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Expression and Relocation of EC-SOD in Neutrophils
against cell injury induced by H2S or oxidative stress. When cells were incubated with hemoglobin, the lactate dehydrogenase release induced by cytotoxic concentrations of free radical generators, NO donors and H2S donors was attenuated, suggesting that hemoglobin uptake may serve to protect the cells. Conclusions: PBMCs in burns upregulate intracellular hemoglobins by a combination of active synthesis and uptake from the extracellular medium during burn-associated hemolysis. We speculate that this process may serve to protect the cells in face of high levels of burn-associated free radicals, oxidants, NO and H2S. doi: 10.1016/j.freeradbiomed.2014.10.382
140 Hydrogen Sulfide Decreases Cytokine Production by Modulation of Histone Deacetylase and Chromatin Remodeling Ester Rios1 and Csaba Szabo1 1 Department of Anesthesiology, University of Texas Medical Branch, Galveston, USA Introduction: H2S is a novel endogenous biological regulator that regulates, among others, the cell's oxidative-nitrative balance in normal physiological conditions as well as in several diseases. In this study we investigated the role of H2S in the chromatin modulation in a lipopolysaccharide (LPS) induced in vitro inflammation model, in conjunction with evaluating its effects on cytokine production. Results: THP1 differentiated in macrophages were pretreated with NaHS (a H2S donor) at 10, 100, 500 or 1000 μM for 30 min. In order to stimulate cytokine production, the cells were challenged with LPS (1 μg/ml) for 1, 4, 8 or 24 hours. Histone H3 acetylation was analyzed by chromatin immunoprecipitation; cytokine production was measured by ELISA; histone deacetylase (HDAC) activity was analyzed by a standard biochemical assay. H2S inhibited IL-6 and TNF production, most robustly at the two highest concentrations used. This effect was associated with a concomitant reduction of histone H3 acetylation at IL-6 and TNF-Į SURPRWHUV LQ WKH FHOOV H[SRVHG ZLWK +2S or H2S+LPS. Conclusion: We interpret the current results as follows: H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in gene transcription of various pro-inflammatory cytokines. We speculate that this mechanism may contribute to the previously demonstrated antiinflammatory effect of H2S and various H2S donors. doi: 10.1016/j.freeradbiomed.2014.10.383
141 Cellulose Nanocrystal (CNC) Cationic Derivative Induces NLRP3 S-Glutathionylation and IL-1ȕ Secretion in a ROS-Dependent Manner Bruno Cesar Toledo da Silva1, Erinolaoluwa Araoye1, Usha Hemraz2, Rajesh Sunasee1, and Karina Ckless1 1 SUNY Plattsburgh, USA, 2National Research Council of Canada, Crystalline cellulose nanocrystal (CNC) has emerged as a novel material for a wide variety of important applications, including gene delivery. In another study we found that some cationic CNC derivatives activate NLRP3/IL-ȕ LQIOammatory pathway. This is not a surprise since these compounds are a fiber-like and therefore potential activators of this pathway. We also found that these compounds increase mitochondrial and non-mitochondrial reactive oxygen species (ROS), which may be associated with
their inflammatory activity. However the precise mechanisms by which ROS participate in the NLRP3/IL-ȕVHFUHWLRQLVQRWWRWDOO\ elucidated. It has been postulated that NLRP3, a cysteine-rich SURWHLQFRXOGDFWDV³UHGR[VHQVRU´ of this inflammatory pathway, and therefore target for oxidative posttranslational modifications, such as S-glutathionylation. In this study we instigated whether or not NLRP3 is target for S-glutathionylation induced by CNC-gpoly(AEMA)-2, a CNC cationic derivative, and the impact on its inflammatory activity. Mouse macrophages (J774.A1) were stimulated for 24h with 25, 50 and 100 μg/mL with CNC-gpoly(AEMA)-2, in presence or absence of ROS inhibitors, DPI and MitoTEMPO. Mitochondrial ROS was evaluted by Mitosox staining, IL-1E secretion and intracellular NLRP3 were analyzed by ELISA and Western blotting, respectively. S-glutathionylation of NLRP3 was assessed by immunoprecipitation with glutathione antibody followed by Western blot analysis using NLRP3 antibody. IL-ȕ VHFUHWLRQ FDXVHG E\ CNC-g-poly(AEMA)-2 was completely abolished in presence of DPI and MitoTEMPO. NLRP3 S-glutathionyltion pattern as well as MitoSox staining were consistent with these findings, although both effects were not completely abolished by MitoTEMPO. When these inhibitors were added after 6h of treatment the inhibitory effect on IL-ȕ secretion was no longer observed. These findings indicate that LQLWLDO LQFUHDVHV LQ 526 LV QHFHVVDU\ IRU ,/ȕ VHFUHWLRQ LQGXFHG CNC-g-poly(AEMA)-2 NLRP3 and suggest that Sglutathionylation is associated with this effect. doi: 10.1016/j.freeradbiomed.2014.10.384
