Epigenetic signatures driving metaplastic repair mechanisms in human alveolar type II cells

University Of California, San Francisco, San Francisco CA

Investigators

Abstract

Project Summary/Abstract: The alveolar epithelium, composed of alveolar type I (AT1) and alveolar type II (AT2) cells, is crucial for performing gas exchange and maintaining normal lung physiology. AT2 cells are known to proliferate and differentiate into AT1 cells under homeostatic conditions and in response to injury to restore alveolar structure and function. At a histologic level, progressive fibrotic lung diseases are characterized by loss of alveolar epithelial cells, replacement of normal lung tissue with extracellular matrix, and formation of “honeycomb” cysts lined by epithelial cells, which are thought to be airway-derived, in a process called “bronchiolization”. Our lab has shown that AT2 cells are capable of trans-differentiation into airway-like basal cells (BCs) under both in vivo and in vitro conditions, raising the possibility that the bronchiolization seen in fibrotic lung diseases may occur at least in part via metaplastic differentiation of AT2 cells. Previous efforts profiling transcriptional and epigenetic variation in alveolar epithelial cells have identified several putative pathways responsible for directing differentiation trajectories of AT2 cells, including the Wnt, TGF-β, and YAP/TAZ pathways. While the underlying pathophysiology is largely disease-specific, evidence suggests activation of shared dysfunctional regenerative pathways in epithelial cells of the distal lung that ultimately lead to fibrosis. The gene regulatory networks mediating AT2-to-BC metaplasia, in particular, are poorly understood. We propose to use single-cell RNA-seq paired with transduction of lentiviral barcodes in primary human AT2 cells to prospectively lineage trace their differentiation into alveolar-basal intermediate (ABI) cells in the organoid model, and subsequently direct them towards a BC, AT1, or AT2 phenotype using specific culture conditions that have been experimentally optimized by our lab and others. In parallel, we will attempt retrospective lineage tracing using mitochondrial somatic mutations recovered with a modified single-cell RNA-seq protocol from human lung tissues donated to research after lung transplantation from both healthy and IPF donors. We will profile epigenetic variation using ATAC-seq and ChIP-seq in ABI-derived cells in response to activation of specific signaling pathways to direct their differentiation. We will then compare the profiles thus identified with single-cell ATAC-seq data from donated human lung tissues. By combining robust lineage tracing in human cells, epigenetic profiling, and single-cell transcriptomics, we hope to gain a better understanding of the epithelial differentiation pathways that are dysregulated in lung fibrosis.