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Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed.Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib.Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958–68. ©2017 AACR.

Supplementary Figure S1. Mutations in CCND3 increase sensitivity to mTOR inhibition. Supplementary Figure S2. Dose-response plots for everolimus and palbociclib. Supplementary Figure S3. mTOR inhibition with everolimus cross-regulates the CDK/Rb pathway. Supplementary Figure S4. Erk activity is linked to CDK4/6 inhibition. Supplementary Figure S5. Palbociclib treatment decreases the phosphorylation of Erk1/2. Supplementary Figure S6. Cells remain attached throughout the Seahorse metabolic assays. Supplementary Figure S7. Immunohistochemistry staining of tumor harboring mouse brains for GIC markers.

B cell–activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

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-1β, 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.

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use an acute (14 day) and a chronic (28 day) model of adrenergic stimulation to induce heart failure in two novel mouse models: 1) We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP-flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1 MyHC6-Cre ). 2) Using mice expressing the tamoxifen-inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen-inducible deletion of Panx1 in cardiomyocytes (Panx1 Tnnt2-MerCreMer ). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1 MyHC6-Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. Furthermore, after chronic administration of isoproterenol (15 mg/kg/day, osmotic pump) Panx1 MyHC6-Cre mice were still protected from cardiac hypertrophy compared to Panx1 fl/fl littermates. In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1-dependent release of ATP in a dose-dependent manner. This was demonstrated with both pharmacological blockade by spironolactone and siRNA-mediated knock-down of Panx1. Taken together, these data demonstrates that Panx1 deficiency in cardiomyocytes protects against isoproterenol-induced cardiac dysfunction and that ATP is released through Panx1 in response to isoproterenol. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify Panx1 as a novel therapeutic target to prevent heart failure.

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.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI., Debilitating anemias in chronic diseases can result from deficient iron delivery to red cell precursors. Here, the authors show how this deficiency damages the cytoskeletal framework of progenitor cells and identify a targeted strategy for cytoskeletal repair, leading to anemia correction.

The role of extracellular purine nucleotides, including adenosine triphosphate (ATP) and adenosine, as modulators of post-transplantation 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 post-transplant 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 post-transplant inflammation, and minimizing complications including rejection, graft failure and associated comorbidities.

Rationale: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. Objectives: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. Methods and Results: We generated endothelial Fto -deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet–induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto -deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D 2 levels. Blockade of prostaglandin D 2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto -deficient mice on high-fat diet; conversely, direct addition of prostaglandin D 2 rescued myogenic tone in high-fat diet–fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D 2 application. Conclusions: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

The objective of this study was to test if a low-calorie diet plus interval exercise (LCD+INT) reduced oxidized and non-oxidized phospholipids in relation to improved weight-related quality of life (QoL) to a greater extent than an energy-deficit matched LCD in obese females. Subjects (age: 47.2±2.6 years, body mass index: 37.5±1.3 kg/m(2)) were randomized to a 13-day LCD (n = 12; mixed meals of ~1200 kcal/day) or LCD+INT (n = 13; 12 sessions of 60 min/day alternating 3 min at 50% and 90% peak heart rate plus an additional 350 kcal shake fed after exercise to match energy availability between groups). Weight-related QoL (Laval Questionnaire) as well as oxidized (POVPC, HOOA-PC, HPETE-PC, HETE-PC, PEIPC, KOOA-PC) and non-oxidized (PAPC and lysoPC) phospholipids were assessed pre- and post-intervention. Fitness (VO(2)peak), body composition (BodPod), and clinical bloods were also tested. LCD+INT significantly increased VO(2)peak (mL/kg/min, P=0.03) compared to LCD despite similar fat loss, blood glucose, insulin sensitivity, and inflammatory responses. LCD+INT had significantly greater increases in QoL sexual life domain (P=0.05) and tended to have a greater increase in the emotions domain (P=0.09) and total score (P=0.10) compared to LCD. There were no significant differences between treatments for changes in phospholipids despite LCD+INT increasing measured oxidized and non-oxidized phospholipids while LCD decreased POVPC, HOOA-PC, and PEIPC as well as non-oxidized PAPC and lysoPC. Interestingly, the rise in PEIPC correlated with elevated VO(2)peak (mL/kg/min r=0.42, P=0.05). Decreased caloric intake was, however, linked to a decrease in PAPC (r=0.53, P=0.01), lysoPC (r=0.52, P=0.02), POVPC (r=0.43, P=0.05), and HPETE-PC (r=0.43, P=0.05). The decrease in HETE-PC also correlated with increases in the QoL domains symptoms (r=−0.46, P=0.04), hygiene/clothing (r=−0.53, P=0.01), emotions (r=−0.53, P=0.01), social interactions (r=−0.49, P=0.02), and total score (r=−0.52, P=0.02). In conclusion, although LCD and LCD+INT improved weight related QoL over 13 days in females with obesity, LCD+INT tended to improve sexual life, emotions as well as total QoL score more than LCD. These data suggest caloric restriction and fitness may act through different mechanisms to support QoL.

