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.

cfDNA as a surrogate marker for COVID-19 severity in patients with influenza-like symptoms with and without SARS-CoV-2 infections in general practice: a study protocol for a prospective cohort study.

INTRODUCTION: The clinical course of patients with a SARS-CoV-2 (COVID-19) infection varies widely, from symptom-free to severe courses that can lead to death. Laboratory values of SARS-CoV-2 patients such as lymphocyte counts or C-reactive protein (CRP) do not allow a prediction of the actual course of the disease. To identify a possible predictive marker for the differentiation and prognosis of illness with influenza-like symptoms with and without SARS-CoV-2 infections in general practice, we will analyse the concentrations of cell-free DNA (cfDNA) levels, laboratory and clinical parameters, temperature, oxygen saturation, breathing rate and concomitant symptoms in patients with flu-like symptoms with and without a SARS-CoV-2 infection. METHODS AND ANALYSIS: This is a single-centre, two-arm, parallel longitudinal cohort study with a total of 44 patients. 22 patients with flu-like symptoms without a SARS-CoV-2 infection and 22 patients with flu-like symptoms with a SARS-CoV-2 infection will be recruited. The primary objective is to compare cfDNA levels in ambulatory patients in general practice with flu-like symptoms with SARS-CoV-2 infection with those with influenza like symptoms without a SARS-CoV-2 infection during the disease (day 7 and day 14). The secondary objective is to determine whether there is a correlation between cfDNA concentrations on the one hand, and laboratory and clinical parameters on the other hand. cfDNA, differential blood count, high-sensitive CRP and erythrocyte sedimentation rate will be measured in blood samples, concomitant symptoms will be surveyed via a self-assessment questionnaire, and oxygen saturation, breathing rate and examination of the lungs will be reported by treating physicians. ETHICS AND DISSEMINATION: Ethical approval was issued on 1 March 2021 by the Ethics Committee Essen under the number 21-9916-BO. Findings will be published in peer-reviewed open-access journals and presented at national and international conferences. TRIAL REGISTRATION NUMBER: DRKS00024722.

Association of Innate and Acquired Aerobic Capacity With Resilience in Healthy Adults: Protocol for a Randomized Controlled Trial of an 8-Week Web-Based Physical Exercise Intervention.

BACKGROUND: Physical activity alleviates chronic stress. The latest research suggests a relationship between resilience and physical fitness. Beneficial adaptations of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system, endocannabinoid system, and tryptophan pathway, which are induced by an active lifestyle, are considered to be conducive to resilience. However, detailed knowledge on the molecular link between the effects of acute and chronic physical exercise and improved resilience to stress in humans is missing. Moreover, the relationship between innate and acquired aerobic capacity and resilience is poorly understood. OBJECTIVE: The aim of this study is to implement a human exercise intervention trial addressing the following main hypotheses: a high innate aerobic capacity is associated with high resilience to stress, and web-based physical exercise training improves aerobic capacity of physically inactive adults, which is accompanied by improved resilience. In this setting, we will analyze the relationship between resilience parameters and innate and acquired aerobic capacity as well as circulating signaling molecules. METHODS: A total of 70 healthy, physically inactive (<150 minutes/week of physical activity) adults (aged 18-45 years) will be randomly assigned to an intervention or control group. Participants in the intervention group will receive weekly training using progressive endurance and interval running adapted individually to their remotely supervised home training performance via web-based coach support. A standardized incremental treadmill exercise test will be performed before and after the intervention period of 8 weeks to determine the innate and acquired aerobic capacity (peak oxygen uptake). Before and after the intervention, psychological tests and questionnaires that characterize parameters implicated in resilience will be applied. Blood and saliva will be sampled for the analysis of cortisol, lactate, endocannabinoids, catecholamines, kynurenic acid, and further circulating signal transducers. Statistical analysis will provide comprehensive knowledge on the relationship between aerobic capacity and resilience, as well as the capacity of peripheral factors to mediate the promoting effects of exercise on resilience. RESULTS: The study was registered in October 2019, and enrollment began in September 2019. Of the 161 participants who were initially screened via a telephone survey, 43 (26.7%) fulfilled the inclusion criteria and were included in the study. Among the 55% (17/31) of participants in the intervention group and 45% (14/31) of participants in the control group who completed the study, no serious adverse incidents were reported. Of 43 participants, 4 (9%) withdrew during the program (for individual reasons) and 8 (19%) have not yet participated in the program; moreover, further study recruitment was paused for an indeterminate amount of time because of the COVID-19 pandemic. CONCLUSIONS: Our study aims to further define the physiological characteristics of human resilience, and it may offer novel approaches for the prevention and therapy of mental disorders via an exercise prescription. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/29712.