Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients.

In the early phase of the pandemic, we were among the first to postulate that neutrophil extracellular traps (NETs) play a key role in COVID-19 pathogenesis. This exploratory prospective study based on 279 individuals showed that plasma levels of neutrophil elastase, myeloperoxidase and circulating DNA of nuclear and mitochondrial origins in nonsevere (NS), severe (S) and postacute phase (PAP) COVID-19 patients were statistically different as compared to the levels in healthy individuals, and revealed the high diagnostic power of these NETs markers in respect to the disease severity. The diagnostic power of NE, MPO, and cir-nDNA as determined by the Area Under Receiver Operating Curves (AUROC) was 0.95, 097, and 0.64; 0.99, 1.0, and 0.82; and 0.94, 1.0, and 0.93, in NS, S, and PAP patient subgroups, respectively. In addition, a significant fraction of NS, S as well as of PAP patients exhibited aCL IgM/IgG and anti-B2GP IgM/IgG positivity. We first demonstrate persistence of these NETs markers in PAP patients and consequently of sustained innate immune response imbalance, and a prolonged low-level pro-thrombotic potential activity highlighting the need to monitor these markers in all COVID-19 PAP individuals, to investigate postacute COVID-19 pathogenesis following intensive care, and to better identify which medical resources will ensure complete patient recovery.

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA.

BACKGROUND: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. METHODS: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). RESULTS: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. CONCLUSIONS: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Host/genetic factors associated with COVID-19 call for precision medicine.

If the current rate of infection are to be better managed, and future waves of infection kept at bay, it is absolutely necessary that the conditions and mechanisms of exposure to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) be better understood, as well as the downstream severe or lethal clinical complications. While the identification of notable comorbidities has now helped to define broad risk groups, the idiosyncratic responses of individual patients can generate unexpected clinical deterioration that is difficult to predict from initial clinical features. Thus, physicians caring for patients with COVID-19 face clinical dilemmas on a daily basis. The ability to decipher individual predispositions to SARS-CoV-2 infection or severe illness, in light of variations in host immunological and inflammatory responses, in particular as a result of genetic variations, would be of great benefit in infection management. To this end, this work associates the description of COVID-19 clinical complications, comorbidities, sequelae, and environmental and genetic factors. We also give examples of underlying genomic susceptibility to COVID-19, especially with regard to the newly reported link between the disease and the unbalanced formation of neutrophil extracellular traps. As a consequence, we propose that the host/genetic factors associated with COVID-19 call for precision medicine in its treatment. This is to our knowledge the first article describing elements towards precision medicine for patients with COVID-19.

Neutrophil Extracellular Traps and By-Products Play a Key Role in COVID-19: Pathogenesis, Risk Factors, and Therapy.

Understanding of the pathogenesis of the coronavirus disease-2019 (COVID-19) remains incomplete, particularly in respect to the multi-organ dysfunction it may cause. We were the first to report the analogous biological and physiological features of COVID-19 pathogenesis and the harmful amplification loop between inflammation and tissue damage induced by the dysregulation of neutrophil extracellular traps (NETs) formation. Given the rapid evolution of this disease, the nature of its symptoms, and its potential lethality, we hypothesize that COVID-19 progresses under just such an amplifier loop, leading to a massive, uncontrolled inflammation process. Here, we describe in-depth the correlations of COVID-19 symptoms and biological features with those where uncontrolled NET formation is implicated in various sterile or infectious diseases. General clinical conditions, as well as numerous pathological and biological features, are analogous with NETs deleterious effects. Among NETs by-products implicated in COVID-19 pathogenesis, one of the most significant appears to be elastase, in accelerating virus entry and inducing hypertension, thrombosis and vasculitis. We postulate that severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) may evade innate immune response, causing uncontrolled NETs formation and multi-organ failure. In addition, we point to indicators that NETS-associated diseases are COVID-19 risk factors. Acknowledging that neutrophils are the principal origin of extracellular and circulating DNA release, we nonetheless, explain why targeting NETs rather than neutrophils themselves may in practice be a better strategy. This paper also offers an in-depth review of NET formation, function and pathogenic dysregulation, as well as of current and prospective future therapies to control NETopathies. As such, it enables us also to suggest new therapeutic strategies to fight COVID-19. In combination with or independent of the latest tested approaches, we propose the evaluation, in the short term, of treatments with DNase-1, with the anti-diabetic Metformin, or with drugs targeting elastase (i.e., Silvelestat). With a longer perspective, we also advocate a significant increase in research on the development of toll-like receptors (TLR) and C-type lectin-like receptors (CLEC) inhibitors, NET-inhibitory peptides, and on anti-IL-26 therapies.

SARS-CoV2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure.

We demonstrate that the general clinical conditions, risk factors and numerous pathological and biological features of COVID-19 are analogous with various disorders caused by the uncontrolled formation of neutrophil extracellular traps and their by-products. Given the rapid evolution of this disease's symptoms and its lethality, we hypothesize that SARS-CoV2 evades innate immune response causing COVID-19 progresses under just such an amplifier loop, leading to a massive, uncontrolled inflammation process. This work allows us to propose new strategies for treating the pandemic.