A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility.

The cellular lipidome comprises thousands of unique lipid species. Here, using mass spectrometry-based targeted lipidomics, we characterize the lipid landscape of human and mouse immune cells ( www.cellularlipidatlas.com ). Using this resource, we show that immune cells have unique lipidomic signatures and that processes such as activation, maturation and development impact immune cell lipid composition. To demonstrate the potential of this resource to provide insights into immune cell biology, we determine how a cell-specific lipid trait-differences in the abundance of polyunsaturated fatty acid-containing glycerophospholipids (PUFA-PLs)-influences immune cell biology. First, we show that differences in PUFA-PL content underpin the differential susceptibility of immune cells to ferroptosis. Second, we show that low PUFA-PL content promotes resistance to ferroptosis in activated neutrophils. In summary, we show that the lipid landscape is a defining feature of immune cell identity and that cell-specific lipid phenotypes underpin aspects of immune cell physiology.

ACAD10 is not required for metformin's metabolic actions or for maintenance of whole-body metabolism in C57BL/6J mice.

AIM: Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. MATERIALS AND METHODS: We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. RESULTS: Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. CONCLUSIONS: Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.

Administration of an LXR agonist promotes atherosclerotic lesion remodelling in murine inflammatory arthritis.

Objectives: The leading cause of mortality in patients with rheumatoid arthritis is atherosclerotic cardiovascular disease (CVD). We have shown that murine arthritis impairs atherosclerotic lesion regression, because of cellular cholesterol efflux defects in haematopoietic stem and progenitor cells (HSPCs), causing monocytosis and impaired atherosclerotic regression. Therefore, we hypothesised that improving cholesterol efflux using a Liver X Receptor (LXR) agonist would improve cholesterol efflux and improve atherosclerotic lesion regression in arthritis. Methods: Ldlr -/- mice were fed a western-type diet for 14 weeks to initiate atherogenesis, then switched to a chow diet to induce lesion regression and divided into three groups; (1) control, (2) K/BxN serum transfer inflammatory arthritis (K/BxN) or (3) K/BxN arthritis and LXR agonist T0901317 daily for 2 weeks. Results: LXR activation during murine inflammatory arthritis completely restored atherosclerotic lesion regression in arthritic mice, evidenced by reduced lesion size, macrophage abundance and lipid content. Mechanistically, serum from arthritic mice promoted foam cell formation, demonstrated by increased cellular lipid accumulation in macrophages and paralleled by a reduction in mRNA of the cholesterol efflux transporters Abca1, Abcg1 and Apoe. T0901317 reduced lipid loading and increased Abca1 and Abcg1 expression in macrophages exposed to arthritic serum and increased ABCA1 levels in atherosclerotic lesions of arthritic mice. Moreover, arthritic clinical score was also attenuated with T0901317. Conclusion: Taken together, we show that the LXR agonist T0901317 rescues impaired atherosclerotic lesion regression in murine arthritis because of enhanced cholesterol efflux transporter expression and reduced foam cell development in atherosclerotic lesions.

Macrophage polarization state affects lipid composition and the channeling of exogenous fatty acids into endogenous lipid pools.

Adipose-tissue-resident macrophages (ATMs) maintain metabolic homeostasis but also contribute to obesity-induced adipose tissue inflammation and metabolic dysfunction. Central to these contrasting effects of ATMs on metabolic homeostasis is the interaction of macrophages with fatty acids. Fatty acid levels are increased within adipose tissue in various pathological and physiological conditions, but appear to initiate inflammatory responses only upon interaction with particular macrophage subsets within obese adipose tissue. The molecular basis underlying these divergent outcomes is likely due to phenotypic differences between ATM subsets, although how macrophage polarization state influences the metabolism of exogenous fatty acids is relatively unknown. Herein, using stable isotope-labeled and nonlabeled fatty acids in combination with mass spectrometry lipidomics, we show marked differences in the utilization of exogenous fatty acids within inflammatory macrophages (M1 macrophages) and macrophages involved in tissue homeostasis (M2 macrophages). Specifically, the accumulation of exogenous fatty acids within triacylglycerols and cholesterol esters is significantly higher in M1 macrophages, while there is an increased enrichment of exogenous fatty acids within glycerophospholipids, ether lipids, and sphingolipids in M2 macrophages. Finally, we show that functionally distinct ATM populations in vivo have distinct lipid compositions. Collectively, this study identifies new aspects of the metabolic reprogramming that occur in distinct macrophage polarization states. The channeling of exogenous fatty acids into particular lipid synthetic pathways may contribute to the sensitivity/resistance of macrophage subsets to the inflammatory effects of increased environmental fatty acid levels.

