Resolvin D1 primes the resolution process initiated by calorie restriction in obesity-induced steatohepatitis.

Insulin resistance and nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis combined with inflammation, are major sequelae of obesity. Currently, lifestyle modification (i.e., weight loss) is the first-line therapy for NASH. However, weight loss resolves steatosis but not inflammation. In this study, we tested the ability of resolvin D1 (RvD1), an anti-inflammatory and proresolving molecule, to promote the resolution initiated by calorie restriction in obese mice with NASH. Calorie restriction reduced adipose and liver weight (-56 and -13%, respectively; P<0.001), serum leptin and resistin levels, hepatic steatosis, and insulin resistance. In addition to these, mice receiving RvD1 during the dietary intervention showed increased adiponectin expression at both the mRNA and protein levels and reduced liver macrophage infiltration (-15%, P<0.01). Moreover, RvD1 skewed macrophages from an M1- to an M2-like anti-inflammatory phenotype, induced a specific hepatic miRNA signature (i.e., miR-219-5p and miR-199a-5p), and reduced inflammatory adipokine mRNA and protein expression and macrophage innate immune response. In precision-cut liver slices (PCLSs), which override the influence of circulating factors, RvD1 attenuated hypoxia-induced mRNA and protein expression of COX-2, IL-1ß, IL-6, and CCR7. Of note, RvD1 anti-inflammatory actions were absent in macrophage-depleted PCLSs. In summary, RvD1 acts as a facilitator of the hepatic resolution process by reducing the inflammatory component of obesity-induced NASH.

Molecular interplay between Δ5/Δ6 desaturases and long-chain fatty acids in the pathogenesis of non-alcoholic steatohepatitis.

OBJECTIVE: The mechanisms underlying non-alcoholic steatohepatitis (NASH) are not completely elucidated. In the current study we integrated gene expression profiling of liver biopsies from NASH patients with translational studies in mouse models of steatohepatitis and pharmacological interventions in isolated hepatocytes to identify new molecular targets in NASH. DESIGN AND RESULTS: Using oligonucleotide microarray analysis we identified a significant enrichment of genes involved in the multi-step catalysis of long-chain polyunsaturated fatty acids, namely, Δ-5 desaturase (Δ5D) and Δ6D in NASH. Increased expression of Δ5D and Δ6D at both mRNA and protein level were confirmed in livers from mice with high-fat diet-induced obesity and NASH. Gas chromatography analysis revealed impaired desaturation fluxes toward the ω-6 and ω-3 pathways resulting in increased ω-6 to ω-3 ratio and reduced ω-3 index in human and mouse fatty livers. Restoration of hepatic ω-3 content in transgenic fat-1 mice expressing an ω-3 desaturase, which allows the endogenous conversion of ω-6 into ω-3 fatty acids, produced a significant reduction in hepatic insulin resistance, steatosis, macrophage infiltration, necroinflammation and lipid peroxidation, accompanied by attenuated expression of genes involved in inflammation, fatty acid uptake and lipogenesis. These results were mostly reproduced by feeding obese mice with an exogenous ω-3-enriched diet. A combined Δ5D/Δ6D inhibitor, CP-24879, significantly reduced intracellular lipid accumulation and inflammatory injury in hepatocytes. Interestingly, CP-24879 exhibited superior antisteatotic and anti-inflammatory actions in fat-1 and ω-3-treated hepatocytes. CONCLUSIONS: These findings indicate that impaired hepatic fatty acid desaturation and unbalanced ω-6 to ω-3 ratio play a role in the pathogenesis of NASH.

Coordinate functional regulation between microsomal prostaglandin E synthase-1 (mPGES-1) and peroxisome proliferator-activated receptor γ (PPARγ) in the conversion of white-to-brown adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.

Cell-specific PPARγ deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells.

BACKGROUND & AIMS: PPARγ plays an essential role in the transcriptional regulation of genes involved in lipid and glucose metabolism, insulin sensitivity, and inflammation. We recently demonstrated that PPARγ plays a causative role in hepatocyte lipid deposition, contributing to the pathogenesis of hepatic steatosis. In this study, we investigated the role of PPARγ in the inflammatory and fibrogenic response of the liver. METHODS: Heterozygous floxed/null Cre/LoxP mice with targeted deletion of PPARγ in either hepatocytes (Alb-Cre), macrophages (LysM-Cre) or hepatic stellate cells (HSCs) (aP2-Cre) were submitted to carbon tetrachloride (CCl4) liver injury. Further analyses were performed in precision-cut liver slices (PCLS) and primary cultures of hepatocytes, macrophages, and HSCs. RESULTS: LysM-Cre mice displayed an exacerbated response to chronic CCl4 injury and showed higher necroinflammatory injury, lipid peroxidation, inflammatory infiltrate, cleaved-caspase-3 and caspase 3/7 activity, and COX-2, TNF-α, CXCL2, and IL-1ß expression than Alb-Cre and control mice. The deleterious effects of PPARγ disruption in liver macrophages were confirmed in an acute model of CCl4 injury as well as in PCLS incubated with LPS. Moreover, LysM-Cre mice showed an aggravated fibrogenic response to CCl4, as revealed by more prominent Sirius Red and Masson's trichrome staining, elevated hydroxyproline content and induced α-SMA and TIMP-1 expression. Importantly, aP2-Cre mice with specific disruption of PPARγ in HSCs, as confirmed by immunocytochemical analysis of individual liver cells, also showed exacerbated liver damage and fibrogenic response to CCl4. CONCLUSIONS: These data unveil anti-inflammatory and anti-fibrogenic roles for PPARγ in non-parenchymal liver cells.

Resolvin D1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an M2-like phenotype.

We recently demonstrated that ω-3-polyunsaturated fatty acids ameliorate obesity-induced adipose tissue inflammation and insulin resistance. In this study, we report novel mechanisms underlying ω-3-polyunsaturated fatty acid actions on adipose tissue, adipocytes, and stromal vascular cells (SVC). Inflamed adipose tissue from high-fat diet-induced obese mice showed increased F4/80 and CD11b double-positive macrophage staining and elevated IL-6 and MCP-1 levels. Docosahexaenoic acid (DHA; 4 µg/g) did not change the total number of macrophages but significantly reduced the percentage of high CD11b/high F4/80-expressing cells in parallel with the emergence of low-expressing CD11b/F4/80 macrophages in the adipose tissue. This effect was associated with downregulation of proinflammatory adipokines in parallel with increased expression of IL-10, CD206, arginase 1, resistin-like molecule α, and chitinase-3 like protein, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. This shift was confined to the SVC fraction, in which secretion of Th1 cytokines (IL-6, MCP-1, and TNF-α) was blocked by DHA. Notably, resolvin D1, an anti-inflammatory and proresolving mediator biosynthesized from DHA, markedly attenuated IFN-γ/LPS-induced Th1 cytokines while upregulating arginase 1 expression in a concentration-dependent manner. Resolvin D1 also stimulated nonphlogistic phagocytosis in adipose SVC macrophages by increasing both the number of macrophages containing ingested particles and the number of phagocytosed particles and by reducing macrophage reactive oxygen species production. No changes in adipocyte area and the phosphorylation of hormone-sensitive lipase, a rate-limiting enzyme regulating adipocyte lipolysis, were observed. These findings illustrate novel mechanisms through which resolvin D1 and its precursor DHA confer anti-inflammatory and proresolving actions in inflamed adipose tissue.

An investigation of the resolution of inflammation (catabasis) in COPD.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is characterized by an enhanced inflammatory response to smoking that persists despite quitting. The resolution of inflammation (catabasis) is a complex and highly regulated process where tissue resident macrophages play a key role since they phagocytose apoptotic cells (efferocytosis), preventing their secondary necrosis and the spill-over of their pro-inflammatory cytoplasmic content, and release pro-resolution and tissue repair molecules, such as TGFß, VEGF and HGF. Because inflammation does not resolve in COPD, we hypothesized that catabasis may be abnormal in these patients. METHODS: To explore this hypothesis, we studied lung tissue samples obtained at surgery from 21 COPD patients, 22 smokers with normal spirometry and 13 non-smokers controls. In these samples we used: (1) immunohistochemistry to assess the expression of CD44, CD36, VEGF and TGFß in lung macrophages; (2) real time PCR to determine HGF, PPARγ, TGFß, VEGF and MMP-9 gene expression; and, (3) ELISA to quantify lipoxin A4, a lipid mediator of catabasis. RESULTS: We found that current and former smokers with COPD showed: (1) more inflammation (higher MMP-9 expression); (2) reduced macrophage surface expression of CD44, a key efferocytosis receptor; and, (3) similar levels of TGFß, VEGF, HGF, PPARγ, and lipoxin A4 than smokers with normal spirometry, despite the presence of inflammation and disease. CONCLUSIONS: These results identify several potential abnormalities of catabasis in patients with COPD.

Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts.

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and ß-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.

5-lipoxygenase activating protein signals adipose tissue inflammation and lipid dysfunction in experimental obesity.

The presence of the so-called low-grade inflammatory state is recognized as a critical event in adipose tissue dysfunction, leading to altered secretion of adipokines and free fatty acids (FFAs), insulin resistance, and development of hepatic complications associated with obesity. This study was designed to investigate the potential contribution of the proinflammatory 5-lipoxygenase (5-LO) pathway to adipose tissue inflammation and lipid dysfunction in experimental obesity. Constitutive expression of key components of the 5-LO pathway, as well as leukotriene (LT) receptors, was detected in adipose tissue as well as in adipocyte and stromal vascular fractions. Adipose tissue from obese mice, compared with that from lean mice, exhibited increased 5-LO activating protein (FLAP) expression and LTB(4) levels. Incubation of adipose tissue with 5-LO products resulted in NF-kappaB activation and augmented secretion of proinflammatory adipokines such as MCP-1, IL-6, and TNF-alpha. In addition, LTB(4), but not LTD(4), reduced FFA uptake in primary adipocytes, whereas 5-LO inhibition suppressed isoproterenol-induced adipose tissue lipolysis. In mice with dietary obesity, elevated FLAP expression in adipose tissue was paralleled with macrophage infiltration, increased circulating FFA levels, and hepatic steatosis, phenomena that were reversed by FLAP inhibition with Bay-X-1005. Interestingly, FLAP inhibition induced AMP-activated protein kinase phosphorylation in parallel with decreases in hormone-sensitive lipase activity and the expression and secretion of TNF-alpha and IL-6. Similar effects were observed in differentiated 3T3-L1 adipocytes incubated with either Bay-X-1005 or the selective LTB(4) receptor antagonist U-75302. Taken together, these findings indicate that the 5-LO pathway signals the adipose tissue low-grade inflammatory state and steatogenic potential in experimental obesity.

Disruption of the 12/15-lipoxygenase gene (Alox15) protects hyperlipidemic mice from nonalcoholic fatty liver disease.

UNLABELLED: We have shown that Alox15, the gene encoding for 12/15-lipoxygenase (12/15-LO), is markedly up-regulated in livers from apolipoprotein E-deficient (ApoE(-/-)) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE(-/-) mice with a targeted disruption of the Alox15 gene to assess the role of 12/15-LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE(-/-) mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15-LO double-knockout (ApoE(-/-)/12/15-LO(-/-)) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-18, and IL-6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high-fat diet-induced insulin resistance, up-regulated insulin receptor substrate-2, and exerted opposite effects on hepatic c-Jun amino-terminal kinase and adenosine monophosphate-activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin-resistant adipokines MCP-1, TNFα, and resistin while increasing the expression of glucose transporter-4. Interestingly, compared with ApoE(-/-) mice, which exhibited increased hepatic caspase-3 staining, ApoE(-/-)/12/15-LO(-/-) mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE(-/-) mice were more vulnerable to TNFα-induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. CONCLUSION: Collectively, our data suggest a potentially relevant mechanism linking 12/15-LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin.

5-lipoxygenase deficiency reduces hepatic inflammation and tumor necrosis factor alpha-induced hepatocyte damage in hyperlipidemia-prone ApoE-null mice.

UNLABELLED: The actual risk factors that drive hepatic inflammation during the transition from steatosis to steatohepatitis are unknown. We recently demonstrated that hyperlipidemia-prone apolipoprotein E-deficient (ApoE(-/-)) mice exhibit hepatic steatosis and increased susceptibility to hepatic inflammation and advanced fibrosis. Because the proinflammatory 5-lipoxygenase (5-LO) pathway was found to be up-regulated in these mice and given that 5-LO deficiency confers cardiovascular protection to ApoE(-/-) mice, we determined the extent to which the absence of 5-LO would alter liver injury in these mice. Compared with ApoE(-/-) mice, which showed expected hepatic steatosis and inflammation, ApoE/5-LO double-deficient (ApoE(-/-)/5-LO(-/-)) mice exhibited reduced hepatic inflammation, macrophage infiltration, tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-18 expression, caspase-3 and nuclear factor-kappaB (NF-kappaB) activities, and serum alanine aminotransferase levels in the absence of changes in hepatic steatosis. The lack of 5-LO produced a remarkable insulin-sensitizing effect in the adipose tissue because peroxisome proliferator-activated receptor gamma, insulin receptor substrate-1, and adiponectin were up-regulated, whereas c-Jun amino-terminal kinase phosphorylation and MCP-1 and IL-6 expression were down-regulated. On the other hand, hepatocytes isolated from ApoE(-/-)/5-LO(-/-) mice were more resistant to TNF-alpha-induced apoptosis. The 5-LO products leukotriene (LT) B(4), LTD(4), and 5-HETE consistently triggered TNF-alpha-induced apoptosis and compromised hepatocyte survival by suppressing NF-kappaB activity in the presence of actinomycin D. Moreover, ApoE(-/-)/5-LO(-/-) mice were protected against sustained high-fat diet (HFD)-induced liver injury and hepatic inflammation, macrophage infiltration and insulin resistance were significantly milder than those of ApoE(-/-) mice. Finally, pharmacological inhibition of 5-LO significantly reduced hepatic inflammatory infiltrate in the HFD and ob/ob models of fatty liver disease. CONCLUSION: These combined data indicate that hyperlipidemic mice lacking 5-LO are protected against hepatic inflammatory injury, suggesting that 5-LO is involved in mounting hepatic inflammation in metabolic disease.

Obesity-induced insulin resistance and hepatic steatosis are alleviated by omega-3 fatty acids: a role for resolvins and protectins.

Omega-3-polyunsaturated fatty acids (omega-3-PUFAs) have well-documented protective effects that are attributed not only to eicosanoid inhibition but also to the formation of novel biologically active lipid mediators (i.e., resolvins and protectins). In this study, we examined their effects on ob/ob mice, an obesity model of insulin resistance and fatty liver disease. Dietary intake of omega-3-PUFAs had insulin-sensitizing actions in adipose tissue and liver and improved insulin tolerance in obese mice. Genes involved in insulin sensitivity (PPARgamma), glucose transport (GLUT-2/GLUT-4), and insulin receptor signaling (IRS-1/IRS-2) were up-regulated by omega-3-PUFAs. Moreover, omega-3-PUFAs increased adiponectin, an anti-inflammatory and insulin-sensitizing adipokine, and induced AMPK phosphorylation, a fuel-sensing enzyme and a gatekeeper of the energy balance. Concomitantly, hepatic steatosis was alleviated by omega-3-PUFAs. A lipidomic analysis with liquid chromatography/mass spectrometry/mass spectrometry revealed that omega-3-PUFAs inhibited the formation of omega-6-PUFA-derived eicosanoids, while triggering the formation of omega-3-PUFA-derived resolvins and protectins. Moreover, representative members of these lipid mediators, namely resolvin E1 and protectin D1, mimicked the insulin-sensitizing and antisteatotic effects of omega-3-PUFAs and induced adiponectin expression to a similar extent that of rosiglitazone, a member of the thiazolidinedione family of antidiabetic drugs. Taken together, these findings uncover beneficial actions of omega-3-PUFAs and their bioactive lipid autacoids in preventing obesity-induced insulin resistance and hepatic steatosis.

Resolution of adipose tissue inflammation.

The presence of the so-called "low-grade" inflammatory state is recognized as a critical event in adipose tissue dysfunction in obesity. This chronic "low-grade" inflammation in white adipose tissue is powerfully augmented through the infiltration of macrophages, which, together with adipocytes, perpetuate a vicious cycle of macrophage recruitment and secretion of free fatty acids and deleterious adipokines that predispose the development of obesity-related comorbidities, such as insulin resistance and nonalcoholic fatty liver disease. In the last decade, many factors have been identified that contribute to mounting uncontrolled inflammation in obese adipose tissue. Among them, bioactive lipid mediators derived from the cyclooxygenase and 5-lipoxygenase pathways, which convert the omega-6-polyunsaturated fatty acid (PUFA) arachidonic acid into potent proinflammatory eicosanoids (i.e., prostaglandins [PGs] and leukotrienes), have emerged. Interestingly, the same lipid mediators that initially trigger the inflammatory response also signal the termination of inflammation by stimulating the biosynthesis of anti-inflammatory and proresolving lipid autacoids. This review discusses the current status, characteristics, and progress in this class of "stop signals", including the lipoxins, which were the first identified omega-6 PUFA-derived lipid mediators with potent anti-inflammatory properties; the recently described omega-3 PUFA-derived lipid mediators resolvins and protectins; and the cyclopentenone PGs of the D series. Special emphasis is given to the participation of these bioactive lipid autacoids in the resolution of adipose tissue inflammation and in preventing the development of obesity-related complications.

[Basic mechanisms of hepatocellular injury. Role of inflammatory lipid mediators]. / Mecanismos básicos de lesión hepatocelular. Papel de los mediadores lipídicos de inflamación.

The presence of a lesion in the cellular parenchyma is common to a large number of chronic liver diseases, such as viral hepatitides, alcoholic hepatitis, chronic cholestasis and steatohepatitis. Although the pathogenesis may vary according to the etiological agent, a series of mechanisms is common to all. Notable among these mechanisms are Kupffer cell activation and inflammatory cell recruitment, free oxygen radical formation and the development of oxidative stress, cytokine production, mainly TNFa and TGFb, and inflammatory mediator release due to arachidonic acid oxidation through the COX-2 and 5-LO pathways.

Comparative protection against liver inflammation and fibrosis by a selective cyclooxygenase-2 inhibitor and a nonredox-type 5-lipoxygenase inhibitor.

In this study, we examined the relative contribution of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), two major proinflammatory pathways up-regulated in liver disease, to the progression of hepatic inflammation and fibrosis. Separate administration of 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), a selective COX-2 inhibitor, and CJ-13,610, a 5-LO inhibitor, to carbon tetrachloride-treated mice significantly reduced fibrosis as revealed by the analysis of Sirius Red-stained liver sections without affecting necroinflammation. Conversely, combined administration of SC-236 and 4-[3-[4-(2-methylimidazol-1-yl)-phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (CJ-13,610) reduced both necroinflammation and fibrosis. These findings were confirmed in 5-LO-deficient mice receiving SC-236, which also showed reduced hepatic monocyte chemoattractant protein 1 expression. Interestingly, SC-236 and CJ-13,610 significantly increased the number of nonparenchymal liver cells with apoptotic nuclei (terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive). Additional pharmacological profiling of SC-236 and CJ-13,610 was performed in macrophages, the primary hepatic inflammatory cell type. In these cells, SC-236 inhibited prostaglandin (PG) E2 formation in a concentration-dependent manner, whereas CJ-13,610 blocked leukotriene B4 biosynthesis. Of note, the simultaneous addition of SC-236 and CJ-13,610 resulted in a higher inhibitory profile on PGE2 biosynthesis than the dual COX/5-LO inhibitor licofelone. These drugs differentially regulated interleukin-6 mRNA expression in macrophages. Taken together, these findings indicate that both COX-2 and 5-LO pathways are contributing factors to hepatic inflammation and fibrosis and that these two pathways of the arachidonic acid cascade represent potential targets for therapy.

Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA.

Docosahexaenoic acid (DHA) is a omega-3 essential fatty acid that reduces the incidence and severity of a number of diseases. Recently, a novel series of DHA-derived lipid mediators with potent protective actions has been identified. In this study we demonstrate that dietary amplification of these DHA-derived products protects the liver from necroinflammatory injury. In vitro, supplementation of hepatocytes with DHA significantly reduced hydrogen peroxide-induced DNA damage, evaluated by the "comet assay," and oxidative stress, determined by measurement of malondialdehyde levels. In vivo, dietary supplementation of mice with DHA ameliorated carbon tetrachloride-induced necroinflammatory damage. In addition, hepatic cyclooxygenase-2 expression and PGE2 levels were significantly reduced in mice fed DHA-enriched diets. In these animals, increased hepatic formation of DHA-derived lipid mediators (i.e., 17S-hydroxy-DHA (17S-HDHA) and protectin D1) was detected by HPLC-gas chromatography/mass spectrometry analysis. Consistent with these findings, synthetic 17-HDHA abrogated genotoxic and oxidative damage in hepatocytes and decreased TNF-alpha release and 5-lipoxygenase expression in macrophages. In a transactivation assay, 17-HDHA acted in a concentration-dependent manner as a PPARgamma agonist. Taken together, these findings identify a potential role for DHA-derived products, specifically 17S-HDHA and protectin D1, in mediating the protective effects of dietary DHA in necroinflammatory liver injury.

Association of a variant in the gene encoding for ERV1/ChemR23 with reduced inflammation in visceral adipose tissue from morbidly obese individuals.

Obesity comorbidities are closely associated with chronic low-grade adipose tissue inflammation. A number of SNPs associated with inflammation has been identified, underscoring the impact of genetic determinants on this process. Here, we screened SNPs in genes with pro-inflammatory (IL-1ß, IL-6, STAT3 and JAK2), anti-inflammatory (IL-10 and SOCS3) and pro-resolving (ERV1/ChemR23) properties in 101 obese and 99 non-obese individuals. Among the SNPs analyzed, we identified that individuals carrying a C allele in the rs1878022 polymorphism of the ERV1/ChemR23 gene, which encodes for the receptor of the pro-resolving mediator RvE1, had increased ERV1/ChemR23 protein expression and reduced levels of the inflammatory cytokine IL-6 in adipose tissue. Moreover, patients carrying the C allele in homozygosity had lower plasma levels of IL-6, IFN-α2, IL-15, IL-1ra, IL-10, GM-CSF, G-CSF and VEGF and enhanced leukocyte responsiveness to RvE1. C-carriers also exhibited decreased TAG to HDL ratio, a surrogate marker of insulin resistance and a predictor of incident fatty liver. Finally, we confirmed in vivo that the ERV1/ChemR23 receptor regulates systemic and tissue inflammation since mice lacking ERV1/ChemR23 expression showed increased IL-6 levels in adipose tissue and peritoneal macrophages. Together, our study identified an ERV1/ChemR23 variant that protects patients with obesity from excessive inflammatory burden.

Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells.

Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl(4))-induced damage. Rats treated with CCl(4) showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl(4) treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B(4) and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A(4), an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.

Resolution of inflammation in obesity-induced liver disease.

Low-grade inflammation in adipose tissue is recognized as a critical event in the development of obesity-related co-morbidities. This chronic inflammation is powerfully augmented through the infiltration of macrophages, which together with adipocytes, perpetuate a vicious cycle of inflammatory cell recruitment and secretion of free fatty acids and deleterious adipokines that predispose to greater incidence of metabolic complications. In the last decade, many factors have been identified to contribute to mounting unresolved inflammation in obese adipose tissue. Among them, pro-inflammatory lipid mediators (i.e., leukotrienes) derived from the omega-6 polyunsaturated arachidonic acid have been shown to play a prominent role. Of note, the same lipid mediators that initially trigger the inflammatory response also signal its termination by stimulating the formation of anti-inflammatory signals. Resolvins and protectins derived from the omega-3 polyunsaturated docosahexaenoic and eicosapentaenoic acids have emerged as a representative family of this novel class of autacoids with dual anti-inflammatory and pro-resolving properties that act as "stop-signals" of the inflammatory response. This review discusses the participation of these endogenous autacoids in the resolution of adipose tissue inflammation, with a special emphasis in the amelioration of obesity-related metabolic dysfunctions, namely insulin resistance and non-alcoholic fatty liver disease.

New insights into the role of macrophages in adipose tissue inflammation and Fatty liver disease: modulation by endogenous omega-3 Fatty Acid-derived lipid mediators.

Obesity is causally linked to a chronic state of "low-grade" inflammation in adipose tissue. Prolonged, unremitting inflammation in this tissue has a direct impact on insulin-sensitive tissues (i.e., liver) and its timely resolution is a critical step toward reducing the prevalence of related co-morbidities such as insulin resistance and non-alcoholic fatty liver disease. This article describes the current state-of-the-art knowledge and novel insights into the role of macrophages in adipose tissue inflammation, with special emphasis on the progressive changes in macrophage polarization observed over the course of obesity. In addition, this article extends the discussion to the contribution of Kupffer cells, the liver resident macrophages, to metabolic liver disease. Special attention is given to the modulation of macrophage responses by omega-3-PUFAs, and more importantly by resolvins, which are potent anti-inflammatory and pro-resolving autacoids generated from docosahexaenoic and eicosapentaenoic acids. In fact, resolvins have been shown to work as endogenous "stop signals" in inflamed adipose tissue and to return this tissue to homeostasis by inducing a phenotypic switch in macrophage polarization toward a pro-resolving phenotype. Collectively, this article offers new views on the role of macrophages in metabolic disease and their modulation by endogenously generated omega-3-PUFA-derived lipid mediators.

Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols.

The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE-/-) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CCl4)-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE-/- mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-beta1, MCP-1, and TIMP-1 expression than wild-type control mice. After CCl4 challenge, ApoE-/- mice exhibited exacerbated steatosis (Oil Red O staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis (Sirius red staining and alpha-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct correlations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE-/- mice, since exacerbated liver injury was not present in untreated age-paired ApoE-/- mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE-/- mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE-/- mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-kappaB-independent mechanism and upregulated TIMP-1 expression. In macrophages, oxysterols increased TGF-beta1 secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce proinflammatory and profibrogenic mechanisms in liver cells.