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The role of alternatively activated (M2) monocytes in limiting NO bioavailability in falciparum malaria
Abstracts/Nitric Oxide 42 (2014) 99–153
vascular HbCO levels reached a steady state of 60% of total hemoglobin content (in an average of 50 min) one group of animals was given an injection of phosphate buffered saline (PBS) and another an injection of Ngb partially ligated with oxygen over 5 min. In the mice that received Ngb HbCO levels dropped by 34% ± 7% (n = 3) immediately after the infusion as compared to 12% ± 1% (n = 4) for mice which received PBS. Because the autoxidation lifetime of our Ngb is 5 hrs at 37 °C, Ngb saturated with molecular oxygen may also be given to increase oxygen delivery at the same time as it scavenges CO. We observed that the Ngb is excreted through the urinary system in mice with urine Ngb content as high as 2.4 mM and up to 94% saturated with CO after the animals are sacrificed. We are currently evaluating the effects of Ngb on COpoisoned mitochondria and in mouse neurocognitive and survival studies. We plan to proceed to large animal trials followed by clinical trials for CO poisoning in the field, where access to fast hyperbaric oxygen therapy is not possible. Keywords: Carbon monoxide; Poisoning; Neuroglobin.
Oral 1731-1. The role of alternatively activated (M2) monocytes in limiting NO bioavailability in falciparum malaria http://dx.doi.org/10.1016/j.niox.2014.09.012 J. Brice Weinberg a, Alicia D. Volkheimer a, Youwei Chen a, Matthew P. Rubach a, Esther D. Mwaikambo b, Jackson Mukemba b, Salvatory Florence b, Tsin W. Yeo c, Donald L. Granger d,e, Nicholas M. Anstey c a Duke and VA Medical Centers, Durham, NC, USA b Hubert Kairuki Memorial University, Dar es Salaam, Tanzania c Menzies School for Health Research, Darwin, Australia d University of Utah, Salt Lake City, UT, USA e VA Medical Centers, Salt Lake City, UT, USA
Background: We established earlier that NO has potent antimalaria disease activity in humans. We have discovered a variety of mechanisms by which host NO bioavailability is decreased in children and adults with falciparum malaria (Weinberg et al., Curr. Opin. Infect. Dis. 21 (2008) 468–475, for review). The underlying process(es) causing changes in these factors with resultant low NO in malaria is not known. Monocytes-macrophages can be activated through the classical pathway (“M1”) with cytokines such as IFN-g and TNF, or through the alternative pathway (“M2”) with cytokines such as IL-4, IL-10, and IL-13. M1 cells are characterized by increased NOS2, NO, and superoxide production, while M2 cells have decreased NOS2 and NO production, increased arginase 1 expression, and increased expression of the scavenger receptor CD163 and the mannose receptor CD206 by monocytes. Hypothesis: Monocytes in falciparum malaria patients are alternatively activated, and this activation contributes to the low NO bioavailability in malaria. Objective: Determine the status of monocyte activation in Tanzanian children with falciparum malaria. Methods: We studied the categories healthy control (HC, n = 62), moderately severe malaria (MSM, n = 54), and severe malaria (SM, n = 50) (total n = 166). We used real-time RT-PCR to measure PBMC mRNA for arginase 1 & 2, NOS2, and GTP cyclohydrolase 1; flow cytometry for monocyte surface CD163 & CD206; ELISA for plasma soluble CD163; and ELISA for plasma cytokine levels. Results: Compared to HC subjects, patients with malaria had significantly higher PBMC arginase 1 mRNA, lower NOS2 mRNA, slightly higher GTPCH-1 mRNA, and unchanged arginase 2 mRNA; higher PBMC arginase 1 protein by immunoblot; higher plasma arginase enzyme activity and soluble CD163; higher
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monocyte surface CD206 and CD163; and higher plasma IL10, IL-4, and IL-13. Overall, this pattern of changes we see in malaria patients is characteristic of alternatively activated (M2) monocytes. Conclusions: Our results indicate presence of alternatively activated (M2) monocytes in children with falciparum malaria. We speculate that the M2 status of mononuclear phagocytes is a major factor underlying certain causes of low NO bioavailability in malaria. Preventing M2 development or reversing M2 status to either no activation or M1 activation status will likely be useful in preventing and treating malaria. Keywords: Malaria; Monocyte; Alternative activation; M2; Nitric oxide; Arginase; Arginine.
