<i>Streptococcus pneumoniae</i> colonization modulates human nasal epithelial responses to respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality in infants globally. Specific nasal bacterial genera are differentially associated with RSV severity in infants: <i>Haemophilus</i> and <i>Streptococcus</i> with more severe disease and <i>Dolosigranulum</i> with healthy controls or milder outcomes. We hypothesized these differential bacterial effects begin at the epithelial level. Therefore, we established human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) as a model system to assess effects of individual nasal microbionts on the epithelial response to subsequent RSV infection and of RSV on those microbionts. Infant-derived HNO-ALI were monocolonized with either <i>Streptococcus pneumoniae,</i> nontypeable <i>Haemophilus influenzae,</i> or <i>Dolosigranulum pigrum</i> one day before viral infection. RSV reduced colonizing <i>S. pneumoniae</i> and <i>D. pigrum</i> levels without affecting <i>H. influenzae. S. pneumoniae</i> precolonization uniquely reduced RSV levels during infection. <i>S. pneumoniae</i> precolonization also modulated the epithelial transcriptional response to RSV infection more so than <i>H. influenzae</i> or <i>D. pigrum</i>, with a pronounced effect on genes involved in immune response, cell cycle, stress, and growth signaling. Gene set enrichment analysis showed <i>S. pneumoniae</i> precolonization blunted RSV-induced increase in inflammatory and immune responses, consistent with <i>S. pneumoniae</i> also modulating RSV-induced cytokine production. Furthermore, <i>S. pneumoniae</i> precolonization blocked RSV-mediated dysregulation of cell-cycle genes, consistent with preventing arrest. Bacterial rescue of cell-cycle progression is a potential mechanism for reduced infectious virion production, since cell-cycle arrest enhances RSV replication. HNO-ALI facilitated elucidation of bacterial-viral-epithelial interplay at a frequent site of viral infection, directly linking nasal bacterial colonization to RSV infection dynamics.