Angiopoietin-Like4 Is a Novel Marker of COVID-19 Severity.

Vascular dysfunction and capillary leak are common in critically ill COVID-19 patients, but identification of endothelial pathways involved in COVID-19 pathogenesis has been limited. Angiopoietin-like 4 (ANGPTL4) is a protein secreted in response to hypoxic and nutrient-poor conditions that has a variety of biological effects including vascular injury and capillary leak. OBJECTIVES: To assess the role of ANGPTL4 in COVID-19-related outcomes. DESIGN SETTING AND PARTICIPANTS: Two hundred twenty-five COVID-19 ICU patients were enrolled from April 2020 to May 2021 in a prospective, multicenter cohort study from three different medical centers, University of Washington, University of Southern California and New York University. MAIN OUTCOMES AND MEASURES: Plasma ANGPTL4 was measured on days 1, 7, and 14 after ICU admission. We used previously published tissue proteomic data and lung single nucleus RNA (snRNA) sequencing data from specimens collected from COVID-19 patients to determine the tissues and cells that produce ANGPTL4. RESULTS: Higher plasma ANGPTL4 concentrations were significantly associated with worse hospital mortality (adjusted odds ratio per log2 increase, 1.53; 95% CI, 1.17-2.00; p = 0.002). Higher ANGPTL4 concentrations were also associated with higher proportions of venous thromboembolism and acute respiratory distress syndrome. Longitudinal ANGPTL4 concentrations were significantly different during the first 2 weeks of hospitalization in patients who subsequently died compared with survivors (p for interaction = 8.1 × 10-5). Proteomics analysis demonstrated abundance of ANGPTL4 in lung tissue compared with other organs in COVID-19. ANGPTL4 single-nuclear RNA gene expression was significantly increased in pulmonary alveolar type 2 epithelial cells and fibroblasts in COVID-19 lung tissue compared with controls. CONCLUSIONS AND RELEVANCE: ANGPTL4 is expressed in pulmonary epithelial cells and fibroblasts and is associated with clinical prognosis in critically ill COVID-19 patients.

Evidence of premature lymphocyte aging in people with low anti-spike antibody levels after BNT162b2 vaccination.

SARS-CoV-2 vaccines have unquestionably blunted the overall impact of the COVID-19 pandemic, but host factors such as age, sex, obesity, and other co-morbidities can affect vaccine efficacy. We identified individuals in a relatively healthy population of healthcare workers (CORALE study cohort) who had unexpectedly low peak anti-spike receptor binding domain (S-RBD) antibody levels after receiving the BNT162b2 vaccine. Compared to matched controls, "low responders" had fewer spike-specific antibody-producing B cells after the second and third/booster doses. Moreover, their spike-specific T cell receptor (TCR) repertoire had less depth and their CD4+ and CD8+T cell responses to spike peptide stimulation were less robust. Single cell transcriptomic evaluation of peripheral blood mononuclear cells revealed activation of aging pathways in low responder B and CD4+T cells that could underlie their attenuated anti-S-RBD antibody production. Premature lymphocyte aging may therefore contribute to a less effective humoral response and could reduce vaccination efficacy.

Admission respiratory status predicts mortality in COVID-19.

COVID-19 has significant case fatality. Glucocorticoids are the only treatment shown to improve survival, but only among patients requiring supplemental oxygen. WHO advises patients to seek medical care for "trouble breathing," but hypoxemic patients frequently have no respiratory symptoms. Our cohort study of hospitalized COVID-19 patients shows that respiratory symptoms are uncommon and not associated with mortality. By contrast, objective signs of respiratory compromise-oxygen saturation and respiratory rate-are associated with markedly elevated mortality. Our findings support expanding guidelines to include at-home assessment of oxygen saturation and respiratory rate in order to expedite life-saving treatments patients to high-risk COVID-19 patients.

Sample processing and single cell RNA-sequencing of peripheral blood immune cells from COVID-19 patients.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) allows in-depth assessment of transcriptional changes in immune cells of patients with COVID-19. However, collecting, processing, and analyzing samples from patients with COVID-19 pose many challenges because blood samples may contain infectious virus, identification of immune cell subtypes can be difficult, and biological interpretation of analytical results is complex. To address these issues, we describe a protocol for sample processing, sorting, methanol fixation, and scRNA-seq analysis of PBMCs from frozen buffy coat samples from patients with COVID-19. For complete details on the use and execution of this protocol, please refer to (Yao et al., 2021).

Cell-Type-Specific Immune Dysregulation in Severely Ill COVID-19 Patients.

Recent studies have demonstrated immunologic dysfunction in severely ill coronavirus disease 2019 (COVID-19) patients. We use single-cell RNA sequencing (scRNA-seq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMCs) from healthy (n = 3) and COVID-19 patients with moderate disease (n = 5), acute respiratory distress syndrome (ARDS, n = 6), or recovering from ARDS (n = 6). Our data reveal transcriptomic profiles indicative of defective antigen presentation and interferon (IFN) responsiveness in monocytes from ARDS patients, which contrasts with higher responsiveness to IFN signaling in lymphocytes. Furthermore, genes involved in cytotoxic activity are suppressed in both natural killer (NK) and CD8 T lymphocytes, and B cell activation is deficient, which is consistent with delayed viral clearance in severely ill COVID-19 patients. Our study demonstrates that COVID-19 patients with ARDS have a state of immune imbalance in which dysregulation of both innate and adaptive immune responses may be contributing to a more severe disease course.