Integrated histopathology, spatial and single cell transcriptomics resolve cellular drivers of early and late alveolar damage in COVID-19

The most common cause of death due to COVID-19 remains respiratory failure. Yet, our understanding of the precise cellular and molecular changes underlying lung alveolar damage is limited. Here, we integrate single cell transcriptomic data of COVID-19 donor lungs with spatial transcriptomic data stratifying histopathological stages of diffuse alveolar damage (DAD). We identify changes in cellular composition across progressive DAD, including waves of molecularly distinct macrophages and depleted epithelial and endothelial populations throughout different types of tissue damage. Predicted markers of pathological states identify immunoregulatory signatures, including IFN-alpha and metallothionein signatures in early DAD, and fibrosis-related collagens in organised DAD. Furthermore, we predict a fibrinolytic shutdown via endothelial upregulation of SERPINE1/PAI-1. Cell-cell interaction analysis revealed macrophage-derived SPP1/osteopontin signalling as a key regulator during early DAD. These results provide the first comprehensive, spatially resolved atlas of DAD stages, highlighting the cellular mechanisms underlying pro-inflammatory and pro-fibrotic pathways across alveolar damage progression. ### Competing Interest Statement In the past three years, S.A.T. has consulted for or been a member of scientific advisory boards at Qiagen, Sanofi, GlaxoSmithKline and ForeSite Labs. She is a consultant and equity holder for TransitionBio and EnsoCell. J.M. holds equity in Glencoe Software which builds products based on OME-NGFF. The remaining authors declare no competing interests.