Development of asthma therapies through bilirubin nanoparticles or mutant allergen

Abstract

Part 1. Although asthma, a chronic inflammatory airway disease, is relatively well-managed by inhaled corticosteroids, the side effects associated with the long-term use of these agents precipitate the need for alternative therapeutic options based on different modes of action. Bilirubin, a potent endogenous antioxidant and anti-inflammatory molecule, have been shown to ameliorate asthmatic symptoms, however, its clinical translation has been limited owing to its water insolubility and associated potential toxicity. Here we report the first application of bilirubin-based nanoparticles (BRNPs), composed of PEGylated bilirubin, as a nanomedicine for the treatment of allergic lung inflammatory disease. In the present study, the anti-asthmatic effects of BRNPs were assessed in a mouse model of fungal allergen-induced asthma. Because allergen-specific type 2 T-helper (Th2) cells play a key role in the pathogenesis and progression of allergic asthma, the effects of BRNPs on Th2 immune responses were investigated both in vivo and in vitro. Compared with unconjugated bilirubin (UCB), treatment with BRNPs in vivo suppressed the symptoms of experimental asthma and dramatically ameliorated Th2-related allergic lung inflammation. Consistent with these results, BRNPs mediated a reduction of Th2 cell populations and the expression of related cytokines by antibody-stimulated CD4+ T cells in vitro. Therefore, our results establish BRNPs as an important immunomodulatory agent that may be useful as a therapeutic for allergic lung inflammatory disease and other immune-mediated disorders.

Part 2. Asthma is a chronic inflammatory disease of the lower airways characterized by recurrent airway obstruction and wheezing. There are several causes of asthma, however, in an immunological aspect, the main cause of asthma is an allergen such as house dust mite, fungal protease, and pollen. One of the most important characteristics of allergens is a protease from fungi and the enzyme activity of the protease is thought to be critical to induce the Th2-mediated immune responses. Generally, protease activity of Aspergillus oryzae is strong enough to develop allergic asthma. By manipulating protease activity, we secured a fundamental know-how to develop asthma vaccine, which can also control the differentiation and function of Th cells and induce immune stimulation and/or tolerance. Thus, we cloned the cDNA of protease-inactive mutant A. oryzae protease and optimized codons for bacterial expression. Expressed and purified mutant A. oryzae protease was being lost protease activity and administrated into mice. In the group of mice administrated with a mutant protease, allergic responses were not induced compared to a group of mice administrated with wild-type A. oryzae protease. Immune tolerance rather than immune stimulation was observed in mutant A. oryzae protease administrated group. Additionally, we observed a decreased asthma phenotypes by pre-treatment of mutant A. oryzae protease in allergen-induced asthmatic mice. Through this study, we will develop asthma vaccines for the prevention and/or therapeutics for the disease and validate the effectiveness of the newly developed asthma vaccines in allergic asthma models.