Functional evaluation of fibroblast-to-myofibroblast transition as an in vitro screening tool for pulmonary fibrosis

Polyhexamethylene guanidine (PHMG), a major component of humidifier disinfectants (HDs) linked to fatal lung injuries, has been implicated in the development of pulmonary fibrosis. However, most previous studies have primarily focused on epithelial injury, with limited attention to fibroblast activation - a central mechanism in fibrogenesis. In this study, we present an integrated testing strategy (ITS) designed for the early in vitro screening of fibrogenic agents, centered on fibroblast-to-myofibroblast transition (FMT) as a core functional and mechanistic endpoint. Human lung fibroblasts (MRC-5) were exposed to PHMG-HCl, and fibrotic responses were assessed across four key events (KEs): (1) TGF-β1 signaling activation, (2) myofibroblast differentiation, (3) extracellular matrix (ECM) deposition, and (4) enhanced cell migration and contraction. PHMG-HCl exposure led to increased reactive oxygen species (ROS) generation and upregulation of TGF-β1, α-SMA, and COL1A, consistent with FMT induction. Functional assays further demonstrated elevated cell motility and collagen gel contraction. These in vitro finding were corroborated by in vivo rat models and 3D EpiAirway™ systems, which exhibited inflammation, collagen deposition, and epithelial barrier disruption. Collectively, this FMT-based ITS provides a mechanistically grounded and functionally predictive platform for early identification of pulmonary fibrogenic potential, offering an efficient and ethical alternative to traditional toxicity testing methods.