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Phosphatidylethanolamine N-Modified by Lipid Aldehydes Activate Endothelial Cells
binding of p47phox with gp91 phox and increased protein radicals. NADPH oxidase activation and protein radical formation were significantly reduced in leptin knockout mice. Isolated Kupffer cells from DIO mice with steatohepatitic lesions had significantly high DMPO-nitrone adducts, TNF-α, CCL8 (MCP-2) • and NO production when compared to leptin KO mice and were inhibited by SOD mimetic Tempol or NADPH oxidase inhibitor, apocynin or high (100 mM) concentrations of DMPO. To investigate whether the free radical production by leptin influenced the antigen presentation capacity of Kupffer cells, flow cytometric analysis of MHC Class II and CD-80 expression was performed. Results indicated that administration of apocynin, Tempol or DMPO, or use of leptin KO mice significantly decreased MHC Class II and CD-80 expression, indicating a prominent role for leptin and leptin-induced free radicals in antigen presentation. Possible involvement of downstream pathways for free radical generation by leptin was based on our observation of higher incidence of purinergic (P2X7) receptors in wild-type DIO kupffer cells compared to the leptin KO group. When treated with a specific P2X7 antagonist, isolated Kupffer cells showed decreased protein radical adducts, TNF-α and chemokine release, and reduced ability to present antigens in spite of higher leptin levels. Results from studies with P2X7 KO mice fed with a high fat diet will also be discussed. Thus, in one such mechanism, increased leptin levels may induce higher P2X7 receptors on Kupffer cells and synergize free radical formation. This event may result in downstream effects related to increased macrophage activation and antigen presentation and worsen steatohepatitis of obesity. doi: 10.1016/j.freeradbiomed.2010.10.389
380 NLRP3 is a Target for Redox Regulation of SAA induced Inflammasome Activation
381 Phosphatidylethanolamine NModified by Lipid Aldehydes Activate Endothelial Cells Sean S. Davies1, Zhongyi Chen1, Lilu Guo1, Brian E. Cox1, and Venkataraman Amarnath1 1 Vanderbilt University Peroxidation of plasma lipoproteins and vascular cells has been implicated in atherosclerosis. Lipid peroxidation generates a number of reactive lipid aldehydes which activate cultured vascular cells, but the exact mechanisms underlying this activation remain unclear. The potential contribution of protein modification by lipid aldehydes to their pro-inflammatory effects have been relatively well-studied; in contrast, very little is known about the contribution of phosphatidylethanolamine (PE) headgroup modification to these effects. The potential relevance of PE modification was suggested by the recent finding that treatment of human umbilical vein endothelial cells (HUVEC) with a lipid aldehyde (isoketal) resulted in more modified PE than protein. Plasma levels of PE N-modified by isoketal (N-isoketalPE) also significantly increase during oxidative stress. To assess whether PEs that were N-modified by various known reactive oxidized lipids (oxN-PEs) had pro-inflammatory activity that might contribute to atherosclerosis, we synthesized several oxN-PEs, incubated them with HUVEC, and then measured the ability of treated HUVEC to bind THP-1 monocytes. A subset of oxN-PEs, including N-isoketal-PE, N-oxononenal-PE, and N-dodecanoyl-PE activated HUVEC to bind THP-1 monocytes, while other oxN-PE such as N-glutaryl-PE had no effect. Fluorescently labeled Nisoketal-PE was rapidly internalized by HUVEC to punctuate structures consistent with mitochondria, but was internalized by THP-1 macrophages to structures consistent with lysosomes. To explore the mechanism of oxN-PE activation of HUVEC, we treated HUVEC with N-isoketal-PE and found that it induced p38 activation and ICAM-1 expression. Inhibiting p38 blocked induction of ICAM-1. In summary, oxN-PEs recapitulate key proinflammatory features of reactive oxidized lipids and may therefore be an important mediator of their effects.
