Cellular and molecular heterogeneities and signatures, and pathological trajectories of fatal COVID-19 lungs defined by spatial single-cell transcriptome analysis (preprint)

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. Here, we examined postmortem COVID-19 lung tissues by spatial single-cell transcriptome analysis (SSCTA). We identified 18 major parenchymal and immune cell types, all of which are infected by SARS-CoV-2. Compared to the non-COVID-19 control, COVID-19 lungs have reduced alveolar cells (ACs), and increased innate and adaptive immune cells. Additionally, 19 differentially expressed genes in both infected and uninfected cells across the tissues mirror the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that are correlated with high cell densities (HIHD). The HIHD regions express high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2, and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration that have increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, echoing the tissue damage and wound healing processes. Sparse non-negative matrix factorization (SNMF) analysis of neighborhood cell type composition (NCTC) features identified 7 signatures that capture structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A progresses with primarily increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B is marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions are marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations primarily consisting of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-{beta}-SMAD2/3 pathways in these cells, which likely mediate the upregulation of COL1A1 and COL1A2, and excessive fibrosis in the lung tissues. Together, this study provides an SSCTA atlas of cellular and molecular signatures of fatal COVID-19 lungs, revealing the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells (preprint)

The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signaling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), which contains gene cis-regulatory information. We apply SPaRTAN to CITE-seq datasets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq datasets with a toolset for user-friendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each dataset, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.

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

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--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 (CD3{varepsilon}). 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 CD3{varepsilon}+ 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.

Sensing of COVID-19 Antibodies in Seconds via Aerosol Jet Printed Three Dimensional Electrodes (preprint)

Rapid diagnosis is critical for the treatment and prevention of diseases. In this research, we report sensing of antibodies specific to SARS-CoV-2 virus in seconds via an electrochemical platform consisting of gold micropillar array electrodes decorated with reduced graphene oxide and functionalized with recombinant viral antigens. The array electrodes are fabricated by Aerosol Jet (AJ) nanoparticle 3D printing, where gold nanoparticles (3-5nm) are assembled in 3D space, sintered, and integrated with a microfluidic device. The device is shown to detect antibodies to SARS-CoV-2 spike S1 protein and its receptor-binding-domain (RBD) at concentrations down to 1pM via electrochemical impedance spectroscopy and read by a smartphone-based user interface. In addition, the sensor can be regenerated within a minute by introducing a low-pH chemistry that elutes the antibodies from the antigens, allowing successive testing of multiple antibody samples using the same sensor. The detection time for the two antibodies tested in this work is 11.5 seconds. S1 protein sensing of its antibodies is specific, which cross-reacts neither with other antibodies nor with proteins such as Nucleocapsid antibody and Interleukin-6 protein. The proposed sensing platform is generic and can also be used for the rapid detection of biomarkers for other infectious agents such as Ebola, HIV, and Zika, which will benefit the public health.