Tissue-specific expression of Cas9 has no impact on whole-body metabolism in four transgenic mouse lines.

OBJECTIVE: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. METHODS: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. RESULTS: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. CONCLUSIONS: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.

Oral Supplementation of an Alkylglycerol Mix Comprising Different Alkyl Chains Effectively Modulates Multiple Endogenous Plasmalogen Species in Mice.

Plasmalogens or alkenylphospholipids are a sub-class of glycerophospholipids with numerous biological functions and are thought to have protective effects against metabolic disease. Dietary supplementation with alkylglycerols (AKGs) has been shown to increase endogenous plasmalogen levels, however effective modulation of different molecular plasmalogen species has not yet been demonstrated. In this study, the effects of an orally-administered AKG mix (a mixture of chimyl, batyl and selachyl alcohol at a 1:1:1 ratio) on plasma and tissue lipids, including plasmalogens, was evaluated. Mice on a Western-type diet were treated with either an AKG mix or vehicle (lecithin) for 1, 2, 4, 8 and 12 weeks. Treatment with the AKG mix significantly increased the total plasmalogen content of plasma, liver and adipose tissue as a result of elevations in multiple plasmalogen species with different alkenyl chains. Alkylphospholipids, the endogenous precursors of plasmalogens, showed a rapid and significant increase in plasma, adipose tissue, liver and skeletal muscle. A significant accumulation of alkyl-diacylglycerol and lyso-ether phospholipids was also observed in plasma and tissues. Additionally, the dynamics of plasmalogen-level changes following AKG mix supplementation differed between tissues. These findings indicate that oral supplementation with an AKG mix is capable of upregulating and maintaining stable expression of multiple molecular plasmalogen species in circulation and tissues.

Shark liver oil supplementation enriches endogenous plasmalogens and reduces markers of dyslipidemia and inflammation.

Plasmalogens are membrane glycerophospholipids with diverse biological functions. Reduced plasmalogen levels have been observed in metabolic diseases; hence, increasing their levels might be beneficial in ameliorating these conditions. Shark liver oil (SLO) is a rich source of alkylglycerols that can be metabolized into plasmalogens. This study was designed to evaluate the impact of SLO supplementation on endogenous plasmalogen levels in individuals with features of metabolic disease. In this randomized, double-blind, placebo-controlled cross-over study, the participants (10 overweight or obese males) received 4-g Alkyrol® (purified SLO) or placebo (methylcellulose) per day for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. SLO supplementation led to significant changes in plasma and circulatory white blood cell lipidomes, notably increased levels of plasmalogens and other ether lipids. In addition, SLO supplementation significantly decreased the plasma levels of total free cholesterol, triglycerides, and C-reactive protein. These findings suggest that SLO supplementation can enrich plasma and cellular plasmalogens and this enrichment may provide protection against obesity-related dyslipidemia and inflammation.

Characterization of the circulating and tissue-specific alterations to the lipidome in response to moderate and major cold stress in mice.

