RSV-NTHi Co-Infection Skews Host Immunity by Suppressing Type I IFN Responses and Enhancing Pro-Inflammatory Responses

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease in young children, yet determinants of disease severity in otherwise healthy infants remain poorly understood. RSV disease severity has been linked to temporal differences in airway epithelial type I/III IFN and inflammatory responses, with defective viral genomes (DVGs) acting as potent inducers of type I/III IFNs. Additionally, an increased abundance of nontypeable <i>Haemophilus influenzae</i> (NTHi) is associated with more severe RSV disease, although the mechanisms underlying this association and the impacts of NTHi on DVG replication and DVG-driven host responses remain unclear. To address this knowledge gap, we modeled a clinically relevant but underexplored scenario of simultaneous transmission by performing concurrent RSV-NTHi co-infection experiments. Co-infection reduced titers of RSV but not of NTHi. Mechanistically, extracellular NTHi inhibited RSV particle binding to host cells. Using A549 cells and human precision-cut lung slices, we found that NTHi alone was a weak inducer of type I/III IFNs but a strong inducer of pro-inflammatory cytokines. Accordingly, RSV-NTHi co-infection suppressed DVG replication and DVG-driven IFN responses while enhancing inflammatory signaling, potentially driven by increased cell-associated NTHi. Together, these findings provide a mechanistic basis for how NTHi exacerbates RSV disease severity through dysregulated host immune responses rather than increased viral burden.