Objective Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. Methods In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. Results In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. Conclusions These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications., Highlights • Panx1 channel activation plays a critical role in thermogenic regulation in brown adipocytes. • Panx1 channel activity in brown adipocytes was induced by β3 adrenergic receptor stimulation. • Inactivation of Panx1 channels in cultured brown adipocytes resulted in defective cellular thermogenesis. • In mice, genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis. • Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis and aggravated diet-induced obesity and insulin resistance in mice.

Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of β-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process., eLife digest Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the β-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when β-cells become exhausted from increased insulin demand and stop producing insulin. β-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing β-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy β-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2—that is, ‘smell’ (‘find-me’ signals or sensing factors released by apoptotic cells), ‘taste’ (phagocyte–apoptotic cell contact) and ‘ingestion’ (corpse internalization)—activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells. Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway.

Objective— Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1β in the formation of murine experimental AAAs. Here, the hypothesis that IL-1β–induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested. Approach and Results— NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1β. In vitro, IL-1RA attenuated IL-1β–induced NETosis in human neutrophils. Mechanistically, IL-1β treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-β–induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage–day 3 of aneurysm induction. IL-1β–knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1β–knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation. Conclusions— Altogether, the results suggest a dominant role of IL-1β–induced NETosis in AAA formation.

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Oxidized phospholipids are products of lipid oxidation that are found on oxidized low-density lipoproteins and apoptotic cell membranes. These biologically active lipids were shown to affect a variety of cell types and attributed pro-as well as anti-inflammatory effects. In particular, macrophages exposed to oxidized phospholipids drastically change their gene expression pattern and function. These ‘Mox,’macrophages were identified in atherosclerotic lesions, however, it remains unclear how lipid oxidation products are sensed by macrophages and how they influence their biological function. Here, we review recent developments in the field that provide insight into the structure, recognition, and downstream signaling of oxidized phospholipids in macrophages.

Redox signaling is one of the key elements involved in cardiovascular diseases. Two important molecules are the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the oxidoreductase thioredoxin-1 (Trx-1). Recent Advances: During the previous years, a lot of studies investigated Nrf2 and Trx-1 as protective proteins in cardiovascular disorders. Moreover, post-translational modifications of those molecules were identified that play an important role in the cardiovascular system. This review will summarize changes in the vasculature in atherosclerosis and ischemia reperfusion injury of the heart and the newest findings achieved with Nrf2 and Trx-1 therein. Interestingly, Nrf2 and Trx-1 can act together as well as independently of each other in protection against atherosclerosis and ischemia and reperfusion injury.In principle, pharmacological activation of a transcription factor-like Nrf2 can be dangerous, since a transcription regulator has multiple targets and the pleiotropic effects of such activation should not be ignored. Moreover, overactivation of Nrf2 as well as long-term treatment with Trx-1 could be deleterious for the cardiovascular system.Therefore, the length of treatment with Nrf2 activators and/or Trx-1 has first to be studied in more detail in cardiovascular disorders. Moreover, a combination of Nrf2 activators and Trx-1 should be investigated and taken into consideration. Antioxid. Redox Signal. 26, 630-644.

