The clinical relevance of heme detoxification by the macrophage heme oxygenase system.

Heme degradation by the heme oxygenase (HMOX) family of enzymes is critical for maintaining homeostasis and limiting heme-induced tissue damage. Macrophages express HMOX1 and 2 and are critical sites of heme degradation in healthy and diseased states. Here we review the functions of the macrophage heme oxygenase system and its clinical relevance in discrete groups of pathologies where heme has been demonstrated to play a driving role. HMOX1 function in macrophages is essential for limiting oxidative tissue damage in both acute and chronic hemolytic disorders. By degrading pro-inflammatory heme and releasing anti-inflammatory molecules such as carbon monoxide, HMOX1 fine-tunes the acute inflammatory response with consequences for disorders of hyperinflammation such as sepsis. We then discuss divergent beneficial and pathological roles for HMOX1 in disorders such as atherosclerosis and metabolic syndrome, where activation of the HMOX system sits at the crossroads of chronic low-grade inflammation and oxidative stress. Finally, we highlight the emerging role for HMOX1 in regulating macrophage cell death via the iron- and oxidation-dependent form of cell death, ferroptosis. In summary, the importance of heme clearance by macrophages is an active area of investigation with relevance for therapeutic intervention in a diverse array of human diseases.

Pannexin 1 Channels Control Cardiomyocyte Metabolism and Neutrophil Recruitment During Non-Ischemic Heart Failure.

Pannexin 1 (PANX1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, a possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 ). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism both in vivo and in vitro . In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knock-down of PANX1. To investigate non-ischemic heart failure and the preceding cardiac hypertrophy we administered isoproterenol, and we demonstrate that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases and cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + , Ly6g + ), to the myocardium. Together these data demonstrate that PANX1 deficiency in cardiomyocytes impacts glycolytic metabolism and protects against cardiac hypertrophy in non-ischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.

Macrophage acetyl-CoA carboxylase regulates acute inflammation through control of glucose and lipid metabolism.

Acetyl-CoA carboxylase (ACC) regulates lipid synthesis; however, its role in inflammatory regulation in macrophages remains unclear. We generated mice that are deficient in both ACC isoforms in myeloid cells. ACC deficiency altered the lipidomic, transcriptomic, and bioenergetic profile of bone marrow-derived macrophages, resulting in a blunted response to proinflammatory stimulation. In response to lipopolysaccharide (LPS), ACC is required for the early metabolic switch to glycolysis and remodeling of the macrophage lipidome. ACC deficiency also resulted in impaired macrophage innate immune functions, including bacterial clearance. Myeloid-specific deletion or pharmacological inhibition of ACC in mice attenuated LPS-induced expression of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß, while pharmacological inhibition of ACC increased susceptibility to bacterial peritonitis in wild-type mice. Together, we identify a critical role for ACC in metabolic regulation of the innate immune response in macrophages, and thus a clinically relevant, unexpected consequence of pharmacological ACC inhibition.

Targeting oxidized phospholipids by AAV-based gene therapy in mice with established hepatic steatosis prevents progression to fibrosis.

Oxidized phosphatidylcholines (OxPCs) are implicated in chronic tissue damage. Hyperlipidemic LDL-R--deficient mice transgenic for an OxPC-recognizing IgM fragment (scFv-E06) are protected against nonalcoholic fatty liver disease (NAFLD). To examine the effect of OxPC elimination at different stages of NAFLD progression, we used cre-dependent, adeno-associated virus serotype 8-mediated expression of the single-chain variable fragment of E06 (AAV8-scFv-E06) in hepatocytes of albumin-cre mice. AAV8-induced expression of scFv-E06 at the start of FPC diet protected mice from developing hepatic steatosis. Independently, expression of scFv-E06 in mice with established steatosis prevented the progression to hepatic fibrosis. Mass spectrometry-based oxophospho-lipidomics identified individual OxPC species that were reduced by scFv-E06 expression. In vitro, identified OxPC species dysregulated mitochondrial metabolism and gene expression in hepatocytes and hepatic stellate cells. We demonstrate that individual OxPC species independently affect disease initiation and progression from hepatic steatosis to steatohepatitis, and that AAV-mediated expression of scFv-E06 is an effective therapeutic intervention.

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

Macrophage metabolic adaptation to heme detoxification involves CO-dependent activation of the pentose phosphate pathway.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Extracellular nucleotide signaling in solid organ transplantation.

The role of extracellular purine nucleotides, including adenosine triphosphate (ATP) and adenosine, as modulators of posttransplantation outcome and ischemia-reperfusion injury is becoming increasingly evident. Upon pathological release of ATP, binding and activation of P2 purinergic surface receptors promote tissue injury and inflammation, while the expression and activation of P1 receptors for adenosine have been shown to attenuate inflammation and limit ischemia-induced damage, which are central to the viability and long-term success of allografts. Here we review the current state of the transplant field with respect to the role of extracellular nucleotide signaling, with a focus on the sources and functions of extracellular ATP. The connection between ischemia reperfusion, purinergic signaling, and graft preservation, as well as the role of ATP and adenosine as driving factors in the promotion and suppression of posttransplant inflammation and allograft rejection, are discussed. We also examine novel therapeutic approaches that take advantage of the ischemia-reperfusion-responsive and immunomodulatory roles for purinergic signaling with the goal of enhancing graft viability, attenuating posttransplant inflammation, and minimizing complications including rejection, graft failure, and associated comorbidities.

Application of vinylogous carbamates and vinylogous aminonitriles to the regiospecific synthesis of uniquely functionalized pyrroles and quinolones.

Pyrroles and quinolones represent core structures, which are routinely found in both natural and synthetic bioactive substances. Consequently, the development of efficient and regiospecific methods for the preparation of such heterocycles with unique functionality is of some importance. We describe herein the regiospecific synthesis of 1,2,3,4-tetrasubstituted pyrroles containing polar substituents and such products are prepared from vinylogous carbamates and vinylogous aminonitriles. We also describe the regiospecific synthesis of 3-aryl containing 1,3,6trisubstituted quinolones from vinylogous carbamates. The use of an amine exchange reaction to prepare precursors for the pyrrole and quinolone forming cyclizations represents a key factor in the strategy.

Ortho group activation of a bromopyrrole ester in Suzuki-Miyaura cross-coupling reactions: Application to the synthesis of new microtubule depolymerizing agents with potent cytotoxic activities.

New microtubule depolymerizing agents with potent cytotoxic activities have been prepared with a 5-cyano or 5-oximino group attached to a pyrrole core. The utilization of ortho activation of a bromopyrrole ester to facilitate successful Suzuki-Miyaura cross-coupling reactions was a key aspect of the synthetic methodology. This strategy allows for control of regiochemistry with the attachment of four completely different groups at the 2, 3, 4 and 5 positions of the pyrrole scaffold. Biological evaluations and molecular modeling studies are reported for these examples.

The application of formyl group activation of bromopyrrole esters to formal syntheses of lycogarubin C, permethyl storniamide A and lamellarin G trimethyl ether.

Lycogarubin C, permethyl storniamide A and lamellarin G trimethyl ether are pyrrole containing, natural products, which exhibit interesting biological properties. Such properties include anti-tumor activity on a variety of cancer cell lines including those that confer drug resistance, inhibition of HIV integrase and vascular disrupting activity. We now describe the use of methyl and ethyl 3-bromo-2-formylpyrrole-5-carboxylate as building blocks for the formal synthesis of these three highly functionalized, bioactive pyrroles. These new building blocks will now provide ready access to the natural products and many novel analogs due to the ability to easily modify positions 2,3,4 and 5 of the pyrrole core.