142 Expression and Relocation of EC-SOD in Neutrophils Randi Heidemann Gottfredsen1, Ulrike G. Larsen1, Jeppe Praetorius1, Jan J. Enghild2, Niels Borregaard3, and Steen Vang Petersen1 1 Department of Biomedicine, Aarhus University, Denmark, 2 Department of Molecular Biology and Genetics, Aarhus University, Denmark, 3Department of Hematology, National University Hospital, University of Copenhagen, Denmark At inflammatory conditions, neutrophils play a central role in generating reactive oxygen species and proteases to combat invading microorganisms as well as producing a range of cytokines that further mature the inflammatory response. The antioxidant protein extracellular superoxide dismutase (EC-SOD) is the only extracellular protein with the capacity to remove interstitial superoxide. Studies have shown that EC-SOD is associated with both macrophages and neutrophils and that the protein has the capacity to modulate the inflammatory response. We have recently shown that activation of macrophages stimulates the release of EC-SOD into the extracellular space. To further characterize the role of EC-SOD at inflammation, we have analyzed the expression and distribution of the protein in isolated neutrophils. Analyses by flow cytometry and electron microscopy show that EC-SOD is present on the cell surface as well as in intracellular compartments. This observation was further corroborated by sub-cellular fractionation. Interestingly, we show that the protein is redistributed upon cellular activation induced by phorbol myristate acetate (PMA). In line with our results obtained from bone marrow-derived macrophages, we show that the protein is un-detectable in the supernatant of resting neutrophils whereas PMA-induced activation relocates the protein to the extracellular space. Moreover, we provide evidence for the association between neutrophil extracellular traps (NETs) and EC-SOD. In concert, our data indicate that activated neutrophils may provide EC-SOD to the site of inflammation, suggesting an active role of the protein in establishing an adequate inflammatory response. doi: 10.1016/j.freeradbiomed.2014.10.385
S66
SFRBM 2014
140 Hydrogen Sulfide Decreases Cytokine Production by Modulation of Histone Deacetylase and Chromatin Remodeling Ester Rios1 and Csaba Szabo1 1 Department of Anesthesiology, University of Texas Medical Branch, Galveston, USA Introduction: H2S is a novel endogenous biological regulator that regulates, among others, the cell's oxidative-nitrative balance in normal physiological conditions as well as in several diseases. In this study we investigated the role of H2S in the chromatin modulation in a lipopolysaccharide (LPS) induced in vitro inflammation model, in conjunction with evaluating its effects on cytokine production. Results: THP1 differentiated in macrophages were pretreated with NaHS (a H2S donor) at 10, 100, 500 or 1000 μM for 30 min. In order to stimulate cytokine production, the cells were challenged with LPS (1 μg/ml) for 1, 4, 8 or 24 hours. Histone H3 acetylation was analyzed by chromatin immunoprecipitation; cytokine production was measured by ELISA; histone deacetylase (HDAC) activity was analyzed by a standard biochemical assay. H2S inhibited IL-6 and TNF production, most robustly at the two highest concentrations used. This effect was associated with a concomitant reduction of histone H3 acetylation at IL-6 and TNF-Į SURPRWHUV LQ WKH FHOOV H[SRVHG ZLWK +2S or H2S+LPS. Conclusion: We interpret the current results as follows: H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in gene transcription of various pro-inflammatory cytokines. We speculate that this mechanism may contribute to the previously demonstrated antiinflammatory effect of H2S and various H2S donors. doi: 10.1016/j.freeradbiomed.2014.10.383
