Sanjoy Roychowdhury

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

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. (HEPATOLOGY 2013)

Complement and alcoholic liver disease: Role of C1q in the pathogenesis of ethanol-induced liver injury in mice

BACKGROUND & AIMS Complement is involved in the development of alcoholic liver disease in mice; however, the mechanisms for complement activation during ethanol exposure have not been identified. C1q, the recognition subunit of the first complement component, binds to apoptotic cells, thereby activating the classical complement pathway. Because ethanol exposure increases hepatocellular apoptosis, we hypothesized that ethanol-induced apoptosis would lead to activation of complement via the classical pathway. METHODS Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets or pair-fed control diets for 4 or 25 days. RESULTS Ethanol feeding for 4 days increased apoptosis of Kupffer cells in both wild-type and C1qa-/- mice. Ethanol-induced deposition of C1q and C3b/iC3b/C3c was colocalized with apoptotic Kupffer cells in wild-type, but not C1qa-/-, mice. Furthermore, ethanol-induced increases in tumor necrosis factor-alpha and interleukin-6 expression at this early time point were suppressed in C1q-deficient mice. Chronic ethanol feeding (25 days) increased steatosis, hepatocyte apoptosis, and activity of serum alanine and aspartate aminotransferases in wild-type mice. These markers of hepatocyte injury were attenuated in C1qa-/- mice. In contrast, chronic ethanol (25 days)-induced increases in cytochrome P450 2E1 expression and oxidative stress did not differ between wild-type and C1qa-/- mice. CONCLUSIONS For the first time, these data indicate that ethanol activates the classical complement pathway via C1q binding to apoptotic cells in the liver and that C1q contributes to the pathogenesis of ethanol-induced liver injury.

An early complement-dependent and TLR-4-independent phase in the pathogenesis of ethanol-induced liver injury in mice.

The innate immune system has been implicated in the pathogenesis of alcoholic liver disease. Although innate immunity is usually considered an early response to injury, previous work implicating innate immunity in ethanol‐induced liver injury focuses primarily on long‐term ethanol exposure. We investigated the early period of ethanol exposure to determine whether there were temporal associations between activation of innate immune responses and known correlates of liver injury. Female C57BL/6 mice were allowed free access to an ethanol‐containing Lieber‐DeCarli diet or were pair‐fed a control diet. Within 4 days of ethanol exposure, we observed a striking spike in expression of hepatic proinflammatory cytokines—including tumor necrosis factor α (TNF‐α), interleukin‐6, and interferon‐γ—prior to hepatic triglyceride accumulation or increased plasma alanine aminotransferase activities, as well as before the induction of cytochrome P450 2E1 or oxidative stress. This early spike in inflammatory cytokines coincided with deposition of C3b‐iC3b/C3c (C3b) in the liver. This deposition, resulting from the cleavage of the third component of the complement system (C3), is evidence for activation of complement in response to ethanol. C3−/− mice were protected from the early, ethanol‐induced increase in hepatic TNF‐α expression. Ethanol increased C3b deposition in mice deficient in C3a receptor or C5a receptor, as well as in wild‐type mice depleted of hepatic macrophages; however, there was no increase in hepatic TNF‐α in the absence of C3a receptor, C5a receptor, or hepatic macrophages. In contrast, the absence of Toll‐like receptor 4 (TLR‐4) had no effect on the early, ethanol‐induced increase in either C3b or TNF‐α. Conclusion: We have identified a complement‐ and macrophage‐dependent, but TLR‐4 independent, phase in the pathogenesis of ethanol‐induced liver injury. (HEPATOLOGY 2009.)

Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver.

Ethanol‐induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol‐induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber‐deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair‐fed control diet. Hepatic Trx‐1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol‐induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol‐induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate‐buffered saline (PBS). Conclusion: Ethanol feeding increased accumulation of hepatic 4‐hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor α, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol‐fed mice, treatment with rhTrx reduced 4‐hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase‐mediated dUTP‐biotin nick‐end labeling‐positive cells, caspase‐3 activity, and cytokeratin‐18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol‐induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis. (HEPATOLOGY 2009.)