This study analyzed the effects of 24 h of cold stress (22°C or 5°C vs. mice maintained at 30 °C) on the plasma, brown adipose tissue (BAT), subcutaneous (SubQ) and epididymal (Epi) white adipose tissue (WAT), liver, and skeletal muscle lipidome of mice. Using mass spectrometry-lipidomics, 624 lipid species were detected, of which 239 were significantly altered in plasma, 134 in BAT, and 51 in the liver. In plasma, acylcarnitines and free fatty acids were markedly increased at 5°C. Plasma triacylglycerols (TGs) were reduced at 22°C and 5°C. We also identified ether lipids as a novel, cold-induced lipid class. In BAT, TGs were the principal lipid class affected by cold stress, being significantly reduced at both 22°C and 5°C. Interestingly, although BAT TG species were uniformly affected at 5°C, at 22°C we observed species-dependent effects, with TGs containing longer and more unsaturated fatty acids particularly sensitive to the effects of cold. In the liver, TGs were the most markedly affected lipid class, increasing in abundance at 5 °C. TGs containing longer and more unsaturated fatty acids accumulated to a greater degree. Our work demonstrates the following: 1) acute exposure to moderate (22°C) cold stress alters the plasma and BAT lipidome; although this effect is markedly less pronounced than at 5°C. 2) Cold stress at 5°C dramatically alters the plasma lipidome, with ether lipids identified as a novel lipid class altered by cold exposure. 3) Cold-induced alterations in liver and BAT TG levels are not uniform, with changes being influenced by acyl chain composition.

Inhibition of interleukin-1ß signalling promotes atherosclerotic lesion remodelling in mice with inflammatory arthritis.

OBJECTIVES: Rheumatoid arthritis (RA), an inflammatory joint disorder, independently increases the risk of cardiovascular disease (CVD). IL-1ß contributes to both RA and CVD. We hypothesised that inhibiting IL-1 signalling with the IL-1R antagonist, anakinra, would dampen inflammation and promote resolution of atherosclerosis in arthritic mice. METHODS: Low-density lipoprotein receptor (Ldlr)-deficient mice were fed a Western-type diet for 14 weeks to develop atherosclerotic plaques. Mice were then switched to a chow diet, promoting lesion regression, and randomised to a control group or into groups where arthritis was induced by passive transfer of K/BxN arthritogenic serum. The arthritic mice were further randomised to vehicle or anakinra. RESULTS: Arthritis impaired atherosclerotic lesion regression when cholesterol was lowered. This was associated with a higher burden of plaque macrophages, likely due to monocytosis, driven by myelopoiesis in the bone marrow and spleen. Interestingly, delayed intervention with anakinra had no effect on arthritis in these mice. However, a significant improvement in atherosclerotic plaque remodelling to a more stable phenotype was observed. This was associated with fewer circulating monocytes, caused by a reduction in splenic extramedullary myelopoiesis. CONCLUSION: We show that inhibiting IL-1 signalling in arthritic mice with pre-existing atherosclerosis promotes lesion remodelling to a more stable phenotype, that is less likely to rupture and cause ischemic events such as myocardial infarction. This suggests that IL-1R antagonism may suppress CVD complications in patients with RA. Furthermore, inhibiting IL-1ß signalling in other patients with inflammatory diseases that also predispose to CVD may also benefit from anti-IL-1 therapy.

Transient Intermittent Hyperglycemia Accelerates Atherosclerosis by Promoting Myelopoiesis.

RATIONALE: Treatment efficacy for diabetes mellitus is largely determined by assessment of HbA1c (glycated hemoglobin A1c) levels, which poorly reflects direct glucose variation. People with prediabetes and diabetes mellitus spend >50% of their time outside the optimal glucose range. These glucose variations, termed transient intermittent hyperglycemia (TIH), appear to be an independent risk factor for cardiovascular disease, but the pathological basis for this association is unclear. OBJECTIVE: To determine whether TIH per se promotes myelopoiesis to produce more monocytes and consequently adversely affects atherosclerosis. METHODS AND RESULTS: To create a mouse model of TIH, we administered 4 bolus doses of glucose at 2-hour intervals intraperitoneally once to WT (wild type) or once weekly to atherosclerotic prone mice. TIH accelerated atherogenesis without an increase in plasma cholesterol, seen in traditional models of diabetes mellitus. TIH promoted myelopoiesis in the bone marrow, resulting in increased circulating monocytes, particularly the inflammatory Ly6-Chi subset, and neutrophils. Hematopoietic-restricted deletion of S100a9, S100a8, or its cognate receptor Rage prevented monocytosis. Mechanistically, glucose uptake via GLUT (glucose transporter)-1 and enhanced glycolysis in neutrophils promoted the production of S100A8/A9. Myeloid-restricted deletion of Slc2a1 (GLUT-1) or pharmacological inhibition of S100A8/A9 reduced TIH-induced myelopoiesis and atherosclerosis. CONCLUSIONS: Together, these data provide a mechanism as to how TIH, prevalent in people with impaired glucose metabolism, contributes to cardiovascular disease. These findings provide a rationale for continual glucose control in these patients and may also suggest that strategies aimed at targeting the S100A8/A9-RAGE (receptor for advanced glycation end products) axis could represent a viable approach to protect the vulnerable blood vessels in diabetes mellitus. Graphic Abstract: A graphic abstract is available for this article.

