The long-term goal of this grant project is to understand the roles and mechanisms of the airway epithelium in Th2-type immune responses. Human airways are constantly exposed to the products of environmental allergens and microbes. Airway inflammation in patients with asthma is characterized by increased production of Th2 cytokines. However, the immunological mechanisms to explain the relationship between the environmental exposure and Th2-type inflammation in asthma are poorly understood. IL-33 is a new and potent Th2-driving cytokine; it is constitutively produced and stored in the nucleus of airway epithelial cells. We recently found that exposure to common aeroallegens, such as Alternaria and cockroach, induces a rapid release of ATP from airway epithelial cells, produces a sustained increase in intracellular calcium concentration ([Ca2+]i), and stimulates IL-33 secretion. Importantly, suppressing expression of P2 purinergic receptors, either P2Y2R or P2X7R, abolishes the increase in [Ca2+]i and IL-33 release. Therefore, we hypothesize that exposure to airborne allergens stimulates ATP secretion across the apical membrane of airway epithelial cells, resulting in concerted activation of purinergic receptors, increased [Ca2+]i and mobilization and release of stored IL-33. We also hypothesize that asthma is associated with an altered purinergic signaling response to airborne allergens, leading to enhanced epithelial release of IL-33. To test these hypotheses, in Aim 1, we will determine the mechanisms involved in allergen-evoked ATP release from the airway epithelium. In Aim 2, we will examine the roles of P2Y2R and P2X7R in regulating the release of IL-33 from airway epithelial cells. In Aim 3, we will investigate the molecular basis for differences in allergen-sensitivity in the epithelium between patients with asthma and normal individuals. We have developed robust in vitro models to dissect the epithelial cell responses to environmental allergens. A combination of complementary molecular, cell biological and pharmacological approaches will be employed. The technical expertise and equipment required for these studies are readily available in our laboratories. Therefore, proposed studies are likely to provide a better understanding of how airway epithelia respond to environmental exposure to allergens and will define the molecular mechanisms responsible for epithelial cell secretion of a potent Th2-driving cytokine, IL-33. The studies will also address a fundamental question of whether airway epithelial cells from patients with asthma behave differently from those of healthy individuals when they are exposed to airborne allergens. Ultimately, these studies will provide an important characterization of key signaling pathway(s) and molecule(s) involved in allergen-induced airway inflammation, allowing for identification of critical targets for development of novel therapeutic strategies to treat or to prevent asthma and related airway disorders.