Michele T. Pritchard

Chronic ethanol feeding increases activation of NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role of increased reactive oxygen in LPS-stimulated ERK1/2 activation and TNF-α production

Reactive oxygen species (ROS) contribute to the development of chronic ethanol‐induced liver injury. Although ROS modulate the activity of many signal transduction pathways, the molecular targets of ROS during ethanol exposure are not well understood. Here, we investigated whether specific ROS‐sensitive signal transduction pathways contribute to increased tumor necrosis factor α (TNF‐α) production by Kupffer cells after chronic ethanol feeding to rats. Lipopolysaccharide (LPS) rapidly increased ROS production, measured by dihydrorhodamine fluorescence, in Kupffer cells from ethanol‐ and pair‐fed rats, and ROS production was 2.5‐fold greater in ethanol‐fed compared with pair‐fed. Pretreatment with diphenyleneiodonium (DPI), which inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalized ROS production in Kupffer cells from ethanol‐fed rats. LPS rapidly increased Rac1‐guanosinetriphosphatase (GTPase) activity and p67phox translocation to the plasma membrane in Kupffer cells from pair‐fed rats. After ethanol feeding, Rac1‐GTPase activity was already increased over pair‐fed at baseline and remained elevated over pair‐fed after LPS stimulation. Further, LPS‐stimulated p67phox translocation to the plasma membrane was enhanced after chronic ethanol feeding. LPS‐stimulated extracellular signal‐regulated kinase (ERK)1/2 and p38 phosphorylation, two signaling pathways regulated by ROS, were increased twofold in Kupffer cells from ethanol‐fed rats compared with pair‐fed controls. However, only LPS‐stimulated ERK1/2 phosphorylation was inhibited by DPI, which also reduced LPS‐stimulated TNF‐α production in Kupffer cells from pair‐ and ethanol‐fed rats. These results demonstrate that chronic ethanol feeding increases LPS‐stimulated NADPH oxidase‐dependent production of ROS in Kupffer cells. Further, ERK1/2 is an important target of NADPH oxidase‐derived ROS in Kupffer cells, contributing to enhanced LPS‐stimulated TNF‐α production by Kupffer cells after chronic ethanol feeding.

Obesity, diabetes mellitus, and liver fibrosis

Obesity is a global epidemic with more than 1 billion overweight adults and at least 300 million obese patients worldwide. Diabetes is characterized by a defect in insulin secretion or a decrease in sensitivity to insulin, which results in elevated fasting blood glucose. Both obesity and elevated fasting glucose are risk factors for nonalcoholic fatty liver disease, a disease spectrum that includes hepatic steatosis (nonalcoholic fatty liver), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Increased adiposity and insulin resistance contribute to the progression from NASH to fibrosis through the development of a profibrotic mileau in the liver, including increased hepatocellular death, increased reactive oxygen species generation, and an altered adipokine/cytokine balance. This review will summarize recent advances in our understanding of the pathological interactions among excessive fat accumulation, insulin resistance, and hepatic fibrogenesis and discuss specific molecular pathways that may be of interest in the development of therapeutic interventions to prevent and/or reverse hepatic fibrosis.

Kupffer Cells in the Liver

Kupffer cells are a critical component of the mononuclear phagocytic system and are central to both the hepatic and systemic response to pathogens. Kupffer cells are reemerging as critical mediators of both liver injury and repair. Kupffer cells exhibit a tremendous plasticity; depending on the local metabolic and immune environment, then can express a range of polarized phenotypes, from the proinflammatory M1 phenotype to the alternative/M2 phenotype. Multiple M2 phenotypes can be distinguished, each involved in the resolution of inflammation and wound healing. Here, we have provided an update on recent research that has contributed to the developing delineation of the contribution of Kupffer cells to different types of liver injury, with an emphasis on alcoholic and nonalcoholic liver diseases. These recent advances in our understanding of Kupffer cell function and regulation will likely provide new insights into the potential for therapeutic manipulation of Kupffer cells to promote the resolution of inflammation and enhance wound healing in liver disease.

Differential Contributions of C3, C5, and Decay-Accelerating Factor to Ethanol-Induced Fatty Liver in Mice

BACKGROUND AND AIMS The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.

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.)

