A subset of Kupffer cells regulates metabolism through the expression of CD36.

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206loESAM- population (KC1) and a minor CD206hiESAM+ population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.

Impaired Kupffer Cell Self-Renewal Alters the Liver Response to Lipid Overload during Non-alcoholic Steatohepatitis.

Kupffer cells (KCs) are liver-resident macrophages that self-renew by proliferation in the adult independently from monocytes. However, how they are maintained during non-alcoholic steatohepatitis (NASH) remains ill defined. We found that a fraction of KCs derived from Ly-6C+ monocytes during NASH, underlying impaired KC self-renewal. Monocyte-derived KCs (MoKCs) gradually seeded the KC pool as disease progressed in a response to embryo-derived KC (EmKC) death. Those MoKCs were partly immature and exhibited a pro-inflammatory status compared to EmKCs. Yet, they engrafted the KC pool for the long term as they remained following disease regression while acquiring mature EmKC markers. While KCs as a whole favored hepatic triglyceride storage during NASH, EmKCs promoted it more efficiently than MoKCs, and the latter exacerbated liver damage, highlighting functional differences among KCs with different origins. Overall, our data reveal that KC homeostasis is impaired during NASH, altering the liver response to lipids, as well as KC ontogeny.

Pivotal role of NAMPT in the switch of melanoma cells toward an invasive and drug-resistant phenotype.

In BRAFV600E melanoma cells, a global metabolomic analysis discloses a decrease in nicotinamide adenine dinucleotide (NAD+) levels upon PLX4032 treatment that is conveyed by a STAT5 inhibition and a transcriptional regulation of the nicotinamide phosphoribosyltransferase (NAMPT) gene. NAMPT inhibition decreases melanoma cell proliferation both in vitro and in vivo, while forced NAMPT expression renders melanoma cells resistant to PLX4032. NAMPT expression induces transcriptomic and epigenetic reshufflings that steer melanoma cells toward an invasive phenotype associated with resistance to targeted therapies and immunotherapies. Therefore, NAMPT, the key enzyme in the NAD+ salvage pathway, appears as a rational target in targeted therapy-resistant melanoma cells and a key player in phenotypic plasticity of melanoma cells.

Macrophage ontogeny and functional diversity in cardiometabolic diseases.

Macrophages are the dominant immune cell types in the adipose tissue, the liver or the aortic wall and they were originally believed to mainly derived from monocytes to fuel tissue inflammation in cardiometabolic diseases. However, over the last decade the identification of tissue resident macrophages (trMacs) from embryonic origin in these metabolic tissues has provided a breakthrough in the field forcing to better comprehend macrophage diversity during pathological states. Infiltrated monocyte-derived macrophages (moMacs), similar to trMacs, adapt to the local metabolic environment that eventually shapes their functions. In this review, we will summarize the emerging versatility of macrophages in cardiometabolic diseases with a focus in the control of adipose tissue, liver and large vessels homeostasis.

Granulopoiesis and Neutrophil Homeostasis: A Metabolic, Daily Balancing Act.

Granulopoiesis is part of the hematopoietic hierarchic architecture, where hematopoietic stem cells give rise to highly proliferative multipotent and lineage-committed granulocytic progenitor cells that differentiate into unipotent neutrophil progenitors. Given their short lifespan, neutrophils are rapidly cleared from circulation through specialized efferocytic macrophages. Together with an intrinsic clock, these processes contribute to circadian fluctuations, preserving self-tolerance and protection against invading pathogens. However, metabolic perturbation of granulopoiesis and neutrophil homeostasis can result in low-grade chronic inflammation, as observed with aging. During acute pathogenic infections, hematopoiesis can also be switched into emergency mode, which has been recently associated with significant neutrophil functional heterogeneity. This review focuses on a new reassessment of regulatory mechanisms governing neutrophil production, life-cycle, and diversity in health and disease.

REDD1 deficiency protects against nonalcoholic hepatic steatosis induced by high-fat diet.

Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

Mesothelial cell CSF1 sustains peritoneal macrophage proliferation.

