed to evaluate the change of mucus hypersecretion.6.Sirius red staining and collagen assay was used to detect the subepithelial fibrosis.Western blotting was used to detect the expression of TGF-?1,Smad2 and p-Smad2.7.Lung sections were immunostained with antibodies against ?-SMA and PCNA to assess smooth muscle hypertrophy and hyperplasia.8.VEGF was detected in BALF and lung,and lung sections were immunostained with anti-vWF to measure the angiogenesis in the lung.Results:1.Characteristic features of asthma,including marked elevat ion of airway infiltration of inflammatory cells and subepithelial fibrosis,were induced by OVA.The m RN A and protein expression of IL-13 and IL-25 was significantly upregulated.2.The co-blockade of IL-13 and IL-25 could significantly attenuate the count of eosinophils in BALF,peribronchial accumulation of inflammatory cells and airway hyperresponsiveness(Ah R).3.The level of IL-4,IL-5,IL-13,IL-25,IL-33 and TSLP in BALF or lung supernatant was reduced significantly by co-blockade of IL-13 and IL-25.4.The expression of MUC5 AC protein was downregulated,while PAS scoring and area/Pbm were decreased after co-blockade of IL-13 and IL-25.5.The expression of TGF-?1 and p-Smad2 was significantly downregulated and the total amount of collagen was significantly decreased after co-blockade of IL-13 and IL-25.6.Anti-?-SMA and anti-PCNA immunostaining shows smooth muscle hypertrophy and hyperplasia was ameliorated by co-blockade of IL-13 and IL-25.7.The co-blockade of IL-13 and IL-25 could significantly attenuate the expression of VEGF and the number of v WF-positive vessels.Conclusion:1.Asthma model can be established by OVA.The m RNA and protein expression of IL-13 and IL-25 was significantly upregulated in this model.2.The co-blockade of IL-13 and IL-25 significantly attenuates airway inflammation and airway hyperresponsiveness(AhR).3.The co-blockade of IL-13 and IL-25 significantly ameliorates airway remodeling including mucus production,extracellular collagen deposition,peribronchial smooth muscle hyperplasia and neovascularization.