Combined reduction in the expression of MCL-1 and BCL-2 reduces organismal size in mice.

The intrinsic apoptotic pathway is controlled by the BCL-2 family of proteins, which exhibit either a pro-death or pro-survival function. Gene knockout studies revealed that different pro-survival BCL-2 proteins are critical for the survival of distinct cell types, although overlapping functions amongst such proteins have also been identified. In the process of studying mice lacking single alleles of Mcl-1 (Mcl-1+/-), Bcl-2 (Bcl-2+/-), or both in combination (Mcl-1+/-Bcl-2+/-), we observed that Mcl-1+/-Bcl-2+/- mice weighed less when compared with their wild-type littermates as they aged. Body composition analysis demonstrated that while fat mass was similar to wild-type controls, lean mass was significantly reduced in Mcl-1+/-, Bcl-2+/-, and, most strikingly in Mcl-1+/-Bcl-2+/- mice. The weights of several tissues including the heart, tibialis anterior, and kidney were likewise reduced in Mcl-1+/-Bcl-2+/- mice. When lean mass and specific tissue weights were expressed relative to body weight, these differences were no longer significant, indicating that that Mcl-1+/-Bcl-2+/- mice, and to a lesser extent Mcl-1+/- and Bcl-2+/- mice, are smaller than their wild-type counterparts. Consistently, the anal-naso length was reduced in Mcl-1+/-Bcl-2+/- mice. While minor reductions in size were observed in female Mcl-1+/-Bcl-2+/- mice, these effects were most prominent in males. Notably, Mcl-1+/-Bcl-2+/- males had markedly smaller testes even after accounting for differences in body weight. Collectively, these data reveal that combined loss of a single allele of Mcl-1 and Bcl-2, while not overtly impairing organismal development, leads to a reduction in animal size.

Hematopoiesis is regulated by cholesterol efflux pathways and lipid rafts: connections with cardiovascular diseases.

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.jlr;61/5/667/F1F1f1.

Glycolysis Is Required for LPS-Induced Activation and Adhesion of Human CD14+CD16- Monocytes.

Monocytes in humans consist of 3 subsets; CD14+CD16- (classical), CD14+CD16+ (intermediate) and CD14dimCD16+ (non-classical), which exhibit distinct and heterogeneous responses to activation. During acute inflammation CD14+CD16- monocytes are significantly elevated and migrate to the sites of injury via the adhesion cascade. The field of immunometabolism has begun to elucidate the importance of the engagement of specific metabolic pathways in immune cell function. Yet, little is known about monocyte metabolism and the role of metabolism in mediating monocyte activation and adherence to vessels. Accordingly, we aimed to determine whether manipulating the metabolism of CD14+CD16- monocytes alters their ability to become activated and adhere. We discovered that LPS stimulation increased the rate of glycolysis in human CD14+CD16- monocytes. Inhibition of glycolysis with 2-deoxy-D-glucose blunted LPS-induced activation and adhesion of monocytes. Mechanistically, we found that increased glycolysis was regulated by mTOR-induced glucose transporter (GLUT)-1. Furthermore, enhanced glycolysis increased accumulation of reactive oxygen species (ROS) and activation of p38 MAPK, which lead to activation and adhesion of monocytes. These findings reveal that glycolytic metabolism is critical for the activation of CD14+CD16- monocytes and contributes to our understanding of the interplay between metabolic substrate preference and immune cell function.

