Persistence of Transitional Cell State Without Senescence or Fibrosis During Alveolar Epithelial Regeneration in Human Acute Respiratory Distress Syndrome

ABSTRACT

RATIONALE Acute respiratory distress syndrome (ARDS) results from injury to the alveolar epithelial cell (AEC) barrier leading to pulmonary edema;recovery requires epithelial regeneration. However, the specific defect resulting in ongoing barrier permeability in fatal early ARDS is unknown. In mouse models of ARDS, we found that AEC2s assume a transitional state characterized by transient cell cycle arrest during differentiation towards AEC1s. Transitional cells persist and are senescent in human idiopathic pulmonary fibrosis (IPF), leading some to speculate that persistence of transitional cells is pathognomonic of fibrosis in humans. We hypothesized that transitional cells also arise early in human ARDS and that incomplete AEC1 differentiation from the transitional state underlies barrier permeability and death from respiratory failure in early ARDS. We speculated that in contrast to IPF, transitional cells in early ARDS are in transient cell cycle arrest but not senescent and maintain capacity for an AEC1 fate. METHODS AND RESULTS: Lung tissue was obtained from patients who died within two weeks of hospitalization of ARDS due to COVID-19 or other etiologies, and from patients with IPF. Histology revealed diffuse alveolar damage without fibrosis in patients with ARDS. Immunostaining demonstrated AEC damage and abundant transitional cells in both ARDS and IPF. In ARDS, transitional cells existed in a monolayer on alveolar septa, filling gaps denuded of AEC1s and displaying spread morphologies without AEC1 marker expression, suggesting ongoing but incomplete differentiation. In fibrosis, transitional cells existed on a background of architectural distortion and fibrosis. Meta-analysis of single cell RNA sequencing (scRNAseq) datasets demonstrated that transitional cells were transcriptionally highly similar. However, the senescence marker p16 was expressed in transitional cells in human IPF but not in mouse models. Immunostaining confirmed that transitional cells in IPF but not human ARDS expressed p16. CONCLUSION: We conclude that transitional cells arise in early human ARDS without fibrosis. We propose that incomplete AEC1 differentiation from the transitional state is the specific defect in epithelial regeneration underlying barrier permeability and respiratory failure. We speculate that in early human ARDS, as in mouse models, transitional cells retain the capacity to differentiate into AEC1s, restoring alveolar architecture without fibrosis. However, in IPF and fibroproliferative ARDS, transitional cells become senescent, lose capacity for AEC1 differentiation, and fibrosis ensues. Evolution of transitional cells from a transient cell cycle arrest to a permanent cell cycle arrest (senescence) may be the key defect driving the pathogenesis of fibrosis after injury.