Oral 1731-2. Novel targets for reversing endothelial dysfunction in atherogenesis: Mitochondrial processing peptidase and HDAC2 http://dx.doi.org/10.1016/j.niox.2014.09.013 Deepesh Pandey, Gautam Sikka, Anil Bhunia, Fumin Chang, Yehudis Bergman, Maggie Kuo, Janna Serbo, Dan Berkowitz, Lew Romer Johns Hopkins University School of Medicine
OxLDL activation of Arginase 2 (Arg2) induces endothelial dysfunction during early atherogenesis. The intriguing timeline of this effect is bimodal: Increases in arginase activity in minutes to hours are attributable to post-translational modifications of Arg2 and resulting changes in its subcellular compartmentalization; a delayed peak in Arg2 protein levels beginning at 12 hours is driven by changes in transcriptional regulation. The molecular bases for this bimodal Arg2 regulation are the focus points of this report. Our time course, subcellular fractionation, and immunofluorescence data support the hypothesis that Arg2 is a dual-targeted single gene and single mRNA product found in both mitochondrial and cytosolic fractions of HAEC – a novel pattern of effector system activation in mammalian vascular injury. These data indicate that OxLDL-mediated activation of Arg2 in HAEC and in mouse aortic intima occurs via posttranslational modification and subcellular translocation of preexisting pools of Arg2 from mitochondria to the cytosol. Inhibition or siRNA knockdown of the mitochondrial processing peptidase (MPPα) reduced the cytosolic abundance and activity of Arg2 following OxLDL stimulation, and attenuated OxLDL-mediated changes in HAEC production of ROS and NO. Knockdown of MPPα by adenovirally delivered MPP shRNA interference in isolated mice aortas was associated with improved endothelium-dependent vascular relaxation as assessed by wire myography following OxLDL exposure. Secondly, using classspecific biochemical histone deacetylase (HDAC) inhibitors and siRNA-mediated gene knockdown, we have identified HDAC2 as a specific, critical regulator of homeostasis in the vascular endothelial cells (EC) and Arg2 as a major transcriptional target for HDAC2 that contributes to intimal dysfunction. Additionally, inhibition of neddylation, a post-translational modification that enhances ubiquitin-mediated proteosomal degradation, increased the abundance of HDAC2 and caused decreased Arg2 activity. We therefore hypothesize that atherogenic stimuli such as OxLDL and inflammation downregulate HDAC2 levels in vascular EC through NEDD8-mediated enhancement of the proteosomal degradation of HDAC2, and that this reduction in HDAC2 results in the following cascade. Increased Arg2 expression in EC leads to decreased concentrations of L-arginine with subsequent impairment of NO bioavailability via eNOS substrate depletion, and an increase ROS production with resulting EC dysfunction and atherogenesis.
vascular HbCO levels reached a steady state of 60% of total hemoglobin content (in an average of 50 min) one group of animals was given an injection of phosphate buffered saline (PBS) and another an injection of Ngb partially ligated with oxygen over 5 min. In the mice that received Ngb HbCO levels dropped by 34% ± 7% (n = 3) immediately after the infusion as compared to 12% ± 1% (n = 4) for mice which received PBS. Because the autoxidation lifetime of our Ngb is 5 hrs at 37 °C, Ngb saturated with molecular oxygen may also be given to increase oxygen delivery at the same time as it scavenges CO. We observed that the Ngb is excreted through the urinary system in mice with urine Ngb content as high as 2.4 mM and up to 94% saturated with CO after the animals are sacrificed. We are currently evaluating the effects of Ngb on COpoisoned mitochondria and in mouse neurocognitive and survival studies. We plan to proceed to large animal trials followed by clinical trials for CO poisoning in the field, where access to fast hyperbaric oxygen therapy is not possible. Keywords: Carbon monoxide; Poisoning; Neuroglobin.
