Inflammation and Immunity 376 Effects of Selenoprotein Knockdown on Vascular Inflammatory Responses Stacey L. Aitken1, Sarah A. Mattmiller1, and Lorraine M. Sordillo1 1Michigan State University The initial stages of inflammation require upregulation of adhesion molecules and cytokines for the transmigration of leukocytes into underlying tissue. Overexpression of pro-inflammatory mediators is associated with the development of vascular inflammatory diseases, such as atherosclerosis. Oxidative stress secondary to antioxidant imbalance contributes to vascular inflammation. Selenium (Se) supplementation in vitro is able to reduce vascular adhesion molecule expression likely through its incorporation into selenoproteins and antioxidant functions. However, the exact role of individual selenoenzymes during vascular inflammatory challenges is unknown. Using siRNA technology to knockdown several key selenoproteins in bovine aortic endothelial cells (BAEC), we sought to determine whether specific selenoproteins contribute to the differential regulation of adhesion molecules and cytokines during pro-inflammatory challenge. Initial results using TrxR1 siRNA showed that this selenoporotein is at least partially responsible for controlling adhesion molecule expression and cytokine production in BAEC. These data provide evidence for the involvement of certain selenoenzymes in the regulation of adhesion molecule expression and cytokine production in BAEC during pro-inflammatory challenge.

377 Intraphagosomal Oxidants in the Control of T. Cruzi Infection: Experimental and Theoretical Analysis in Murine and Human Macrophages María Noel Alvarez1, Carolina Prolo1, Natalia Romero1, Lucía Piacenza1, Gonzalo Peluffo1, and Rafael Radi1 1Facultad de Medicina- Universidad de la República Macrophages play a central role in the control of acute Chagas infection. Trypanosoma cruzi must survive antimicrobial responses of host macrophages to establish infection. Phagocytic NADPH-oxidase (NOX2) has been shown to be involved in microbicidal activity. NOX2-generated superoxide (O2

•–) can then be converted into microbicidal oxidants, including hydrogen peroxide (H2O2) and peroxynitrite (ONOO–), which are important components of the killing activity. Macrophage oxidative responses have been shown to diminish in the presence of several intracellular microorganisms. However, during phagocytosis of T.cruzi trypomastigotes by murine macrophages (J774A-1) and human monocytes-derived macrophages (MDMs), respiratory burst was detected using several experimental approaches. In spite of the known importance of cytokine-dependent nitric oxide (•NO) production in the control of murine infection, its role in human macrophages is still debated. We detected NO2

–/NO3– in supernatants from murine, but not human,

macrophages infected with T.cruzi. Nevertheless, the expression of iNOS in human macrophages was detected by flow cytometry and their infection by trypomastigotes was strongly inhibited when nitric oxide synthase 2 was previously induced, with a ~70 % decrease on intracellular parasites. ONOO– contribution to intracellular killing was demonstrated using T. cruzi over-expressers of cytosolic tryparedoxin peroxidase (CPX), an enzyme previously demonstrated to readily decompose ONOO–, these parasites were able to survive into oxidative phagosome microenvironment. Computer-assisted simulations based on experimental data of oxidant formation rates strongly support intraphagosomal peroxynitrite production at concentrations

compatible with its role as microbicidal agent. Overall, these data suggest that both O2

•– and •NO contribute to intracellular killing of T.cruzi in human and murine macrophages. doi: 378 Reactive Oxygen Species Support the Normal Inflammatory Process and Promote Pathological Inflammation in the TNF Receptor­Associated Periodic Syndrome (TRAPS) Ariel C. Bulua1, Martin Pelletier1, Anna Simon1, Ravikanth Maddipati1, Heiyoung Park1, Daniel L. Kastner1, and Richard M. Siegel1 1NIAMS, NIH Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autosomal dominantly inherited autoinflammatory disorder characterized by recurrent fevers with abdominal pain, migratory rash, and inflammation. The disorder, which often occurs in the absence of an obvious trigger, is caused by missense mutations in the type 1 TNF receptor (TNFR1). We have previously shown that TRAPS associated mutant receptors do not bind TNF, do not traffic to the cell surface, and instead are retained at high levels in the endoplasmic reticulum in cells of TRAPS patients and mice with mutations homologous to two TRAPS associated mutants (TRAPS “knock-in” mice). Intracellular accumulation of TNFR1 appears to sensitize cells to LPS, leading to abnormal TNF-independent receptor signaling and enhanced production of inflammatory cytokines. Analysis of signaling events downstream of TNFR1 revealed elevated activation of p38 and JNK MAP kinases both at baseline and in response to LPS stimulation. Reactive Oxygen Species (ROS) have previously been shown to potentiate MAPK activation through inactivation of MAPK phosphatases. For this reason, we sought to determine whether elevated levels of ROS play a role in abnormal signaling and inflammation in cells harboring TNFR1 mutations. We measured ROS levels in both mouse embryonic fibroblasts (MEFs) and human immune cells from TRAPS samples and healthy controls. Our results show elevated baseline ROS but not PMA-induced ROS in TRAPS. Antioxidant treatment decreased the sustained MAPK phosphorylation and inflammatory cytokine production in TRAPS cells. NOX enzymes are thought to be important in generating the ROS that contributes to inflammation. However, mice lacking NOX2 and p22 phox still produced inflammatory cytokines after LPS stimulation. Thus, the NOX family enzymes are not the source of the ROS responsible for these effects. These results suggest that signaling from mutant TNFR1 potentiates TLR signaling through the induction of ROS from a source other than NADPH oxidases. ROS may be a novel therapeutic target in TRAPS. doi:

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P2X7 Receptor­mediated Free Radical Production by Leptin is key to Kupffer cell Activation and MHC Class II Expression in Worsening Steatohepatitis of Obesity Saurabh Chatterjee1, Maria Kadiiska1, and Ronald P Mason1 1NIEHS, National Institutes of Health, USA Steatohepatitis of obesity is characterized by high circulating levels of leptin. We hypothesized that purinergic receptors mediate leptin-induced formation of free radicals in Kupffer cells of diet-induced obese (DIO) mice with steatohepatitic lesions and trigger innate and adaptive immune responses. A low dose (0.8mM/kg) of carbontetrachloride was administered to induce stetatohepatitic lesions in DIO mice. Steatohepatitic DIO mice had increased perivenular aggregation of Kupffer cells, increased

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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:

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NLRP3 is a Target for Redox Regulation of SAA­induced Inflammasome Activation Karina Ckless1, Jennifer L. Ather2, and Matthew E. Poynter2 1SUNY Plattsburgh, 2University 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.

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381 Phosphatidylethanolamine N­Modified by Lipid Aldehydes Activate Endothelial Cells Sean S. Davies1, Zhongyi Chen1, Lilu Guo1, Brian E. Cox1, and Venkataraman Amarnath1 1Vanderbilt 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-isoketal-PE) 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 N-isoketal-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. doi:

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Free Radical­Producing Myeloid­Derived Regulatory Cells: Potent Activators and Suppressors of Lung Inflammation and Airway Hyper­responsiveness Jessy Deshane1,2, Jaroslaw Zmijewski1,2, Amit Gaggar1, Marion Spell1, Kim Estell1, Edward Abraham1,2, Lisa Schwiebert1, and David Chaplin1,2 1 2University 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 myeloid-derived 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:

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