Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury

Abstract

Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Because receptor-interacting protein kinase (RIP) 3-mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3.

Conclusion: CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.

Figure 1. RIP3 was induced in mouse liver following ethanol feeding

(A) C57BL/6J wild-type (WT) mice were allowed free access to ethanol (4d, 11%, 4d,32% or 25d, 32%) or pair-fed control diets, as described in methods. Paraffin-embedded livers were de-paraffinized followed by immuno-detection for RIP3. (B) 129S1/Svlmj (wild-type for CYP2E1-/-) and CYP2E1-deficient mice were allowed free access to ethanol (25d, 32%) or pair-fed control diets. (A/B) Paraffin-embedded livers were de-paraffinized followed by RIP3 immunostaining. Nuclei were counterstained with hematoxylin. All images were acquired using a 20X objective. (C) RIP3-stained areas were quantified using Image Pro-Plus software and analyzed. (D) AST activity was determined in plasma samples from ethanol- and pair-fed mice. (C/D) Values with different alphabetical superscripts were significantly different from each other, p< 0.05. All images were acquired using 20X objective. (E) RIP3 and FADD association was detected using DuoLink proximity ligation assay. Images were acquired using 40X objective.

Figure 2. RIP3 was detected in liver biopsies from patients with alcoholic liver disease

Paraffin-embedded human liver biopsies were de-paraffinized followed by RIP3 immunostaining using standard immunohistochemistry technique. Nuclei were counterstained with hematoxylin. (A) Images were acquired using a 20X objective. (B) RIP3 staining were scored on a scale of 0 – 3. n = 8 for control, n = 20 for ALD (C) RIP3-stained areas were quantified using Image Pro-Plus software and analyzed. * p< 0.05. n = 6 for control, n = 11 for ALD.

Figure 3. Ethanol-induced liver injury and steatosis were attenuated in RIP3-deficient mouse livers

C57BL/6J wild-type (WT) and RIP3-deficient mice were allowed free access to ethanol (4d, 32%) or pair-fed control diets. (A) ALT and AST activities were measured in plasma. Hepatic triglyceride content was measured in whole liver homogenate. Values with different alphabetical superscripts were significantly different from each other, p< 0.05. (B) Frozen liver sections were subjected to Oil-Red O-staining. All images were acquired using a 20X objective.

Figure 4. RIP3-deficiency reduced ethanol-mediated hepatic pro-inflammatory cytokine expression in mouse liver

C57BL/6J wild-type (WT) and RIP3-deficient mice were allowed free access to ethanol (4d, 32%) or pair-fed control diets. (A) Paraffin-embedded liver sections were stained with hematoxylin and eosin. All images were acquired using a 10X objective. Area under the gray box is enlarged to visualize an inflammatory focus. (B) Expression of MCP-1, IL-6 and TNFα mRNA was detected in mouse livers using qRT-PCR measurement. * p< 0.05 for ethanol-fed compared to pair-fed within each genotype. (C) Immunoreactive TNFα protein was evaluated by immunohistochemistry in frozen livers. All images were acquired using a 40X objective. (D) TNFα-positive areas were quantified using Image Pro-Plus software and analyzed. (E) Plasma TNFα concentrations were measured by enzyme-linked immunosorbent assay. * p< 0.05 for ethanol-fed compared to pair-fed within each genotype.

Figure 5. RIP3-deficiency prevented chronic ethanol-induced liver injury and hepatic pro-inflammatory cytokine expression in mice

C57BL/6J wild-type (WT) and RIP3-deficient mice were allowed free access to ethanol (25d, 32%) or pair-fed control diets. (A) ALT and AST activities were measured in plasma. Hepatic triglyceride content was measured in whole liver homogenates. Values with different alphabetical superscripts were significantly different from each other, p< 0.05. (B) Expression of MCP-1, IL-6 and TNFα mRNA was detected in mouse livers using qRT-PCR measurement. * p< 0.05 for ethanol-fed between genotypes. (C) Immunoreactive TNFα protein was evaluated by immunohistochemistry in frozen livers. All images were acquired using a 40X objective. TNF-positive areas were quantified using Image Pro-Plus software and analyzed. Values with different alphabetical superscripts were significantly different from each other, p< 0.05. (D–E) Infiltration of CD45- and Ly6c-positive cells were evaluated by immunohistochemistry in frozen livers. Numbers of foci, as well as the total number of CD45⁺ or Ly6C⁺ cells were counted. * p< 0.05 for comparison between pair-fed and ethanol-fed mice.

Figure 6. RIP3-deficiency did not prevent markers of ethanol-induced apoptosis in mouse liver

C57BL/6J wild-type (WT) and RIP3-deficient mice were allowed free access to ethanol (4d, 32% or 25d, 32%) or pair-fed control diets. Paraffin-embedded livers were de-paraffinized followed by (A/B) TUNEL or (C/D) CK18 staining. (A/B) Images were acquired using 40X objective for TUNEL and 20X for CK18 staining. TUNEL-positive cells were counted and expressed as percent positive of total number of cells per 40X frame. (C/D) CK18-positive cells were counted and expressed as total number of cells per 20X frame. Values with different alphabetical superscripts were significantly different from each other, p< 0.05.

Figure 7. Necrostatin-1 treatment did not reduce ethanol-induced liver injury

C57BL/6J wild-type (WT) mice were allowed free access to ethanol (4d, 32%) or pair-fed control diets. Mice received a once daily injection of necrostatin-1 (1.65mg/Kg body weight) intraperitoneally just prior to the daily provision of ethanol- and control-diet. ALT and AST activities were measured in plasma. Hepatic triglyceride content was measured in whole liver homogenates. Values with different alphabetical superscripts were significantly different from each other, p< 0.05.

Figure 8. JNK activation was reduced in RIP3-deficient mouse livers following chronic ethanol feeding

C57BL/6J wild-type (WT) and RIP3-deficient mice were allowed free access to ethanol (4d, 32%) or pair-fed control diets. Paraffin-embedded livers were de-paraffinized followed by pJNK-immunodetection. (B) Higher magnification/cropped image of pJNK immunohistochemistry. Black arrows indicate positive staining for pJNK and open arrows indicate cells void of pJNK-positive staining. (C) pJNK-positive cells were counted and expressed as total number of cells per 20X frame. All images were acquired using a 20X objective. Values with different alphabetical superscripts were significantly different from each other, p< 0.05.