No abstract

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti–β2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer’s threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. Elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have recently been described in patients with COVID-19. However, their potential role in COVID-19-associated thrombosis remains incompletely understood. In order to elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis, we conducted a case–control study of patients hospitalized with COVID-19 who developed thrombosis, as compared with gender- and age-matched COVID-19 patients without clinical thrombosis. We found that remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera were associated with higher risk of morbid thrombotic events in spite of prophylactic anticoagulation. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19, as well as novel approaches for thrombosis prevention. Electronic supplementary material The online version of this article (10.1007/s11239-020-02324-z) contains supplementary material, which is available to authorized users.

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. Although 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.

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.

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.

BackgroundAdult-onset Still’s disease (AOSD) is a systemic inflammatory disease characterized by neutrophilia and NLRP3 inflammasome and macrophage activation. We investigated the role of neutrophil extracellular traps (NETs) in the pathogenesis of AOSD, and explored the effect of NETs on activating NLRP3 inflammasome and proinflammatory macrophages.MethodsThe sera of 73 AOSD patients and 40 healthy controls were used to detect the level of cell-free DNA and NET-DNA complexes. NET formation ex vivo was analyzed using immunofluorescence and flow plates. The activation of NLRP3 inflammasome in THP-1 cells and proinflammatory macrophages stimulated with DNA purified from NETs was measured using RT-PCR, ELISA, Western blotting and flow cytometry.ResultsThe levels of cell-free DNA and NET-DNA complexes were significantly increased in the circulation of patients with AOSD compared with healthy controls, and freshly isolated neutrophils from patients with AOSD were predisposed to high levels of spontaneous NET release. Interestingly, enhanced NET release was abrogated with NADPH oxidase inhibitors and a mitochondrial scavenger. Furthermore, DNA purified from AOSD NETs activated NLRP3 inflammasomes. NET DNA from AOSD also exerted a potent capacity to accelerate the activation of CD68+CD86+ macrophages and increased the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Finally, the copy number of mitochondrial DNA (mtDNA) in NETs and plasma was significantly increased in AOSD patients, suggesting that mtDNA may be involved in the activation of NLRP3 and inflammatory macrophages.ConclusionsThese findings implicate accelerated NET formation in AOSD pathogenesis through activation of NLRP3 and proinflammatory macrophages, and identify a novel link between neutrophils and macrophages by NET formation in AOSD.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1800-z) contains supplementary material, which is available to authorized users.

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.