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Nox1 Exacerbates Colitis Induced by Salmonella Typhimurium Infection in Mice
tissue compared to control. Tissue activity of PLA2, COX and PGE2 levels were significantly increased in post-ischemic liver tissue compared to non-ischemic controls. Current results suggest that increased hydrolysis of fatty acids via PLA2 triggers activity of COX and leads to increased PGE2 levels. Future studies evaluating agents which block formation of eicosanoids derived from n-6 PUFAs can facilitate the development and application of treatment strategies in liver I/R injury. Acknowledgement: This study was supported by a grant (#113S999) from TUBITAK. doi: 10.1016/j.freeradbiomed.2014.10.364
122 AQP8-Mediated H2O2 Transport: Regulation and Function in B Cells Milena Bertolotti1,2 and Roberto Sitia1,2 1 San Raffaele Scientific Institute, Italy, 2Università Vita-Salute San Raffaele, Italy At low concentrations reactive oxygen species (ROS) are essential for cell function and survival. In fact H 2O2, one of the most physiologically important ROS, inhibits phosphatases via sulfenylation of their active site cysteines, potentiating kinase signaling. How it can diffuse in and between cells is thus a fundamental question in pathophysiology and the question arises of whether the permeability to exogenous H2O2 varies in particular conditions, like our models for physiologically (B to plasma cell differentiation) or pharmacologically induced cellular stress. Many pathways can generate H2O2 and in most of the cases this molecule must cross a membrane to reach its cytosolic targets (phosphatases). Despite H2O2 has long been thought to cross lipid bilayers freely, some members of the AQP family were already shown to be necessary for its transport, and AQP8 was one of them. An AQP8-low expressing I29 μ+ mouse B lymphoma cell line was generated using shRNA specific for AQP8, and exogenous H2O2 transport turned out to be impaired: vice versa the expression of a Halo-AQP8 chimeric protein rescued it. Moreover, Toll-like receptor and B-cell receptor signaling pathways were negatively affected by blocking H2O2 passage, either via AQP8 downregulation or catalase-dependent scavenging of extracellular H2O2 or inhibition of its production in cells lacking a plasma membrane±localized NOX subunit. As a consequence, preventing H2O2 entry inside B lymphocytes impaired membrane-IgM loss and immunoglobulins secretion during plasma cell differentiation process. In conclusion, we have identified a central role for the aquaporin channel AQP8 in modulating not only BCR and TLR signaling but also other downstream cellular functions: AQP8 may therefore represent a new important therapeutic target for all those pathological conditions in which redox signaling is deregulated. doi: 10.1016/j.freeradbiomed.2014.10.365
123 Nox1 Exacerbates Colitis Induced by Salmonella Typhimurium Infection in Mice Steven Esworthy1, Ursula Cota1, Binghui Shen1, Qiang Gao2, James H Doroshow3, and Fong-Fong Chu1 1 Beckman Research Institute of City of Hope, USA, 2The 1st Affiliated Hospital to Henan Univ of Sci and Tech, Luoyang, Henan, China, 3National Cancer Institute, USA Reactive oxygen species (ROS) can alter gut microbe
S60
composition and play an important role in gut inflammation. We have reported that mice deficient in antioxidant enzymes, glutathione peroxidase (GPx)-1 and -2, have very-early-onset spontaneous ileocolitis. A major source of ROS that is associated with colitis in the GPx1/2-double knockout (DKO) mice is Nox1, because the Nox1- and GPx1/2-triple KO (TKO) mice are disease free (FRBM, 2014). To test whether Nox1 plays a role against pathogenic bacterial infection, we used Salmonella enterica serovar Typhimurium (S. Tm) to serve as a mouse model to study bacterial induce inflammation in the cecum. Unexpectedly, we found Nox1-KO mice were more resistant to S. Tm colonization and infection than wild-type (WT) C57BL6/J mice when all mice were on conventional rodent chow. This conclusion is based on the following observations: 1) S. Tm-infection induced 13% weight loss in Nox1-KO mice compared to 23% weight loss in WT mice (P=0.026) analyzed at 4 days postinoculation. 2) Nine of ten (90%) WT mice had high levels (2.5E7 to 2E9 cfu/ gm cecum contents) of S. Tm colonization, while only 50% (6 out of 12) Nox1-KO mice had elevation (>2.5E6 cfu/ gm cecum contents) of S. Tm colonization. 