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. 2018 Jan 8;5(3):399-413.
doi: 10.1016/j.jcmgh.2018.01.003. eCollection 2018 Mar.

Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis

Zhou Zhou  1 , Ming-Jiang Xu  1 , Yan Cai  1 , Wei Wang  1 , Joy X Jiang  2 , Zoltan V Varga  3 , Dechun Feng  1 , Pal Pacher  3 , George Kunos  4 , Natalie J Torok  2 , Bin Gao  1
Affiliations

Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis

Zhou Zhou et al. Cell Mol Gastroenterol Hepatol. .

Abstract

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.

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Figures

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Graphical abstract
Figure 1
Figure 1
Acute ethanol binge and 3-month HFD feeding synergistically activate hepatic fibrosis and neutrophilic pathways. Microarray analysis was performed in the liver tissues from mice subjected to chow diet plus maltose gavage (chow), acute ethanol binge (5 g/kg, 1B), high-fat diet plus maltose gavage (HFD), or HFD-plus-ethanol binge (HFD+1B). (A) Heat map and (B) interactive Venn diagram analyses for genes showing ≥2 fold up-regulation or down-regulation compared with control in microarray data were shown. (C) Top 5 most highly increased pathways in HFD+1B vs chow control mice were listed. N = 4–5 in each group. LXR, liver X receptor; RXR, retinoid X receptor.
Figure 2
Figure 2
HFD-plus-one binge ethanol challenge induces liver fibrosis. Mice were subject to chow diet plus maltose gavage (chow), acute ethanol binge (5 g/kg, 1B), high-fat diet plus maltose gavage (HFD), or HFD-plus-ethanol binge (HFD+1B). Mice were euthanized 9 hours after gavage, and then liver samples were collected and analyzed. (A) Representative images of H&E staining, Sirius Red and Masson Trichrome staining of liver tissues. (B) Sirius Red–positive area was quantified, and body weight was measured. (C) Liver tissues were collected for quantitative RT-PCR analyses of fibrogenic genes. N = 6–7 in each group. *P < .05, **P < .01, ***P < .001.
Figure 3
Figure 3
HFD-plus-multiple binges of ethanol challenge induces liver fibrosis. (A–C) Male C57BL/6J mice were fed an HFD for 3 months, followed by 8 gavages of 5 g/kg ethanol (twice a week) (HFD+mB group), or isocaloric dextrin-maltose (HFD group), while continuing on HFD for an additional month. (A) Representative images are shown. (B and C) Serum ALT and AST levels, liver tissue collagen content, percentage of Sirius Red–positive area, as well as body weight were examined and are shown. (D) Liver tissues from different groups were subject to quantitative RT-PCR analyses of Ly6g mRNA. N = 6–7 in each group. *P < .05, **P < .01, ***P < .001.
Figure 4
Figure 4
CXCL1 plays an important role in promoting liver fibrosis induced by HFD-plus-binge ethanol feeding model. (A) Male C57BL/6J mice were fed an HFD for 3 months and then injected intraperitoneally with control IgG or CXCL1 neutralizing antibody. Fifteen minutes later, mice were gavaged with 5 g/kg ethanol. Nine hours later the tissue samples were analyzed with Sirius Red staining and quantitative RT-PCR for fibrosis. (B) WT C57B/6J mice and Cxcl1 KO mice were subject to HFD+1B ethanol challenge, and mice were euthanized 9 hours after gavage. The liver samples were analyzed with Sirius Red staining and quantitative RT-PCR. Acta2 gene: encodes α-smooth muscle actin. Images represent 1 of 5 fields in each group. (C) Percentage of Sirius Red–positive area and body weight in panels A and B were statistically analyzed. (D) HSCs were isolated from C57BL/6J mice and then cultured, and subject to quantitative RT-PCR analysis of Cxcr2 mRNA. Neutrophils were used as a positive control analysis of Cxcr2 mRNA. (E) Cultured HSCs were incubated with 100 ng/mL CXCL1 or vehicle. Five days later, HSCs were collected and subject to quantitative RT-PCR analyses of fibrosis-related genes. (A–C) N = 5–6. (D) Data were from 2 independent experiments. (E) N = 3. *P < .05, **P < .01.
Figure 5
Figure 5