Regulation of macrophage activation in alcoholic liver disease

Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of tumor necrosis factor α (TNFα). The regulation of TNFα synthesis is controlled by both transcriptional and post‐transcriptional mechanisms via the integration of complex signal transduction pathways activated in response to LPS exposure. Recent data has shown that increased LPS‐stimulated phosphorylation of extracellular signal‐regulated kinase pathway 1/2 (ERK1/2) is one of the important molecular targets of chronic ethanol in Kupffer cells. This increased activation of ERK1/2 after chronic ethanol is associated with increased expression of Egr‐1, a transcription factor required for enhanced LPS‐stimulated TNFα mRNA expression after chronic ethanol exposure. egr‐1 null mice are protected from the development of fatty liver injury in response to chronic ethanol feeding, identifying an essential role for Egr‐1 in the development of chronic ethanol‐induced liver injury. Here we review recent studies aimed at understanding the mechanisms by which chronic ethanol enhances the LPS→ERK1/2→Egr‐1→ΤNFα pathway in Kupffer cells. These studies identify a critical role for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐derived reactive oxygen species in the activation of ERK1/2 and subsequent production of TNFα in Kupffer cells after chronic ethanol feeding.

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.

PAI-1 plays a protective role in CCl4-induced hepatic fibrosis in mice: role of hepatocyte division

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein that has been shown to play a role in experimental fibrosis caused by bile duct ligation (BDL) in mice. However, its role in more severe models of hepatic fibrosis (e.g., carbon tetrachloride; CCl(4)) has not been determined and is important for extrapolation to human disease. Wild-type or PAI-1 knockout mice were administered CCl(4) (1 ml/kg body wt ip) 2x/wk for 4 wk. Plasma (e.g., transaminase activity) and histological (e.g., Sirius red staining) indexes of liver damage and fibrosis were evaluated. Proliferation and apoptosis were assessed by PCNA and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively, as well as by indexes of cell cycle (e.g., p53, cyclin D1). In contrast to previous studies with BDL, hepatic fibrosis was enhanced in PAI-1(-/-) mice after chronic CCl(4) administration. Indeed, all indexes of liver damage were elevated in PAI-1(-/-) mice compared with wild-type mice. This enhanced liver damage correlated with impaired hepatocyte proliferation. A similar effect on proliferation was observed after one bolus dose of CCl(4), without concomitant increases in liver damage. Under these conditions, a decrease in phospho-p38, coupled with elevated p53 protein, was observed; these results suggest impaired proliferation and a potential G(1)/S cell cycle arrest in PAI-1(-/-) mice. These data suggest that PAI-1 may play multiple roles in chronic liver diseases, both protective and damaging, the latter mediated by its influence on inflammation and fibrosis and the former via helping maintain hepatocyte division after an injury.

Hepatic Fibrosis Is Enhanced and Accompanied by Robust Oval Cell Activation after Chronic Carbon Tetrachloride Administration to Egr-1-Deficient Mice

The transcription factor early growth response (Egr)-1 regulates the expression of genes required for execution of the wound healing response. Multiple cycles of injury, coupled to incomplete wound healing, lead to fibrosis. Therefore, we hypothesized that Egr-1 is required for the development of hepatic fibrosis. To test this hypothesis, we exposed wild-type and egr-1(-/-) mice to acute or chronic carbon tetrachloride (CCl(4)). Acute CCl(4) exposure established a profibrotic milieu in the liver, including activation of hepatic stellate cells as well as expression of type 1 collagen genes and tissue inhibitor of matrix metalloproteinase 1 in both wild-type and egr-1(-/-) mice. This response was exacerbated in egr-1(-/-) mice. After chronic CCl(4) exposure, hepatic fibrosis was established in both genotypes; however, the fibrotic response was profoundly worsened in Egr-1-deficient mice. Importantly, enhanced fibrosis in egr-1(-/-) mice was accompanied by a robust activation of the oval cell response, suggesting more severe liver injury and/or reduced hepatocyte proliferation when compared with wild-type mice. Hepatic expression of genes indicative of oval cell activation, as well as the number of cells expressing A6, a mouse oval cell marker, was greater in egr-1(-/-) mice. Taken together, these data reveal novel roles for Egr-1 as a negative regulator of both CCl(4)-induced hepatic fibrosis and the oval cell response.

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.