Intrahepatic neutrophil accumulation and extracellular trap formation are associated with posthepatectomy liver failure

Abstract

Background: Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model.

Methods: We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease.

Results: We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration.

Conclusions: Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.

Graphical abstract

FIGURE 1

Neutrophils and NETs in PHLF. (A) Experimental scheme. Liver biopsies were collected before (Pre) and 2 hours after (Reg) PHx from 25 patients without PHLF and 10 patients with PHLF. (B, C) Immunofluorescence staining of DNA (Hoechst33342), neutrophils (CD66b), and NETs (CitH3) of 35 hepatic resection biopsies taken before resection (Pre) and 2 hours after resection (Reg). Pictures are representative of (B) 25 patients without PHLF or (C) 10 patients with PHLF. (D, E) Relative quantification of (D) neutrophils and (E) NETs in patients with PHLF or without PHLF (two-way ANOVA with Tukey correction for multiple comparisons: *p<0.05, **p<0.01). Regeneration-induced effects depicted as difference after-before resection (∆Reg-Pre) (unpaired t test: **p <0.01). Abbreviations: CitH3, citrullinated histone H3; MPO, myeloperoxidase; NET, neutrophil extracellular trap; PHLF, posthepatectomy liver failure; PHx, partial hepatectomy.

FIGURE 2

Extracellular traps derived from mast cells (MCETs), macrophages (METs), and eosinophils (EETs) in liver regeneration. Relative quantification of immunofluorescence staining of DNA (Hoechst33342). (A) Mast cells (AA1), (B) macrophages (CD68), or (C) eosinophils (EMBP) and their respective extracellular traps via CitH3 of 25 hepatic resection biopsies taken before (Pre) or after resection (Reg). Representative pictures given. (D) Comparison of NETs, MCETs, METs, and EETs Pre vs. Reg in percentage of all extracellular traps (unpaired t test). Abbreviations: CitH3, citrullinated histone H3; EET, eosinophil extracellular trap; MCET, mast cell extracellular trap; MET, macrophage extracellular trap; NET, neutrophil extracellular trap.

FIGURE 3

Plasma levels of CitH3, MPO, and NE are regulated in liver regeneration. (A) Experimental scheme. Plasma was collected 1 day before (POD−1), 1 (POD1) and 5 days after (POD5) PHx from 75 patients without PHLF and 24 patients with PHLF. (B) Upper panel: CitH3, MPO, and NE levels on POD−1, POD1 and POD5 (one-way ANOVA: *p<0.05, ***p<0.001). Lower panel: CitH3, MPO, and NE levels in patients with vs. without PHLF (two-way ANOVA: *p<0.05). (C) ROC curve comparing the predictive potential of MPO POD1 with the prevalence of PHLF (left panel); incidence [%] of PHLF (middle panel) and 90-day mortality (right panel) in high-risk patient groups (defined by a cutoff of MPO POD1 levels of above 160 ng/mL). (D–F) Correlation of plasma CitH3, MPO, and NE POD1 with AST, ALT, and bilirubin POD1 plasma levels. Abbreviations: ALT, alanine-aminotransferase; AST, aspartate aminotransferase; CitH3, citrullinated histone H3; MPO, myeloperoxidase; NE, neutrophil elastase; PHLF, post hepatectomy liver failure; PHx, partial hepatectomy; ROC, receiver operating characteristic.

FIGURE 4

Reduction of NETs through DNase I in an FFD PHx mouse model. (A) Experimental scheme. Fifteen-week-old WT mice on FFD were treated with PBS or DNase I before being subjected to 70% PHx. (B) Remnant liver recovery rate. (C) AST and ALT measurements after PHx. (D) Immunofluorescence staining and relative quantifications of CitH3 and MPO in mouse hepatic sections before and after PHx. (E) Gene expression levels of KI67, PCNA, and Cyclin D1 in harvested mouse liver tissues before and 48 hours after PHx. (F, G) Immunofluorescence staining and relative quantifications of (F) p21 and (G) KI67 in mouse hepatic sections before and after PHx. (one-way ANOVA: *p <0.05, **p <0.01; n=6–7). Abbreviations: ALT, alanine-aminotransferase; AST, aspartate aminotransferase; CitH3, citrullinated histone H3; FFD, fast-food diet; MPO, myeloperoxidase; NET, neutrophil extracellular trap; PHx, partial hepatectomy; WT, wild type.