NETosis and Nucleosome Biomarkers in Septic Shock and Critical COVID-19 Patients: An Observational Study

Laure Morimont, Mélanie Dechamps, Clara David, Céline Bouvy, Constant Gillot, Hélène Haguet, Julien Favresse, Lorian Ronvaux, Julie Candiracci, Marielle Herzog, Pierre-François Laterre, Julien De Poortere, Sandrine Horman, Christophe Beauloye, Jonathan Douxfils, Laure Morimont, Mélanie Dechamps, Clara David, Céline Bouvy, Constant Gillot, Hélène Haguet, Julien Favresse, Lorian Ronvaux, Julie Candiracci, Marielle Herzog, Pierre-François Laterre, Julien De Poortere, Sandrine Horman, Christophe Beauloye, Jonathan Douxfils

Abstract

Background: Neutrophil extracellular traps’ (NETs’) formation is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of microorganisms. Aim: The aim was to evaluate biomarkers of NETs’ formation in a patient cohort admitted to intensive care unit (ICU) due to infection. Methods: Forty-six septic shock patients, 22 critical COVID-19 patients and 48 matched control subjects were recruited. Intact nucleosomes containing histone 3.1 (Nu.H3.1), or citrullinated histone H3R8 (Nu.Cit-H3R8), free citrullinated histone (Cit-H3), neutrophil elastase (NE) and myeloperoxidase (MPO) were measured. Results: Significant differences in Nu.H3.1 and NE levels were observed between septic shock and critical COVID-19 subjects as well as with controls (p-values < 0.05). The normalization of nucleosome levels according to the neutrophil count improved the discrimination between septic shock and critical COVID-19 patients. The ratio of Nu.Cit-H3R8 to Nu.H3.1 allowed the determination of nucleosome citrullination degree, presumably by PAD4. Conclusions: H3.1 and Cit-H3R8 nucleosomes appear to be interesting markers of global cell death and neutrophil activation when combined. Nu.H3.1 permits the evaluation of disease severity and differs between septic shock and critical COVID-19 patients, reflecting two distinct potential pathological processes in these conditions.

Keywords: COVID-19; NETs’ formation; SARS-CoV-2; nucleosomes; septic shock.

Conflict of interest statement

Among the authors, M.H., L.R. and J.C. are employees of Belgian Volition SRL and L.M., C.D., C.B. and J.D. are employees of QUALIblood s.a. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Levels of circulating nucleosomes and neutrophil activation biomarkers in control, septic shock and critical COVID-19 populations. Nu.H3.1, Nu.Cit-H3R8, Cit-H3, NE and MPO were compared. Results are expressed as absolute value or normalized by neutrophils level for each individual. All markers were statistically different in septic shock and critical COVID-19 compared to controls. Only Nu.H3.1 and NE were different between septic shock and critical COVID-19 patients. Boxes represent 25th–75th percentile with median. Whiskers represent min to max variation. Squares represent patients with a thromboembolic event, and non-transparent symbols represent dead patients. *, *** and **** represent p-values ≤ 0.05, ≤0.001 and ≤0.0001, respectively. Only differences that are statistically significant are reported. Abbreviations: Cit-H3, citrullinated histone H3 (citrullinated in R2, R8 and R17); MPO, myeloperoxidase; NE, neutrophil elastase; Nu.Cit-H3R8, citrullinated H3R8-nucleosome; Nu.H3.1, H3.1-nucleosome.
Figure 2
Figure 2
Ratio of circulating nucleosomes and neutrophil activation parameters in control, septic shock and critical COVID-19 subjects. The following ratios were proposed: (i) ratio of Cit-H3 on Nu.H3.1, (ii) ratio of Cit-H3 (citrullinated in R2, R8 and R17) on Nu.Cit-H3R8 and (iii) ratio of Nu.Cit-H3R8 on Nu.H3.1. Boxes represent 25th–75th percentile with median. Whiskers represent min to max variation. Squares represent patients with a thromboembolic event, and non-transparent symbols represent dead patients. *, *** and **** represent p-values ≤ 0.05, ≤0.001 and ≤0.0001, respectively. Only differences that are statistically significant are reported. Abbreviations: Cit-H3, citrullinated histone H3 (citrullinated in R2, R8 and R17); Nu.Cit-H3R8, citrullinated H3R8-nucleosome; Nu.H3.1, H3.1-nucleosome.
Figure 3
Figure 3
Circulating nucleosomes and neutrophil activation parameters in septic shock and critical COVID-19 subjects according to the APACHE-II score. Boxes represent 25th–75th percentile with median. Whiskers represent min to max variation. Squares represent patients with a thromboembolic event, and non-transparent symbols represent dead patients. *, ** represent p-values ≤ 0.05 and ≤0.01, respectively. Only differences that are statistically significant are reported. Abbreviations: Cit-H3, citrullinated histone H3 (citrullinated in R2, R8 and R17); MPO, myeloperoxidase; NE, neutrophil elastase; Nu.Cit-H3R8, citrullinated H3R8-nucleosome; Nu.H3.1, H3.1-nucleosome.
Figure 4
Figure 4
Circulating nucleosomes and neutrophil activation parameters in sepsis and COVID-19 subjects according to the SOFA score. Boxes represent 25th–75th percentile with median. Whiskers represent min to max variation. Squares represent patients with a thromboembolic event, and non-transparent symbols represent dead patients. *, ** and *** represent p-values ≤ 0.05, ≤0.01 and ≤0.001, respectively. Only differences that are statistically significant are reported. Abbreviations: Cit-H3, citrullinated histone H3 (citrullinated in R2, R8 and R17); MPO, myeloperoxidase; NE, neutrophil elastase; Nu.Cit-H3R8, citrullinated H3R8-nucleosome; Nu.H3.1, H3.1-nucleosomes.
Figure 5
Figure 5
Comparison of nucleosome markers and derived ratio calculation according to APACHE-II and SOFA scores in septic shock and critical COVID-19 subjects. Boxes represent 25th–75th percentile with median. Whiskers represent min to max variation. Squares represent patients with a thromboembolic event, and non-transparent symbols represent dead patients. *, **, *** and **** represent p-values ≤ 0.05, ≤0.01, ≤0.001 and ≤0.0001, respectively. Only differences that are statistically significant are reported. Abbreviations: Cit-H3, citrullinated histone H3 (citrullinated in R2, R8 and R17); MPO, myeloperoxidase; NE, neutrophil elastase; Nu.Cit-H3R8, citrullinated H3R8-nucleosome; Nu.H3.1, H3.1-nucleosome.

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