BACKGROUND & AIMS The NLRP3 inflammasome induces inflammation in response to organ injury, but little is known about its regulation. Toll-like receptors (TLRs) provide the first signal required for activation of the inflammasome and stimulate aerobic glycolysis to generate lactate. We examined whether lactate and the lactate receptor, GPR81, regulate TLR induction of signal 1 and limit inflammasome activation and organ injury. METHODS Primary mouse macrophages and human monocytes were incubated with TLR4 agonists and lactate and assayed for levels of pro-IL1β, NLRP3, and CASP1; release of IL1β; and activation of NFκB and caspase 1. Small interfering (si)RNAs were used to reduce levels of GPR81andARRB2, and an NFκB luciferase reporter transgene was transfected in RAW 264.7 cells. Cell lysates were analyzed by immunoprecipitation with an antibody against GPR81. Acute hepatitis was induced in C56BL/6N mice by administration of lipopolysaccharaide (LPS) and D-galactosamine. Acute pancreatitis was induced by administration of LPS and caerulein. Some mice were given intraperitoneal injections of sodium lactate or siRNA against Gpr81. Activation of NFκB in tissue macrophages was assessed in mice that express a reporter transgene. RESULTS In macrophages and monocytes, increasing concentrations of lactate reduced TLR4-mediated induction of Il1B, Nlrp3, and Casp1; activation of NFκB; release of IL1β; and cleavage of CASP1. GPR81 and ARRB2 physically interacted and were required for these effects. Administration of lactate reduced inflammation and organ injury in mice with immune hepatitis; this reduction required Gpr81 dependence in vivo. Lactate also prevented activation of NFκB in macrophages of mice, and when given following injury, reduced the severity of acute pancreatitis and acute liver injury. CONCLUSIONS Lactate negatively regulates TLR induction of the NLRP3 inflammasome and production of IL1β, via ARRB2 and GPR81. Lactate could be a promising immunomodulatory therapy for patients with acute organ injury.

Nonalcoholic steatohepatitis (NASH) is the most common liver disease in industrialized countries. NASH is a progressive disease that can lead to cirrhosis, cancer, and death, and there are currently no approved therapies. The development of NASH in animal models requires intact TLR9, but how the TLR9 pathway is activated in NASH is not clear. Our objectives in this study were to identify NASH-associated ligands for TLR9, establish the cellular requirement for TLR9, and evaluate the role of obesity-induced changes in TLR9 pathway activation. We demonstrated that plasma from mice and patients with NASH contains high levels of mitochondrial DNA (mtDNA) and intact mitochondria and has the ability to activate TLR9. Most of the plasma mtDNA was contained in microparticles (MPs) of hepatocyte origin, and removal of these MPs from plasma resulted in a substantial decrease in TLR9 activation capacity. In mice, NASH development in response to a high-fat diet required TLR9 on lysozyme-expressing cells, and a clinically applicable TLR9 antagonist blocked the development of NASH when given prophylactically and therapeutically. These data demonstrate that activation of the TLR9 pathway provides a link between the key metabolic and inflammatory phenotypes in NASH.

BACKGROUND & AIMS Acute pancreatitis is characterized by early activation of intracellular proteases followed by acinar cell death and inflammation. Activation of damage-associated molecular pattern (DAMP) receptors and a cytosolic complex termed the inflammasome initiates forms of inflammation. In this study, we examined whether DAMP-receptors and the inflammasome provide the link between cell death and the initiation of inflammation in pancreatitis. METHODS Acute pancreatitis was induced by caerulein stimulation in wild-type mice and mice deficient in components of the inflammasome (ASC, NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X7. Resident and infiltrating immune cell populations and pro-IL-1β expression were characterized in control and caerulein-treated adult murine pancreas. TLR9 expression was quantified in pancreatic cell populations. Additionally, wild-type mice were pretreated with a TLR9 antagonist prior to induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate (TLCS). RESULTS Caspase-1, ASC, and NLPR3 were required for inflammation in acute pancreatitis. Genetic deletion of Tlr9 reduced pancreatic edema, inflammation, and pro-IL-1β expression in pancreatitis. TLR9 was expressed in resident immune cells of the pancreas, which are predominantly macrophages. Pretreatment with the TLR9 antagonist IRS954 reduced pancreatic edema, inflammatory infiltrate, and apoptosis. Pretreatment with IRS954 reduced pancreatic necrosis and lung inflammation in TLCS-induced acute pancreatitis. CONCLUSIONS Components of the inflammasome, specifically ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X7 are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.

BACKGROUND & AIMS Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with CF. Liver damage is thought to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. METHODS Dextran sodium sulfate (DSS) was used to induce colitis C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin19 and CD-45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of NF- κB, phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase (SEAP) reporter and human TLR4 were transfected with CFTR cDNAs. RESULTS DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with nor-ursodeoxycholic acid, but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF- κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF- κB. CONCLUSIONS CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in a Src-dependent inflammatory response mediated by TLR4 and NF- κB. These findings might be used to develop therapies for CF-associated cholangiopathy.

