Wajahat Z. Mehal

Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in the Western world. Twenty per cent of NAFLD individuals develop chronic hepatic inflammation (non-alcoholic steatohepatitis, NASH) associated with cirrhosis, portal hypertension and hepatocellular carcinoma, yet the causes of progression from NAFLD to NASH remain obscure. Here, we show that the NLRP6 and NLRP3 inflammasomes and the effector protein IL-18 negatively regulate NAFLD/NASH progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different mouse models reveal that inflammasome-deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, leading to enhanced hepatic tumour-necrosis factor (TNF)-α expression that drives NASH progression. Furthermore, co-housing of inflammasome-deficient mice with wild-type mice results in exacerbation of hepatic steatosis and obesity. Thus, altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of heretofore seemingly unrelated systemic auto-inflammatory and metabolic disorders.

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.

Sterile Inflammation in the Liver

Inflammation In the absence of pathogens occurs in all tissues in response to a wide range of stimuli that cause tissue stress and injury. Such sterile inflammation (SI) is a key process in drug-induced liver injury, nonalcoholic steatohepatitis, and alcoholic steatohepatitis and is a major determinant of fibrosis and carcinogenesis. In SI, endogenous damage-associated molecular patterns (DAMPS), which are usually hidden from the extracellular environment, are released on tissue injury and activate receptors on immune cells. More than 20 such DAMPS have been identified and activate cellular pattern recognition receptors, which were originally identified as sensors of pathogen-associated molecular patterns. Activation of pattern recognition receptors by DAMPS results in a wide range of immune responses, including production of proinflammatory cytokines and localization of immune cells to the site of injury. DAMPS result in the assembly of a cytosolic protein complex termed the inflammasome, which activates the serine protease caspase-1, resulting in activation and secretion of interleukin-1β and other cytokines. SI-driven liver diseases are responsible for the majority of liver pathology in industrially developed countries and lack specific therapy. Identification of DAMPS, their receptors, signaling pathways, and cytokines now provides a wide range of therapeutic targets for which many antagonists are already available.

The liver as a site of T-cell apoptosis: graveyard, or killing field?

Acknowledgments:

Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9†

Apoptosis of hepatocytes results in the development of liver fibrosis, but the molecular signals mediating this are poorly understood. Degradation and modification of nuclear DNA is a central feature of apoptosis, and DNA from apoptotic mammalian cells is known to activate immune cells via Toll‐like receptor 9 (TLR9). We tested if DNA from apoptotic hepatocytes can induce hepatic stellate cell (HSC) differentiation. Our data show that apoptotic hepatocyte DNA and cytidine‐phosphate‐guanosine oligonucleotides induced up‐regulation of transforming growth factor β1 and collagen 1 messenger RNA both in the human HSC line LX‐2 and in primary mouse HSCs. These effects were opposed by TLR9 antagonists. We have recently shown that adenosine inhibits HSC chemotaxis, and we now show that apoptotic hepatocyte DNA also inhibits platelet‐derived growth factor (PDGF)‐mediated HSC chemotaxis. Inhibition of HSC chemotaxis by PDGF was blocked by TLR9 antagonists, and was absent in primary HSCs from mice deficient in TLR9 or the TLR adaptor molecule MyD88. Stimulation of TLR9 on HSCs blocked signaling by the PDGF signaling molecule inositol 1,4,5‐triphosphate and reduced PDGF‐mediated increase in cytosolic Ca2+. Conclusion: DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by (1) providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and (2) inducing a stationary phenotype‐associated up‐regulation of collagen production. (HEPATOLOGY 2007.)

Strange brew: T cells in the liver.

Abstract The liver is a meeting place for thymus-dependent circulating T cells and thymus-independent T cells with natural-killer (NK)-cell markers. Here, Nick Crispe and Waj Mehal argue that interactions between these two T-cell pools might account for the distinctive immunological role of the liver as a focus of peripheral tolerance induction.

Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.

A prognostic rule for elderly patients admitted with community-acquired pneumonia

PURPOSE We sought to identify admission characteristics predicting mortality in elderly patients hospitalized with community-acquired pneumonia and to develop a prognostic staging system and discriminant rule. PATIENTS AND METHODS We retrospectively analyzed data from 2,356 patients aged > or = 65 years admitted with community-acquired pneumonia. Multivariable analyses of a derivation cohort (n = 1,000) identified characteristics associated with hospital mortality. A staging system and discriminant rule based on these characteristics were tested in a validation cohort (n = 1,356). Our discriminant rule was compared with a rule formulated from a heterogeneous adult population with community-acquired pneumonia. RESULTS Hospital mortality rates were 9% (derivation cohort) and 12% (validation cohort). We identified five independent predictors of mortality: age > or = 85 years [odds ratio 1.8 (95% confidence interval 1.1-3.1)], comorbid disease [odds ratio 4.1 (2.1-8.1)], impaired motor response [odds ratio 2.3 (1.4-3.7)], vital sign abnormality [odds ratio 3.4 (2.1-5.4)], and creatinine level > or = 1.5 mg/dL [odds ratio 2.5 (1.5-4.2)]. These variables stratified patients into four distinct stages with increasing mortality in the derivation cohort (Stage 1, 2%; Stage 2, 7%; Stage 3, 22%; Stage 4, 45%; P = 0.001) as well as in the validation cohort (Stage 1, 4%; Stage 2, 11%; Stage 3, 23%; Stage 4, 41%; P = 0.001). The discriminant rule developed from the derivation cohort had greater overall accuracy (77.1%) in the validation cohort than a rule formulated from a heterogeneous adult population (68.0%, P = 0.001). CONCLUSION Elderly patients with community-acquired pneumonia have characteristics at admission that can predict mortality. Our staging system and discriminant rule improve prognostic stratification of these patients.

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.