The lungs are the main site that is affected in severe COVID-19, and post-mortem lung tissue provides crucial insights into the pathophysiology of severe disease. From basic histology to state-of-the-art multiparameter digital pathology technologies, post-mortem lung tissue provides snapshots of tissue architecture, and resident and inflammatory cell phenotypes and composition at the time of death. Contrary to early assumptions that COVID-19 in the lungs is a uniform disease, post-mortem findings have established a high degree of disease heterogeneity. Classic diffuse alveolar damage represents just one phenotype, with disease divisible by early and late progression as well as by pathophysiological process. A distinct lung tissue state occurs with secondary infection; extrapulmonary causes of death might also originate from a pathological process in the lungs linked to microthrombosis. This heterogeneity of COVID-19 lung disease must be recognised in the management of patients and in the development of novel treatment strategies.

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.

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Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression.