The Application And Therapeutic Potential Of Anti-interleukin-33 Active Immunization In Asthma Pathological Mechanism Research

The morbidity and mortality of asthma, which is one of the most common illnesses that seriously endanger human health, are increasing worldwide in the recent decade. Asthma is characterized by chronic inflammation, airway remolding (including mucus hypersecretion, airway obstruction, tissues fibrosis, smooth muscle cells proliferation, airway wall thickening, blood vessel bed amplification and so on) and airway hyper-responsiveness. The current treatment of asthma relies mainly on anti-inflammatory medicines and bronchodilators, which can effectively ameliorate disease onset and severity but not really cure. Quite a number of patients are not sensitive to the treatment and will eventually develop into refractory asthma. Moreover, airway remodeling and persistent pulmonary function injury once establish, present therapeutic approaches can not reverse the symptoms. Therefore, it is urgently required to investigate in depth asthma pathogenesis, especially the molecular mechanism of chronic asthmatic response such as airway remodeling, and to develop new therapeutic targets and strategies for the intervention of the disease process.Generally, airway inflammation is the critical pathogenesis of allergic asthma, typically accompanied by an excessive Th2 immune response. IL-33 is abundantly expressed in human bronchial epithelial cells, smooth muscle cells and dendritic cells, etc. IL-33 can significantly promote Type 2 immune responses, mainly stimulating the differentiation, proliferation and activation of type 2 innate lymphoid cells and Th2 cells. In addition, IL-33 is also a potential chemokine for inflammatory cells like Th2 cell and granulocytes. Previous studies strongly suggested that IL-33 played a key role in the inflammatory responses in allergic asthma. However, the role of IL-33 in pathogenesis of chronic asthma has not been well studied. Whether IL-33 is directly or indirectly involved in the development of chronic asthmatic symptoms, including chronic inflammation, airway remodeling, persistent airway hyperresponsiveness, as well as the related cellular and molecular mechanism are not well known.This current study sought to investigate the possible roles of IL-33 in asthma pathogenesis, through using a new intervention strategy of active immunization to induce a persistent anti-IL33 antibody response and intervene IL-33 signaling, and assess thepotentials of exploring IL-33 as a new therapeutic target and developing active immunization of anti-IL-33 vaccine as a new therapeutic strategy for the clinical treatment of asthma. Firstly, ovalbumin (OVA) induced acute and chronic allergic airway inflammation models of asthma were set up in Balb/c mice. Typical features of airway inflammatory responses were induced, including airway and lung tissue inflammation, airway mucus overproduction, elevated serum OVA-specific IgE, and airway subepithelial collagen deposition. Secondly, the IL-33 epitope or mature molecule-based vaccine were obtained by gene recombiannt techniques. Twelve possible antigenic epitopes were predicted by bioinformatic approaches. The encoding DNA fragments for IL-33 epitopes or mature molecules were cloned into immunodominant domain of hepatitis B core antigen. The recombinant chimeric protein was expressed efficiently in E.coli cells and presented as virus-like particles (VLPs) under observation using an electron microscopy. To be noted, full-length molecules of putative mature IL-33 was successfully presented on the surface of VLPs. The chemeric VLPs were effectively purified and then used for mouse immunization. Thirdly, the immune charateristics of VLPs were investigated. ELISA and Western-blot analyses indicated that VLPs with peptide epitopes or full-length IL-33 molecules could break through the immune tolerance and induce anti-IL-33 antibodies. The effects of different vaccine doses, immunization routes and applied adjuvants on antibody response profiles were investigated, including antibody titers, antibody duration and antibody types (IgA and IgG) or subtypes (IgG1 and IgG2a).Fourthly, the VLPs presenting mature IL-33 molecules were used in an acute asthma model. The mice were preventively immunized with VLPs and then challenged with repeated OVA administrations. The results showed that vaccination effectively reduced the total inflammatory cells, eosinophils and IL-33 levels in BALF. In addition, lung tissue inflamamtion and airway goblet cell hyperplasia were significantly suppressed in vaccinated mice, analyzed through H&E or PAS staining of lung tissue sections followwed by semi-quantitative scoring. Finally, the VLPs presenting mature IL-33 molecules were used in a chronic asthma model. The VLPs immunization was therapeutically conducted when the airway inflammatory responses has been established. The results showed that, VLPs immunization suppressed the accumulation of inflammatory cells in BALF, reduced serum OVA-IgE levels, and elevated Thl-type cytokines IFN-γ and inhibited Th2-type cytokine IL-5 expressions by splenocytes which were isolated from the experimental mice and stimulated in vitro. However, inflammatory cellular infiltration in the lung tissue, airway goblet cell hyperplasia and lung collagen accumulation have no obvious changes, comparing the vaccinated mice with the controls.The results demonstrated that IL-33 plays a key role in the acute airway inflammatory responses, but in chronic response stage, once the airway remodeling has been established, the effects of regulating IL-33 signaling were limited. The results indicated that IL-33 signaling pathway is a possible therapeutic target for asthma treatment and the intervention would better be conducted at the early stage of the disease. The results added our knowledge of the complexity and the possible role of IL-33 in asthma pathogenesis, and also provided useful information for evaluating the potentials of targeting IL-33 in the clinical treatment of asthma.