Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPK␣␤␥ complex or HEK 293 cells to H 2 O 2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPK␣ and AMPK␤ subunits. In experiments using C-terminal truncation mutants of AMPK␣ (amino acids 1-312), we found that mutation of cysteine 299 to alanine diminished the ability of H 2 O 2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H 2 O 2 on kinase activity. Similar to the results obtained with H 2 O 2 -treated HEK 293 cells, activation and S-glutathionylation of the AMPK␣ subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H 2 O 2 are increased. These results demonstrate that physiologically relevant concentrations of H 2 O 2 can activate AMPK through oxidative modification of the AMPK␣ subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. AMPK3 is a serine/threonine kinase that consists of three subunits, of which the ␣ subunit has inducible kinase activity and the ␤ and ␥ subunits have regulatory function. Formation of the ␣␤␥ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the ␥ subunit (1-4). In addition to activation by AMP, phosphorylation of the Thr 172 residue of the ␣ subunit enhances kinase activity (5, 6). Recent studies have shown that the autoinhibitory domain (AID), located between amino acids 312 and 335 of the AMPK␣ subunit, is responsible for the lack of kinase activity under basal conditions (7-9), whereas AMP-induced conformational changes within the ␣␤␥ complex diminish function of the AID and lead to kinase activation.The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2, 10 -12). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13-15). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4,16). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of a...

Although neutrophil extracellular traps (NETs) form to prevent dissemination of pathogenic microorganisms, excessive release of DNA and DNA-associated proteins can also perpetuate sterile inflammation. In this study, we found that the danger-associated molecular pattern protein high-mobility group box 1 (HMGB1) can induce NET formation. NET formation was found after exposure of wild-type and receptor for advanced glycation end products-deficient neutrophil to HMGB1, whereas deficiency of Toll-like receptor (TLR)4 diminished the ability of neutrophils to produce NETs. Incubation of neutrophils with HMGB1 significantly increased the amount of DNA and histone 3 released as well as intracellular histone 3 citrullination, a signaling event that precedes chromatin decondensation. In vivo, neutrophils isolated from bronchoalveolar lavages of mice exposed to LPS and HMGB1 showed consistently greater ability to produce NETs compared with pulmonary neutrophils from mice that received LPS alone. In contrast, mice treated with LPS and neutralizing antibody to HMGB1 had decreased amounts of the inflammatory cytokines TNF-α and macrophage inflammatory protein 2, as well as of free DNA and histone 3 in bronchoalveolar lavage fluids. Airway neutrophils from LPS-exposed mice that had been treated with anti-HMGB1 antibodies showed decreased citrullination of histone 3. These results demonstrate that interactions between HMGB1 and TLR4 enhance the formation of NETs and provide a novel mechanism through which HMGB1 may contribute to the severity of neutrophil-associated inflammatory conditions.

Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46 ± 7.8 or 85 ± 26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21 ± 1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.

The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE ϩ/ϩ ) neutrophils demonstrated significantly greater ability to kill Eschericia coli compared with RAGE Ϫ/Ϫ neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE Ϫ/Ϫ as compared with RAGE ϩ/ϩ mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NA-DPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coliinduced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40 phox subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.receptor for advanced glycation end products; nicotinamide adenine dinucleotide phosphate oxidase; peritonitis; sepsis; inflammation; Eschericia coli NEUTROPHILS play central roles in acute inflammatory and innate immune responses through producing anti-bacterial peptides, cytokines, and other proinflammatory mediators, including reactive oxygen intermediates, and contributing to the formation of extracellular traps (8,22,33). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a high-output, superoxide-generating system, is an important source of the reactive oxygen intermediates produced by activated neutrophils. Deficiency in NADPH oxidase is associated with diminished ability of neutrophils to effectively kill bacteria. There is increased susceptibility to infection with Staphylococci and other extracellular bacteria in patients with chronic granulomatous disease, a condition associated with a hereditary defect of NADPH oxidase (14,15,17,18).The receptor for advanced glycation end products (RAGE) recognizes a diverse spectrum of ligands, including nonenzymatically modified glycoproteins such as advanced glycation end products (AGE) (31, 32), as well as high mobility group box 1 protein (HMGB1) and calgranulin (calcium binding cellular factors, S100B) (19,20). Cellular activation through RAGE engagement i...