Oral 1731-1. The role of alternatively activated (M2) monocytes in limiting NO bioavailability in falciparum malaria http://dx.doi.org/10.1016/j.niox.2014.09.012 J. Brice Weinberg a, Alicia D. Volkheimer a, Youwei Chen a, Matthew P. Rubach a, Esther D. Mwaikambo b, Jackson Mukemba b, Salvatory Florence b, Tsin W. Yeo c, Donald L. Granger d,e, Nicholas M. Anstey c a Duke and VA Medical Centers, Durham, NC, USA b Hubert Kairuki Memorial University, Dar es Salaam, Tanzania c Menzies School for Health Research, Darwin, Australia d University of Utah, Salt Lake City, UT, USA e VA Medical Centers, Salt Lake City, UT, USA
Background: We established earlier that NO has potent antimalaria disease activity in humans. We have discovered a variety of mechanisms by which host NO bioavailability is decreased in children and adults with falciparum malaria (Weinberg et al., Curr. Opin. Infect. Dis. 21 (2008) 468–475, for review). The underlying process(es) causing changes in these factors with resultant low NO in malaria is not known. Monocytes-macrophages can be activated through the classical pathway (“M1”) with cytokines such as IFN-g and TNF, or through the alternative pathway (“M2”) with cytokines such as IL-4, IL-10, and IL-13. M1 cells are characterized by increased NOS2, NO, and superoxide production, while M2 cells have decreased NOS2 and NO production, increased arginase 1 expression, and increased expression of the scavenger receptor CD163 and the mannose receptor CD206 by monocytes. Hypothesis: Monocytes in falciparum malaria patients are alternatively activated, and this activation contributes to the low NO bioavailability in malaria. Objective: Determine the status of monocyte activation in Tanzanian children with falciparum malaria. Methods: We studied the categories healthy control (HC, n = 62), moderately severe malaria (MSM, n = 54), and severe malaria (SM, n = 50) (total n = 166). We used real-time RT-PCR to measure PBMC mRNA for arginase 1 & 2, NOS2, and GTP cyclohydrolase 1; flow cytometry for monocyte surface CD163 & CD206; ELISA for plasma soluble CD163; and ELISA for plasma cytokine levels. Results: Compared to HC subjects, patients with malaria had significantly higher PBMC arginase 1 mRNA, lower NOS2 mRNA, slightly higher GTPCH-1 mRNA, and unchanged arginase 2 mRNA; higher PBMC arginase 1 protein by immunoblot; higher plasma arginase enzyme activity and soluble CD163; higher
101
monocyte surface CD206 and CD163; and higher plasma IL10, IL-4, and IL-13. Overall, this pattern of changes we see in malaria patients is characteristic of alternatively activated (M2) monocytes. Conclusions: Our results indicate presence of alternatively activated (M2) monocytes in children with falciparum malaria. We speculate that the M2 status of mononuclear phagocytes is a major factor underlying certain causes of low NO bioavailability in malaria. Preventing M2 development or reversing M2 status to either no activation or M1 activation status will likely be useful in preventing and treating malaria. Keywords: Malaria; Monocyte; Alternative activation; M2; Nitric oxide; Arginase; Arginine.
Oral 1731-2. Novel targets for reversing endothelial dysfunction in atherogenesis: Mitochondrial processing peptidase and HDAC2 http://dx.doi.org/10.1016/j.niox.2014.09.013 Deepesh Pandey, Gautam Sikka, Anil Bhunia, Fumin Chang, Yehudis Bergman, Maggie Kuo, Janna Serbo, Dan Berkowitz, Lew Romer Johns Hopkins University School of Medicine
OxLDL activation of Arginase 2 (Arg2) induces endothelial dysfunction during early atherogenesis. The intriguing timeline of this effect is bimodal: Increases in arginase activity in minutes to hours are attributable to post-translational modifications of Arg2 and resulting changes in its subcellular compartmentalization; a delayed peak in Arg2 protein levels beginning at 12 hours is driven by changes in transcriptional regulation. The molecular bases for this bimodal Arg2 regulation are the focus points of this report. Our time course, subcellular fractionation, and immunofluorescence data support the hypothesis that Arg2 is a dual-targeted single gene and single mRNA product found in both mitochondrial and cytosolic fractions of HAEC – a novel pattern of effector system activation in mammalian vascular injury. These data indicate that OxLDL-mediated activation of Arg2 in HAEC and in mouse aortic intima occurs via posttranslational modification and subcellular translocation of preexisting pools of Arg2 from mitochondria to the cytosol. Inhibition or siRNA knockdown of the mitochondrial processing peptidase (MPPα) reduced the cytosolic abundance and activity of Arg2 following OxLDL stimulation, and attenuated OxLDL-mediated changes in HAEC production of ROS and NO. Knockdown of MPPα by adenovirally delivered MPP shRNA interference in isolated mice aortas was associated with improved endothelium-dependent vascular relaxation as assessed by wire myography following OxLDL exposure. Secondly, using classspecific biochemical histone deacetylase (HDAC) inhibitors and siRNA-mediated gene knockdown, we have identified HDAC2 as a specific, critical regulator of homeostasis in the vascular endothelial cells (EC) and Arg2 as a major transcriptional target for HDAC2 that contributes to intimal dysfunction. Additionally, inhibition of neddylation, a post-translational modification that enhances ubiquitin-mediated proteosomal degradation, increased the abundance of HDAC2 and caused decreased Arg2 activity. We therefore hypothesize that atherogenic stimuli such as OxLDL and inflammation downregulate HDAC2 levels in vascular EC through NEDD8-mediated enhancement of the proteosomal degradation of HDAC2, and that this reduction in HDAC2 results in the following cascade. Increased Arg2 expression in EC leads to decreased concentrations of L-arginine with subsequent impairment of NO bioavailability via eNOS substrate depletion, and an increase ROS production with resulting EC dysfunction and atherogenesis.