Despite growing acknowledgement of the role of oxidized fatty acids (oxFA) as cellular signaling molecules and in the pathogenesis of disease, developing methods to measure these species in biological samples has proven challenging. Here we describe a novel method utilizing HPLC-ESI-MS/MS to identify and quantify multiple full-length oxFA species in a regioisomer-independent manner without the need for time-consuming sample preparation or derivatization. Building on recent progress in the characterization of FA and their oxidation products by MS/MS, we employed positive-ion ionization by measuring sodium adducts in conjunction with Differential Energy Qualifier Ion Monitoring to unequivocally verify the presence of the hydroperoxide, hydroxide, and ketone oxidation products of linoleic and arachidonic acid. Our HPLC method achieved separation of these oxidized species from their unoxidized counterparts while maintaining regioisomer-independent elution, allowing quantification over a 5 log10 range with a lower limit of quantification of 0.1 picomoles. With a simple sample preparation and a runtime as low as 11 minutes, our method allows the rapid and facile detection and measurement of full-length oxFA in biological samples. We believe this approach will allow for new insight and further investigation into the role of oxFA in metabolic disease.

Nonalcoholic steatohepatitis (NASH) affects 3%-5% of the U.S. population, having severe clinical complications to the development of fibrosis and end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma. A critical cause of NASH is chronic systemic inflammation promoted by innate immune cells, such as liver macrophages (Mϕ) and natural killer (NK) cells. However, little is known about how the crosstalk between Mϕ and NK cells contributes to regulate NASH progression to fibrosis. In this report, we demonstrate that NKp46+ cells play an important role in preventing NASH progression to fibrosis by regulating M1/M2 polarization of liver Mϕ. Using a murine model of NASH, we demonstrate that DX5+NKp46+ NK cells are increased during disease and play a role in polarizing Mϕ toward M1-like phenotypes. This NK's immunoregulatory function depends on the production of interferon-gamma (IFN-γ), but not by granzyme-mediated cytolytic activity. Notably, depletion of NKp46+ cells promotes the development of fibrosis with increased expression of profibrogenic genes as well as skewed M2 Mϕ phenotypes in hepatic tissues. Conclusions: NK cell-derived IFN-γ may be essential for maintaining a balanced inflammatory environment that promotes tissue integrity and limiting NASH progression to fibrosis. (Hepatology 2016;63:799–812)

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., Author summary Fighting infection requires that immune cells synthesize antimicrobial peptides and antibodies and carry out cellular processes like phagocytosis to destroy microbes and clear infected cells. During infection, metabolic processes support and direct immune function. Here, we use the fruit fly Drosophila melanogaster as a model system to understand the interaction between immunity and lipid metabolism. In Drosophila larvae, infection leads to tremendous production of antimicrobial peptides that destroy invading microbes. These peptides are made in the fat body, an organ that is also the site of fat storage. Activating the immune response reduces lipid storage but increases the production of phospholipids that form the membranes of organelles such as the endoplasmic reticulum. This organelle is the starting point for synthesis and secretion of antimicrobial peptides, and its volume is increased in response to immune activation. Shifting lipid metabolism to membrane phospholipid synthesis supports the immune response. However, this comes at the expense of the ability to withstand other types of stress such as desiccation at later stages in life. These findings are important because they suggest that some of the metabolic changes induced by fighting an infection may become detrimental if they are maintained over long periods of time.

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P, Inflammasome activation may contribute to type 2 diabetes, but whether targeting inflammasome is beneficial is unclear. Here the authors show that repurposing nucleoside reverse transcriptase inhibitors for inhibiting inflammasome activation is associated with reduced diabetes development in people and improves insulin sensitivity in experimental settings.

