Identifying the roles of mitochondrial reactive oxygen species and NLRC4 inflammasome in Aspergillus protease-induced airway epithelium inflammation

Abstract

Epidemiological studies have shown that fungal infections are among the main causes of respiratory tract diseases. Fungi such as Aspergillus and Candida species have become increasingly important pathogens as the global climate changes. Aspergillus species are major indoor fungi, and they produce enzymes that contribute to the pathogenesis of Aspergillus-induced diseases. Thus, Aspergillus proteases can cause invasive pulmonary aspergillosis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, and atopic asthma and are involved in a complex interplay between the airway epithelium and host immune responses to inhaled spores. Since the airway epithelium is in continuous contact with foreign organisms, including pathogens, and is the first line of host defense, the physical barrier functions of the epithelium are tightly regulated. Proteolytic allergens such as Aspergillus proteases have the ability to disrupt epithelial barrier functions and cause inflammatory responses due to a disproportion between tolerogenicity and increasing permeability. Additionally, allergenic proteases generate reactive oxygen species (ROS), which cause toxic effects on epithelial tissues and affect cell-signaling mediators involved in immune responses. Mitochondrial ROS are especially important because they directly affect the production of inflammatory cytokines. Furthermore, recent studies have suggested that mitochondrial ROS could induce persistent inflammation via inflammasomes. However, the underlying mechanisms behind the generation of mitochondrial ROS and their role in allergenic protease-induced epithelial inflammation are not fully understood. Therefore, the present study aimed to investigate the effects of Aspergillus proteases on airway inflammation using human primary bronchial epithelial cells and A549 cells, in which we evaluated the inflammatory responses mediated by mitochondrial ROS and by the nucleotide-binding oligomerization domain-like receptor family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome. In addition, we investigated the IL-1ß and caspase1 activation in vivo. Our results demonstrated that mitochondria were important sources of ROS in the context of cell stimulation with Aspergillus proteases. We showed that regulation of mitochondrial ROS could modulate the production of proinflammatory mediators by preventing the activation of mitochondrial ROS-induced activator protein 1 (AP-1) in airway epithelial cells. In addition, the results of this study indicated that uncoupling protein 2 (UCP2) played a role in the regulation of the mitochondrial ROS formation under conditions of cell stimulation with Aspergillus protease and that the NLRC4 inflammasome might be important for fungal protease-induced epithelial inflammation. These findings suggest that targeted suppression of mitochondrial ROS and of the NLRC4 inflammasome may be useful for the development of effective therapeutics to block inflammatory responses caused by fungal proteases in the airways. In the second part, we aimed to evaluate the effects of 18ß-glycyrrhetinic acid (GA) on airway epithelial inflammation using a fungal allergen and the inhibitory effects of GA in the production of mitochondrial ROS. Our results demonstrated that GA was a potent inhibitor of Aspergillus protease-induced inflammatory mediator expression. In addition, in vivo epithelial inflammation against Aspergillus protease stimulation was inhibited by GA. These inhibitory effects were dependent on the mitochondrial ROS/MAPK axis and regulation of UCP2 expression. Therefore, GA may represent a potential effective therapeutic agent for blocking inflammatory responses in the airways.