Karina Ckless1, Jennifer L. Ather2, and Matthew E. Poynter2 1 2 SUNY Plattsburgh, University of Vermont NLRP3 inflammasome activation facilitates the formation of a molecular platform for caspase1-dependent secretion of IL-1β. While the precise mechanisms of NLRP3 inflammasome activation remains obscure, it has been postulated to be mediated by reactive oxygen species (ROS). We have demonstrated that serum amyloid A (SAA), a biomarker of inflammation, induces NLRP3 inflammasome activation in an ATP-ROS-dependent manner. Since NLRP3 is a cysteine-rich protein, the hypothesis of this study was that changes in ROS production by SAA participate in inflammasome activation via redox modulation of NLRP3. Mouse peritoneal exudate macrophages were treated with ROS inhibitors for 20 min before adding 1 µg/mL rhSAA for 16hr. IL-1β secretion, intracellular NLRP3 and pro-IL-1β, were analyzed by ELISA and Western blot, respectively. Ebselen, a peroxide scavenger, and indomethacin, a cycloxygenase inhibitor, strongly inhibited IL-1β secretion, whereas the NADPH oxidase inhibitor, DPI, increased IL-1β secretion. All drugs decreased intracellular pro-IL-1β, and to various degrees, NLRP3 when compared with SAA alone. In additional studies, ROS inhibitors were added at 8h of SAA incubation (at which time secreted IL-1β was not yet detectable,) and the cells were analyzed after 16h of total SAA treatment. In comparison to SAA alone, DPI, Ebselen and the SOD mimetic, MnTBAP, increased IL-1β secretion and decreased intracellular pro-IL-1β levels without affecting NLRP3. A time course study using a reduced cysteine labeling approach reveled that NLRP3 is initially oxidized at 2hr of SAA treatment and gradually reduced at 24hr of SAA treatment. Addition of MnTBAP at 8hr of SAA treatment enhanced the reduced status of NLPR3 and its ability to associate with caspase1. Consistent with some reports, these data suggest that NLRP3 is redox modulated by SAA in a biphasic manner. Moreover, NLRP3 itself is a target for cysteine oxidation that may impact upon inflammasome activity.
Jessy Deshane1,2, Jaroslaw Zmijewski1,2, Amit Gaggar1, Marion Spell1, Kim Estell1, Edward Abraham1,2, Lisa Schwiebert1, and David Chaplin1,2 12 University of Alabama at Birmingham Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloidderived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation. We demonstrate that these free radical producing myeloid-derived cells are master regulators of the airway inflammatory response. The profiles of free radicals they produce depend on induction of the iNOS, arginase, and NADPH + – oxidase pathways. The nitric oxide-producing Ly-6C Ly-6G cells down-modulate T cell activation and dramatically down-regulate antigen-induced airway hyper-responsiveness. The superoxide– + producing Ly-6C Ly-6G cells are potently pro-inflammatory and exacerbate airway hyper-responsiveness. These free radicals not only control the pro- and anti-inflammatory potential of these cells, but also regulate the reciprocal pattern of their infiltration into the lung. These regulatory myeloid cells thus represent important targets for asthma therapy.
doi: 10.1016/j.freeradbiomed.2010.10.390
doi: 10.1016/j.freeradbiomed.2010.10.392
doi: 10.1016/j.freeradbiomed.2010.10.391
382 Free RadicalProducing MyeloidDerived Regulatory Cells: Potent Activators and Suppressors of Lung Inflammation and Airway Hyperresponsiveness
SFRBM/SFRRI 2010
S141
380 NLRP3 is a Target for Redox Regulation of SAA induced Inflammasome Activation
381 Phosphatidylethanolamine NModified by Lipid Aldehydes Activate Endothelial Cells Sean S. Davies1, Zhongyi Chen1, Lilu Guo1, Brian E. Cox1, and Venkataraman Amarnath1 1 Vanderbilt University Peroxidation of plasma lipoproteins and vascular cells has been implicated in atherosclerosis. Lipid peroxidation generates a number of reactive lipid aldehydes which activate cultured vascular cells, but the exact mechanisms underlying this activation remain unclear. The potential contribution of protein modification by lipid aldehydes to their pro-inflammatory effects have been relatively well-studied; in contrast, very little is known about the contribution of phosphatidylethanolamine (PE) headgroup modification to these effects. The potential relevance of PE modification was suggested by the recent finding that treatment of human umbilical vein endothelial cells (HUVEC) with a lipid aldehyde (isoketal) resulted in more modified PE than protein. Plasma levels of PE N-modified by isoketal (N-isoketalPE) also significantly increase during oxidative stress. To assess whether PEs that were N-modified by various known reactive oxidized lipids (oxN-PEs) had pro-inflammatory activity that might contribute to atherosclerosis, we synthesized several oxN-PEs, incubated them with HUVEC, and then measured the ability of treated HUVEC to bind THP-1 monocytes. A subset of oxN-PEs, including N-isoketal-PE, N-oxononenal-PE, and N-dodecanoyl-PE activated HUVEC to bind THP-1 monocytes, while other oxN-PE such as N-glutaryl-PE had no effect. Fluorescently labeled Nisoketal-PE was rapidly internalized by HUVEC to punctuate structures consistent with mitochondria, but was internalized by THP-1 macrophages to structures consistent with lysosomes. To explore the mechanism of oxN-PE activation of HUVEC, we treated HUVEC with N-isoketal-PE and found that it induced p38 activation and ICAM-1 expression. Inhibiting p38 blocked induction of ICAM-1. In summary, oxN-PEs recapitulate key proinflammatory features of reactive oxidized lipids and may therefore be an important mediator of their effects.
