Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Formation and Clearance of NETs in Health and Disease.

Neutrophils are the most abundant innate immune cells in humans and the first line of defense against invading pathogens [...].

A Pleomorphic Puzzle: Heterogeneous Pulmonary Vascular Occlusions in Patients with COVID-19.

Vascular occlusions in patients with coronavirus diseases 2019 (COVID-19) have been frequently reported in severe outcomes mainly due to a dysregulation of neutrophils mediating neutrophil extracellular trap (NET) formation. Lung specimens from patients with COVID-19 have previously shown a dynamic morphology, categorized into three types of pleomorphic occurrence based on histological findings in this study. These vascular occlusions in lung specimens were also detected using native endogenous fluorescence or NEF in a label-free method. The three types of vascular occlusions exhibit morphology of DNA rich neutrophil elastase (NE) poor (type I), NE rich DNA poor (type II), and DNA and NE rich (type III) cohort of eleven patients with six males and five females. Age and gender have been presented in this study as influencing variables linking the occurrence of several occlusions with pleomorphic contents within a patient specimen and amongst them. This study reports the categorization of pleomorphic occlusions in patients with COVID-19 and the detection of these occlusions in a label-free method utilizing NEF.

NET Formation in Systemic Lupus Erythematosus: Changes during the COVID-19 Pandemic.

The severity of the coronavirus disease in 2019 (COVID-19) is strongly linked to a dysregulated immune response. This fuels the fear of severe disease in patients with autoimmune disorders continuously using immunosuppressive/immunomodulating medications. One complication of COVID-19 is thromboembolism caused by intravascular aggregates of neutrophil extracellular traps (NETs) occluding the affected vessels. Like COVID-19, systemic lupus erythematosus (SLE) is characterized by, amongst others, an increased risk of thromboembolism. An imbalance between NET formation and clearance is suggested to play a prominent role in exacerbating autoimmunity and disease severity. Serologic evidence of exposure to SARS-CoV-2 has a minor impact on the SLE course in a Swedish cohort reportedly. Herein, we assessed NET formation in patients from this cohort by neutrophil elastase (NE) activity and the presence of cell-free DNA, MPO-DNA, and NE-DNA complexes and correlated the findings to the clinical parameters. The presence of NE-DNA complexes and NE activity differed significantly in pre-pandemic versus pandemic serum samples. The latter correlated significantly with the hemoglobin concentration, blood cell counts, and complement protein 3 and 4 levels in the pre-pandemic but only with the leukocyte count and neutrophil levels in the pandemic serum samples. Taken together, our data suggest a change, especially in the NE activity independent of exposure to SARS-CoV-2.

Periodontitis-Derived Dark-NETs in Severe Covid-19.

The frequent severe COVID-19 course in patients with periodontitis suggests a link of the aetiopathogenesis of both diseases. The formation of intravascular neutrophil extracellular traps (NETs) is crucial to the pathogenesis of severe COVID-19. Periodontitis is characterised by an increased level of circulating NETs, a propensity for increased NET formation, delayed NET clearance and low-grade endotoxemia (LGE). The latter has an enormous impact on innate immunity and susceptibility to infection with SARS-CoV-2. LPS binds the SARS-CoV-2 spike protein and this complex, which is more active than unbound LPS, precipitates massive NET formation. Thus, circulating NET formation is the common denominator in both COVID-19 and periodontitis and other diseases with low-grade endotoxemia like diabetes, obesity and cardiovascular diseases (CVD) also increase the risk to develop severe COVID-19. Here we discuss the role of propensity for increased NET formation, DNase I deficiency and low-grade endotoxaemia in periodontitis as aggravating factors for the severe course of COVID-19 and possible strategies for the diminution of increased levels of circulating periodontitis-derived NETs in COVID-19 with periodontitis comorbidity.

Neutrophil Extracellular Trap-Driven Occlusive Diseases.

The enlightenment of the formation of neutrophil extracellular traps (NETs) as a part of the innate immune system shed new insights into the pathologies of various diseases. The initial idea that NETs are a pivotal defense structure was gradually amended due to several deleterious effects in consecutive investigations. NETs formation is now considered a double-edged sword. The harmful effects are not limited to the induction of inflammation by NETs remnants but also include occlusions caused by aggregated NETs (aggNETs). The latter carries the risk of occluding tubular structures like vessels or ducts and appear to be associated with the pathologies of various diseases. In addition to life-threatening vascular clogging, other occlusions include painful stone formation in the biliary system, the kidneys, the prostate, and the appendix. AggNETs are also prone to occlude the ductal system of exocrine glands, as seen in ocular glands, salivary glands, and others. Last, but not least, they also clog the pancreatic ducts in a murine model of neutrophilia. In this regard, elucidating the mechanism of NETs-dependent occlusions is of crucial importance for the development of new therapeutic approaches. Therefore, the purpose of this review is to address the putative mechanisms of NETs-associated occlusions in the pathogenesis of disease, as well as prospective treatment modalities.

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to an adaptive immune response in the host and the formation of anti-SARS-CoV-2 specific antibodies. While IgG responses against SARS-CoV-2 have been characterized quite well, less is known about IgA. IgA2 activates immune cells and induces inflammation and neutrophil extracellular trap (NET) formation which may contribute to organ injury and fatal outcome in SARS-CoV-2-infected patients. SARS-CoV-2 spike protein specific antibody levels were measured in plasma samples of 15 noninfected controls and 82 SARS-CoV-2-infected patients with no or mild symptoms, moderate symptoms (hospitalization) or severe disease (intensive care unit, ICU). Antibody levels were compared to levels of C-reactive protein (CRP) and circulating extracellular DNA (ecDNA) as markers for general inflammation and NET formation, respectively. While levels of SARS-CoV-2-specific IgG were similar in all patient groups, IgA2 antibodies were restricted to severe disease and showed the strongest discrimination between nonfatal and fatal outcome in patients with severe SARS-CoV-2 infection. While anti-SARS-CoV-2 IgG and IgA2 levels correlated with CRP levels in severely diseased patients, only anti-SARS-CoV-2 IgA2 correlated with ecDNA. These data suggest that the formation of anti-SARS-CoV-2 IgA2 during SARS-CoV-2 infection is a marker for more severe disease related to NET formation and poor outcome.

Vascular occlusion by neutrophil extracellular traps in COVID-19.

BACKGROUND: Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood. METHODS: Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry. PATIENT FINDINGS: Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage. INTERPRETATION: These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage. FUNDING: Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung.