The Pediatric Airway Epithelium Generates Heavy Chain 3‐Modified Hyaluronan in Response to RSV Infection

ABSTRACT Respiratory syncytial virus (RSV) infection induces significant structural remodeling of the pulmonary extracellular matrix (ECM). While in vivo models demonstrate increased tissue synthesis of pre‐alpha‐inhibitor (P⍺I) and heavy chain 3 (HC3), which covalently modifies and stabilizes hyaluronan (HA), the primary cellular source of this modification remains unclear. We investigated whether the pediatric airway epithelium directly drives the generation of HC3‐modified HA in response to RSV. Primary pediatric airway epithelial cells (AECs) were differentiated at an air‐liquid interface and infected with RSV. Temporal changes in HA distribution, expression of the heavy chain transferase TSG‐6, and HC3 accumulation were assessed over 96 h post‐infection (hpi) via binding assays, RT‐qPCR, Western blotting, and immunofluorescence. RSV infection induced a progressive, time‐dependent accumulation of cell‐associated HA, independent of transcriptional changes in canonical HA synthesis or degradation enzymes. This accumulation was preceded by a rapid, transient upregulation of TSG‐6 mRNA that peaked at 6 hpi. Furthermore, RSV infection significantly increased cellular pre‐α‐inhibitor expression and the apical deposition of HC3, which paralleled viral progression. Functionally, this remodeled ECM significantly enhanced HA‐dependent leukocyte adhesion to the infected epithelial surface. The pediatric airway epithelium actively orchestrates early ECM remodeling during RSV infection. The rapid, TSG‐6‐mediated accumulation of HC3‐modified HA generates a highly stable, pro‐adhesive microenvironment. These findings establish AECs as a critical driver of local matrix dynamics and highlight early HA modifications as a potential therapeutic target in RSV pathogenesis.