TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration

Abstract

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

Keywords: AH; ALD; MyD88; binge ethanol feeding; chemokine.

Fig. 1.

TLR2- and TLR9-deficient mice exhibit reduced liver injury and neutrophil-recruiting chemokine expression in chronic plus binge ethanol (EtOH)-induced alcoholic liver disease (ALD). A–E: wild-type (WT), TLR2-deficient, and TLR9-deficient female mice were subjected to control and Lieber-DeCarli diet (n = 10 per group for pair-fed, n = 14–16 per group for EtOH fed; each experiment was performed with 5–8 mice per group and repeated 2 times). A: liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. B: hepatocyte death was analyzed by TUNEL staining and TUNEL-positive cells were counted. Hepatic steatosis was determined by measuring hepatic triglyceride (TG) levels. The results are expressed as mg TG/g liver. HPF, high-power field. C: expression of proinflammatory cytokines (Il1b, Il6, and Tnf) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and shown as fold change compared with pair-fed WT mice. D: expression of neutrophil-recruiting chemokines (Cxcl1, Cxcl2, Cxcl5, and Ccl2) was determined by qRT-PCR and shown as fold change compared with pair-fed WT mice. E: hepatic expression of CXCL1 was assessed by immunohistochemistry and quantified by measuring CXCL1-positive area. Serum CXCL1 levels were measured by ELISA. KO, knockout. F: hepatic neutrophil infiltration was determined by immunohistochemistry for Ly-6G and the number of Ly-6G-positive cells was counted. Data are presented as means ± SE per group. *P < 0.05; n.s., not significant; n.d., not detected. Original magnification, ×100 [hematoxylin and eosin (H&E)], ×200 (TUNEL, CXCL1, and Ly-6G).

Fig. 2.

Hepatic expression of neutrophil-recruiting chemokines induced by alcohol is independent of Kupffer cells (KCs). A–E: in vivo depletion of KCs was achieved by injection of clodronate liposome (Clod.; 200 μl/mouse) for 2 consecutive days (n = 6 per group). A: KC depletion was confirmed by hepatic expression of F4/80 by using qRT-PCR. B: liver injury was assessed by measuring serum ALT levels. C: expression of proinflammatory cytokines (Il1b, Il6, and Tnf) was determined by qRT-PCR and shown as fold change compared with vehicle-treated pair-fed mice. D: expression of neutrophil-recruiting chemokines (Cxcl1, Cxcl2, and Cxcl5) was determined by qRT-PCR and shown as fold change compared with vehicle-treated pair-fed mice. E: KCs and neutrophils were stained by immunohistochemistry for F4/80 and Ly-6G, respectively. F: quantification was done by measuring F4/80-positive area or counting Ly-6G-positive cells. Data are presented as means ± SE per group. *P < 0.05; **P < 0.01, ***P < 0.001; n.s., not significant. Original magnification, ×200 (F4/80 and Ly-6G).

Fig. 3.

Hepatocytes and hepatic stellate cells (HSCs) are the major cell types for the production of CXCL1. Each liver fraction (hepatocytes, HSCs, and KCs) was isolated from livers of EtOH- and pair-fed mice. Representative results are presented from 2 independent experiments (each isolation was assayed in triplicate). A: Cxcl1 expression in each fraction was determined by qRT-PCR and shown as fold change compared with those of pair-fed mice. B and C: hepatocytes and HSCs were pretreated with Pam3CSK4 (200 ng/ml), and then ODN1668 (5 μg/ml) was added for 30 min (qRT-PCR) and 6 h (ELISA). The Cxcl1 mRNA and CXCL1 protein levels in hepatocytes (B) and HSCs (C) were determined by qRT-PCR and ELISA, respectively. Data are presented as means ± SE per group. *P < 0.05, n.s., not significant.

Fig. 4.

Treatment with a CXCR2 antagonist attenuates alcohol-induced neutrophil-mediated liver injury. A: correlation between serum CXCL1 and ALT levels in mice with or without ALD was analyzed by Spearman correlation test. B–D: CXCR2 blockade was accomplished by an oral administration of a selective antagonist, SB225002 (n = 16 per group; each experiment was performed with 8 mice per group and repeated 2 times). B: liver injury was assessed by measuring serum ALT levels. C: expression of neutrophil markers (Ly6g and Elane) was determined by qRT-PCR and shown as fold change compared with vehicle-treated mice. D: neutrophil infiltration was determined by immunohistochemistry for Ly-6G and its quantification was done by counting Ly-6G-positive cells. Data are presented as means ± SE per group. *P < 0.05. Original magnification, ×200 (Ly-6G).

Fig. 5.

Blocking MyD88 protects against alcohol-mediated CXCL1 production and liver injury. A–D: a MyD88 inhibitory peptide and a control peptide (100 μg/mouse) were administered with intraperitoneal injection to EtOH-fed WT mice (n = 16 per group; each experiment was performed with 8 mice per group and repeated 2 times). A: liver injury was assessed by measuring serum ALT levels. B: serum CXCL1 levels were measured by ELISA. C: expression of Cxcl1, neutrophil markers (Ly6g and Elane), and proinflammatory cytokines (Il1b, Il6, and Tnf) was determined by qRT-PCR and shown as fold change compared with vehicle-treated mice. D: hepatic expression of CXCL1 and neutrophil infiltration were determined by immunohistochemistry for CXCL1 and Ly-6G, respectively. Their quantification was performed. Ctrl Peptide, control peptide; MyD88 i, MyD88 inhibitory peptide. Data are presented as means ± SE per group. *P < 0.05, **P < 0.01; n.s., not significant. Original magnification, ×200 (CXCL1 and Ly-6G).

Fig. 6.

Hepatic CXCL1 expression is upregulated in patients with alcoholic hepatitis. A: hepatic CXCL1 expression was determined by immunohistochemistry on liver biopsy specimens from control individuals or patients with alcoholic hepatitis (AH; n = 5 for control, n = 10 for patient with AH). Quantification was done by measuring CXCL1-positive area. B: neutrophil infiltration was analyzed by immunohistochemistry for Ly-6G and its quantification on liver biopsy specimens of patients with AH. Data are presented as means ± SE per group. *P < 0.05; **P < 0.01. Original magnification, ×200 (CXCL1), ×400 (Ly-6G).