Cystic fibrosis (CF) lungs exhibit mucoinflammatory responses soon after birth, likely triggered by viral infections and/or aspiration. Respiratory syncytial virus (RSV) causes bronchiolitis leading to airway muco- obstruction in young CF children, who exhibit increases in MUC5B and MUC5AC mucins in their airways. Because no treatments are available for CF airway mucus overproduction, there is a clear unmet medical need for therapies that target mucin synthesis in CF airways. CF airway epithelial inflammation triggers endoplasmic reticulum (ER) stress and activates the inositol requiring enzyme 1 (IRE1), which exists in two isoforms, α and β. IRE1α is ubiquitous, but IRE1β is only expressed in mucous cells of the respiratory and GI tracts. We have shown that IRE1β (but not IRE1α) is required for airway mucin production. IRE1 is an ER transmembrane protein with a lumenal domain (sensor of unfolded proteins) and a cytoplasmic domain (effector) with kinase and RNase activities. It is unknown whether the IRE1 lumenal domain senses unfolded mucins and whether its cytoplasmic domains mediate mucin production; however, our previous studies suggested that mucin production triggers ER stress and activates IRE1β kinase-induced RNase activation. Because the activated IRE1 RNase splices the mRNA of X-box binding protein-1 (XBP-1s), a transcription factor that up-regulates mucin production, this may provide a mechanism for CF airway epithelial mucin overproduction. In non-mucous cells, IRE1 kinase activates JNK, p38 MAP kinase and NF-B via protein interactions, but it is unknown whether the IRE1 kinase activates these pathways, which are relevant to CF airways because they can promote mucin production. Our preliminary data indicate that IRE1β, MUC5AC and MUC5B levels are up-regulated in native CF human airways and in freshly isolated CF human distal airway epithelia. Over-expression of wild type IRE1 in primary human bronchial epithelia (HBE) increased mucin production, whereas over-expression of IRE1 mutants that lack kinase and/or RNase activities decreased mucin production. IL-1 and TNF, predominant CF airway cytokines, differently affected XBP-1s (only IL-1 increased XBP-1s) and mucin production (IL-1 > TNF) in HBE, suggesting that they up-regulate mucin production via IRE1β RNase-dependent and independent mechanisms. KIRA6, an IRE1 kinase + RNase inhibitor, blunted IL-1-increased XBP-1s and mucins in CF HBE. Notably, a combination of CFTR modulators (VX-445, VX-661 and VX-770) had no effect on mucin production. RSV infection of HBE increased XBP-1s and mucin production/secretion, and these responses were blunted by KIRA6. Finally, murine parainfluenza virus type 1-infected mice developed viral bronchiolitis and airway muco-obstruction, modeling the bronchiolitis in CF infants infected by RSV. Our aims will test the role of IRE1β protein domains in CF airway cytokine-increased mucin production in CF HBE. We will also evaluate the therapeutic ...