Background & aims: Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown.
Methods: A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils.
Results: HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B-induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis.
Conclusions: The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI's Gene Expression Omnibus (GEO accession number: GSE98153).
Keywords: 4-HNE, 4-hydroxynonenal; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Alcohol; CXCL1, chemokine (C-X-C motif) ligand 1; Csf, colony-stimulating factor gene; FBS, fetal bovine serum; Fatty Liver; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HFD+1B, high-fat diet feeding plus 1 binge of ethanol; HFD+mB, high-fat diet plus multiple binges; HFD, high-fat diet; HSC, hepatic stellate cell; High-Fat Diet; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; Inflammation; KO, knockout; MPO, myeloperoxidase; PCR, polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; Reactive Oxygen Species; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type; cDNA, complementary DNA; mRNA, messenger RNA.