3) Gross inflammation analyzed by cecum histology was detected only in 6 mice, which also had S. Tm levels over 2.5E7 cfu/gm; five were WT and one was a Nox1-KO mice. One additional Nox1-KO mouse had one inflammatory focus and lost only 4.5% of its weight. Based on these findings, we conclude that Nox1 does not inhibit S. Tm invasion or colonization. Instead, Nox1-produced ROS exacerbates S. Tm-induced inflammation presumably by eliciting responses from professional inflammatory cells. This result suggests that using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection. doi: 10.1016/j.freeradbiomed.2014.10.366
124 Hemoglobin Induces Lipid Peroxidation and TLR9 Activation in Vitro Paul Eigenberger1, David Irwin1, Zoe Loomis1, Joanne Maltzhan1, Malcolm Anderson2, and Christina Lisk3 1 Cardiovascular Pulmonary Research Group, University of Colorado, Anschutz Medical Campus, USA, 2Department of Critical Care, Children's Hospital of Colorado, USA, 3Program of Biological Sciences, Boston University School of Medicine, USA Introduction: Free Hb increases ROS and lipid peroxidation through Fenton chemistry reactions, which can be more robust in pro-oxidative microenvironments. It is unclear, however, how Hb induced oxidative stress in the microenvironment contributes to the vascular inflammation and the increased morbidity associated with hemolytic anemia syndromes. Recently, oxidative stress has been shown to activate NFkB through a Toll like receptor 9 (TLR9) mediated pathway. The mechanism behind this interaction is proposed to be either nuclear or mitochondrial DNA, liberated by stress induced cellular trauma. We applied this knowledge to investigate whether hemoglobin induced oxidative stress triggers TLR9 mediated inflammation. We hypothesized that Hb induced lipid peroxidation is linked to TLR9 activation either by cell death or mitochondrial damage and the Hb binding protein, haptoglobin, would attenuate lipid peroxidation and TLR9 activity. Methods: Rat pulmonary artery endothelial cells (RPAEC), or HEK TLR9 reporter cells were incubated for 6 h with HbF 2+ and HbFe3+in the presence or absence of glucose oxidase (GOX) and haptoglobin. Lipid peroxidation, TLR9 activity and cell death were determined using CLICK chemistry, NFkB inducible enzymatic assay and flow cytometry. Results: Data show that HbFe2+ and HbFe3+ (with or without GOX) induced lipid peroxidation in RPAEC and reporter cell TLR9 activation, which were attenuated with haptoglobin, Cell death in
SFRBM 2014
122 AQP8-Mediated H2O2 Transport: Regulation and Function in B Cells Milena Bertolotti1,2 and Roberto Sitia1,2 1 San Raffaele Scientific Institute, Italy, 2Università Vita-Salute San Raffaele, Italy At low concentrations reactive oxygen species (ROS) are essential for cell function and survival. In fact H 2O2, one of the most physiologically important ROS, inhibits phosphatases via sulfenylation of their active site cysteines, potentiating kinase signaling. How it can diffuse in and between cells is thus a fundamental question in pathophysiology and the question arises of whether the permeability to exogenous H2O2 varies in particular conditions, like our models for physiologically (B to plasma cell differentiation) or pharmacologically induced cellular stress. Many pathways can generate H2O2 and in most of the cases this molecule must cross a membrane to reach its cytosolic targets (phosphatases). Despite H2O2 has long been thought to cross lipid bilayers freely, some members of the AQP family were already shown to be necessary for its transport, and AQP8 was one of them. An AQP8-low expressing I29 μ+ mouse B lymphoma cell line was generated using shRNA specific for AQP8, and exogenous H2O2 transport turned out to be impaired: vice versa the expression of a Halo-AQP8 chimeric protein rescued it. Moreover, Toll-like receptor and B-cell receptor signaling pathways were negatively affected by blocking H2O2 passage, either via AQP8 downregulation or catalase-dependent scavenging of extracellular H2O2 or inhibition of its production in cells lacking a plasma membrane±localized NOX subunit. As a consequence, preventing H2O2 entry inside B lymphocytes impaired membrane-IgM loss and immunoglobulins secretion during plasma cell differentiation process. In conclusion, we have identified a central role for the aquaporin channel AQP8 in modulating not only BCR and TLR signaling but also other downstream cellular functions: AQP8 may therefore represent a new important therapeutic target for all those pathological conditions in which redox signaling is deregulated. doi: 10.1016/j.freeradbiomed.2014.10.365
