The Immunoregulation And Targeted Therapy Of Asthma

Asthma is a chronic airway inflammation involved in numerous inflammatory cells, structural cells (such as eosinophils, mast cells, lymphocytes, neutrophils, smooth muscle cells, airway epithelial cells, etc.) and cellular components, which always leading to reversible airflow obstruction in association with airway hyper-responsiveness (AHR). The asthmatics show the recurrent episodes of wheezing, shortness of breath, chest tightness or cough and other symptoms, most patients can be relieved or were improved after treatment. This disease is affecting more than 300 million all over the world, and 30 million in China, the prevalence of asthma in the world ranged from 1% to 18%, and China is also the one of the highest death rate of asthma in worldwild. With the development of global industrialization and the deterioration of environmental pollution, the morbidity and mortality of asthma is still in high level. Asthma will not only cause a great economic burden to the patients and society, but also seriously affect the life quality of the patients. Therefore, it has important significance for the exploration of the underlying pathogenesis and an effective therapeutic strategy for the treatment of asthma.Since the development of the field of pulmonary immunology, asthma has been seen as the hallmark Th2 disorder of the lungs, Th2 cells and the Th2-type cytokines such as IL-4, IL-5, IL-13 participate in allergic asthma existence, and also related to the severity of symptoms, and which was regard as Th2-dominated eosinophilic asthma. On the contrary, expression of IFN-γ has been shown to prevent development of allergen-induced airway inflammation. IFN-γ is mainly produced by CD4+Thl cells and suppresses Th2 responses. Therefore, allergic airway inflammation appears to be regulated by a sensitive balance between an overactive Th2 response and an inhibitory Thl response. Although asthma is classically associated with eosinophilia and Th2 cytokines, some patients show a neutrophil-predominant disease with an absence of Th2 cytokines, and this theory cannot explain the pathogenesis of all types of asthma in clinics. Recent studies based on sputum induction and/or bronchoalveolar lavage and bronchial biopsy, we define eosinophilic asthma as symptomatic airway inflammation characterised by the presence of eosinophils in the airways, non-eosinophilic asthma then represents symptomatic asthma in the absence of eosinophilic airway inflammation. The weighed mean proportion of subjects with eosinophilic asthma was 49%, and 51% had non-eosinophilic asthma. The discovery and study of Th17/IL-17 improved the traditional view that the imbalance of Th1/Th2 axis is the pathogenesis of asthma. IL-17-producing Th17 cells home to the lung and enhance not only neutrophilic, but also eosinophilic airway inflammation, and increasing evidences also show that elevated IL-17 levels are correlated with increased neutrophilic inflammation, a characteristic of severe and steroid-resistant asthma. However, the effect of IL-17 in the immune regulation of asthma is not uniform, and even controversial. It has been suggested that IL-17 enhances the production of cytokines from eosinophils, and amplifies eosinophil activation in the Th2-mediated eosinophilic inflammatory response, possibly by modulating eosinophil function. Conversely, treatment of ovalbumin-sensitized asthmatic mice with exogenous IL-17 inhibits airway inflammation, and reduces peribronchial mucus hypersecretion, as well as decreasing AHR, thus having an inhibitory effect during the course of asthma.Asthma can be controlled with low doses of inhaled corticosteroids, which have now become first-line treatment for most patients. However, approximately 10% of asthmatics need the maximum inhaled dose, and around 1% need regular treatment with oral glucocorticoids (glucocorticoid-dependent asthma). Very rarely, asthmatic patients are completely resistant to corticosteroids, even in high oral doses (steroid-resistant asthma). Although these patients only accounted for 5%-10% of the entire group of asthma, the cost of medical resources occupied about 50%. The proportion of steroid resistant asthma is small, but the treatment rate for emergency medication and hospitalization rate are 15 times and 20 times of patients with mild and moderate asthma, and thus leading to one of the major reasons for the increase in expenditure for asthma treatment. A recent cohort analysis has revealed that eosinophilic inflammation is predominant in patients with mild-to-moderate asthma and that neutrophilic inflammation is dominant in patients with severe refractory asthma, the number of neutrophil increased in patients correlated with a poor response to inhaled steroids.Part I Exogenous Interleukin-17A Inhibits Eosinophil Differentiation and Alleviates Allergic Airway InflammationObjective:To investigate the effects of exogenous IL-17 on allergic airway inflammation, and explored the underlying molecular mechanisms through bone marrow eosinophil differentiation, as well as to detect the influence of IL-17 on the survival and function of eosinophil.Methods:We have established eosinophilic asthma model with C56BL/6 mice, and which were treated with exogenous IL-17 or anti-IL-17 neutralizing antibody through airway instillation. The bronchoalveolar lavage fluid cells was harvested