iron overload and hemolysis modulate monocytes and inflammation in β-thalassemia
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137 The 7 Nicotinic Acetylcholine Receptor Agonist GTS-21 Improves Bacterial Clearance in Mice by Restoring Hyperoxia-Compromised Macrophage Function Ravi Sitapara1, Daniel J Antoine2, Lokesh Sharma1, Vivek S Patel1, Charles R Ashby, Jr1, Samir Gorasiya1, Huan Yang3, Michelle Zur1, and Lin L Mantell1,4 1Department of Pharmaceutical Sciences, St. John's University, USA, 2Department of Molecular and Clinical Pharmacology, University of Liverpool, UK, 3Laboratory of Biomedical Science, Feinstein Institute of Medical Research, USA, 4Center for Inflammation and Immunology, Feinstein Institute of Medical Research, USA
phagocytose and clear bacteria. We have shown that hyperoxia induces the accumulation of HMGB1 in the airways. Extracellular HMGB1 can impair macrophage phagocytosis and increase mortality of mice infected with Pseudomonas aeruginosa (PA). GTS-
endotoxin-induced HMGB1 release from macrophages. The aim of this study was to determine whether GTS-21 could inhibit hyperoxia-induced HMGB1 accumulation in the airways, enhance macrophage function and improve bacterial clearance from lungs in a mouse model of ventilator-associated pneumonia. GTS-21 (0.04, 0.4, and 4 mg/kg) or saline were i.p. administered to mice
2 and subsequently challenged with PA. Levels of extracellular HMGB1 in the airways were determined by western blot analysis and viable bacteria were quantified by colony assay. We found that 4 mg/kg of GTS-21 significantly increased bacterial clearance, decreased acute lung injury and accumulation of airway HMGB1 in these animals. To investigate the underlying cellular mechanisms, RAW 264.7 macrophages were incubated with different concentrations of GTS-21 prior to exposure to 95% O2. Phagocytosis assay was performed to assess macrophage function. We found that GTS-
significantly enhanced macrophage phagocytic activity. Together these results indicate that GTS-21 is effective in inhibiting hyperoxia-induced accumulation of extracellular HMGB1, leading to an enhanced macrophage function, bacterial clearance under
a possible pharmacological target to improve the clinical outcome of patients on ventilators by augmenting host defense against bacterial infections.
138 Iron Overload and Hemolysis Modulate Monocytes and Inflammation in -Thalassemia Nicholas Richard Slater1, Paul Harmatz2, Annie Higa 2, Vivian Ng2, Marcela G G Weyhmiller2, Patricia Evans 2, John B Porter 3, Nancy Sweeters 2, Jackson Price 1, Alisha Manji 1, Angela Manocha1, David W Killilea2, Ashutosh Lal2, Lynne Neumayr2, Elliot Vichinsky2, and Patrick B Walter1,2 1University of Victoria, Canada, 2UCSF Benioff Children's Hospital Oakland, USA, 3University College London, UK
-thalassemia major (TM) patients experience iron overload and hemolysis resulting in oxidative stress, chronic inflammation and an increased risk for infection. Monocytes of the innate immune system rely on pattern recognition receptors (PRR; e.g., toll-like
Recently, PRR ligands have been found to include damage-associated molecular patterns (DAMPs). We hypothesize that
increased iron and heme will generate reactive oxygen species (ROS) that induce lipid peroxidation creating malondialdehyde (MDA) and the DAMP 4-hydroxynonenal (4-HNE) that will modulate monocyte TLR4 activity. We cultured THP-1 monocytic cells with increasing concentrations of ferric citrate, heme, or 4-HNE for 24 hours to model iron overloaded monocytes of TM. ROS and TLR4 expression were quantified using flow cytometry while MDA and IL-6 were measured using a colorimetric assay and ELISA. The THP-1 model was compared to peripheral blood monocytes (PBM) from 13 TM patients. Exposure of THP-1 cells to iron resulted in a dose-dependent formation of ROS, whereas only high concentrations of heme generated ROS (p < 0.001 and p = 0.060). Conversely, heme induced a dose-response generation of MDA but only high concentrations of ferric iron increased MDA production (p < 0.0001 and p = 0.278). 4-HNE added to the culture media negatively correlated to IL-6 production (r = -0.94, p = 0.018). Lastly, larger more granular THP-1 cells expressed more TLR4 and ROS than smaller less granular cells (p < 0.001). PBMs from TM patients displayed a similar TLR4 heterogenic pattern. Our in vitro model demonstrates how iron and heme stimulate production of ROS and MDA, while DAMPs like 4-HNE inhibit TLR4, reducing Il-6 production. Finally, we identified a novel sub-population of monocytic cells that appear to be greater contributors to the inflammatory response.
139 Upregulation of Intracellular Hemoglobin in Peripheral Blood Mononuclear Cells during Burn Injury Serves as an Antioxidant and Cytoprotectant Csaba Szabo1, Attila Brunyanszki1, Katalin Erdelyi1, Gabor Olah1, Ciro Coletta1, Kazunori Yanagi1, Bartosz Szczesny1, and David Herndon2 1Department of Anesthesiology, University of Texas Medical Branch, Galveston, USA, 2Department of Surgery, University of Texas Medical Branch, Galveston, USA Introduction: The classical physiological role of hemoglobin is to carry oxygen from the lungs to the parenchymal tissues via the circulatory system. Recently a few studies have proposed that hemoglobin may also have functional roles intracellularly that are unrelated to oxygen transport. Here we demonstrate the upregulation of hemoglobins in human peripheral blood mononuclear cells after burn injury and present evidence in U937 cells that intracellular hemoglobin may play a role as an antioxidant and cytoprotectant. Results: We isolated human peripheral blood mononuclear cells (PBMCs) from patients with severe third-degree burn injury at admission time and 3 weeks and 2 months later. We have detected a significant accumulation of alpha, beta and delta variations of globin proteins in PBMCs by Western blotting, when compared to healthy volunteers, where these proteins were undetectable. This was associated with increases in the mRNA levels of alpha, beta and delta globin subunits, suggesting that, at least in part, the increase is related to novel protein synthesis. An additional potential for the increased cellular hemoglobins may be an active cellular uptake. Therefore, next, we investigated hemoglobin uptake by cells using monocytic cell line U937. Immuno-cytochemistry and in situ protein/protein co-localization assay (PLA) confirmed that U937 cell take up hemoglobin spiked into the tissue culture medium via CD163 receptors; blocking CD163 by specific peptide or silencing by siRNA inhibited the bind and accumulation of hemoglobins. Because burn injury is associated with increased plasma levels of oxidants, free radicals, nitric oxide (NO) and hydrogen sulfide (H2S), and hemoglobins have the potential to bind these species, we next tested whether hemoglobin uptake has any advantage
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receptor 4 [TLR4]) to provide defense against pathogens.