The Regulatory Role And Mechanism Of IL-17 On Airway Inflammatory Phenotype In An OVA-LPS-induced Murine Neutrophilic Asthma Model

Background and objectiveBronchial asthma is defined as a chronic airway inflammatory disease.Both genetic factors and environmental factors play roles in the incidence of asthma.There are about three hundred million asthma patients in the world.To date,China has about thirty million asthma patients and is one of the countries with the highest asthma mortality rate.Bronchial asthma also has obvious heterogeneity.Different patients or the same patient in different periods of asthma may have different clinical manifestations,disease severity,treatment response,etc..This kind of "difference" is actually the outcome of the interaction of individual internal factors(genetic factors)and external factors(environmental factors).In the past,the infiltration of eosinophils in the airways was considered as the main inflammatory characteristic or phenotype of asthma.However,some new researches found that the infiltration of eosinophils is not the only inflammation phenotype.There are a large number of neutrophils in the airways of some asthmatic patients with acute exacerbation or fatal asthma attacks.Studies have shown that neutrophils infiltration is one of the airway inflammatory phenotypes in severe asthma.And this phenotype is not sensitive to glucocorticoid treatment.Although severe asthma patients accounted for only 5% to 10% of asthma cases,they constituted a huge burden of medical expenditure on asthma prevention and control,because these patients have poor response to current treatment,such as inhaled corticosteroids and long-acting ?2 adrenoceptor agonist(ICS+LABA),leukotriene receptor antagonist(LTRA),theophylline,and long-acting muscarinic receptor antagonist(LAMA),etc..Therefore,it is of great significance to study the pathogenesis of neutrophilic asthma in the prevention and treatment of asthma,especially in severe asthma and steroid-resistant asthma.Th1 / Th2 imbalance has been thought in previous studies the most important immunological mechanism of allergic asthma.Enhanced Th2-response,such as increased Th2 cytokines interleukin(IL)-4,IL-5 and IL-13 and eosinophilic inflammation,can be observed in the asthmatic lungs.On the basis of this theory,the researchers developed targeted therapies to Th2 cytokines,such as monoclonal antibodies against IL-5 and IL-13 and recombinant human IL-4 receptor,trying to reverse the Th1 / Th2 imbalance.But the outcome of some clinical trials was not as good as they seem.On the other hand,Th1-cytokine treatment of asthma has not achieved good results.Therefore,the Th1 / Th2 imbalance can not fully explain the pathogenesis of eosinophilic asthma,nor explain the pathogenesis of neutrophilic asthma.Neutrophilic asthma may have different immunological mechanisms.In subsequent clinical studies,elevated expression of IL-17,IL-1?,IL-6,IL-8 and other inflammatory cytokines were observed in induced sputum supernatant,bronchoalveolar lavage fluid(BALF)and bronchial lung tissues in neutrophilic asthma patients.IL-17 may be positive correlated with the airway hyperresponsiveness(AHR)in asthmatic patients.However,the exact mechanism is still unknown.CD4+ helper T lymphocyte(Th)is one of the most important immune cells involved in immune responses.CD4+ Th17 cells and their secreted cytokine IL-17 are involved in the elimination of pathogenic microorganisms such as bacteria,fungi,mycoplasma,chlamydia and so on,and can cause neutrophil and / or monocyte inflammation.Therefore,we hypothesized that CD4+ Th17 cells and IL-17 play important role in the pathogenesis of airway neutrophilic inflammation.Further research on this mechanism may provide a new therapeutic strategy for the prevention and treatment of severe asthma.ObjectiveIn this study,we established a murine neutrophilic asthma model sensitized by ovalbumin(OVA)combined with lipopolysaccharide(LPS).The characteristics of airway inflammation,AHR,the expression of inflammatory cytokines in BALF,and the differentiation of CD4+ Th cells in spleen and lung draining lymph nodes were compared with those of OVA-induced eosinophilic inflammation model.We studied the role of Th17 / IL-17 in the pathogenesis of neutrophilic inflammation in asthmatic mice using IL-17 gene knockout mice.MethodsThirty C57BL/6J mice were randomly subgrouped into three groups: control group(NS