Muhammad A. Sohail

Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome

Hepatocyte death results in a sterile inflammatory response that amplifies the initial insult and increases overall tissue injury. One important example of this type of injury is acetaminophen-induced liver injury, in which the initial toxic injury is followed by innate immune activation. Using mice deficient in Tlr9 and the inflammasome components Nalp3 (NACHT, LRR, and pyrin domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1, we have identified a nonredundant role for Tlr9 and the Nalp3 inflammasome in acetaminophen-induced liver injury. We have shown that acetaminophen treatment results in hepatocyte death and that free DNA released from apoptotic hepatocytes activates Tlr9. This triggers a signaling cascade that increases transcription of the genes encoding pro-IL-1beta and pro-IL-18 in sinusoidal endothelial cells. By activating caspase-1, the enzyme responsible for generating mature IL-1beta and IL-18 from pro-IL-1beta and pro-IL-18, respectively, the Nalp3 inflammasome plays a crucial role in the second step of proinflammatory cytokine activation following acetaminophen-induced liver injury. Tlr9 antagonists and aspirin reduced mortality from acetaminophen hepatotoxicity. The protective effect of aspirin on acetaminophen-induced liver injury was due to downregulation of proinflammatory cytokines, rather than inhibition of platelet degranulation or COX-1 inhibition. In summary, we have identified a 2-signal requirement (Tlr9 and the Nalp3 inflammasome) for acetaminophen-induced hepatotoxicity and some potential therapeutic approaches.

TLR9 and the NLRP3 Inflammasome Link Acinar Cell Death With Inflammation in Acute Pancreatitis

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 initiate 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 (apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC], NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X(7). Resident and infiltrating immune cell populations and pro-interleukin-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 before induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate. RESULTS Caspase-1, ASC, and NLRP3 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 taurolithocholic acid 3-sulfate-induced acute pancreatitis. CONCLUSIONS Components of the inflammasome, ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X(7) are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.

Inflammasome-mediated regulation of hepatic stellate cells

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 microg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with alpha-smooth muscle actin and visualized by confocal microscopy, and TGF-beta and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca(2+)) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP(3)). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl(4)) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-beta and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca(2+) via IP(3) in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl(4) and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.

Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition

The Rho/ROCK pathway is activated in differentiated hepatic stellate cells (HSCs) and is necessary for assembly of actin stress fibers, contractility, and chemotaxis. Despite the importance of this pathway in HSC biology, physiological inhibitors of the Rho/ROCK pathway in HSCs are not known. We demonstrate that adenosine induces loss of actin stress fibers in the LX‐2 cell line and primary HSCs in a manner indistinguishable from Rho/ROCK inhibition. Loss of actin stress fibers occurs via the A2a receptor at adenosine concentrations above 10 μM, which are present during tissue injury. We further demonstrate that loss of actin stress fibers is due to a cyclic adenosine monophosphate, protein kinase A–mediated pathway that results in Rho inhibition. Furthermore, a constitutively active Rho construct can inhibit the ability of adenosine to induce loss of actin stress fibers. Actin stress fibers are required for HSC contraction, and we demonstrate that adenosine inhibits endothelin‐1 and lysophosphatidic acid–mediated HSC contraction. We propose that adenosine is a physiological inhibitor of the Rho pathway in HSCs with functional consequences, including loss of HSC contraction. (HEPATOLOGY 2009;49:185‐194)

Adenosine Inhibits Chemotaxis and Induces Hepatocyte-Specific Genes in Bone Marrow Mesenchymal Stem Cells

