Megan R. McMullen

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)

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.

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.

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.

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression ROLE OF TNF-α IN ADIPONECTIN-STIMULATED INTERLEUKIN-10 PRODUCTION

Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-α expression in RAW264.7 macrophages; this TNF-α then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-α mRNA accumulation were associated with increased TNF-α promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFκB; mutation of either the Egr-1 or NFκB binding sites in the TNF-α promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IκB super-repressor prevented gAcrp-stimulated TNF-α promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-α promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-α production by macrophages via ERK1/2→Egr-1 and NFκB-dependent mechanisms; these increases in TNF-α in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.

The anti‐inflammatory effects of adiponectin are mediated via a heme oxygenase‐1–dependent pathway in rat Kupffer cells

Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor alpha (TNF‐α) expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with adiponectin, an anti‐inflammatory adipokine. Here we tested the hypothesis that adiponectin‐mediated suppression of LPS signaling in Kupffer cells is mediated via an interleukin‐10 (IL‐10)/heme oxygenase‐1 (HO‐1) pathway after chronic ethanol feeding. Knockdown of IL‐10 expression in primary cultures of Kupffer cells with small interfering RNA (siRNA) prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS‐stimulated TNF‐α expression. gAcrp increased IL‐10 mRNA and protein expression, as well as expression of the IL‐10 inducible gene, HO‐1; expression was higher in Kupffer cells from ethanol‐fed rats compared with pair‐fed controls. Although IL‐10 receptor surface expression on Kupffer cells was not affected by ethanol feeding, IL‐10–mediated phosphorylation of STAT3 and expression of HO‐1 was higher in Kupffer cells after ethanol feeding. Inhibition of HO‐1 activity, either by treatment with the HO‐1 inhibitor zinc protoporphyrin or by siRNA knockdown of HO‐1, prevented the inhibitory effect of gAcrp on LPS‐stimulated TNF‐α expression in Kupffer cells. LPS‐stimulated TNF‐α expression in liver was increased in mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO‐1 expression, ethanol‐induced sensitivity to LPS was ameliorated. Conclusion: gAcrp prevents LPS‐stimulated TNF‐α expression in Kupffer cells through the activation of the IL‐10/STAT3/HO‐1 pathway. Kupffer cells from ethanol‐fed rats are highly sensitive to the anti‐inflammatory effects of gAcrp; this sensitivity is associated with both increased expression and sensitivity to IL‐10. (HEPATOLOGY 2010.)

Short-term treatment of RAW264.7 macrophages with adiponectin increases TNF-α expression via ERK1/2 activation and EGR-1 expression: Role of TNF-α in adiponectin-stimulated IL-10 production

Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-α expression in RAW264.7 macrophages; this TNF-α then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-α mRNA accumulation were associated with increased TNF-α promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFκB; mutation of either the Egr-1 or NFκB binding sites in the TNF-α promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IκB super-repressor prevented gAcrp-stimulated TNF-α promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-α promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-α production by macrophages via ERK1/2→Egr-1 and NFκB-dependent mechanisms; these increases in TNF-α in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.

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.)

Mechanisms for the anti-inflammatory effects of adiponectin in macrophages.

Adiponectin is an adipokine with potent anti‐inflammatory properties. The development of alcoholic liver disease is thought to involve increased pro‐inflammatory activity, mediated in part by the activation of hepatic macrophages (Kupffer cells). Chronic ethanol feeding sensitizes hepatic macrophages to activation by lipopolysaccharide (LPS), leading to increased production of reactive oxygen species and tumor necrosis factor‐α (TNF‐α). Adiponectin can normalize Toll‐like receptor‐4 (TLR‐4) mediated signaling in hepatic macrophages after ethanol feeding, likely contributing to the hepatoprotective effect of adiponectin in the progression of alcoholic liver disease. However, the mechanisms by which adiponectin suppress TLR‐4 mediated responses are not well understood. Using the macrophage‐like cell line, RAW264.7, we have investigated the molecular mechanisms by which adiponectin suppresses LPS‐stimulated TNF‐α production. Globular adiponectin (gAcrp)‐mediated desensitization of LPS‐stimulated responses in RAW264.7 macrophages was dependent on the production of the anti‐inflammatory cytokine interleukin (IL)‐10. gAcrp initially increased TNF‐α expression in RAW264.7 macrophages; this TNF‐α then contributed to increased expression of IL‐10. This initial gAcrp‐mediated increase in TNF‐α production by macrophages was mediated via activation of ERK1/2→Egr‐1 and nuclear factor (NF)‐κB‐dependent mechanisms. gAcrp‐stimulated IL‐10 expression was also dependent on the phosphorylation of cAMP response element‐binding protein and the cAMP response element in the IL‐10 promoter. In summary, these studies reveal a complex, integrated response of macrophages to gAcrp. gAcrp initially activated signaling pathways considered to be pro‐inflammatory, with a subsequent increase in the expression of the potent, anti‐inflammatory cytokine, IL‐10. Increased IL‐10 expression was ultimately required for the suppression of TLR4‐mediated signaling by gAcrp.