The dauer larva is an alternative larval stage in Caenorhabditis elegans which allows animals to survive through periods of low food availability. Well-fed worms live for about three weeks, but dauer larvae can live for at least two months without affecting post-dauer lifespan. Mutations in daf-2 and age-1, which produce a dauer constitutive (Daf-C) phenotype, and in clk-1, which are believed to slow metabolism, markedly increase adult lifespan. Here we show that a ctl-1 mutation reduces adult lifespan in otherwise wild-type animals and eliminates the daf-c and clk-1-mediated extension of adult lifespan. ctl-1 encodes an unusual cytosolic catalase; a second gene, ctl-2, encodes a peroxisomal catalase. ctl-1 messenger RNA is increased in dauer larvae and adults with the daf-c mutations. We suggest that the ctl-1 catalase is needed during periods of starvation, as in the dauer larva, and that its misexpression in daf-c and clk-1 adults extends lifespan. Cytosolic catalase may have evolved to protect nematodes from oxidative damage produced during prolonged dormancy before reproductive maturity, or it may represent a general mechanism for permitting organisms to cope with the metabolic changes that accompany starvation.

SC, Mehal WZ. P2x7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice. Am J Physiol Gastrointest Liver Physiol 302: G1171-G1179, 2012. First published March 1, 2012; doi:10.1152/ajpgi.00352.2011.-Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7Ϫ/Ϫ), and in wild-type mice. P2X7Ϫ/Ϫ mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre-or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7Ϫ/Ϫ mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ectoNTPDase CD39 (CD39Ϫ/Ϫ) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39Ϫ/Ϫ mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1␤ release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAPinduced hepatotoxicity.CD39; nod-like receptor family, pryin domain containing-3, caspase-1; inflammasome; damage-associated molecular pattern ACETAMINOPHEN (APAP) overdose is the most common cause of acute liver failure in the Unites States and Europe (15,18,24). Furthermore, cases of acute hepatic failure due to APAP continue to rise (24). Toxicity is initiated by metabolism of APAP via reductive pathways to reactive metabolites. An antidote exists for patients early in the course of poisoning during this metabolism phase. However, with increased delays in administration of therapy, the frequency with which patients develop hepatocellular injury worsens.The initial, direct toxic injury induces an area of necrosis in the centrilobular regions. Liver injury is propagated by an inflammatory response after the drug has been metabolized and the initial centrilobular injury has occurred (23). The nature of this inflammatory response has been the subject of significant investigation in the past several years.Prior research by our group indicates that inflammasome activation is a crucial initiating step in the propagation of APAP-induced hepatic injury (10). Proinflammatory cytokines IL-1␤ and IL-18 are thought to be crucial to the prop...

The initial injury in acute pancreatitis (AP) is characteristically sterile and results in acinar cells necrosis. Intracellular contents released from damaged cells into the extracellular space serve as damage associated molecular patterns (DAMPs) that trigger inflammation. There is increasing evidence that this sterile inflammatory response mediated through DAMPs released from necrotic acinar cells is a key determinant of further pancreatic injury, remote organ injury, and disease resolution in experimental models. A number of DAMPS, including high-mobility group box protein 1 (HMGB1), DNA, ATP, and heat shock protein 70 (hsp70), have been shown to have a role in experimental pancreatitis. Many of these DAMPs are also detectable in the human pancreatitis. Genetic deletion and pharmacologic antagonism demonstrate that specific DAMP receptors, including TOLL-like receptor 4 (TLR4), TOLL-like receptor 9 (TLR9) and P2X7, are also required for inflammation in experimental AP. Down-stream DAMP sensing components include NLRP3, caspase1, interleukin-1β (IL-1), interleukin-18 (IL-18), and IL-1 receptor (IL-1R), and also are required for full experimental pancreatitis. These DAMP-mediated pathways provide novel therapeutic targets using antagonists of TLR’s and other receptors.

Balancing systemic iron levels within narrow limits is critical for maintaining human health. There are no known pathways to eliminate excess iron from the body and therefore iron homeostasis is maintained by modifying dietary absorption so that it matches daily obligatory losses. Several dietary factors can modify iron absorption. Polyphenols are plentiful in human diet and many compounds, including quercetin – the most abundant dietary polyphenol – are potent iron chelators. The aim of this study was to investigate the acute and longer-term effects of quercetin on intestinal iron metabolism. Acute exposure of rat duodenal mucosa to quercetin increased apical iron uptake but decreased subsequent basolateral iron efflux into the circulation. Quercetin binds iron between its 3-hydroxyl and 4-carbonyl groups and methylation of the 3-hydroxyl group negated both the increase in apical uptake and the inhibition of basolateral iron release, suggesting that the acute effects of quercetin on iron transport were due to iron chelation. In longer-term studies, rats were administered quercetin by a single gavage and iron transporter expression measured 18 h later. Duodenal FPN expression was decreased in quercetin-treated rats. This effect was recapitulated in Caco-2 cells exposed to quercetin for 18 h. Reporter assays in Caco-2 cells indicated that repression of FPN by quercetin was not a transcriptional event but might be mediated by miRNA interaction with the FPN 3′UTR. Our study highlights a novel mechanism for the regulation of iron bioavailability by dietary polyphenols. Potentially, diets rich in polyphenols might be beneficial for patients groups at risk of iron loading by limiting the rate of intestinal iron absorption.