Transcriptomic signature-guided depletion of intermediate alveolar epithelial cells ameliorates pulmonary fibrosis in mice

Abstract Single-cell RNA sequencing (scRNA-seq) analyses of human and murine models of pulmonary fibrosis have uncovered multiple novel cell states involved in lung repair and regeneration, including an intermediate epithelial cell state identified as the KRT5-/KRT17+ aberrant basaloid cells in humans and the cytokeratin 8-positive alveolar differentiation intermediate (Krt8+ ADI) in mice. However, the specific contributions of these transitional cells to fibrogenesis remain poorly understood. In this study, we introduce a novel RNA-sensing-dependent protein translation technology that enables selective targeting and functional interrogation of Krt8+ ADI cells both in vitro and in vivo . Through transcriptomic analysis, we identified small proline-rich protein 1A ( SPRR1A ) mRNA as a highly specific marker shared by mouse Krt8+ ADI cells and human KRT5-/KRT17+ aberrant basaloid cells, effectively distinguishing them from other lung cell populations during lung injury and repair. Leveraging this transcriptomic specificity, we deployed programmable RNA sensors to drive EGFP reporter expression selectively in Krt8+ ADI cells in vivo . EGFP-labeled cells isolated from mouse lungs closely recapitulated the transcriptomic and phenotypic features of Krt8+ ADI cells, validating efficient and specific targeting. To assess the functional role of these cells, we established an RNA-sensing-dependent diphtheria toxin receptor (DTR) system to selectively ablate Sprr1a-expressing cells following diphtheria toxin (DT) administration. Targeted depletion of Sprr1a + cells significantly reduced fibrosis in bleomycin-injured mice, underscoring the pathogenic role of these transitional epithelial cells in lung fibrogenesis. Collectively, our findings define intermediate alveolar epithelial cells as key drivers of fibrosis and suggest that targeting this population may offer new therapeutic avenues for pulmonary fibrosis.