Icam-1–deficient mice have reduced hepatic neutrophil infiltration, liver injury, and fibrosis induced by HFD+1B ethanol feeding. WT and Icam-1 KO mice were subject to HFD+1B ethanol challenge, and mice were euthanized 9 hours after gavage. (A) Liver sections were stained with MPO, Sirius Red, and Masson Trichrome. Images represent 1 of 5 fields in each group. Arrows indicate MPO+ cells. (B) Quantitative RT-PCR analyses of neutrophil marker gene Ly6g. (C) Serum ALT levels were analyzed. (D) Percentage of Sirius Red–positive area and body weight were analyzed. (E) Quantitative RT-PCR analyses of fibrosis-related genes. (B–E) N = 5–7 in each group. *P < .05.
Figure 6
Figure 6
P47phoxKO mice have reduced liver damage and fibrosis in the HFD+1B ethanol model. (A) C57BL/6J mice were subject to HFD+1B ethanol or HFD-plus-maltose (HFD group) challenge. Liver tissues were collected and subject to 4-HNE staining. (B–D) WT and p47phox KO mice were subject to HFD+1B ethanol challenge. Malondialdehyde Sirius Red, and MPO staining were performed on the liver tissue sections. (B) Representative images are shown, arrows indicate MPO+ cells). (C) Serum ALT and AST levels, percentage of Sirius Red–positive area, as well as body weight were measured. (D) Quantitative RT-PCR analyses of liver fibrosis-related genes and Ly6g mRNA. N = 4–7 in each group, *P < .05.
Figure 7
Figure 7
Engulfment of neutrophil apoptotic body (AB) does not activate HSCs. Neutrophils from the bone marrow and HSCs from the liver were isolated. The neutrophils were labeled with 5-Carboxytetramethylrhodamine Succinimidyl Ester and exposed to UV to generate apoptotic bodies. These apoptotic bodies were incubated with HSCs for 24 hours. (A and B) Engulfment was evaluated under a microscope. (A) Representative images are shown. (B) The phagocytosis/engulfment rate was calculated as the percentage of cells with intracellular 5-Carboxytetramethylrhodamine Succinimidyl Ester-labeled apoptotic bodies. (C) HSCs from panel B then were collected for quantitative RT-PCR assay to evaluate the expression of Col1a1, α-smooth muscle actin, and transforming growth factor β (Tgfb).
Figure 8
Figure 8
Co-culture with HSCs reduces apoptosis and promotes survival of neutrophils. (A) Confocal immunofluorescence staining of MPO and desmin (a marker for HSCs) in the liver sections of HFD+1B or HFD+mB ethanol-fed mice. Only a few number of MPO+ cells were observed in this figure owing to extremely high magnification from confocal microscope analysis. (B) Mouse bone marrow neutrophils were cultured alone or with 1-day cultured HSCs (left panel) or 5-day cultured HSCs (right panel). The number of live neutrophils was counted and statistically analyzed. (C) Wright–Giemsa staining of the neutrophils cultured for 2 days with or without HSCs. Arrows indicate apoptotic neutrophils determined according to the morphology. (D) Statistical analysis of TUNEL+ neutrophils after culture for 1 or 2 days with or without HSCs. (A and C) Images represent 1 of at least 5 randomly selected fields. n = 3–4 in each group, *P < .05, **P < .01, ***P < .001. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 9
Figure 9
HSCs delay the spontaneous apoptosis of cultured neutrophils via the secretion of GM-CSF and IL-15. (A) Quantitative RT-PCR analyses of mRNA expression levels of cytokines and growth factors in 1-day or 5-day cultured HSCs. N = 3 in each group. (B) Concentrations of GM-CSF from cultured HSCs were analyzed with an enzyme-linked immunosorbent assay. Values were obtained from 4 independent experiments. (C and D) Quantitative RT-PCR analyses of Csf2 and Il15 mRNA expression levels from HFD+1B vs HFD groups. N = 4–6 in each group. (E) Csf2 and Il15 expression in primary hepatocytes and HSCs after 1-day culture. N = 3–7 in each group. (F) Neutrophils were cultured with 5-day cultured HSCs or HSC-conditioned medium as well as neutralizing antibodies against GM-CSF or IL-15 as indicated. The number of live cells was counted and statically analyzed. N = 4–8 in each group. *P < .05, **P < .01, ***P < .001 vs control medium group, #P < .05 vs neutrophils + HSCs group. Egf, epidermal growth factor; Hgf, hepatocyte growth factor.
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