Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and nonsmokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium. Overall design: For the in vivo dataset, we sequenced a total of 68,370 (36,248 after QC) epithelial cells from across 15 human tracheal donors (6 heavy smokers, 6 never-smokers, 2 light smokers, and 1 pediatric subject). For the in vitro dataset we sequenced a total of 8,722 (5,976 after QC) epithelial cells sampled from 20 time points along the differentiation process taken by air-liquid interface (ALI) cultures seeded from primary basal cells of 3 human tracheal donors (from ALI seed day to day 32 post-airlift)