for counting the numbers of total cell and the classified cell such as eosinophil, airway inflammatory cells recruitment and mucus overexpression were assessed by HE and PAS staining, and cytokines levels in serum were detected by ELISA. We also investigated the effect of IL-17 on eosinophil differentiation and its progenitor formation with FACS analysis, and the transcript factors or chemokines receptors for eosinophil differentiation were examined. In vito, we explored the influence of IL-17 on eosinophil differentiation, survival and cytokines secretion.Results:Exogenous IL-17 significantly attenuated the features of allergic inflammation induced by ovalbumin in mice. It inhibited eosinophil differentiation both in vivo and in vitro, accompanied by down-regulated expression of CC chemokine receptor 3(CCR3), GATA-1, and GATA-2, as well as reduced formation of common myeloid progenitors and eosinophil progenitors, but without influencing eosinophil apoptosis. In addition, IL-17 had little effect on secretion of the inflammatory cytokines by eosinophils. Neutralization of endogenous IL-17 significantly augmented eosinophil recruitment in the airways.Conclusion:Together, these findings suggest that exogenous IL-17 protects against allergic airway inflammation most likely through inhibition of the eosinophil differentiation in bone marrow, but not via the influence of survival and function for eosinophil. This project emphasize the promising strategy for asthma control based on inhibition of bone marrow eosinophil differentiation.Part II Interleukin-17A Promotes Epithelial-Mesenchymal Transition via ROS-Mediated TGF-β1 Production in Chronic Airway InflammationObjective:We thought to explore the role of IL-17A (IL-17) in epithelial-mesenchymal transition (EMT) process and the potential mechanisms in allergic airway inflammation, and search the new theory for pathogenesis and treatment of asthma.Methods:We have established asthma model with wild type and IL-17 deficient mice, airway inflammation levels were assessed by BALF cell counts and HE and PAS staining of lung tissues. IHC analysis for EMT changes(a-SMA< N-Cadherin) and ROS marker( 8-OHdG)in lung were determined. We have detected IL-17 and TGF-β1 in lung homogenate, and their correlations were also assessed. In vitro, the regulatory role for IL-17 in EMT, reactive oxygen species (ROS) and TGF-β1 production were applied on human bronchial epithelial (HBE) cells with Q-PCR, IF or FACS. Besides, the correlations between IL-17 and these biomarkers were also assessed in bronchial biopsies from asthmatics.Results:IL-17 deficiency attenuated inflammatory response and expression of collagen, EMT markers a-smooth muscle actin (SMA), N-Cadherin, TGF-β1, and ROS maker 8-OHdG, positive correlations were observed between IL-17 and TGF-β1 levels in mice. IL-17 enhanced a-SMA, N-Cadherin and ROS-mediated TGF-β1 expression in HBE cells. Based on the findings with ROS inhibitor and TGF-β1, we suggest the up-regulatory effect of IL-17 for EMT biomarkers mediated at partially by ROS-TGF-β1 pathway in vitro. Besides, IL-17 was also positively related to EMT markers, 8-OHdG and TGF-β1 in asthmatics.Conclusion:We presented here that IL-17A could aggravate airway inflammation and epithelial-mesenchymal transition. This effect may be mediated, at partially, through the increased local expression of ROS-mediated TGF-β1 production. This novel pathway involved in asthma might guide a new direction on the worthy therapeutic strategies.Part Ⅲ Induction of Granulocyte Apoptosis by Bcl-2 Inhibitors Reduces Steroid-insensitive Airway InflammationObjective:We thought to explore the underlying mechanisms of airway inflammation persistence as well as corticosteroid resistance, and to investigate a new strategy of effective treatment against corticosteroid-insensitive neutrophilic asthma.Methods:Mouse models of either eosinophil-dominated or neutrophil-dominated airway inflammation were used in this study to test corticosteroid sensitivity in vivo and in vitro. We also used vav-Bcl-2 transgenic mice to confirm the importance of granulocytes apoptosis in the clearance of airway inflammation. To explore the effects of ABT-737 or ABT-199 on immune cells in lung of asthma. Finally, Bcl-2 inhibitors ABT-737 or ABT-199 was tested for their therapeutic effects against eosinophilic or neutrophilic airway inflammation and airway hyperresponsiveness.Results:Overexpression of Bcl-2 protein was found to be responsible for persistence of granulocytes in bronchoalveolar lavage fluid following allergic challenge. This was important as allergen-induced airway inflammation aggravated and persisted in vav-Bcl-2 transgenic mice, where nucleated hematopoietic cells were over-expressed with Bcl-2 and resistant to apoptosis. Bcl-2 inhibitors, ABT-737 or ABT-199 play efficient roles in alleviation of either eosinophilic or corticosteroid resistant-neutrophilic airway inflammation, by inducing granulocytes apoptosis. Moreover, these inhibitors were found to be more efficient than steroid to induce granulocytes apoptosis ex vivo from severe asthma patients.Conclusion:Apoptosis of granulocytes is essential for the clearance of allergen-induced airway inflammation. Bcl-2 inhibitors ABT-737 or ABT-199 may be promising drugs for the treatment against airway inflammation, especially for corticosteroid-insensitive neutrophilic airway inflammation.