Macrophages play a central role during infection, inflammation and tissue homeostasis maintenance. Macrophages have been identified in all organs and their core transcriptomic signature and functions differ from one tissue to another. Interestingly, macrophages have also been identified in the peritoneal cavity and these cells have been extensively used as a model for phagocytosis, efferocytosis and polarization. Peritoneal macrophages are involved in B-cell IgA production, control of inflammation and wound healing following thermal-induced liver surface injury. These cells presumably require and interact with the omentum, where milky spot stromal cells have been proposed to secrete CSF1 (colony stimulating factor 1). Peritoneal macrophages depend on CSF1 for their generation and survival, but the identity of CSF1 producing cells inside the large peritoneal cavity remains unknown. Here we investigated peritoneal macrophage localization and their interaction with mesothelial cells, the major cell type predicted to secrete CSF1. Our data revealed that mesothelial cells produce membrane bound and secreted CSF1 that both sustain peritoneal macrophage growth.

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Cholesterol Mass Efflux Capacity, Incident Cardiovascular Disease, and Progression of Carotid Plaque.

Objective- To assess the role of HDL (high-density lipoprotein)-mediated cholesterol mass efflux capacity (CMEC) in incident cardiovascular disease and carotid plaque progression. Approach and Results- We measured CMEC in 2 cohorts aged 45 to 84 years at baseline derived from the MESA (Multi-Ethnic Study of Atherosclerosis). Cohort 1 comprised 465 cases with incident cardiovascular disease events during 10 years of follow-up and 465 age- and sex-matched controls; cohort 2 comprised 407 cases with progression of carotid plaque measured by ultrasonography at 2 exams >10 years and 407 similarly matched controls. Covariates and outcome events were ascertained according to the MESA protocol. CMEC level was modestly correlated with HDL cholesterol ( R=0.13; P<0.001) but was not associated with age, sex, race/ethnicity, body mass index, diabetes mellitus, alcohol use, smoking status, or statin use. Higher CMEC level was significantly associated with lower odds of cardiovascular disease (odds ratio, 0.82 per SD of CMEC [95% CI, 0.69-0.98; P=0.031] in the fully adjusted model) in cohort 1 but higher odds of carotid plaque progression (odds ratio, 1.24 per SD of CMEC [95% CI, 1.04-1.48; P=0.018] in the fully adjusted model) in cohort 2 but without dose-response effect. In subgroup analysis within cohort 1, higher CMEC was associated with lower risk of incident coronary heart disease events (odds ratio, 0.72 per SD of CMEC (95% CI, 0.5-0.91; P=0.007) while no association was found with stroke events. Conclusions- These findings support a role for HDL-mediated cholesterol efflux in an atheroprotective mechanism for coronary heart disease but not stroke.

The modern interleukin-1 superfamily: Divergent roles in obesity.

Obesity is now recognised as a chronic, low-grade inflammatory disease contributing to insulin resistance, type 2 diabetes (T2D) and cardiovascular disease (CVD). Multiple mechanisms leading to the low grade inflammation in this setting have been suggested. Due to the complexity and interconnection of inflammatory and metabolic responses, there also remains a need to fully elucidate the inflammatory mechanisms that control obesity and associated metabolic disorders. One important avenue in the field that has gained great attention is the interleukin (IL)-1 superfamily of cytokines that consist of IL-1ß, IL-18 and IL-33. IL-1ß is well known for its contribution as an inflammatory mediator in obesity contributing to insulin resistance and T2D, whereas the IL-18 and IL-33 cytokines have been shown to oppose metabolic dysregulation. This review will focus on the current understanding of the IL-1 superfamily of cytokines in the setting of obesity and discuss how endogenous feedback loops can be exploited for therapeutic approaches to fight obesity and subsequent cardiometabolic disorders.

Poststatin era in atherosclerosis management: lessons from epidemiologic and genetic studies.

PURPOSE OF REVIEW: Cardiovascular diseases (CVD) are the leading cause of death worldwide with over 17 million deaths every year and represent a major public health challenge. The last decade has seen the emergence of novel antiatherogenic therapies. RECENT FINDINGS: Despite intensive lipid and blood pressure interventions, the burden of CVD is expected to markedly progress because of the global aging of the population and increasing exposure to detrimental lifestyle-related risk. Epidemiologic and genetic studies helped to better apprehend the biology of atherosclerosis and allowed pharmaceutical innovation and recent translational successes. This includes the development of novel lipid and glucose-lowering therapies and the leverage of anti-inflammatory therapies. SUMMARY: Here, we discuss promises and expectations of emerging scientific and pharmaceutical innovations and translational successes to meet the global therapeutic demand.

Disruption of Glut1 in Hematopoietic Stem Cells Prevents Myelopoiesis and Enhanced Glucose Flux in Atheromatous Plaques of ApoE(-/-) Mice.