SUMMARY Phagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca2+ (phase 1), and by concurrently activating the mitochondrial Ca2+ (mCa2+) uniporter (MCU), they trigger a rapid influx of Ca2+ into the mitochondria (phase 2). mCa2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa2+ signaling, Mcu−/− macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca2+ signaling is an electrometabolic switch to fuel phagosome killing., Graphical Abstract, In Brief The signaling mechanisms that rapidly reallocate metabolic resources to meet the bioenergetic demands of microbial killing are not understood. Seegren et al. show that mitochondrial Ca2+ signaling serves as a fast electrometabolic switch to fuel microbial killing by phagocytes. This study identifies the mitochondrial Ca2+ channel MCU as a critical component of cell-intrinsic antimicrobial responses.

Adipose tissue macrophages (ATMs) adapt their metabolic phenotype either to maintain lean tissue homeostasis or drive inflammation and insulin resistance in obesity. However, the factors in the adipose tissue microenvironment that control ATM phenotypic polarization and bioenergetics remain unknown. We have recently shown that oxidized phospholipids (OxPL) uniquely regulate gene expression and cellular metabolism in Mox macrophages, but the presence of the Mox phenotype in adipose tissue has not been reported. Here we show, using extracellular flux analysis, that ATMs isolated from lean mice are metabolically inhibited. We identify a unique population of CX3CR1neg/F4/80low ATMs that resemble the Mox (Txnrd1+HO1+) phenotype to be the predominant ATM phenotype in lean adipose tissue. In contrast, ATMs isolated from obese mice had characteristics typical of the M1/M2 (CD11c+CD206+) phenotype with highly activated bioenergetics. Quantifying individual OxPL species in the stromal vascular fraction of murine adipose tissue, using targeted liquid chromatography-mass spectrometry, revealed that high fat diet-induced adipose tissue expansion led to a disproportional increase in full-length over truncated OxPL species. In vitro studies showed that macrophages respond to truncated OxPL species by suppressing bioenergetics and up-regulating antioxidant programs, mimicking the Mox phenotype of ATMs isolated from lean mice. Conversely, full-length OxPL species induce proinflammatory gene expression and an activated bioenergetic profile that mimics ATMs isolated from obese mice. Together, these data identify a redox-regulatory Mox macrophage phenotype to be predominant in lean adipose tissue and demonstrate that individual OxPL species that accumulate in adipose tissue instruct ATMs to adapt their phenotype and bioenergetic profile to either maintain redox homeostasis or to promote inflammation.

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the in vivo scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages in vivo . Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel in vitro model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and de novo lipogenesis-mediated lipid droplet formation in macrophages.

Marginal zone and follicular B cells together constitute the B2 cell population, which is known to promote cardiovascular diseases by secretion of pathogenic antibodies. However, it is not completely understood how B2 cells are activated. Here, we tested the hypothesis that B cell activating factor (BAFF) activates B2 cells and promote abdominal aortic aneurysm (AAA) formation. Since BAFF can either exist as a 3mer or multimerize to a highly active 60mer, we further examined if the 60mer is critical for B2 cell-mediated pathogenicity. Anti-BAFF antibody (Ab) Sandy-2 was injected to C57BL/6 male mice at 1 mg/kg once in every 14 days. AAA was induced by topical elastase model after 14 days of Sandy-2 injection. Native PAGE and ELISA methods were used to determine binding of Abs to recombinant BAFF 3mer and 60mer. For in vitro experiments, B cells were isolated from murine spleens. Activation of B cells was examined by Western blotting and RNA sequencing and by surface expression of CD23 and MHC II by flow cytometry. Metabolic reprogramming of B cells by BAFF was determined by extracellular flux analysis using a Seahorse XF24 Flux Analyzer. Sandy-2 bound to both 3mer and 60mer, resulting in suppressed AAA formation (n=8, pIn vitro , the 60mer, but not the 3mer, significantly activated both NF-kB1 and -kB2 signaling, and induced expression of B2 cell activation markers and anti-apoptotic genes in B cells. Inhibitors of NF-kB signaling decreased B cell activation in response to 60mer. The 60mer treatment significantly increased mitochondrial respiration and glycolysis in B cells, supporting an activated status. An antibody against multimerization site of BAFF (anti-multiBAFF) significantly suppressed B cell activation relative to a control Ab, in a neutrophil:B cell co-culture model. The effect of the anti-multiBAFF Ab on AAA formation is currently being tested in our laboratory. Altogether, our results suggest a critical role for BAFF 60mer in skewing B cells to an activated B2 cell phenotype, supporting a pathogenic role of B2 cells in AAA.

