Autoantibodies stabilize neutrophil extracellular traps in COVID-19 (preprint)

The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p<0.0001). Both anti-NET IgG and IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and to a lesser extent anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID sera. Furthermore, purified IgG from COVID sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.

Endothelial cell-activating antibodies in COVID-19 (preprint)

ObjectiveWhile endothelial dysfunction has been implicated in the widespread thrombo-inflammatory complications of coronavirus disease-19 (COVID-19), the upstream mediators of endotheliopathy remain for the most part cryptic. Our aim was to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. MethodsHuman endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID sepsis. Cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) were quantified by in-cell ELISA. ResultsSerum and plasma from patients with COVID-19 increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and tracked with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly restrained upregulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. ConclusionThese data are the first to suggest that some patients with COVID-19 have potentially diverse antibodies that drive endotheliopathy, adding important context regarding thrombo-inflammatory effects of autoantibodies in severe COVID-19.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism (preprint)

Background: Men are disproportionately affected by the coronavirus disease-2019 (COVID-19) and experience higher mortality as compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial damage are not defined.Methods: Here, we determined the effects of SARS-CoV-2 spike protein-mediated endothelial damage under conditions of exposure to androgens and TNF-α. We tested the therapeutic effects of approved drugs: angiotensin receptor blockade by valsartan and mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in the plasma from men and women diagnosed with COVID-19.Findings: Exposure of endothelial cells (ECs) in vitro to androgen dihydrotestosterone (DHT) exacerbated spike protein S1-mediated endothelial injury transcripts for cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 and anti-fibrinolytic PAI-1 (P<0.05), as well as increased THP-1 monocyte adhesion to ECs (P=0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. Pre-treatment of ECs with valsartan did not increase endothelial activation by S1. TNF-α exacerbated S1-induced EC activation and spironolactone abrogated this effect. Analysis of plasma from patients hospitalized with COVID-19 showed concordant higher VCAM-1 in men as compared to women.Interpretation: Androgen exposure promotes spike protein-mediated endothelial injury by increasing markers of inflammation and thrombosis. A beneficial therapeutic effect of the FDA-approved drug spironolactone was observed, supporting a rationale for further evaluation as an adjunct treatment in COVID-19. Funding: This work was supported by the Michigan Biological Research Initiative on Sex Differences in Cardiovascular Disease, COVID-19 Ignitor Award, and University of Michigan Taubman Medical Research Institute.Declaration of Interests: No conflicts of interest, financial or otherwise, are declared by the author(s).Ethics Approval Statement: Study involving measurement of circulating endothelial injury markers in the plasma of COVID-19 patients was approved by the Institutional Review Board (HUM00180521) at the University of Michigan.

Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients (preprint)

BackgroundPatients with coronavirus disease 19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions, while lung histopathology often reveals fibrin-based occlusion of small vessels in patients who succumb to the disease. At the same time, bleeding complications have been observed in some patients. Better understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. ObjectiveTo evaluate fibrinolysis among a large cohort of hospitalized COVID-19 patients. Patients and methods118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot lysis assays. ResultsWe found markedly elevated levels of tPA and PAI-1 among patients hospitalized with COVID-19. Both factors demonstrated a strong correlation with neutrophil counts and markers of neutrophil activation, but not with D-dimer. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were also strongly correlated with mortality and with a significant enhancement in spontaneous ex vivo clot lysis. ConclusionWhile both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis and suggests that further study of tPA as a potential biomarker is warranted.

Prothrombotic antiphospholipid antibodies in COVID-19 (preprint)

Patients with coronavirus disease 19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. At the same time, lung histopathology often reveals fibrin-based occlusion of small vessels in patients who succumb to the disease. Antiphospholipid syndrome (APS) is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies (aPL) targeting phospholipids and phospholipid-binding proteins. Small case series have recently detected aPL in patients with COVID-19. Here, we measured eight types of aPL (anticardiolipin IgG/IgM/IgA, anti-beta-2 glycoprotein I IgG/IgM/IgA, and anti- phosphatidylserine/prothrombin (PS/PT) IgG/IgM) in the sera of 172 patients hospitalized with COVID-19. We detected anticardiolipin IgM antibodies in 23%, anti-PS/PT IgG in 24%, and anti-PS/PT IgM in 18%. Any aPL was present in 52% of patients using the manufacturer's threshold and in 30% using a more stringent cutoff (>40 units). Higher levels of aPL were associated with neutrophil hyperactivity (including the release of neutrophil extracellular traps/NETs), higher platelet count, more severe respiratory disease, and lower glomerular filtration rates. Similar to patients with known and longstanding APS, IgG fractions isolated from patients with COVID-19 promoted NET release from control neutrophils. Furthermore, injection of these COVID-19 IgG fractions into mice accelerated venous thrombosis. Taken together, these studies suggest that a significant percentage of patients with COVID-19 become at least transiently positive for aPL and that these aPL are potentially pathogenic.

Neutrophil calprotectin identifies severe pulmonary disease in COVID-19 (preprint)

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. While it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n=172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n=94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 +/- 7031 ng/ml, n=32) as compared to those who remained free of intubation (3365 +/- 3146, p<0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Neutrophil extracellular traps and thrombosis in COVID-19 (preprint)

Background: Early studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. While elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have been described in patients with COVID-19, their potential role in COVID-19-associated thrombosis remains unknown. Objectives: To elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis. Patients/Methods: This is a retrospective, case-control study of patients hospitalized with COVID-19 who developed thrombosis (n=11), as compared with gender- and age-matched COVID-19 patients without clinical thrombosis (n=33). In addition to capturing clinical data, we measured remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera. Results: The majority of patients (9/11) were receiving at least prophylactic doses of heparinoids at the time thrombosis was diagnosed. As compared with controls, patients with COVID-19-associated thrombosis had significantly higher blood levels of markers of NETs (cell-free DNA, myeloperoxidase-DNA complexes, citrullinated histone H3) and neutrophil activation (calprotectin). The thrombosis group also had higher levels of D-dimer, CRP, ferritin, and platelets, but not troponin or neutrophils. Finally, there were strong associations between markers of hyperactive neutrophils (calprotectin and cell-free DNA) and D-dimer. Conclusion: Elevated levels of neutrophil activation and NET formation in patients hospitalized with COVID-19 are associated with higher risk of morbid thrombotic complications. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19.

Neutrophil extracellular traps (NETs) as markers of disease severity in COVID-19 (preprint)

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have potential to propagate inflammation and microvascular thrombosis, including in the lungs of patients with acute respiratory distress syndrome. While elevated levels of blood neutrophils predict worse outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera from patients with COVID-19 (n=50 patients, n=84 samples) have elevated levels of cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter two are highly specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute phase reactants including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. In summary, these data reveal high levels of NETs in many patients with COVID-19, where they may contribute to cytokine release and respiratory failure. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts, and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.