P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice

Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 02/2012; 52(9):1666-79. DOI: 10.1016/j.freeradbiomed.2012.02.010
Source: PubMed


While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

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    • "Similar inhibitory effects on inflammasome activation have been reported for ROS inhibitors, N-acetyl-L-cysteine (NAC) and APDC, in response to asbestos, MSU, and ATP (Dostert et al., 2008). P2X7R has been shown to mediate NADPH oxidase-dependent oxidative stress both in vivo (Chatterjee et al., 2012) and in vitro (Korcok et al., 2004; Liu et al., 2011), thereby indicating a critical role for P2X7R in activation of the NLRP3 inflammasome via ROS signaling. "
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    ABSTRACT: Inflammasomes are specialized signaling platforms critical for the regulation of innate immune and inflammatory responses. Various NLR family members (i.e., NLRP1, NLRP3, and IPAF) as well as the PYHIN family member AIM2 can form inflammasome complexes. These multiprotein complexes activate inflammatory caspases (i.e., caspase-1) which in turn catalyze the maturation of select pro-inflammatory cytokines, including interleukin (IL)-1beta and IL-18. Activation of the NLRP3 inflammasome typically requires two initiating signals. Toll-like receptor (TLR) and NOD-like receptor (NLR) agonists activate the transcription of pro-inflammatory cytokine genes through an NF-kB dependent priming signal. Following exposure to extracellular ATP, stimulation of the P2X purinoreceptor-7 (P2X7R), which results in K+ efflux, is required as a second signal for NLRP3 inflammasome formation. Alternative models for NLRP3 activation involve lysosomal destabilization and phagocytic NADPH oxidase and /or mitochondria-dependent reactive oxygen species (ROS) production. In this review we examine regulatory mechanisms that activate the NLRP3 inflammasome pathway. Furthermore, we discuss the potential roles of NLRP3 in metabolic and cognitive diseases, including obesity, type 2 diabetes mellitus, Alzheimer's disease, and major depressive disorder. Novel therapeutics involving inflammasome activation may result in possible clinical applications in the near future.
    Full-text · Article · May 2014 · Molecules and Cells
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    • "Other sources of oxidative stress, such as xenobiotic-induced hepatotoxicity, may very likely potentiate progression of simple steatosis to steatohepatitis. In previous experiments, we have found in accordance to others that rat steatotic hepatocytes exert higher sensitivity to the acute injury caused by hepatotoxins in vivo [14–16] and in vitro [17]. NAFLD, including simple steatosis, predisposes the liver to the increased risk of hepatotoxicity [18]. "
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    ABSTRACT: Oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of nonalcoholic fatty liver disease and toxic liver injury. The present study was designed to evaluate the effect of exogenous inducer of oxidative stress (tert-butyl hydroperoxide, tBHP) on nonfatty and steatotic hepatocytes isolated from the liver of rats fed by standard and high-fat diet, respectively. In control steatotic hepatocytes, we found higher generation of ROS, increased lipoperoxidation, an altered redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates, as compared to intact lean hepatocytes. Fatty hepatocytes exposed to tBHP exert more severe damage, lower reduced glutathione to total glutathione ratio, and higher formation of ROS and production of malondialdehyde and are more susceptible to tBHP-induced decrease in mitochondrial membrane potential. Respiratory control ratio of complex I was significantly reduced by tBHP in both lean and steatotic hepatocytes, but reduction in NADH-dependent state 3 respiration was more severe in fatty cells. In summary, our results collectively indicate that steatotic rat hepatocytes occur under conditions of enhanced oxidative stress and are more sensitive to the exogenous source of oxidative injury. This confirms the hypothesis of steatosis being the first hit sensitizing hepatocytes to further damage.
    Full-text · Article · Mar 2014 · Oxidative medicine and cellular longevity
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    • "The cellular effects of leptin are mediated by the leptin receptor isoforms Ob (long) and Ob (short), which belong to the IL-6 family of receptors and activate the JAK-STAT pathway for their downstream effects (Gorska et al., 2010). Leptin is known to mediate oxidative stress by activating NADPH oxidase and inducing formation of peroxynitrite (Chatterjee et al., 2012a; De Minicis et al., 2008). Leptin also mediates Kupffer cell activation and thus triggers the release of innate immune mediators and T cell proliferation , through its actions in the liver and other peripheral tissues (Kamada et al., 2008; Lafrance et al., 2010; Shen et al., 2005). "
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    ABSTRACT: Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver.
    Full-text · Article · Feb 2013 · Toxicology and Applied Pharmacology
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