Carla Bender Kim

Lung repair in response to viral infection requires integrated communication between epithelial and immune compartments, yet the impact of antiviral mediators on epithelial regenerative capacity remains poorly defined. Here, we demonstrate that type I interferon (IFN-I) signaling primes the lung for alveolar renewal following viral challenge. IFN-I contributes to the induction of an interferon-stimulated gene-high (ISG) ^hi Sca-1 ^Pos population of alveolar type II (ATII) epithelial cells. Sca-1 ^Pos ATIIs exhibit enhanced proliferative capacity and increased organoid-forming efficiency compared with their Sca-1 ^Neg counterparts. Viral challenge concurrently drives phenotypic reprogramming of tissueresident alveolar macrophages (trAMs). Sca-1 ^Pos ATIIs display heightened responsiveness to oncostatin M (OSM) and, following viral challenge, require trAM-derived OSM for their proliferation. Together, these findings reveal that viral stimuli induce coordinated IFN-I-dependent epithelial and macrophage states that poise the lung for regeneration, positioning IFN-I not only as a central antiviral defense mechanism but as a priming signal that prepares lung tissue for renewal.

We provide streamlined protocols to generate both airway and alveolar epithelial organoids from a single, clinical BAL specimen. From standardized specimen collection to organoid plating to passaging and differentiation, we show that rare, primary epithelial cells in BAL can give rise to all the major airway and alveolar cell types. Our serum-free, feeder-free, and sorting-free methods offer a simplified starting point for using patient-derived organoids to model lung disease.

The Impact of the Exposome on Epithelial Barriers: New Approach Methodologies for Translational Research - Thoracic Research and Practice

The lungs are highly susceptible to chronic disease in advanced age, likely due to the uniquely compromised repair function of alveolar type II (AT2) cells, facultative progenitor cells that maintain the gas exchange surface. Using aging mouse models, single-cell sequencing, and ex vivo organoid assays, we found that homeostatic aged AT2 cells exhibited an Interferon γ (IFNγ) response associated with IFNγ+ CD8+ T cells in tertiary lymphoid structures (TLS). Aged AT2 cells exhibit impaired regeneration in organoid assays and lost markers of an IFNγ response outside the lung microenvironment, demonstrating that elevated local IFNγ influences the state of AT2 cells. Neutralization of IFNγ signaling and immunoproteasome knockout mice with attenuated IFNγ levels partially rescued aged AT2 cell regeneration. Our findings demonstrate that local IFN γ signaling in aging lungs actively represses alveolar regeneration, establishing chronic inflammatory signaling as a cause of age-related decline in the lung. Halting chronic inflammatory processes restored alveolar regeneration and may provide a means to improve lung health in old age.

Lung organoids are versatile experimental models, but their broader use in studying human disease is limited by the scarcity of starting material and the complexity of current methods. To align organoid technology with common clinical practice, we developed airway and alveolar organoids using cells obtained from patients' bronchoalveolar lavage (BAL) fluid. Building on existing techniques, we showed that BAL is a reliable, accessible source of primary human epithelial cells, yielding airway and alveolar organoids within 10 days. Organoids can then be expanded over many passages for downstream analysis. Our streamlined methods do not require cell sorting or other complex procedures, all cells are derived from a single patient, and media are based on serum-free, chemically-defined formulations. Here, we present detailed protocols for organoid establishment, standardized passaging and phenotyping, and differentiation of both airway and alveolar models. We provide a time course of BAL-derived airway organoid differentiation at air-liquid interface, and we demonstrate proof of principle for differentiation of BAL-derived alveolar organoids in 3D culture. These methods can be readily adapted to generate and characterize organoids from lung tissue, tracheobronchial specimens, or other primary cells from humans or mice, expanding the potential to use lung organoids for disease modeling.

Cancers display cellular, genetic and epigenetic heterogeneity, complicating disease modeling. Multiple cell states defined by gene expression have been described in lung adenocarcinoma (LUAD). However, the functional contributions of cell state and the regulatory programs that control chromatin and gene expression in the early stages of tumor initiation are not well understood. Using single-cell RNA and ATAC sequencing in Kras/p53-driven tumor organoids, we identified two major cellular states: one more closely resembling alveolar type 2 (AT2) cells (SPC-high), and the other with epithelial-mesenchymal-transition (EMT)-associated gene expression (Hmga2-high). Each state exhibited distinct transcription factor networks, with SPC-high cells associated with TFs regulating AT2 fate and Hmga2-high cells enriched in Wnt- and NFκB-related TFs. CD44 was identified as a marker for the Hmga2-high state, enabling functional comparison of the two populations. Organoid assays and orthotopic transplantation revealed that SPC-high, CD44-negative cells exhibited higher tumorigenic potential within the lung microenvironment. These findings highlight the utility of organoids in understanding chromatin regulation in early tumorigenesis and identifying novel early-stage therapeutic targets in Kras-driven LUAD.

Cell plasticity, changes in cell fate, is crucial for tissue regeneration. In the lung, failure of regeneration leads to diseases, including fibrosis. However, the mechanisms governing alveolar cell plasticity during lung repair remain elusive. We previously showed that PCLAF remodels the DREAM complex, shifting the balance from cell quiescence towards cell proliferation. Here, we find that PCLAF expression is specific to proliferating lung progenitor cells, along with the DREAM target genes transactivated by lung injury. Genetic ablation of Pclaf impairs AT1 cell repopulation from AT2 cells, leading to lung fibrosis. Mechanistically, the PCLAF-DREAM complex transactivates CLIC4, triggering TGF-β signaling activation, which promotes AT1 cell generation from AT2 cells. Furthermore, phenelzine that mimics the PCLAF-DREAM transcriptional signature increases AT2 cell plasticity, preventing lung fibrosis in organoids and mice. Our study reveals the unexpected role of the PCLAF-DREAM axis in promoting alveolar cell plasticity, beyond cell proliferation control, proposing a potential therapeutic avenue for lung fibrosis prevention.