Bone marrow–derived mesenchymal stem cells (MSCs) have therapeutic potential in liver injury, but the signals responsible for MSC localization to sites of injury and initiation of differentiation are not known. Adenosine concentration is increased at sites of cellular injury and inflammation, and adenosine is known to signal a variety of cellular changes. We hypothesized that local elevations in the concentration of adenosine at sites of tissue injury regulate MSC homing and differentiation. Here we demonstrate that adenosine does not induce MSC chemotaxis but dramatically inhibits MSC chemotaxis in response to the chemoattractant hepatocyte growth factor (HGF). Inhibition of HGF‐induced chemotaxis by adenosine requires the A2a receptor and is mediated via up‐regulation of the cyclic adenosine monophosphate (AMP)/protein kinase A pathway. This results in inhibition of cytosolic calcium signaling and down‐regulation of HGF‐induced Rac1. Because of the important role of Rac1 in the formation of actin stress fibers, we examined the effect of adenosine on stress fiber formation and found that adenosine inhibits HGF‐induced stress fiber formation. In addition, we found that adenosine induces the expression of some key endodermal and hepatocyte‐specific genes in mouse and human MSCs in vitro. Conclusion: We propose that the inhibition of MSC chemotaxis at sites of high adenosine concentration results in localization of MSCs to areas of cellular injury and death in the liver. We speculate that adenosine might initiate the process of differentiation of MSCs into hepatocyte‐like cells. (HEPATOLOGY 2010.)

467 Aspirin Blocks Acetaminophen Induced Hepatotoxicity and Mortality in Mice - Dependent On the ASC/Caspase-1 Inflammasome

Acetaminophen (APAP) hepatotoxicity is dependent on the immune system, but the responsible molecular pathways have not been identified. Recently it was reported that IL-1 receptor signaling is required for APAP induced hepatotoxicity (Nature Med. July 2007, vol. 13,7. p851). The protein complex of pattern recognition molecules, the adaptor protein ASC and caspase-1 constitutes the inflammasome. Activation of the inflammasome results in production of IL-1 beta, resulting in IL-1 receptor signaling. Aims: i) Determine the role of the inflammasome in APAP hepatotoxicity. ii) Test if aspirin down-modulates the inflammasome pathway. iii) Test if aspirin blocks APAP hepatotoxicity. Methods: APAP hepatotoxicity (500 mg/kg ip single injection) was induced in wild-type C57BL/6 mice, and mice deficient in inflammasome components ASC, or caspase-1. An In-Vivo assay of inflammation dependent on the ASC/Caspase-1 pathway was used to test if aspirin can reduce inflammation mediated by this pathway. This consisted of intraperitoneal (ip) injection of monosodium urate (MSU) (3mg/mouse), with quantification of peritoneal neutrophils at 3 hrs. The ability of aspirin (approx 6mg/kg in drinking water starting 3 days prior to APAP), the platelet inhibitor clopidogrel (30 mg/kg by gavage every 24 hrs starting 2 days prior to APAP) and a cox-1 inhibitor (SC-560, 5mg/kg by gavage every 12 hrs starting 60 hrs before APAP) to protect from APAP induced injury was tested. Results: Mice deficient in the adaptor protein ASC or caspase-1 had reduced mortality from hepatotoxic doses of APAP (P<0.027 and P<0.013 respectively). In wild-type mice aspirin reduced inflammation mediated by the ASC/Caspase1 inflammasome as demonstrated by reduced ip neutrophil accumulation (P<0.015). In wild-type mice at 12 hrs after APAP injection aspirin improved histology and transaminitis (control group mean ALT 2365 iu/ml SD 1801 vs aspirin group mean ALT 944 iu/ml SD 373 p<0.04). In wild-type mice aspirin also reduced mortality from APAP hepatotoxicity (at 72 hrs after APAP mortality was 10/13 in control group and 3/17 in group receiving aspirin P<0.015). Clopidogrel and the cox-1 inhibitor SC-560 did not reduce mortality from APAP hepatotoxicity, suggesting that the mechanism of action of aspirin was not via inhibition of platelet degranulation or cox-1. Conclusions: The ASC/Caspase-1 inflammasome is required for APAP induced hepatotoxicity, and can be suppressed by aspirin. Aspirin reduces APAP induced mortality. Co-formulation of aspirin with APAP may reduce APAP induced liver failure.