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.

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...

Inflammasome pathways are important in chronic diseases, but it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as LPS and ATP that provide two distinct signals resulting in rapid production of IL-1β, with lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP, but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.

Pathogens and sterile insults both result in an inflammatory response. A significant part of this response is mediated by cytosolic machinery termed as the inflammasome which results in the activation and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Both of these are known to result in the activation of an acute inflammatory response, resulting in the production of downstream inflammatory cytokines such as tumor necrosis factor (TNF-α), interferon-gamma (IFN-γ), chemotaxis of immune cells, and induction of tissue injury. Surprisingly this very acute inflammatory pathway is also vital for the development of a full fibrogenic response in a number of organs including the lung, liver, and skin. There is evidence for the inflammasome having a direct role on tissue specific matrix producing cells such as the liver stellate cell, and also indirectly through the activation of resident tissue macrophage populations. The inflammasome requires stimulation of two pathways for full activation, and initiating stimuli include Toll-like receptor (TLR) agonists, adenosine triphosphate (ATP), particulates, and oxidative stress. Such a role for an acute inflammatory pathway in fibrosis runs counter to the prevailing association of TGF-β driven anti-inflammatory and pro-fibrotic pathways. This identifies new therapeutic targets which have the potential to simultaneously decrease inflammation, tissue injury and fibrosis. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.