RATIONALE: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with (18)F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. OBJECTIVE: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE(-/-) mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE(-/-) hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common ß subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1(+/-), ApoE(-/-), and ApoE(-/-)Glut1(+/-) mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE(-/-) hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE(-/-) mice transplanted with ApoE(-/-) bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. CONCLUSIONS: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE(-/-) mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.

HIF-2α in Resting Macrophages Tempers Mitochondrial Reactive Oxygen Species To Selectively Repress MARCO-Dependent Phagocytosis.

Hypoxia-inducible factor (HIF)-α isoforms regulate key macrophage (MΦ) functions during ischemic inflammation. HIF-2α drives proinflammatory cytokine production; however, the requirements for HIF-2α during other key MΦ functions, including phagocytosis, are unknown. In contrast to HIF-1α, HIF-2α was not required for hypoxic phagocytic uptake. Surprisingly, basal HIF-2α levels under nonhypoxic conditions were necessary and sufficient to suppress phagocytosis. Screening approaches revealed selective induction of the scavenger receptor MARCO, which was required for enhanced engulfment. Chromatin immunoprecipitation identified the antioxidant NRF2 as being directly responsible for inducing Marco Concordantly, Hif-2α-/- MΦs exhibited reduced antioxidant gene expression, and inhibition of mitochondrial reactive oxygen species suppressed Marco expression and phagocytic uptake. Ex vivo findings were recapitulated in vivo; the enhanced engulfment phenotype resulted in increased bacterial clearance and cytokine suppression. Importantly, natural induction of Hif-2α by IL-4 also suppressed MARCO-dependent phagocytosis. Thus, unlike most characterized prophagocytic regulators, HIF-2α can act as a phagocytic repressor. Interestingly, this occurs in resting MΦs through tempering of steady-state mitochondrial reactive oxygen species. In turn, HIF-2α promotes MΦ quiescence by blocking a MARCO bacterial-response pathway. IL-4 also drives HIF-2α suppression of MARCO, leading to compromised bacterial immunosurveillance in vivo.

Understanding macrophage diversity at the ontogenic and transcriptomic levels.

Macrophages are phagocytes characterized by high lysosomal activity and are involved in a wide range of biological processes. Consequently, macrophages have long been recognized for their critical roles in development as well as in healthy and pathological states. Our knowledge about macrophage biology has evolved greatly over the past several years. Significantly, it has now been demonstrated that monocytes are not direct precursors for most tissue-resident macrophages at the steady state. Only few tissue macrophage populations derive from monocytes during homeostasis; rather, monocytes give rise to inflammatory macrophages that infiltrate tissues during inflammation. Tissue-resident macrophages have recently been characterized at the transcriptional level, which provided the basis to uncover the molecular pathways controlling their functional diversity as well as to identify a core signature. Transcription factors controlling specific tissue-resident macrophage populations have been described, suggesting that diversity is under the control of specific regulatory programs. In this review, we discuss and summarize several of the new paradigms emerging in the field of macrophage biology. In particular, we emphasize new findings relevant to macrophage ontogeny, similarities and differences observed across macrophage populations, and gene regulatory programs controlling specialized aspects of tissue macrophage functions.

Plasma metabolite profiles, cellular cholesterol efflux, and non-traditional cardiovascular risk in patients with CKD.

BACKGROUND: Patients with chronic kidney disease (CKD) experience high rates of atherosclerotic cardiovascular disease and death that are not fully explained by traditional risk factors. In animal studies, defective cellular cholesterol efflux pathways which are mediated by the ATP binding cassette transporters ABCA1 and ABCG1 are associated with accelerated atherosclerosis. We hypothesized that cholesterol efflux in humans would vary in terms of cellular components, with potential implications for cardiovascular disease. METHODS: We recruited 120 CKD patients (eGFR<30mL/min/1.73m2) and 120 control subjects (eGFR ≥60mL/min/1.73m2) in order to measure cholesterol efflux using either patients' HDL and THP-1 macrophages or patients' monocytes and a flow cytometry based cholesterol efflux assay. We also measured cell-surface levels of the common ß subunit of the IL-3/GM-CSF receptor (IL-3Rß) which has been linked to defective cholesterol homeostasis and may promote monocytosis. In addition, we measured plasma inflammatory cytokines and plasma metabolite profiles. RESULTS: There was a strong positive correlation between cell-surface IL-3Rß levels and monocyte counts in CKD (P<0.001). ABCA1 mRNA was reduced in CKD vs. control monocytes (P<0.05), across various etiologies of CKD. Cholesterol efflux to apolipoprotein A1 was impaired in monocytes from CKD patients with diabetic nephropathy (P<0.05), but we found no evidence for a circulating HDL-mediated defect in cholesterol efflux in CKD. Profiling of plasma metabolites showed that medium-chain acylcarnitines were both independently associated with lower levels of cholesterol transporter mRNA in CKD monocytes at baseline (P<0.05), and with cardiovascular events in CKD patients after median 2.6years of follow-up. CONCLUSIONS: Cholesterol efflux in humans varies in terms of cellular components. We report a cellular defect in ABCA1-mediated cholesterol efflux in monocytes from CKD patients with diabetic nephropathy. Unlike several traditional risk factors for atherosclerotic cardiovascular disease, plasma metabolites inversely associated with endogenous cholesterol transporters predicted cardiovascular events in CKD patients. (Funded by the National Institute of Diabetes and Digestive and Kidney DiseasesK23DK097288 and others.).

