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

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.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19 (preprint)

Background: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with a novel airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. Methods: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. Results: Forty patients (32M:8F, age:22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. Conclusions: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established.

Down-regulation of MALAT1 is a hallmark of tissue and peripheral proliferative T cells in COVID-19 (preprint)

T cells play key protective but also pathogenic roles in COVID-19. We studied expression of long non-coding RNAs (lncRNAs) in COVID-19 T cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1- anti-correlating gene signature included processes associated with T cell activation such as cell division, oxidative phosphorylation and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T cells marked dividing T cells in both lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

Identification of a protein expression signature distinguishing early from organising diffuse alveolar damage in COVID-19 patients. (preprint)

Diffuse alveolar damage (DAD) is a histopathological finding associated with severe viral infections, including SARS-CoV-2. However, the mechanisms mediating progression of DAD are poorly understood. Applying protein digital spatial profiling to lung tissue obtained from a cohort of 27 COVID-19 autopsy cases from the UK, we identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246), and VISTA that distinguishes early / exudative DAD from late / organising DAD with good predictive accuracy. These proteins warrant further investigation as potential immunotherapeutic targets to modulate DAD progression and improve patient outcome.