PROJECT 2 SUMMARY Mucus plugging is common in severe asthma, is associated with type 2 inflammation and poor lung function and persists despite high dose inhaled corticosteroids. Mucus plugging is also a critical feature of fatal asthma. Consequently, although we have made significant progress in targeting airway inflammation in asthma, there is still an unmet need for therapeutic approaches that directly prevent or treat pathological mucus and mucus plugging. The overall objectives of this project are to understand the molecular mechanisms that regulate the differentiation and function of goblet cells that produce airway mucins and secrete pathological mucus in human asthma and to develop pre-clinical data on two novel therapeutic approaches that target these processes. In Aim 1, we propose to determine the roles of miR-141/200 family members in epithelial cell differentiation, pathological mucus production, and airway mucus plugging in asthma. Of the highly homologous five-member miR-141/200 miRNA family, our preliminary data implicate miR-141 in goblet cell differentiation, mucin production and mucus secretion. However, our data suggest that other members of this microRNA family also play a role in these processes. We propose to test the hypothesis that miR-141/200 family members are important for the transition of basal cells to secretory cells, mucin production and mucus secretion, playing roles that are complementary (or even antagonistic) to miR-141. We will also test the hypothesis that these miR-141/200 family members localize to epithelial cells at sites of type 2 inflammation and mucus plugging in human asthma and are associated with severe disease. In Aim 2 we propose to determine the mechanisms underlying IRE1α regulation of secretory epithelial cell differentiation and pathological mucus production. Our preliminary data show that endoplasmic reticulum (ER) stress, unfolded protein response (UPR) pathway activation and activation of IRE1α kinase endoribonuclease (RNase) are increased during secretory cell differentiation in patients with asthma. We also show that conditional deletion of IRE1α in the murine airway epithelium or inhibition with a novel small molecule inhibitor (KIRA8) in human airway epithelial cells, inhibit goblet cell metaplasia and airway epithelial mucin production. IRE1α may mediate these effects through two distinct outputs: an “adaptive” mode which typically promotes homeostasis, and a “terminal” mode, which leads to maladaptive cellular outcomes. Recently we have developed another novel small molecule inhibitor, PAIR2, which specifically inhibits the terminal mode of UPR activation. In this aim, we propose to test the hypotheses that IRE1α inhibition decreases goblet cell metaplasia by inhibiting the transition of basal cells into a transitional secretory cell state, that terminal UPR activation mechanistically connects IRE1α to goblet cell metaplasia (which can be inhibited by PAIR2), and that terminal UPR si...