Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients

Abstract

Background & aims: Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD.

Methods: MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif^-/- bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH).

Results: HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif^-/-→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif^-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH.

Conclusions: Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.

Keywords: Alcoholic liver disease; Hepatocytes; Inflammation; Innate immune system; MIF; Translational research.

Fig. 1. MIF release from HuH7 hepatocytes, but not THP-1 macrophages, in response to ethanol

(A,D) Expression of MIF mRNA in HuH7 and differentiated THP-1 macrophages was measured by qRT-PCR. MIF and TNF-α accumulation was detected by ELISA in the cell culture medium of (B, C) HuH7 cells and (E, F) differentiated THP-1 macrophages after challenge with 50mM EtOH or 200 ng/mL LPS. Values represent mean ± SEM. * p< 0.05 compared to basal, n= 3.

Fig. 2. MIF-deficiency in non-myeloid cells protected mice from chronic ethanol-induced hepatic injury, inflammation and steatosis

WT→WT, Mif^−/−→WT and WT→Mif ^−/− chimeric mice were allowed free access to diets with increasing concentrations of ethanol or pair-fed a control diet for 25 days. Enzyme activities of (A) ALT and (B) AST were measured in plasma. (C) Hepatic triglyceride content was measured in whole liver homogenates. Values with different alphabetical superscripts were significantly different from each other (p< 0.05). (D) Paraffin-embedded liver sections were stained with hematoxylin and eosin and (E) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was quantified using ImagePro. All images were acquired using a 10X objective. Values represent means ± SEM, n = 4 pair-fed and n = 6 ethanol-fed.

Fig. 3. MIF-deficiency in non-myeloid, but not myeloid cells protected mice from chronic ethanol-induced expression of mRNA for immune cells and cytokines/chemokines

MIF^−/−→WT and WT→MIF ^−/− chimeric mice were allowed free access to diets with increasing concentrations of ethanol or pair-fed a control diet for 25 days. Expression of (A) MCP-1, CXCL10, CXCL1, CXCL2 mRNA was detected in mouse livers using qRT-PCR. Gene expression was normalized to 18S (B/C) Paraffin-embedded liver sections were stained with antibody against (B) F4/80 and (C) Ly6C. Images were acquired with a 20X objective. F4/80 positive staining and Ly6C⁺ cells were quantified using ImagePro. Values represent means ± SEM, n = 4 pair-fed and n = 6 ethanol-fed. Values with different superscripts are significantly different from each other (p< 0.05).

Fig. 4. Expression of MIF in livers and peripheral and suprahepatic serum of patients with alcoholic hepatitis

(A) Paraffin-embedded liver sections were stained for MIF in liver biopsies obtained from patients with alcoholic hepatitis, auto-immune hepatitis or healthy liver controls. All images were acquired using a 40X objective. Images are representative of 5 samples for AH, 5 autoimmune hepatitis and 4 for healthy liver controls. (B) qRT-PCR analysis of MIF mRNA in livers of patients with alcoholic hepatitis and healthy liver controls. Values represent means ± SEM, n = 17 AH and n = 7 healthy liver controls. (C–G) MIF concentration in suprahepatic serum was correlated by regression analysis with (C) peripheral serum, (D) bilirubin, (E) AST, (F) triglycerides and (G) GGT. (H/I) MIF concentration in (H) suprahepatic and (I) peripheral serum was measured by ELISA. p<0.05.