141 Cellulose Nanocrystal (CNC) Cationic Derivative Induces NLRP3 S-Glutathionylation and IL-1ȕ Secretion in a ROS-Dependent Manner Bruno Cesar Toledo da Silva1, Erinolaoluwa Araoye1, Usha Hemraz2, Rajesh Sunasee1, and Karina Ckless1 1 SUNY Plattsburgh, USA, 2National Research Council of Canada, Crystalline cellulose nanocrystal (CNC) has emerged as a novel material for a wide variety of important applications, including gene delivery. In another study we found that some cationic CNC derivatives activate NLRP3/IL-ȕ LQIOammatory pathway. This is not a surprise since these compounds are a fiber-like and therefore potential activators of this pathway. We also found that these compounds increase mitochondrial and non-mitochondrial reactive oxygen species (ROS), which may be associated with
their inflammatory activity. However the precise mechanisms by which ROS participate in the NLRP3/IL-ȕVHFUHWLRQLVQRWWRWDOO\ elucidated. It has been postulated that NLRP3, a cysteine-rich SURWHLQFRXOGDFWDV³UHGR[VHQVRU´ of this inflammatory pathway, and therefore target for oxidative posttranslational modifications, such as S-glutathionylation. In this study we instigated whether or not NLRP3 is target for S-glutathionylation induced by CNC-gpoly(AEMA)-2, a CNC cationic derivative, and the impact on its inflammatory activity. Mouse macrophages (J774.A1) were stimulated for 24h with 25, 50 and 100 μg/mL with CNC-gpoly(AEMA)-2, in presence or absence of ROS inhibitors, DPI and MitoTEMPO. Mitochondrial ROS was evaluted by Mitosox staining, IL-1E secretion and intracellular NLRP3 were analyzed by ELISA and Western blotting, respectively. S-glutathionylation of NLRP3 was assessed by immunoprecipitation with glutathione antibody followed by Western blot analysis using NLRP3 antibody. IL-ȕ VHFUHWLRQ FDXVHG E\ CNC-g-poly(AEMA)-2 was completely abolished in presence of DPI and MitoTEMPO. NLRP3 S-glutathionyltion pattern as well as MitoSox staining were consistent with these findings, although both effects were not completely abolished by MitoTEMPO. When these inhibitors were added after 6h of treatment the inhibitory effect on IL-ȕ secretion was no longer observed. These findings indicate that LQLWLDO LQFUHDVHV LQ 526 LV QHFHVVDU\ IRU ,/ȕ VHFUHWLRQ LQGXFHG CNC-g-poly(AEMA)-2 NLRP3 and suggest that Sglutathionylation is associated with this effect. doi: 10.1016/j.freeradbiomed.2014.10.384
142 Expression and Relocation of EC-SOD in Neutrophils Randi Heidemann Gottfredsen1, Ulrike G. Larsen1, Jeppe Praetorius1, Jan J. Enghild2, Niels Borregaard3, and Steen Vang Petersen1 1 Department of Biomedicine, Aarhus University, Denmark, 2 Department of Molecular Biology and Genetics, Aarhus University, Denmark, 3Department of Hematology, National University Hospital, University of Copenhagen, Denmark At inflammatory conditions, neutrophils play a central role in generating reactive oxygen species and proteases to combat invading microorganisms as well as producing a range of cytokines that further mature the inflammatory response. The antioxidant protein extracellular superoxide dismutase (EC-SOD) is the only extracellular protein with the capacity to remove interstitial superoxide. Studies have shown that EC-SOD is associated with both macrophages and neutrophils and that the protein has the capacity to modulate the inflammatory response. We have recently shown that activation of macrophages stimulates the release of EC-SOD into the extracellular space. To further characterize the role of EC-SOD at inflammation, we have analyzed the expression and distribution of the protein in isolated neutrophils. Analyses by flow cytometry and electron microscopy show that EC-SOD is present on the cell surface as well as in intracellular compartments. This observation was further corroborated by sub-cellular fractionation. Interestingly, we show that the protein is redistributed upon cellular activation induced by phorbol myristate acetate (PMA). In line with our results obtained from bone marrow-derived macrophages, we show that the protein is un-detectable in the supernatant of resting neutrophils whereas PMA-induced activation relocates the protein to the extracellular space. Moreover, we provide evidence for the association between neutrophil extracellular traps (NETs) and EC-SOD. In concert, our data indicate that activated neutrophils may provide EC-SOD to the site of inflammation, suggesting an active role of the protein in establishing an adequate inflammatory response. doi: 10.1016/j.freeradbiomed.2014.10.385
S66
SFRBM 2014