Broad SARS-CoV-2 cell tropism and immunopathology in lung tissues from fatal COVID-19

Summary Background Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection in patients with Coronavirus Disease 2019 (COVID-19) prominently manifests with pulmonary symptoms histologically reflected by diffuse alveolar damage (DAD), excess inflammation, pneumocyte hyperplasia and proliferation, and formation of platelet aggregates or thromboemboli. However, the mechanisms mediating these processes remain unclear. Methods We performed multicolor staining for viral proteins, and lineage cell markers to identify SARS-CoV-2 tropism and to define the lung pathobiology in postmortem tissues from five patients with fatal SARS-CoV-2 infections. Findings The lung parenchyma showed severe DAD with thromboemboli in all cases. SARS-CoV-2 infection was found in an extensive range of cells including alveolar epithelial type II/pneumocyte type II (AT2) cells (HT2-280), ciliated cells (tyr-α-tubulin), goblet cells (MUC5AC), club-like cells (MUC5B) and endothelial cells (CD31 and CD34). Greater than 90% of infiltrating immune cells were positive for viral proteins including macrophages and monocytes (CD68 and CD163), neutrophils (ELA-2), natural killer (NK) cells (CD56), B-cells (CD19 and CD20), and T-cells (CD3e). Most but not all infected cells were positive for the viral entry receptor angiotensin-converting enzyme-2 (ACE2). The numbers of infected and ACE2-positive cells correlated with the extent of tissue damage. The infected tissues exhibited low numbers of B-cells and abundant CD3e+ T-cells consisting of mainly T helper cells (CD4), few cytotoxic T cells (CTL, CD8), and no T regulatory cell (FOXP3). Antigen presenting molecule HLA-DR of B and T cells was abundant in all cases. Robust interleukin-6 (IL-6) expression was present in most uninfected and infected cells, with higher expression levels observed in cases with more tissue damage. Interpretation In lung tissues from severely affected COVID-19 patients, there is evidence for broad SARS-CoV-2 cell tropisms, activation of immune cells, and clearance of immunosuppressive cells, which could contribute to severe tissue damage, thromboemboli, excess inflammation and compromised adaptive immune responses. Funding This work used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource, which is supported in part by award P30CA047904 from the National Cancer Institute, and by UPMC Hillman Cancer Center Startup Fund and Pittsburgh Foundation Endowed Chair in Drug Development for Immunotherapy to S.-J. Gao. HIGHLIGHTS We provide an atlas of lung immunopathology of fatal SARS-CoV-2 infections, revealing: Unexpected broad cell tropism and infection of parenchymal, endothelial and immune cells by SARS-CoV-2, which are associated with massive tissue damage and thromboemboli; Clearance of immunosuppressive T-regulatory cells, and suppression of B cells and cytotoxic T cells; Extensive infiltration and activation of immune cells; Pronounced IL-6 expression in all types of infected and uninfected cells. Research in context Evidence before this study Pulmonary symptoms reflected by diffuse alveolar damage (DAD), excess inflammation, pneumocyte hyperplasia and proliferation, formation of platelet aggregates, and thromboemboli are the pathological features of COVID-19. However, the mechanisms mediating these processes have not been elucidated. We searched PubMed up to September 15, 2020 using the keywords “coronavirus disease 2019”, “COVID-19”, “SARS-CoV-2”, “cell tropism”, “cell markers”, “inflammation”, “interleukin 6”, “immune response”, “immune suppression”, “immunofluorescence” and “immunohistochemistry”, with no language restrictions. Single cell RNA sequencing (scRNA-seq) has revealed extensive expression of SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) in a large variety of cell types. However, only low levels of SARS-CoV-2 infection have been detected in macrophages, neutrophils, type II pneumocytes (AT2), and goblet, club, ciliated and endothelial cells by scRNA-seq and immunohistochemistry. COVID-19 blood samples contain high levels of inflammatory cytokines including interleukin-6 (IL-6), high levels of monocytes and neutrophils, and depletion of lymphocytes. There is no information on the cell types infected by SARS-CoV-2 and extent of infection, the precise producing cells of inflammatory cytokines, and the status of immune cells in lungs from fatal COVID-19 patients. Added value of this study By multicolor staining for viral proteins and lineage markers in lung tissues from five fatal COVID-19 patients, we reveal SARS-CoV-2 infection in an extensive range of cells including type II pneumocytes (HT2-280), and ciliated (tyr-α-tubulin), goblet (MUC5AC), club-like (MUC5B) and endothelial cells (CD31 and CD34), which is correlated with the extent of DAD and thromboemboli. SARS-CoV-2 infection is found in greater than 90% of infiltrating immune cells, including macrophages and monocytes (CD68 and CD163), neutrophils (ELA-2), natural killer cells (CD56), B-cells (CD19 and CD20), and T-cells (CD3e). Most but not all infected cells were positive for ACE2. There are abundant macrophages, monocytes, neutrophils and natural killer cells but low numbers of B-cells and abundant CD3e+ T-cells consisting of mainly T helper cells (CD4), few cytotoxic T cells (CTL, CD8), and no T regulatory cell (FOXP3). Antigen presenting molecule HLA-DR of B and T cells was abundant in all cases. Robust IL-6 expression was present in most uninfected and infected cells, with higher expression levels observed in cases with more tissue damage. Implications of all the available evidence In lung tissues from severely affected COVID-19 patients, there is evidence for broad SARS-CoV-2 cell tropisms, hyperactive immune cells, and clearance of immune cells including immunosuppressive cells, which could contribute to severe tissue damage, thromboemboli, excess inflammation and compromised adaptive immune responses. These results have implications for development of treatments.