Background & Aims Mice exposed to the hepatocellular carcinogen diethylnitrosamine at 2weeks of age have a high risk of developing primary liver tumors later in life. Previous studies have demonstrated that diethylnitrosamine-treated mice have increased tumor burden when fed an obesigenic "Western" diet rich in lard fat and sugar. However, the role of dietary fats vs. sugars in the promotion of liver cancer is poorly understood. The aim of this study was to determine how altering dietary fats vs. sugars affects tumor burden in the diethylnitrosamine model. Methods C57BL/6N mice were treated with diethylnitrosamine at 2weeks of age and, from 6 to 32weeks of age, fed one of five diets that differed in fat and sugar content, including normal chow, ketogenic, and Western diets. Results Mice fed sugar-rich diets had the greatest tumor burden irrespective of dietary fat content. In contrast, mice fed a high-fat low-sugar diet had the least tumor burden despite obesity and glucose intolerance. When evaluated as independent variables, tumor burden was positively correlated with hepatic fat accumulation, postprandial insulin, and liver IL-6, and inversely correlated with serum adiponectin. In contrast, tumor burden did not correlate with adiposity, fasting insulin, or glucose intolerance. Furthermore, mice fed high sugar diets had lower liver expression of p21 and cleaved caspase-3 compared to mice fed low sugar diets. Conclusions These data indicate that dietary sugar intake contributes to liver tumor burden independent of excess adiposity or insulin resistance in mice treated with diethylnitrosamine.

B cell activating factor (BAFF) regulates differentiation and survival of B cells by binding to the surface receptors BAFF receptor (BR3), transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA). During differentiation, intracellular metabolic reprogramming is crucial, such as, naïve B cells are metabolically quiescent, whereas, antibody producing plasma cells are metabolically active. We have reported that depletion of B cells protects mice from abdominal aortic aneurysm (AA), however it is not clear how B cells promote AA growth. BAFF exists as a 3mer (binds only to BR3) or as a 60mer (binds to BR3, TACI and BCMA). Therefore, we hypothesize that BAFF multimerization regulates the immune and metabolic phenotype of B cells by binding to BAFF receptors and modulate AA growth. Immunohistology was performed on AA tissues collected from patients undergoing open AA repair. Experimental AA was induced by elastase perfusion of abdominal aorta or angiotensin II infusion (1000 ng/kg/min) method in 8 weeks old male C57BL/6 or apolipoprotein E knockout mice, respectively. Western blotting, flow cytometry and Seahorse extracellular flux assays were used to determine immune and metabolic changes in B cells in response to recombinant BAFF 3mer and 60mer. BR3+ B cells were detected in the milieu of BAFF in human AAs. Mouse AAs demonstrated significant infiltration (>50/section) of CD138+ plasma B cells, but few (4-10/section) CD20+ B cells. In vitro, BAFF 3mer induced canonical NF-kB, whereas, 60mer induced both canonical and non-canonical NF-kB signaling. Moreover, the 3mer significantly decreased mitochondrial density, oxygen consumption rate, and surface expression of IgD and IgM indicating a metabolically quiescent state of B cells. However, these parameters were significantly increased by the 60mer similar to plasma cells. Anti-BR3 IgG1, but not a control IgG1 antibody decreased BAFF 60mer-induced oxygen consumption rate by 50%. In a pilot study (n=10/group), anti-BR3 IgG1, but not the control IgG1 aggravated angiotensin II-induced AA growth. Altogether, our results suggest that BAFF 3mer and 60mer oppositely regulate immune and metabolic phenotype of B cells and inhibition of BAFF-BR3 signaling is detrimental for AA growth.

Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM. Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed. Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib. Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958–68. ©2017 AACR.