Adiponectin and Heme Oxygenase-1 Suppress TLR4/MyD88-Independent Signaling in Rat Kupffer Cells and in Mice after Chronic Ethanol Exposure

Alcoholic liver disease is mediated via activation of TLR4 signaling; MyD88-dependent and -independent signals are important contributors to injury in mouse models. Adiponectin, an anti-inflammatory adipokine, suppresses TLR4/MyD88-dependent responses via induction of heme oxygenase-1 (HO-1). Here we investigated the interactions between chronic ethanol, adiponectin, and HO-1 in regulation of TLR4/MyD88-independent signaling in macrophages and an in vivo mouse model. After chronic ethanol feeding, LPS-stimulated expression of IFN-β and CXCL10 mRNA was increased in primary cultures of Kupffer cells compared with pair-fed control mice. Treatment of Kupffer cells with globular adiponectin (gAcrp) normalized this response. LPS-stimulated IFN-β/CXCL10 mRNA and CXCL10 protein was also reduced in RAW 264.7 macrophages treated with gAcrp or full-length adiponectin. gAcrp and full-length adiponectin acted via adiponectin receptors 1 and 2, respectively. gAcrp decreased TLR4 expression in both Kupffer cells and RAW 264.7 macrophages. Small interfering RNA knockdown of HO-1 or inhibition of HO-1 activity with zinc protoporphyrin blocked these effects of gAcrp. C57BL/6 mice were exposed to chronic ethanol feeding, with or without treatment with cobalt protoporphyrin, to induce HO-1. After chronic ethanol feeding, mice were sensitized to in vivo challenge with LPS, expressing increased IFN-β/CXCL10 mRNA and CXCL10 protein in liver compared with control mice. Pretreatment with cobalt protoporphyrin 24 h before LPS challenge normalized this effect of ethanol. Adiponectin and induction of HO-1 potently suppressed TLR4-dependent/MyD88-independent cytokine expression in primary Kupffer cells from rats and in mouse liver after chronic ethanol exposure. These data suggest that induction of HO-1 may be a useful therapeutic strategy in alcoholic liver disease.

Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice.

Background: Chronic alcohol consumption leads to inflammation in adipose tissue, disrupting normal metabolic activity of adipocytes. Results: Expression of an alcohol metabolizing enzyme, cytochrome P4502E1, initiates inflammation in adipose. Bid-dependent apoptosis and activation of complement then exacerbate this initial response. Conclusion: Adipose inflammation during alcohol feeding develops in response to cytochrome P450 expression. Significance: Preventing adipose inflammation may prevent the pathphysiological effects of ethanol. Chronic, heavy alcohol exposure results in inflammation in adipose tissue, insulin resistance, and liver injury. Here we have identified a CYP2E1/Bid/C1q-dependent pathway that is activated in response to chronic ethanol and is required for the development of inflammation in adipose tissue. Ethanol feeding for 25 days to wild-type (C57BL/6J) mice increased expression of multiple markers of adipose tissue inflammation relative to pair-fed controls independent of increased body weight or adipocyte size. Ethanol feeding increased the expression of CYP2E1 in adipocytes, but not stromal vascular cells, in adipose tissue and Cyp2e1−/− mice were protected from adipose tissue inflammation in response to ethanol. Ethanol feeding also increased the number of TUNEL-positive nuclei in adipose tissue of wild-type mice but not in Cyp2e1−/− or Bid −/− mice. Apoptosis contributed to adipose inflammation, as the expression of multiple inflammatory markers was decreased in mice lacking the Bid-dependent apoptotic pathway. The complement protein C1q binds to apoptotic cells, facilitating their clearance and activating complement. Making use of C1q-deficient mice, we found that activation of complement via C1q provided the critical link between CYP2E1/Bid-dependent apoptosis and onset of adipose tissue inflammation in response to chronic ethanol. In summary, chronic ethanol increases CYP2E1 activity in adipose, leading to Bid-mediated apoptosis and activation of complement via C1q, finally resulting in adipose tissue inflammation. Taken together, these data identify a novel mechanism for the development of adipose tissue inflammation that likely contributes to the pathophysiological effects of ethanol.

Inhibition of apoptosis protects mice from ethanol-mediated acceleration of early markers of CCl4-induced fibrosis but not steatosis or inflammation

BACKGROUND Correlative evidence indicates that apoptosis is associated with the progression of alcoholic liver disease. If apoptosis contributes to ethanol (EtOH)-induced steatohepatitis and/or fibrosis, then mice deficient in Bid, a key pro-apoptotic Bcl-2 family member, or mice treated with a pan-caspase inhibitor (VX166) should be resistant to EtOH-induced liver injury. METHODS This hypothesis was tested in mice using a model of chronic, heavy EtOH-induced liver injury, as well as in a model in which moderate EtOH feeding accelerated the appearance of early markers of hepatic fibrosis in response to acute carbon tetrachloride (CCl(4) ) exposure. RESULTS Chronic EtOH feeding to mice increased TUNEL- and cytokeratin-18-positive cells in the liver, as well as the expression of receptor-interacting protein kinase 3 (RIP3), a marker of necroptosis. In this model, Bid-/- mice or wild-type mice treated with VX166 were protected from EtOH-induced apoptosis, but not EtOH-induced RIP3 expression. Bid deficiency or inhibition of caspase activity did not protect mice from EtOH-induced increases in plasma alanine and aspartate amino transferase activity, steatosis, or mRNA expression of some inflammatory cytokines. Moderate EtOH feeding to mice enhanced the response of mice to acute CCl(4) exposure, resulting in increased expression of α-smooth muscle actin and accumulation of extracellular matrix protein. VX166-treatment attenuated EtOH-mediated acceleration of these early indicators of CCl(4) -induced hepatic fibrosis, decreasing the expression of α-smooth muscle actin, and the accumulation of extracellular matrix protein. CONCLUSIONS EtOH-induced apoptosis of hepatocytes was mediated by Bid. Apoptosis played a critical role in the accelerating the appearance of early markers of CCl(4) -induced fibrosis by moderate EtOH but did not contribute to EtOH-induced hepatocyte injury, steatosis, or expression of mRNA for some inflammatory cytokines.

