Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain.

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Evaluation of an in vitro coronary stent thrombosis model for preclinical assessment.

Stent thrombosis remains an infrequent but significant complication following percutaneous coronary intervention. Preclinical models to rapidly screen and validate therapeutic compounds for efficacy are lacking. Herein, we describe a reproducible, high throughput and cost-effective method to evaluate candidate therapeutics and devices for either treatment or propensity to develop stent thrombosis in an in vitro bench-top model. Increasing degree of stent malapposition (0.00 mm, 0.10 mm, 0.25 mm and 0.50 mm) was associated with increasing thrombosis and luminal area occlusion (4.1 ± 0.5%, 6.3 ± 0.5%, 19.7 ± 4.5%, and 92.6 ± 7.4%, p < 0.0001, respectively). Differences in stent design in the form of bare-metal, drug-eluting, and bioresorbable vascular scaffolds demonstrated differences in stent thrombus burden (14.7 ± 3.8% vs. 20.5 ± 3.1% vs. 86.8 ± 5.3%, p < 0.01, respectively). Finally, thrombus burden was significantly reduced when healthy blood samples were incubated with Heparin, ASA/Ticagrelor (DAPT), and Heparin+DAPT compared to control (DMSO) at 4.1 ± 0.6%, 6.9 ± 1.7%, 4.5 ± 1.2%, and 12.1 ± 1.8%, respectively (p < 0.01). The reported model produces high throughput reproducible thrombosis results across a spectrum of antithrombotic agents, stent design, and degrees of apposition. Importantly, performance recapitulates clinical observations of antiplatelet/antithrombotic regimens as well as device and deployment characteristics. Accordingly, this model may serve as a screening tool for candidate therapies in preclinical evaluation.

Extracellular Vesicles Secreted by Atherogenic Macrophages Transfer MicroRNA to Inhibit Cell Migration.

OBJECTIVE: During inflammation, macrophages secrete vesicles carrying RNA, protein, and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis; however, the role of macrophage-derived EVs in atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNAs) in EVs to mediate cell-cell communication and promote proinflammatory and proatherogenic phenotypes in recipient cells. APPROACH AND RESULTS: We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized low-density lipoprotein) and characterized the EV miRNA expression profile. We confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365, and miR-503 in atherogenic EVs compared with controls and demonstrate that these EVs are taken up and transfer exogenous miRNA to naive recipient macrophages. Bioinformatic pathway analysis suggests that atherogenic EV miRNAs are predicted to target genes involved in cell migration and adhesion pathways, and indeed delivery of EVs to naive macrophages reduced macrophage migration both in vitro and in vivo. Inhibition of miR-146a, the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1) and HuR (human antigen R or ELAV-like RNA-binding protein 1) in recipient cells, and knockdown of IGF2BP1 and HuR using short interfering RNA greatly reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. CONCLUSIONS: EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.

The walking dead: macrophage inflammation and death in atherosclerosis.

PURPOSE OF REVIEW: To highlight recent studies that describe novel inflammatory and signaling mechanisms that regulate macrophage death in atherosclerosis. RECENT FINDINGS: Macrophages contribute to all stages of atherosclerosis. The traditional dogma states that in homeostatic conditions, macrophages undergo apoptosis and are efficiently phagocytosed to be cleared by a process called efferocytosis. In advanced atherosclerosis, however, defective efferocytosis results in secondary necrosis of these uncleared apoptotic cells, which ultimately contributes to the formation of the characteristic necrotic core and the vulnerable plaque. Here, we outline the different types of lesional macrophage death: apoptosis, autophagic and the newly defined necroptosis (i.e. a type of programmed necrosis). Recent discoveries demonstrate that macrophage necroptosis directly contributes to necrotic core formation and plaque instability. Further, promoting the resolution of inflammation using preresolving mediators has been shown to enhance efferocytosis and decrease plaque vulnerability. Finally, the canonical 'don't eat me' signal CD47 has recently been described as playing an important role in atherosclerotic lesion progression by impairing efficient efferocytosis. Although we have made significant strides in improving our understanding of cell death and clearance mechanisms in atherosclerosis, there still remains unanswered questions as to how these pathways can be harnessed using therapeutics to promote lesion regression and disease stability. SUMMARY: Improving our understanding of the mechanisms that regulate macrophage death in atherosclerosis, in particular apoptosis, necroptosis and efferocytosis, will provide novel therapeutic opportunities to resolve atherosclerosis and promote plaque stability.