ATP-binding cassette transporters, atherosclerosis, and inflammation.

Although recent genome-wide association studies have called into question the causal relationship between high-density lipoprotein (HDL) cholesterol levels and cardiovascular disease, ongoing research in animals and cells has produced increasing evidence that cholesterol efflux pathways mediated by ATP-binding cassette (ABC) transporters and HDL suppress atherosclerosis. These differing perspectives may be reconciled by a modified HDL theory that emphasizes the antiatherogenic role of cholesterol flux pathways, initiated in cells by ABC transporters. ABCA1 and ABCG1 control the proliferation of hematopoietic stem and multipotential progenitor cells in the bone marrow and hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis in the spleen. Thus, activation of cholesterol efflux pathways by HDL infusions or liver X receptor activation results in suppression of hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis, leading to decreased production of monocytes and neutrophils and suppression of atherosclerosis. In addition, macrophage-specific knockout of transporters has confirmed their role in suppression of inflammatory responses in the arterial wall. Recent studies have also shown that ABCG4, a close relative of ABCG1, controls platelet production, atherosclerosis, and thrombosis. ABCG4 is highly expressed in megakaryocyte progenitors, where it promotes cholesterol efflux to HDL and controls the proliferative responses to thrombopoietin. Reconstituted HDL infusions act in an ABCG4-dependent fashion to limit hypercholesterolemia-driven excessive platelet production, thrombosis, and atherogenesis, as occurs in human myeloproliferative syndromes. Activation of ABC transporter-dependent cholesterol efflux pathways in macrophages, hematopoietic stem and multipotential progenitor cells, or platelet progenitors by reconstituted HDL infusion or liver X receptor activation remain promising approaches to the treatment of human atherothrombotic diseases.

Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice.

RATIONALE: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. OBJECTIVE: To assess the role of macrophage cholesterol efflux pathways in atherogenesis. METHODS AND RESULTS: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. CONCLUSIONS: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Deficiency of ATP-binding cassette transporter B6 in megakaryocyte progenitors accelerates atherosclerosis in mice.

OBJECTIVE: The ATP-binding cassette (ABC) transporter B6 (ABCB6) is highly expressed in megakaryocyte progenitors, but its role in platelet production and disease has not been elucidated. APPROACH AND RESULTS: Among various ABC transporters, ABCB6 was highly expressed in megakaryocyte progenitors, exhibiting the same pattern of expression of genes involved in heme synthesis pathway. Transplantation of Abcb6 deficient (Abcb6(-/-)) bone marrow into low density lipoprotein receptor deficient recipient mice resulted in expansion and proliferation of megakaryocyte progenitors, attributable to increased reactive oxygen species production in response to porphyrin loading. The enhanced megakaryopoiesis in Abcb6(-/-) bone marrow-transplanted mice was further illustrated by increased platelet counts, mean platelet volume, and platelet activity. Platelets from Abcb6(-/-) bone marrow-transplanted mice had higher levels of chemokine (C-C motif) ligand 5, which was associated with increased plasma chemokine (C-C motif) ligand 5 levels. There were also increased platelet-leukocyte aggregates, which resulted in leukocyte activation. Abcb6(-/-) bone marrow-transplanted mice had accelerated atherosclerosis which was associated with deposition of the chemotactic agent, chemokine (C-C motif) ligand 5 in atherosclerotic plaques, resulting in increased macrophage accumulation. CONCLUSIONS: Our findings identify a new role of ABCB6 in preventing atherosclerosis development by dampening platelet production, reactivity, and chemokine (C-C motif) ligand 5 deposition in atherosclerotic lesions.