Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use

Abstract

Background & aims: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking.

Methods: Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion.

Results: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice.

Conclusions: Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis.

Lay summary: Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Keywords: Alcoholic hepatitis; Binge drinking; Neutrophil depletion; Neutrophil elastase; Sepsis.

Figure 1:. Binge drinking induces increases in blood alcohol content and serum endotoxin but acute alcohol reduces ex vivo NET formation in human neutrophils

Following binge drinking by healthy human subjects, blood samples were analyzed for alcohol content (A), serum endotoxin (B) and bacterial 16s rDNA (C). Ex vivo NETosis assay of neutrophils with and without acute alcohol exposure followed by PMA stimulation was assessed for extracellular DNA (D) and quantitative NETosis assay (E) methods, respectively. Samples are representative of 7–15 healthy subjects. A p < 0.05 compared to baseline no treatment control (*) or PMA with no alcohol treatment group (#) was considered statistically significant by analysis of variance. NS, not significant.

Figure 2:. Reduced hepatic neutrophil infiltration and NET formation following acute alcohol use associated with sepsis

C57BL/6 mice received sugar or ethanol gavage daily for three days followed by a single i.p. LPS injection (0.05mg/kg). Mice were sacrificed 12h after LPS injection and liver samples analyzed by immunohistology for neutrophil elastase (A). Liver samples from mice were additionally analyzed by western blotting for HMGB1 (B) and citrullinated histone H3 (C) using β-Actin as a protein loading control. ELISA was performed on total liver protein lysate for MCP1 (D). A p < 0.05 compared to baseline sugar gavage (*) or sugar plus i.p. LPS treatment (#) was considered statistically significant using ANOVA for 5–6 mice per experimental group. NS, not significant.

Figure 3:. Binge drinking associated with sepsis is characterized by the sustained hepatic presence of neutrophils, NETosis molecules, and increased cell death in mice.

C57BL/6 mice received three alcohol or sugar gavages with i.p. LPS or saline treatment. Mice were sacrificed after 15h and liver/serum samples analyzed by immunohistology for neutrophil elastase (A), western blot analysis for citrullinated histone H3 (B), RT-qPCR for IL-6 (C) and ELISA for MCP-1(D). TUNEL assay was performed on liver samples from C57BL/6 binge alcohol or sugar gavage mice with or without i.p. LPS or saline treatment as indicated (E). A p < 0.05 compared to baseline sugar gavage (*) or sugar plus i.p. LPS treatment (#) was considered statistically significant using ANOVA for 3–6 mice per experimental group.

Figure 4:. IL6 does not impact neutrophil viability but alcohol use suppresses effective phagocytosis of NETotic neutrophils and promotes macrophage differentiation to a pro-inflammatory phenotype.

(A) Human blood neutrophils were treated with varying doses of IL6 and maintained in culture over 48h. Neutrophil apoptosis was assessed over time using flow cytometry as indicated. (B-E) Human primary macrophages and RAW 264.7 cells were acutely exposed to alcohol (50 mM, 4 h) and cocultured with CFSE labeled NETotic neutrophils (7:1 ratio of dead [NETotic] neutrophils to human macrophages or RAW 264.7) for 3 h or 48 h. Three hours after coculture, RAW 264.7 were harvested and analyzed by flow cytometry for MΦ phagocytosis (B & C). (D & E) Following 48h post co-culture, acute alcohol-exposed RAW 264.7 cells were analyzed by flow cytometry for MΦ differentiation to an M1 (D) versus M2 (E) phenotype. A p < 0.05 compared to baseline sugar gavage controls (*) or sugar plus LPS stimulation (#) was considered statistically significant using ANOVA for three independent repeat experiments.

Figure 5:. Neutrophil depletion in mice alleviates binge alcohol-induced hepatic inflammation and injury.

Mice were untreated or pretreated with anti-mouse Ly6G or IgG control antibody followed by i.p. LPS injection as indicated. Blood (A) and liver (B) samples were collected after 15 h of LPS injection and analyzed for neutrophil content by flow cytometry. Mice were untreated or pretreated with anti-mouse Ly6G or IgG control antibody followed by three alcohol binges or sugar gavage as indicated. Some mice received additional i.p. LPS (0.05mg/kg) as indicated. Mice were sacrificed 15 h after i.p. LPS injection and serum/liver samples collected. Liver samples were analyzed by western blotting for citrullinated histone H3 and β-actin was used as a loading control (C). Liver samples were analyzed by fluorescent immunobiology for neutrophil elastase and Histone H3 as NET markers with dapi as nuclear stain. Serum samples collected from mice were analyzed for MCP-1 (D) and AST (E). A p < 0.05 compared to baseline sugar gavage IgG controls (*) or sugar IgG plus LPS stimulation (#) was considered statistically significant using ANOVA for 3–6 mice per experimental group.

Figure 6:. Illustrations summarizing our findings on the hepatic effects of alcohol on NET formation and clearance during binge-alcohol drinking associated with sepsis.

(A) Acute alcohol induces spontaneous NET formation by neutrophils. In the same cells subsequent PMA stimulation or antigenic challenge (LPS) results in significantly reduced NET formation compared to stimulations/activation of alcohol naïve neutrophils. (B) Acute sepsis in wild type C57BL/6 mice is associated with significant neutrophil hepatic infiltration and NET formation which occurs 9–12h post i.p LPS. Following NET formation, NETosis occurs followed by clearance of NETotic Neutrophils and NETotic DAMPs which occurs 15–20 post LPS injection in mice(C) Acute alcohol treatment of macrophages or binge alcohol use in mice significantly lowers the efferocytosis capacity of macrophages and clearance of NETotic neutrophils-DAMPs. Schematic illustrations made use of some motifolio templates (www.motifolio.com).