“…Moreover, the mortality is significant higher in ARDS patients with impaired AFC than in ARDS patients with normal AFC (22) suggesting that ENaC dysfunction is critical in ARDS pathology. In addition, increased concentration of reactive intermediates following various forms of lung injury have been shown to also inhibit the Na + /K + -ATPase which abolishes active Na + transport leading to alveolar edema (53)(54)(55)(56)(57). However, data presented herein clearly establish that in our models of lung injury, Na + /K + -ATPase is not inhibited and the decrease in short circuit current across cultured human airway cells post heme exposure is due to damage to the apically located Na + conducting pathways, including ENaC.…”
Fax: (205) 934-7476; Tel: (205) 975-9673 Conflict of interest statement: The authors have declared that no conflict of interest exists.Authors Contribution: SA and IA designed and performed studies assessing RBC lysis measurements of heme and heme induced oxidative injury; AL and ZY designed and performed the ion transport experiments; JAM designed, conducted and analyzed the mass spectrometry studies; AB, DF and SM completed the necessary forms to obtain permissions from the UAB and St. Louis University IRBs to perform measurements in human samples; DAF designed and performed all measurements to assess brominated lipids in humans and mice; SA performed the modelling studies; SA, AL and SM wrote all drafts of this manuscript; SM conceived the study, designed and organized experiments, analyzed data, and inspected all primary data.
ABSTRACTWe previously reported that cell-free heme (CFH) is increased in the plasma of patients with acute and chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time the presence of CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl 2 , with plasmalogens) in the plasma of patients and mice exposed to Cl 2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. A single intramuscular injection of the heme-scavenging protein hemopexin (4 µg/kg body weight) in mice, one hour post halogen exposure, reversed RBC fragility and decreased CFH levels to those of air controls. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloridesensitive (ENaC) and cation sodium (Na + ) channels in mouse alveolar cells and transepithelial Na + transport across human airway cells with EC 50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86-1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na + transport across the channel. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.
“…Moreover, the mortality is significant higher in ARDS patients with impaired AFC than in ARDS patients with normal AFC (22) suggesting that ENaC dysfunction is critical in ARDS pathology. In addition, increased concentration of reactive intermediates following various forms of lung injury have been shown to also inhibit the Na + /K + -ATPase which abolishes active Na + transport leading to alveolar edema (53)(54)(55)(56)(57). However, data presented herein clearly establish that in our models of lung injury, Na + /K + -ATPase is not inhibited and the decrease in short circuit current across cultured human airway cells post heme exposure is due to damage to the apically located Na + conducting pathways, including ENaC.…”
Fax: (205) 934-7476; Tel: (205) 975-9673 Conflict of interest statement: The authors have declared that no conflict of interest exists.Authors Contribution: SA and IA designed and performed studies assessing RBC lysis measurements of heme and heme induced oxidative injury; AL and ZY designed and performed the ion transport experiments; JAM designed, conducted and analyzed the mass spectrometry studies; AB, DF and SM completed the necessary forms to obtain permissions from the UAB and St. Louis University IRBs to perform measurements in human samples; DAF designed and performed all measurements to assess brominated lipids in humans and mice; SA performed the modelling studies; SA, AL and SM wrote all drafts of this manuscript; SM conceived the study, designed and organized experiments, analyzed data, and inspected all primary data.
ABSTRACTWe previously reported that cell-free heme (CFH) is increased in the plasma of patients with acute and chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time the presence of CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl 2 , with plasmalogens) in the plasma of patients and mice exposed to Cl 2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. A single intramuscular injection of the heme-scavenging protein hemopexin (4 µg/kg body weight) in mice, one hour post halogen exposure, reversed RBC fragility and decreased CFH levels to those of air controls. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloridesensitive (ENaC) and cation sodium (Na + ) channels in mouse alveolar cells and transepithelial Na + transport across human airway cells with EC 50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86-1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na + transport across the channel. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.
“…The proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity. [32][33][34][35][36] LUBAC is an essential regulator of NF-B activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung IAV infection. 35,36 The respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity.…”
Section: Lubac Regulates the Amplitude Of The Lung Epithelial Driven mentioning
confidence: 99%
“…[32][33][34][35][36] LUBAC is an essential regulator of NF-B activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung IAV infection. 35,36 The respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity. 23,[37][38][39] As IAV replicates within the respiratory epithelium cells, the cytosolic pattern recognition receptor (PRR), RIG-I, is activated and initiates formation of a signaling platform to which LUBAC is recruited.…”
Section: Lubac Regulates the Amplitude Of The Lung Epithelial Driven mentioning
confidence: 99%
“…Instead, dampening of the immune response may be more effective, as was the case with LUBAC destabilization noted above. 36 FDAapproved anti-inflammatory drugs, including corticosteroids and statins, have been proposed for the treatment of "cytokine storm" associated with severe IAV infection. 49 Moreover, current data regarding their efficacy is limited to mouse models and retrospective patient observations.…”
Section: Pharmacological Immunomodulation During Iav Infectionmentioning
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“…The proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity. 32 , 33 , 34 , 35 , 36 LUBAC is an essential regulator of NF-κB activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung IAV infection. 35 , 36 Fig.…”
Influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. While the majority of those exposed have minor symptoms and recover with little morbidity, an estimated 500,000 people succumb to IAV-related complications each year worldwide. In these severe cases, an exaggerated inflammatory response, known as “cytokine storm”, occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ARDS). Data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. Current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. We have reported that the amplitude of the inflammatory response is regulated by Linear Ubiquitin Assembly Complex (LUBAC) activity and that dampening of LUBAC activity is protective during severe influenza virus infection. Therapeutic modulation of LUBAC activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. Here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes.
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