Generation of neutrophil extracellular traps in patients with acute liver failure is associated with poor outcome

Abstract

Background and aims: Acute liver failure (ALF) is characterized by significant changes in the hemostatic system and by systemic inflammation. The formation of neutrophil extracellular traps (NETs), in which an activated neutrophil expels its DNA, histones, and granular enzymes, such as myeloperoxidase (MPO), has been associated with immune-mediated and thrombotic diseases. We hypothesized that formation of NETs in patients with ALF contributes to progression of disease.

Approach and results: A total of 676 patients with ALF (international normalized ratio [INR], ≥1.5) or severe acute liver injury (ALI; INR, ≥2.0) were recruited from the U.S. ALF Study Group Registry between 2011 and 2018, of whom 308 patients (45.6%) had acetaminophen-induced ALF. Up to 21 days after admission, 483 patients (71.5%) survived without liver transplantation (LT). Levels of cell-free DNA (cfDNA) and the specific NET marker MPO-DNA complexes were measured in plasma samples obtained on admission and compared to levels in healthy controls. In addition, liver tissue obtained at transplantation of 20 ALF patients was stained for NETs. Levels of cfDNA were 7.1-fold, and MPO-DNA complexes 2.5-fold, higher in patients with ALF compared to healthy controls. cfDNA levels were not associated with 21-day transplant-free survival, but were higher in those patients with more-severe disease on admission, as reflected by various laboratory and clinical parameters. MPO-DNA levels were 30% higher in patients with ALF who died or required urgent LT. Liver tissue of ALF patients stained positive for NETs in 12 of 18 evaluable patients.

Conclusions: Here, we provide evidence for NET formation in patients with ALF. Elevated plasma levels of MPO-DNA complexes in patients with ALF were associated with poor outcome, which suggests that NET formation contributes to disease progression.

FIGURE 1

Plasma levels of cfDNA were determined in patients with severe ALI or ALF (n = 675) and compared to levels of cfDNA in healthy controls (n = 40). ****p < 0.0001

FIGURE 2

MPO‐DNA complex levels were determined in plasma of patients with severe ALI or ALF (n = 629) and compared to levels of MPO‐DNA complexes in healthy controls (n = 40). ****p < 0.0001, Abbreviation: OD450nm, optical density at 450 nm

FIGURE 3

Liver tissue from ALF patients stained positive for NET marker. Representative images of liver tissue obtained from ALF patients with different degrees of citrullinated histone expression, which is a commonly used marker of NETs. Differences in expression, ranging from no expression (score 0), focal expression (positive in <10% of neutrophils; score 1), moderate expression (10%‐50% of neutrophils; score 2), to extensive expression (>50% of neutrophils; score 3) are depicted