Neutrophil Extracellular Traps Induced by COVID-19 Plasma Persist Longitudinally: A Link to Endothelial Injury

Author(s):
Edward Kelly; Mary Oliver; Bonnie Carney; Lauren Moffatt; Jeffrey Shupp

Background:

Neutrophil extracellular trap (NET) formation is a protective mechanism that neutrophils possess to respond to host infection or inflammation. However, dysregulation of NETosis has been implicated in many disease processes including cancer and autoimmune disease. Most recently, studies have focused on the role of NETosis in the pathogenesis of COVID-19 infection. While the exact mechanisms of their involvement remain largely unknown, this study aimed to elucidate the progression of NET formation over the time course of COVID-19 infection and their possible role in endothelial injury.

Hypothesis:

NET formation induced by Covid-19 will persist over time and COVID-19 plasma will show increased Syndecan-1 levels when compared to controls.

Methods:

Plasma samples were taken from COVID-19 patients at admission, day 3 and day 14 of hospitalization. Neutrophils were extracted from healthy donors and isolated using Ficoll separation. Neutrophils were treated with COVID-19 plasma for 3hrs. Myeloperoxidase (MPO) colorimetric activity assay kit was used as a marker to assess for NET formation. Spectrophotometry was used to obtain optical density measurements in order to quantify MPO. Syndecan-1 (SDC-1) ELISA was run using the same samples, as well as all patients with admission and day 1 plasma.

Results:

Plasma samples from 16 different COVID-19 positive patients were used in this study. 58 yrs ± 13.8 was the average age of the patient population. 30-day mortality was 18.75 percent. There were no significant differences between individual patients and levels persisted throughout the time course. All patient plasma induced upregulation of MPO at all timepoints when compared to cell only controls. All patient samples had significantly higher MPO levels when compared to cells treated with healthy plasma. At admission, 60% of patients had SDC-1 levels significantly higher than controls (p<0.05, n=10) while day 1 samples showed 88% of patients with SDC-1 levels significantly higher than controls (p<0.05, N=8).

Conclusions:

While neutrophils are thought of as part of the protective machinery of the innate immune system, their dysregulation can be detrimental to the host. Our study shows that COVID-19 plasma induces significant amounts of NET formation that persists over the time course of the disease. Furthermore, patients exhibit increased SDC-1 levels implicating endothelial injury in the pathogenesis of COVID-19 infection. Further characterization of this relationship could provide targets for future treatments.