Macrophage miRNAs in atherosclerosis.

The discovery of endogenous microRNAs (miRNAs) in the early 1990s has been followed by the identification of hundreds of miRNAs and their roles in regulating various biological processes, including proliferation, apoptosis, lipid metabolism, glucose homeostasis and viral infection Esteller (2011), Ameres and Zamore (2013) [1,2]. miRNAs are small (~22 nucleotides) non-coding RNAs that function as "rheostats" to simultaneously tweak the expression of multiple genes within a genetic network, resulting in dramatic functional modulation of biological processes. Although the last decade has brought the identification of miRNAs, their targets and function(s) in health and disease, there remains much to be deciphered from the human genome and its complexities in mechanistic regulation of entire genetic networks. These discoveries have opened the door to new and exciting avenues for therapeutic interventions to treat various pathological diseases, including cardiometabolic diseases such as atherosclerosis, diabetes and obesity. In a complex multi-factorial disease like atherosclerosis, many miRNAs have been shown to contribute to disease progression and may offer novel targets for future therapy. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis.

RATIONALE: Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. OBJECTIVE: We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. METHODS AND RESULTS: In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. CONCLUSIONS: This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Therapeutic Inhibition of miR-33 Promotes Fatty Acid Oxidation but Does Not Ameliorate Metabolic Dysfunction in Diet-Induced Obesity.

OBJECTIVE: miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. APPROACH AND RESULTS: High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33-treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33-treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33-treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. CONCLUSIONS: We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.

Protein kinase C controls vesicular transport and secretion of apolipoprotein E from primary human macrophages.

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/ß and not PKCδ, -ε, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

Unlocking the door to new therapies in cardiovascular disease: microRNAs hold the key.

MicroRNAs are the most abundant class of regulatory noncoding RNA and are estimated to regulate over half of all human protein-coding genes. The heart is comprised of some of the most complex and highly conserved genetic networks and is thus under tight regulation by post-transcriptional mechanisms. MicroRNAs (miRNAs) have been found to regulate virtually all aspects of cardiac physiology and pathophysiology, from the development of inflammatory atherosclerosis to hypertrophic remodeling in heart failure. Owing to the wide-spread involvement of miRNAs in the development of and protection from many diseases, there has been increasing excitement surrounding their potential as novel therapeutic targets to treat and prevent the worldwide epidemic of cardiovascular disease.

Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order.

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.

Cathepsin G deficiency decreases complexity of atherosclerotic lesions in apolipoprotein E-deficient mice.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

Histone deacetylase 7: a signalling hub controlling development, inflammation, metabolism and disease.

Histone deacetylases (HDACs) catalyse removal of acetyl groups from lysine residues on both histone and non-histone proteins to control numerous cellular processes. Of the 11 zinc-dependent classical HDACs, HDAC4, 5, 7 and 9 are class IIa HDAC enzymes that regulate cellular and developmental processes through both enzymatic and non-enzymatic mechanisms. Over the last two decades, HDAC7 has been associated with key roles in numerous physiological and pathological processes. Molecular, cellular, in vivo and disease association studies have revealed that HDAC7 acts through multiple mechanisms to control biological processes in immune cells, osteoclasts, muscle, the endothelium and epithelium. This HDAC protein regulates gene expression, cell proliferation, cell differentiation and cell survival and consequently controls development, angiogenesis, immune functions, inflammation and metabolism. This review focuses on the cell biology of HDAC7, including the regulation of its cellular localisation and molecular mechanisms of action, as well as its associative and causal links with cancer and inflammatory, metabolic and fibrotic diseases. We also review the development status of small molecule inhibitors targeting HDAC7 and their potential for intervention in different disease contexts.

The histone deacetylase Hdac7 supports LPS-inducible glycolysis and Il-1ß production in murine macrophages via distinct mechanisms.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1ß and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

GPIbalpha-selective activation of platelets induces platelet signaling events comparable to GPVI activation events.

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

Publisher Correction: RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.