group),eosinophilic asthma group(OVA group)and neutrophilic asthma group(OVA+LPS group),10 mice in each group.On day 1 and 7,the mice of OVA group and OVA+LPS group were sensitized via intraperitoneal injection with 100 ?l OVA(50 ?g)and the mixture of aluminum hydroxide.Additionally,the mice were intranasally instilled with 100 ?g of OVA and 1?g of LPS.Mice of NS group were treated with saline.On days 14 to 18,the mice of OVA group and OVA+LPS group were consecutively exposed to an aerosolized 1% OVA solution for one hour.The mice of NS group were challenged with saline instead of OVA.After the final challenge,the mice were anesthetized for the measurement of AHR and bronchoalveolar lavage(BAL).BAL fluid(BALF)cells were counted using Wright-Giemsa staining.The expression of IL-1?,IL-4,IL-5,IL-17,and IL-8 in BALF supernatant were detected using ELISA kits.The pathological changes of lung and bronchial tissues using hymatoxylin-eosin(H&E)staining and mucus secretion of airway epithelial cells using periodic acid-Schiff(PAS)staining were observed.CD4+ Th cells in spleen and lung draining lymph nodes were detected by flow cytometry.We also sensitized IL-17 gene knockout mice using LPS and OVA to replicate the neutrophilic asthma model.After challenged with OVA,the AHR,inflammatory cells and cytokines in BALF and the pathological damage of lung tissue were detected in IL-17 gene knockout mice.We also observed the differentiation of CD4+ Th cells in spleen and lung draining lymph nodes in IL-17 knockout mice using flow cytometry.Results1.Compared with OVA-induced eosinophilic asthma mice,the mice treated with OVA and LPS had predominant neutrophils infiltration in the airways with higher AHR,more severe lung inflammation,but reduced goblet cells metaplasia.2.The neutrophilic asthma mice were observed with increased expression of IL-1?,IL-8 and IL-17,but reduced expression of Th2 cytokines,IL-4 and IL-5,in BALF compared with those of OVA-induced eosinophilic asthma mice.3.The differentiation of CD4+ Th cells both in spleen and lung draining lymph nodes was different between the two kinds of asthma mice.Compared with the eosinophilic asthma mice,neutrophilic asthma mice had an increased differentiation of CD4+ IL-17+ Th17 cells and a decreased differentiation of CD4+ IL-4+ Th2 cells.However,the differentiation of CD4+ IFN?+ Th1 cells was not significantly changed.4.Compared with wild-type neutrophilic asthma mice,IL-17 gene knockout mice,although sensitized with OVA and LPS,showed decreased AHR and reduced neutrophils in BALF whilst eosinophils in BALF were increased and the goblet cell metaplasia was enhanced.5.In comparison with wild-type neutrophilic asthma mice,the expression of Th2 cytokines,IL-4 and IL-5,was elevated and the expression of IL-1?,IL-8 and IL-17 was significantly decreased in BALF of IL-17 gene knockout mice.6.Compared with wild-type neutrophilic asthma mice,CD4+ IL-17+ Th17 cells of IL-17 gene knockout mice were significantly reduced both in spleen and in lung draining lymph nodes.Interestingly,the differentiation of CD4+ IL-4+ Th2 cells was elevated.The differentiation of CD4+ IFN?+ Th1 cells were decreased in spleen.However,this change of CD4+ IFN?+ Th1 cells was not observed in lung draining lymph nodes.Conclusion1.Compared with eosinophilic asthma mice sensitized with OVA,the neutrophilic asthma mice induced by OVA and LPS show typical airway neutrophils infiltration with higher AHR and more severe lung inflammation.2.The elevated CD4+ IL-17+ Th17 cells and IL-17 cytokine play a key role in neutrophilic asthma.3.The increased CD4+ IL-17+ Th17 cells and IL-17 cytokine inhibit the differentiation of CD4+ IL-4+ Th2 cells,and further downregulate the Th2 responses,such as airway eosinophils infiltration,mucus hypersecretion and Th2 cytokines expression in asthma.Our results proved in animal models that exposure to endotoxin during repeated bacterial infection can shift the asthmatic airway inflammatory phenotype from eosinophilic inflammation to neutrophilic inflammation.The mechanism of the inflammatory phenotype shift depends on the differentiation of CD4+ Th17 cells and the secretion of IL-17.This discovery provides evidence for the immunological mechanism of the "hygiene hypothesis".Th17 cell passway is the key point in the development of neutrophilic asthma,which may be a potential therapeutic target for the treatment of neutrophilic asthma.