Project Abstract: Rhinoviruses (RV) have been impugned in the development of asthma and are the leading cause of acute asthma exacerbations. RV exposure evokes inflammation of the airways, but how RV modulates human airway smooth muscle cell (HASM) function to alter bronchomotor tone is unclear. HASM is the pivotal cell modulating airway tone, shortening in response to contractile agonist stimulation through increases in intracellular calcium, through activation of Rho kinase, and through modulation of actin dynamics. We previously showed that RV exposure evokes AHR in human precision cut lung slices (hPCLS), increases [Ca2+]i in HASM, and increases agonist-induced phosphorylation of myosin light chain in HASM from human airway epithelial cell (HAEC)/HASM co-cultures stimulated with RV-C15. We also demonstrate that RVC exposure attenuates β2 agonist-induced bronchodilation in hPCLS, cAMP production in HASM, and attenuates bronchodilator-induced reversal of HASM contraction. Our preliminary data shows that TFF3, and other inflammatory mediators, are released from HAEC and hPCLS following exposure to RVC, and that TFF3 attenuates bronchodilation of human small airways and production cAMP in HASM in response to bronchodilators. Furthermore, our data demonstrates that inhibition of TFF3 and putative TFF3 receptors can reverse RV-C15-induced attenuation of bronchodilation. Therefore, we posit RV-C15 modifies signaling mechanisms modulating bronchomotor tone in HASM through a TFF3- dependent mechanism, thereby altering responsiveness of the airways to bronchodilators. We also show that signaling downstream of Gαs-coupled receptors is attenuated in HASM following exposure to RV-C15- conditioned HAEC media. We propose a central hypothesis that RVC exposure of HAEC attenuates β2 agonist-induced bronchodilation via modulation of Gαs-coupled receptor function via TFF3-dependent mechanisms. We will utilize HASM cells, air-liquid interface-differentiated HAEC, and hPCLS to examine the aims of this proposal. In Aim 1, we will determine how RV exposure modulates Gαs-coupled receptor-induced signaling to attenuate bronchodilator-induced airway/HASM relaxation and how asthma alters these mechanisms. Aim 2 will examine how RV-C15-induced TFF3 release attenuates agonist-induced bronchodilation/relaxation of HASM and how asthma alters these mechanisms. Utilizing primary HAEC/HASM co-cultures and an ex vivo system of human small airways, we will delineate signaling pathway alterations regulating bronchomotor tone that can be targeted in the treatment of RV-induced exacerbations of asthma to restore bronchodilator responsiveness.