123 Nox1 Exacerbates Colitis Induced by Salmonella Typhimurium Infection in Mice Steven Esworthy1, Ursula Cota1, Binghui Shen1, Qiang Gao2, James H Doroshow3, and Fong-Fong Chu1 1 Beckman Research Institute of City of Hope, USA, 2The 1st Affiliated Hospital to Henan Univ of Sci and Tech, Luoyang, Henan, China, 3National Cancer Institute, USA Reactive oxygen species (ROS) can alter gut microbe
S60
composition and play an important role in gut inflammation. We have reported that mice deficient in antioxidant enzymes, glutathione peroxidase (GPx)-1 and -2, have very-early-onset spontaneous ileocolitis. A major source of ROS that is associated with colitis in the GPx1/2-double knockout (DKO) mice is Nox1, because the Nox1- and GPx1/2-triple KO (TKO) mice are disease free (FRBM, 2014). To test whether Nox1 plays a role against pathogenic bacterial infection, we used Salmonella enterica serovar Typhimurium (S. Tm) to serve as a mouse model to study bacterial induce inflammation in the cecum. Unexpectedly, we found Nox1-KO mice were more resistant to S. Tm colonization and infection than wild-type (WT) C57BL6/J mice when all mice were on conventional rodent chow. This conclusion is based on the following observations: 1) S. Tm-infection induced 13% weight loss in Nox1-KO mice compared to 23% weight loss in WT mice (P=0.026) analyzed at 4 days postinoculation. 2) Nine of ten (90%) WT mice had high levels (2.5E7 to 2E9 cfu/ gm cecum contents) of S. Tm colonization, while only 50% (6 out of 12) Nox1-KO mice had elevation (>2.5E6 cfu/ gm cecum contents) of S. Tm colonization. 3) Gross inflammation analyzed by cecum histology was detected only in 6 mice, which also had S. Tm levels over 2.5E7 cfu/gm; five were WT and one was a Nox1-KO mice. One additional Nox1-KO mouse had one inflammatory focus and lost only 4.5% of its weight. Based on these findings, we conclude that Nox1 does not inhibit S. Tm invasion or colonization. Instead, Nox1-produced ROS exacerbates S. Tm-induced inflammation presumably by eliciting responses from professional inflammatory cells. This result suggests that using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection. doi: 10.1016/j.freeradbiomed.2014.10.366
124 Hemoglobin Induces Lipid Peroxidation and TLR9 Activation in Vitro Paul Eigenberger1, David Irwin1, Zoe Loomis1, Joanne Maltzhan1, Malcolm Anderson2, and Christina Lisk3 1 Cardiovascular Pulmonary Research Group, University of Colorado, Anschutz Medical Campus, USA, 2Department of Critical Care, Children's Hospital of Colorado, USA, 3Program of Biological Sciences, Boston University School of Medicine, USA Introduction: Free Hb increases ROS and lipid peroxidation through Fenton chemistry reactions, which can be more robust in pro-oxidative microenvironments. It is unclear, however, how Hb induced oxidative stress in the microenvironment contributes to the vascular inflammation and the increased morbidity associated with hemolytic anemia syndromes. Recently, oxidative stress has been shown to activate NFkB through a Toll like receptor 9 (TLR9) mediated pathway. The mechanism behind this interaction is proposed to be either nuclear or mitochondrial DNA, liberated by stress induced cellular trauma. We applied this knowledge to investigate whether hemoglobin induced oxidative stress triggers TLR9 mediated inflammation. We hypothesized that Hb induced lipid peroxidation is linked to TLR9 activation either by cell death or mitochondrial damage and the Hb binding protein, haptoglobin, would attenuate lipid peroxidation and TLR9 activity. Methods: Rat pulmonary artery endothelial cells (RPAEC), or HEK TLR9 reporter cells were incubated for 6 h with HbF 2+ and HbFe3+in the presence or absence of glucose oxidase (GOX) and haptoglobin. Lipid peroxidation, TLR9 activity and cell death were determined using CLICK chemistry, NFkB inducible enzymatic assay and flow cytometry. Results: Data show that HbFe2+ and HbFe3+ (with or without GOX) induced lipid peroxidation in RPAEC and reporter cell TLR9 activation, which were attenuated with haptoglobin, Cell death in
SFRBM 2014