Cellular Ensembles in Alveolar Homeostasis and Repair

Lung epithelium, the lining that covers the inner surface of the respiratory tract, is directly exposed to the environment and thus susceptible to airborne toxins, irritants, and pathogen-induced damages. In adult mammalian lungs, epithelial cells are generally quiescent but can respond rapidly to repair damaged tissues. Evidence from experimental injury models in rodents and human clinical samples has led to the identification of these regenerative cells, as well as pathological metaplastic states specifically associated with different forms of damages. The primary alveolar stem cell, alveolar type-2 cells (AT2s) are sparsely distributed and make up only 5% of the surface area. Despite this organization, AT2s are still able to maintain tissue homeostasis and achieve efficient repair after injury. However, the underlying mechanisms of stem cell activation, injury response, and subsequent cell-cell communication signals mediating resolution of injury and restoration of alveolar homeostasis remain elusive. Additionally, modulation of these regenerative cells for therapeutic potential has not been well established, primarily due to a lack of viable gene editing tools and vehicles for gene delivery to the alveolar stem cells, and neighboring niche. First, to better target the lung alveolus, we screened and identified cell-type specific adeno-associated virus (AAV) serotypes, enabling efficient targeting and gene expression of exogenous genes in alveolar stem cells as well neighboring mesenchyme. These tools were also capable for both in vitro and in vivo gene editing, forgoing the need for development of complex genetic mouse models as well as enabling diverse, viral-based screens. Second, using 3-dimensional, thick tissue imaging we reveal that a single AT2 cell can enface multiple alveolar compartments by virtue of a unique, multi-apical domain architecture. Lineage tracing and live imaging coupled with genetic and AAV-mediated selective ablation of AT2s was used to show robust recovery of AT2 numbers and distribution via clonal proliferation and migration, even after three successive rounds of ablation. Clonal tracing revealed that a single AT2 can differentiate to cover multiple alveolar cups. During injury repair, AT2s dynamically reorganize their apical domains to facilitate either migration or differentiation. Single- cell transcriptome profiling, genetic and pharmacologic disruption of actin dynamics, and evaluation of multiple physiologically relevant disease states identified the roles of actin cytoskeleton, cell migration, and multi-apical domains in AT2 recovery and regenerative potency. Lastly, using cell-type specific ablation of alveolar type 1 cells (AT1s), we identified novel mechanisms of epithelium-mediated signaling to mesenchymal fibroblasts, thereby uncovering novel mechanisms of fibrosis initiation and progression. Modulation of AT1 ablation dynamics preferentially drives fibrosis or, in contrast, emphysema, both at histological and physiological levels, as assessed by whole body plethysmography. Single-cell sequencing identified the epithelial and mesenchymal cell identities involved in regenerative processes, as well as identification of a PDGFA signaling axis between AT1s and resident alveolar fibroblasts necessary for fibroblast maintenance. We demonstrate that modulation of these signaling pathways during lung regeneration could enhance fibrosis or convert fibrosis to emphysema. In sum, the work presented herein both develops functional tools for perturbation of alveolar stem cells, as well as an improved understanding of alveolar architecture, stem cell dynamics during injury repair, homeostatic intercellular signaling, and mechanisms of disease progression.