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Plasmalogens: A potential therapeutic target for neurodegenerative and cardiometabolic disease.

Plasmalogens are a class of membrane glycerophospholipids with unique properties. They contain a vinyl-ether linked alkyl chain at the sn-1 position of the glycerol backbone and, typically, a polyunsaturated fatty acyl chain at the sn-2 position. Plasmalogens are critical for human health and have established roles in neuronal development, the immune response and as endogenous antioxidants. However, the mechanistic bases of these and other biological functions of plasmalogens are not well defined. Lipidomic studies have characterised reduced levels of plasmalogens in a number of disease states, including neurodegenerative and cardiometabolic disease, highlighting the potential of plasmalogen modulation as a therapeutic strategy. A number of approaches have been proposed to upregulate plasmalogen levels in different clinical settings; these include dietary intervention with inositol or the naturally occurring metabolic precursors known as alkylglycerols. Plasmalogen modulation has been utilised in both preclinical and clinical studies to prevent onset and/or attenuate progression of neurodegenerative diseases, atherosclerosis, insulin resistance and hepatosteatosis. These studies are providing new insight into the mechanistic role of plasmalogens in disease and their therapeutic potential. In this review, we will examine the strategies for plasmalogen modulation and recent progress toward therapeutic applications with a focus on neurodegenerative and cardiometabolic disease.

Fat for fuel: lipid metabolism in haematopoiesis.

The importance of metabolic regulation in the immune system has launched back into the limelight in recent years. Various metabolic pathways have been examined in the context of their contribution to maintaining immune cell homeostasis and function. Moreover, this regulation is also important in the immune cell precursors, where metabolism controls their maintenance and cell fate. This review will discuss lipid metabolism in the context of haematopoiesis, that is blood cell development. We specifically focus on nonoxidative lipid metabolism which encapsulates the synthesis and degradation of the major lipid classes such as phospholipids, sphingolipids and sterols. We will also discuss how these metabolic processes are affected by haematological malignancies such as leukaemia and lymphoma, which are known to have altered metabolism, and how these different pathways contribute to the pathology.

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis.

Aim: Rheumatoid arthritis (RA) is associated with an approximately two-fold elevated risk of cardiovascular (CV)-related mortality. Patients with RA present with systemic inflammation including raised circulating myeloid cells, but fail to display traditional CV risk-factors, particularly dyslipidaemia. We aimed to explore if increased circulating myeloid cells is associated with impaired atherosclerotic lesion regression or altered progression in RA. Methods and results: Using flow cytometry, we noted prominent monocytosis, neutrophilia, and thrombocytosis in two mouse models of RA. This was due to enhanced proliferation of the haematopoietic stem and progenitor cells (HSPCs) in the bone marrow and the spleen. HSPCs expansion was associated with an increase in the cholesterol content, due to a down-regulation of cholesterol efflux genes, Apoe, Abca1, and Abcg1. The HSPCs also had enhanced expression of key myeloid promoting growth factor receptors. Systemic inflammation was found to cause defective cellular cholesterol metabolism. Increased myeloid cells in mice with RA were associated with a significant impairment in lesion regression, even though cholesterol levels were equivalent to non-arthritic mice. Lesions from arthritic mice exhibited a less stable phenotype as demonstrated by increased immune cell infiltration, lipid accumulation, and decreased collagen formation. In a progression model, we noted monocytosis, enhanced monocytes recruitment to lesions, and increased plaque macrophages. This was reversed with administration of reconstituted high-density lipoprotein (rHDL). Furthermore, RA patients have expanded CD16+ monocyte subsets and a down-regulation of ABCA1 and ABCG1. Conclusion: Rheumatoid arthritis impairs atherosclerotic regression and alters progression, which is associated with an expansion of myeloid cells and disturbed cellular cholesterol handling, independent of plasma cholesterol levels. Infusion of rHDL prevented enhanced myelopoiesis and monocyte entry into lesions. Targeting cellular cholesterol defects in people with RA, even if plasma cholesterol is within the normal range, may limit vascular disease.