Macrophage migration inhibitory factor contributes to ethanol-induced liver injury by mediating cell injury, steatohepatitis, and steatosis

Abstract

Macrophage migration inhibitory factor (MIF), a multipotent protein that exhibits both cytokine and chemotactic properties, is expressed by many cell types, including hepatocytes and nonparenchymal cells. We hypothesized that MIF is a key contributor to liver injury after ethanol exposure. Female C57BL/6 or MIF-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet for 4 (11% total kcal;early response) or 25 (32% kcal; chronic response) days. Expression of MIF messenger RNA (mRNA) was induced at both 4 days and 25 days of ethanol feeding. After chronic ethanol, hepatic triglycerides and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased in wildtype, but not MIF-/-, mice. In order to understand the role of MIF in chronic ethanol-induced liver injury, we investigated the early response of wildtype and MIF-/- to ethanol. Ethanol feeding for 4 days increased apoptosis of hepatic macrophages and activated complement in both wildtype and MIF-/- mice. However, tumor necrosis factor alpha (TNF-α) expression was increased only in wildtype mice. This attenuation of TNF-α expression was associated with fewer F4/80+ macrophages in liver of MIF-/- mice. After 25 days of ethanol feeding, chemokine expression was increased in wildtype mice, but not MIF-/- mice. Again, this protection was associated with decreased F4/80+ cells in MIF-/- mice after ethanol feeding. Chronic ethanol feeding also sensitized wildtype, but not MIF-/-, mice to lipopolysaccharide, increasing chemokine expression and monocyte recruitment into the liver.

Conclusion: Taken together, these data indicate that MIF is an important mediator in the regulation of chemokine production and immune cell infiltration in the liver during ethanol feeding and promotes ethanol-induced steatosis and hepatocyte damage.

Figure 1

Ethanol feeding increased MIF expression in liver and plasma. C57BL/6 mice were allowed free access to an ethanol-containing liquid diet or pair-fed control diet. (A/B) Expression of MIF and CD74 mRNA was measured in liver by qRT-PCR at two time points of ethanol feeding, 4d, 11% and 25d, 32%, or in pair-fed control mice. Expression of the genes of interest was normalized to 18S (n=4 pair-fed and n=6 for ethanol-fed mice). (C) The relative concentration of MIF in plasma was assessed by Western blot. n=3 pair-fed and n=6 for ethanol-fed mice. Immunoreactive MIF was assessed via (D) immunohistochemistry in liver and (E) Western blot in isolated hepatocytes (H) and non-parenchymal cells (N). (E) Relative CD74 was measured by Western blot in hepatocytes and NPCs isolated from pair-fed (P) and 25d, 32% ethanol-fed mice (E). (D) IHC images represent 2 images per liver and were acquired using 20x objective (n=5 pair-fed and n=9 ethanol-fed mice). Values represent means ± SEM. Asterisks represent statistical significance between pair-fed and ethanol-fed groups (P < 0.05).

Figure 2

MIF−/− mice were protected from chronic ethanol-induced liver injury. C57BL/6 and MIF−/− mice were allowed free access to ethanol containing diets or pair-fed control diets. Liver injury was characterized at 25d, 32% ethanol feeding compared to pair-fed controls. (A) Hepatic triglycerides were measured by biochemical assay and plasma ALT and AST were quantified enzymatically. (B) TUNEL positive nuclei were visualized and semi-quantified in paraffin-embedded liver sections. (C) Induction of CYP2E1 protein was measured by immunoblot and quantified via densitometry. (D) Immunoreactive 4HNE adducts were visualized by immunohistochemistry and semi-quantified in paraffin-embedded liver sections. Images were acquired using 10x (4HNE) or 20x (TUNEL) objective. Figures represent 2 images per liver (n=9 pair-fed and n=11 ethanol-fed mice). Values represent means ± SEM, n=4 pair-fed and n=6 for ethanol-fed mice. Values with different superscripts are significantly different from each other (P < 0.05).

Figure 3

Early immune responses in liver after 4d, 11% ethanol feeding. C57BL/6 and MIF−/−mice were allowed free access to ethanol containing diets or pair-fed control diets. (A) TUNEL positive nuclei, immunoreactive (B) C3b/iC3b/C3c and (C) TNFα were visualized in paraffin-embedded or OCT-frozen liver sections. Values indicate percentage of TUNEL⁺ nuclei/Dapi (A), total number of positive punctae for C3c/iC3b/C3c (B) and F4/80 (D), and mean fluorescence intensity for TNFα (C). Black arrows indicate zoom of white boxes to represent sinusoidal TUNEL⁺ nuclei (A) or deposition of C3b/iC3b/C3c (B). (D) F4/80 mRNA expression was measured via qRT-PCR and (E) immunoreactive F4/80 was visualized in OCT-frozen liver sections. F4/80 mRNA expression was normalized to 18S. Images were acquired using 10x (TNFα) or 20x (TUNEL, C3b/iC3b/C3c, F4/80) objective. Figures represent 2 images per liver. 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).

Figure 4

Leukocyte recruitment after chronic ethanol feeding in wild-type and MIF−/− mice. Leukocyte phenotype in liver of C57BL/6 and MIF−/− mice was analyzed after 25d, 32% ethanol feeding. (A) Immunoreactive F4/80 was visualized and semi-quantified in liver sections frozen in OCT. Figures represent 2 images per liver. (B) F4/80 mRNA expression in liver was measured via qRT-PCR. Images were acquired using 10x objective. F4/80 mRNA expression was normalized to 18S. n=4 pair-fed and n=6 ethanol-fed. (C) Total CD45⁺ leukocytes and monocyte cell markers, CD11c and Ly6C, were quantified in isolated liver non-parenchymal cells via flow cytometry. CD11c and Ly6c graphs represent fold change compared to wild-type pair-fed mice. (D) Ly6c was assessed by immunohistochemistry and semi-quantified in liver sections frozen in OCT. Black arrows indicate zoom of white boxes to represent clusters of Ly6c staining. Values represent means ± SEM, n=10 pair-fed and n=10 ethanol-fed. Values with different superscripts are significantly different from each other (P < 0.05).

Figure 5

MIF−/− mice were protected from increased expression of pro-inflammatory mediators after chronic ethanol feeding. Expression of (A) TLR4, TNFα, (B) MCP-1, CXCL10 and MIP2 mRNA was quantified in liver via qRT-PCR. Expression of the genes of interest was normalized to 18S. 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).

Figure 6

MIF−/− mice were protected from ethanol-sensitized LPS-induced inflammation. C57BL/6 and MIF−/− mice were allowed free access to ethanol containing diets or pair-fed control diets. After 25d, 32% ethanol feeding, mice were challenged with LPS via intraperitoneal injection 4hr prior to euthanasia. (A) Hematoxylin and eosin stained liver sections were examined and (B) assigned an inflammatory score by a pathologist. Inflammatory score was based on mononuclear cell and lobular inflammation. Arrows indicate foci of infiltrating cells (inset represent zoom of lower black arrow). (C) Expression of MCP-1, ICAM-1 and CD62E mRNA was quantified in liver by qRT-PCR. Expression of the genes of interest was normalized to 18S. Images were acquired using 10x objective. Figures represent 2 images per liver. 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).

Figure 7

Proposed interaction between ethanol, MIF and innate immune responses. Ethanol feeding in mice results in activation of Kupffer cells and increased MIF production. These two phenomena initiate a complex positive feedback loop involving monocyte infiltration, increased production of pro-inflammatory mediators and chemokines, ultimately leading to chronic inflammation and injury.