Abstract Rationale Respiratory Syncytial Virus (RSV) bronchiolitis is the leading cause of respiratory morbidity and mortality in young children under two years of age and is characterized by airway obstruction poorly responsive to conventional bronchodilators, such as β2-adrenergic receptor (β2AR) agonists. We and others have demonstrated that RSV-induced epithelial inflammation leads to β2AR desensitization in infant airway smooth muscle cells (ASMCs), thereby contributing to this relaxation defect. Therefore, this study aims to determine how RSV infection affects the alternative ASMC relaxant pathway mediated by Nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP signaling, and whether therapeutic targeting of this pathway can restore ASMC relaxation and airway patency in infant RSV bronchiolitis. Methods We established three early-life RSV infection models using infant precision-cut lung slices (PCLSs), coculture of neonatal epithelial cells at the air-liquid interface with infant primary ASMCs, and BALB/c pups (7 days old). Utilizing these in vivo and ex vivo models, we assessed the impact of RSV infection on NOC-5 (a NO donor)-induced airway relaxation, epithelial nitric oxide synthase (iNOS) expression, and NO production. Following coculture with RSV-infected neonatal epithelia, infant ASMCs were detected for changes in the sGC abundance, S-nitrosylation (SNO), and enzymatic activity. To evaluate therapeutic potentials, we tested whether 8-Br-cGMP (a membrane-permeable cGMP analog) and Bay 60-2770 (a NO-independent SNO-sGC activator) could rescue ASMC relaxation through the cGMP-mediated pathway in RSV-infected infant PCLSs. Results RSV infection significantly reduced NOC-5-induced airway relaxation in infant PCLSs infected with RSV (1x106 PFU, 72 hours) and in PCLSs from RSV (1x106 PFU/10 μL)-infected mouse pups, but not in their adult counterparts. Indeed, RSV infection elicited markedly higher iNOS expression and NO production in neonatal than in adult epithelial cells. However, the excessive epithelial NO induced S-nitrosylation and dysfunction of sGC, thereby diminishing cGMP production in response to NO stimulation and reducing cGMP-mediated ASMC relaxation. Notably, both 8-Br-cGMP and Bay 60-2770 restored the ASMC relaxation in RSV-infected infant PCLSs, suggesting the cGMP downstream signaling cascade remains functionally intact and that sGC is the critical node of dysfunction. Conclusion Our findings suggest that early-life RSV infection induces excessive epithelial NO, which impairs cGMP-mediated ASMC relaxation through S-nitrosylation and inactivation of sGC. Pharmacological activation of SNO-sGC effectively restores ASMC relaxation and may represent a promising strategy to improve airway patency and clinical outcomes in severe infant RSV bronchiolitis. This abstract is funded by: Charles Hood Foundation; NHLBI
Bai et al. (Fri,) studied this question.