What is the actual relationship between neutrophil extracellular traps and COVID-19 severity? A longitudinal study.

BACKGROUND: Neutrophil extracellular traps (NETs) have repeatedly been related to COVID-19 severity and mortality. However, there is no consensus on their quantification, and there are scarce data on their evolution during the disease. We studied circulating NET markers in patients with COVID-19 throughout their hospitalization. METHODS: We prospectively included 93 patients (201 blood samples), evaluating the disease severity in 3 evolutionary phases (viral, early, and late inflammation). Of these, 72 had 180 samples in various phases. We also evaluated 55 controls with similar age, sex and comorbidities. We measured 4 NET markers in serum: cfDNA, CitH3, and MPO-DNA and NE-DNA complexes; as well as neutrophil-related cytokines IL-8 and G-CSF. RESULTS: The COVID-19 group had higher CitH3 (28.29 vs 20.29 pg/mL, p = 0.022), and cfDNA, MPO-DNA, and NE-DNA (7.87 vs 2.56 ng/mL; 0.80 vs 0.52 and 1.04 vs 0.72, respectively, p < 0.001 for all) than the controls throughout hospitalisation. cfDNA was the only NET marker clearly related to severity, and it remained higher in non-survivors during the 3 phases. Only cfDNA was an independent risk factor for mortality and need for intensive care. Neutrophil count, IL-8, and G-CSF were significantly related to severity. MPO-DNA and NE-DNA showed significant correlations (r: 0.483, p < 0.001), including all 3 phases and across all severity grades, and they only remained significantly higher on days 10-16 of evolution in those who died. Correlations among the other NET markers were lower than expected. CONCLUSIONS: The circulating biomarkers of NETs were present in patients with COVID-19 throughout hospitalization. cfDNA was associated with severity and mortality, but the three other markers showed little or no association with these outcomes. Neutrophil activity and neutrophil count were also associated with severity. MPO-DNA and NE-DNA better reflected NET formation. cfDNA appeared to be more associated with overall tissue damage; previous widespread use of this marker could have overestimated the relationship between NETs and severity. Currently, there are limitations to accurate NET markers measurement that make it difficult to assess its true role in COVID-19 pathogenesis.

IL-6 and cfDNA monitoring throughout COVID-19 hospitalization are accurate markers of its outcomes.

BACKGROUND: Severe COVID-19 entails a dysregulated immune response, most likely inflammation related to a lack of virus control. A better understanding of immune toxicity, immunosuppression balance, and COVID-19 assessments could help determine whether different clinical presentations are driven by specific types of immune responses. The progression of the immune response and tissular damage could predict outcomes and may help in the management of patients. METHODS: We collected 201 serum samples from 93 hospitalised patients classified as moderately, severely, and critically ill. We differentiated the viral, early inflammatory, and late inflammatory phases and included 72 patients with 180 samples in separate stages for longitudinal study and 55 controls. We studied selected cytokines, P-selectin, and the tissue damage markers lactate dehydrogenase (LDH) and cell-free DNA (cfDNA). RESULTS: TNF-α, IL-6, IL-8, and G-CSF were associated with severity and mortality, but only IL-6 increased since admission in the critical patients and non-survivors, correlating with damage markers. The lack of a significant decrease in IL-6 levels in the critical patients and non-survivors in the early inflammatory phase (a decreased presence in the other patients) suggests that these patients did not achieve viral control on days 10-16. For all patients, lactate dehydrogenase and cfDNA levels increased with severity, and cfDNA levels increased in the non-survivors from the first sample (p = 0.002) to the late inflammatory phase (p = 0.031). In the multivariate study, cfDNA was an independent risk factor for mortality and ICU admission. CONCLUSIONS: The distinct progression of IL-6 levels in the course of the disease, especially on days 10-16, was a good marker of progression to critical status and mortality and could guide the start of IL-6 blockade. cfDNA was an accurate marker of severity and mortality from admission and throughout COVID-19 progression.