Karina Ckless1, Jennifer L. Ather2, and Matthew E. Poynter2 1 2 SUNY Plattsburgh, University of Vermont NLRP3 inflammasome activation facilitates the formation of a molecular platform for caspase1-dependent secretion of IL-1β. While the precise mechanisms of NLRP3 inflammasome activation remains obscure, it has been postulated to be mediated by reactive oxygen species (ROS). We have demonstrated that serum amyloid A (SAA), a biomarker of inflammation, induces NLRP3 inflammasome activation in an ATP-ROS-dependent manner. Since NLRP3 is a cysteine-rich protein, the hypothesis of this study was that changes in ROS production by SAA participate in inflammasome activation via redox modulation of NLRP3. Mouse peritoneal exudate macrophages were treated with ROS inhibitors for 20 min before adding 1 µg/mL rhSAA for 16hr. IL-1β secretion, intracellular NLRP3 and pro-IL-1β, were analyzed by ELISA and Western blot, respectively. Ebselen, a peroxide scavenger, and indomethacin, a cycloxygenase inhibitor, strongly inhibited IL-1β secretion, whereas the NADPH oxidase inhibitor, DPI, increased IL-1β secretion. All drugs decreased intracellular pro-IL-1β, and to various degrees, NLRP3 when compared with SAA alone. In additional studies, ROS inhibitors were added at 8h of SAA incubation (at which time secreted IL-1β was not yet detectable,) and the cells were analyzed after 16h of total SAA treatment. In comparison to SAA alone, DPI, Ebselen and the SOD mimetic, MnTBAP, increased IL-1β secretion and decreased intracellular pro-IL-1β levels without affecting NLRP3. A time course study using a reduced cysteine labeling approach reveled that NLRP3 is initially oxidized at 2hr of SAA treatment and gradually reduced at 24hr of SAA treatment. Addition of MnTBAP at 8hr of SAA treatment enhanced the reduced status of NLPR3 and its ability to associate with caspase1. Consistent with some reports, these data suggest that NLRP3 is redox modulated by SAA in a biphasic manner. Moreover, NLRP3 itself is a target for cysteine oxidation that may impact upon inflammasome activity.
Jessy Deshane1,2, Jaroslaw Zmijewski1,2, Amit Gaggar1, Marion Spell1, Kim Estell1, Edward Abraham1,2, Lisa Schwiebert1, and David Chaplin1,2 12 University of Alabama at Birmingham Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloidderived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation. We demonstrate that these free radical producing myeloid-derived cells are master regulators of the airway inflammatory response. The profiles of free radicals they produce depend on induction of the iNOS, arginase, and NADPH + – oxidase pathways. The nitric oxide-producing Ly-6C Ly-6G cells down-modulate T cell activation and dramatically down-regulate antigen-induced airway hyper-responsiveness. The superoxide– + producing Ly-6C Ly-6G cells are potently pro-inflammatory and exacerbate airway hyper-responsiveness. These free radicals not only control the pro- and anti-inflammatory potential of these cells, but also regulate the reciprocal pattern of their infiltration into the lung. These regulatory myeloid cells thus represent important targets for asthma therapy.
doi: 10.1016/j.freeradbiomed.2010.10.390
doi: 10.1016/j.freeradbiomed.2010.10.392
doi: 10.1016/j.freeradbiomed.2010.10.391
382 Free RadicalProducing MyeloidDerived Regulatory Cells: Potent Activators and Suppressors of Lung Inflammation and Airway Hyperresponsiveness
SFRBM/SFRRI 2010
S141