Early growth response-1 attenuates liver injury and promotes hepatoprotection after carbon tetrachloride exposure in mice

BACKGROUND & AIMS Inflammatory gene expression plays a pathological role in acute and chronic hepatic inflammation, yet, inflammation also promotes liver repair by inducing protective mechanisms to limit collateral tissue damage by priming hepatocytes for proliferation. Early growth response (Egr)-1, a transcription factor that regulates inflammatory gene expression, plays a pathological role in many animal models of acute and chronic inflammatory disease. Here, we tested the hypothesis that Egr-1 is beneficial after toxic liver injury. METHODS Acute liver injury was induced in wild-type and egr-1-/- mice by a single injection of carbon tetrachloride (CCl(4)). Liver injury, inflammatory, and hepatoprotective gene expression and signaling events were measured 18, 48, and 72 h after CCl(4) administration. RESULTS Peak liver injury was greater in egr-1-/- mice compared to wild-type mice. Enhanced injury in egr-1-/- mice was associated with reduced tumor necrosis factor (TNF)alpha mRNA and protein expression, reduced Akt phosphorylation and nuclear localization of NFkappaB-p65 in nuclei of cells in the hepatic sinusoid. Expression of inducible nitric oxide synthase and cyclooxygenase-2, TNFalpha-regulated genes that have hepatoprotective function, was attenuated in egr-1-/- mice compared to wild-type mice. Although plasma interleukin (IL)-6 protein and hepatic accumulation of IL-6, glycoprotein 130, and IL-6 receptor alpha mRNA in wild-type and egr-1-/- mice were equivalent, signal transducer and activator of transcription 3 phosphorylation was attenuated in egr-1-/- mice and associated with reduced oncostatin M expression. CONCLUSIONS In contrast to its role in inflammation-mediated tissue injury in other models, Egr-1 expression promotes protection in the liver after CCl(4) exposure.

Protective role of HO-1 and carbon monoxide in ethanol-induced hepatocyte cell death and liver injury in mice

BACKGROUND & AIMS Alcoholic liver disease is associated with inflammation and cell death. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-apoptotic and anti-inflammatory properties. Here we tested the hypothesis that induction of HO-1 or treatment with a carbon monoxide releasing molecule (CORM) during chronic ethanol exposure protects and/or reverses ethanol-induced liver injury. METHODS Female C57BL/6J mice were allowed free access to a complete liquid diet containing ethanol or to pair-fed control diets for 25days. Mice were treated with cobalt protoporphyrin (CoPP) to induce HO-1 expression during ethanol feeding or once liver injury had been established. Mice were also treated with CORM-A1, a CO-releasing molecule (CORM), after ethanol-induced liver injury was established. The impact of HO-1 induction on ethanol-induced cell death was investigated in primary cultures of hepatocytes. RESULTS Induction of HO-1 during or after ethanol feeding, as well as treatment with CORM-A1, ameliorated ethanol-induced increases in AST and expression of mRNAs for inflammatory cytokines. Treatment with CoPP or CORM-A1 also reduced hepatocyte cell death, indicated by decreased accumulation of CK18 cleavage products and reduced RIP3 expression in hepatocytes. Exposure of primary hepatocyte cultures to ethanol increased their sensitivity to TNFα-induced cell death; this response was attenuated by necrostatin-1, an inhibitor of necroptosis, but not by caspase inhibitors. Induction of HO-1 with CoPP or CORM-3 treatment normalized the sensitivity of hepatocytes to TNFα-induced cell death after ethanol exposure. CONCLUSIONS Therapeutic strategies to increase HO-1 and/or modulate CO availability ameliorated chronic ethanol-induced liver injury in mice, at least in part by decreasing hepatocellular death.

Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl4, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl4) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl4, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl4. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis. Conclusion Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl4. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol.