Fahrettin Haczeyni

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

BACKGROUND & AIMS NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Exercise improves adipose function and inflammation and ameliorates fatty liver disease in obese diabetic mice

Adipose inflammation and dysfunction underlie metabolic obesity. Exercise improves glycemic control and metabolic indices, but effects on adipose function and inflammation are less clear. Accordingly, it was hypothesized that exercise improves adipose morphometry to reduce adipose inflammation in hyperphagic obese mice.

Obeticholic acid improves adipose morphometry and inflammation and reduces steatosis in dietary but not metabolic obesity in mice

Nonalcoholic steatohepatitis (NASH) is the outcome of interactions between overnutrition, energy metabolism, and adipose function. Obeticholic acid (OCA) improves steatosis in patients but for unknown reasons does not resolve NASH pathology. This study therefore investigated OCA effects in Wt mice, which develop obesity with atherogenic dietary feeding, and appetite‐dysregulated, Alms1 mutant foz/foz mice fed the same diet, which develop metabolic obesity and diabetes.

The selective peroxisome proliferator–activated receptor-delta agonist seladelpar reverses nonalcoholic steatohepatitis pathology by abrogating lipotoxicity in diabetic obese mice

Lipotoxicity associated with insulin resistance is central to nonalcoholic steatohepatitis (NASH) pathogenesis. To date, only weight loss fully reverses NASH pathology, but mixed peroxisome proliferator–activated receptor‐alpha/delta (PPAR‐α/δ) agonists show some efficacy. Seladelpar (MBX‐8025), a selective PPAR‐δ agonist, improves atherogenic dyslipidemia. We therefore used this agent to test whether selective PPAR‐δ activation can reverse hepatic lipotoxicity and NASH in an obese, dyslipidemic, and diabetic mouse model. From weaning, female Alms1 mutant (foz/foz) mice and wild‐type littermates were fed an atherogenic diet for 16 weeks; groups (n = 8‐12) were then randomized to receive MBX‐8025 (10 mg/kg) or vehicle (1% methylcellulose) by gavage for 8 weeks. Despite minimally altering body weight, MBX‐8025 normalized hyperglycemia, hyperinsulinemia, and glucose disposal in foz/foz mice. Serum alanine aminotransferase ranged 300‐600 U/L in vehicle‐treated foz/foz mice; MBX‐8025 reduced alanine aminotransferase by 50%. In addition, MBX‐8025 normalized serum lipids and hepatic levels of free cholesterol and other lipotoxic lipids that were increased in vehicle‐treated foz/foz versus wild‐type mice. This abolished hepatocyte ballooning and apoptosis, substantially reduced steatosis and liver inflammation, and improved liver fibrosis. In vehicle‐treated foz/foz mice, the mean nonalcoholic fatty liver disease activity score was 6.9, indicating NASH; MBX‐8025 reversed NASH in all foz/foz mice (nonalcoholic fatty liver disease activity score 3.13). Conclusion: Seladelpar improves insulin sensitivity and reverses dyslipidemia and hepatic storage of lipotoxic lipids to improve NASH pathology in atherogenic diet–fed obese diabetic mice. Selective PPAR‐δ agonists act independently of weight reduction, but counter lipotoxicity related to insulin resistance, thereby providing a novel therapy for NASH. (Hepatology Communications 2017;1:663–674)

TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival

Background and aims: TLR9 deletion protects against steatohepatitis due to choline-amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods: We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type (Wt), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9-/-, and Tlr9-/-foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results: Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9-/- mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9-/-foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9+/+.foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9-/- mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt Livers from Ath-fed Tlr9-/- mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt, and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology. CONCLUSION Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis.

Causes and mechanisms of adipocyte enlargement and adipose expansion

Adipose tissue plays a significant role in whole body energy homeostasis. Obesity‐associated diabetes, fatty liver and metabolic syndrome are closely linked to adipose stress and dysfunction. Genetic predisposition, overeating and physical inactivity influence the expansion of adipose tissues. Under conditions of constant energy surplus, adipocytes become hypertrophic and adipose tissues undergo hyperplasia so as to increase their lipid storage capacity, thereby keeping circulating blood glucose and fatty acids below toxic levels. Nonetheless, adipocytes have a saturation point where they lose capacity to store more lipids. At this stage, when adipocytes are fully lipid‐engorged, they express stress signals. Adipose depots (particularly visceral compartments) from obese individuals with a severe metabolic phenotype are characterized by the high proportion of hypertrophic adipocytes. This review focuses on the mechanisms of adipocyte enlargement in relation to adipose fatty acid and cholesterol metabolism, and considers how this may be related to adipose dysfunction.

Mouse models of non-alcoholic steatohepatitis: A reflection on recent literature: Systematic review of mouse NASH models

Non‐alcoholic steatohepatitis (NASH) is strongly associated with overnutrition, insulin resistance, and predisposition to type 2 diabetes. To critically analyze the translational significance of currently used animal models of NASH, we reviewed articles published during the last 3 years that studied NASH pathogenesis using mouse models. Among 146 articles, 34 (23%) used models in which overnutrition was reported, and 36 (25%) demonstrated insulin resistance, with or without glucose intolerance. Half the articles contained no information on whether mice exhibited overnutrition or insulin resistance. While 75 papers (52%) reported > 2‐fold increase of serum/plasma alanine aminotransferase (ALT) compared with controls, ALT levels were near normal or not reported in 48%. Liver pathology was assessed by a pathologist with an interest in liver pathology in 53% of articles published in gastroenterology/hepatology journals, versus 43–44% in other journals. While there appears to be a trend to use models that are potentially relevant to the pathogenesis of human NASH, journals currently publish data on mouse models in which overnutrition and insulin resistance do not occur, without ALT increase or appropriate analysis of NASH pathology. We recommend that investigators, reviewers, and journal editors carefully consider the validity of NASH models in current use and that moves are made to reach a consensus on what the minimal criteria should be.