Effects of dyslipidaemia on monocyte production and function in cardiovascular disease

Shamim Rahman1, Andrew J. Murphy2,3 and Kevin J. Woollard1

1Renal & Vascular Inflammation Section, Division of Immunology and Inflammation, Imperial

College London, Du Cane Road, London W12 0NN, UK.

2Haematopoiesis and leukocyte biology lab, Baker IDI Heart & Diabetes Research Institute,

75 Commercial Rd, Melbourne VIC 3004 Australia.

3Department of Immunology, Monash University, Melbourne, Victoria, 3004, Australia.

Correspondence to K.J.W. k.woollard@imperial.ac.uk

Abstract | Monocytes are heterogeneous effector cells involved in the maintenance and

restoration of tissue integrity. Hyperlipidaemia can accelerate cardiovascular disease

progression. Monocytes and macrophages are involved in cardiovascular disease

progression, and are associated with the development of unstable atherosclerotic plaques.

However, monocyte responses to hyperlipidaemia are poorly understood. In the past

decade, accumulating data describe the relationship between the dynamic, blood lipid

environment and the heterogeneous circulating monocyte pool, which might have profound

consequences for cardiovascular disease. In this Review, we explore the updated view of

monocytes in cardiovascular disease and their relationship with macrophages in promoting

the homeostatic and inflammatory responses related to atherosclerosis. We describe the

different definitions of dyslipidaemia, highlight current theories on the ontogeny of

monocyte heterogeneity, discuss how dyslipidaemia might alter monocyte production, and

explore the mechanistic interface linking dyslipidaemia with monocyte effector functions,

such as migration and the inflammatory response. Finally, we discuss the role of dietary and

endogenous lipid species in mediating dyslipidaemic responses, and the role of these lipids

in promoting the risk of cardiovascular disease through modulation of monocyte behaviour.

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Investigation of monocytes and macrophages has had a resurgence of activity over the past

10 years. This increase in monocyte and macrophage research is a result of a refinement of

the definition and characterization of the mononuclear phagocyte system. These

phagocytes make up diverse populations of cells with tissue-specific specialization to

maintain homeostasis, such as Kupffer cells in the liver and alveolar macrophages in the

lung1. Some of these tissue-specific macrophages can maintain their population levels

through in situ proliferation, and do not require continuous replenishment via blood

monocytes2. Therefore, the role of blood monocytes as short-lived macrophage precursors

might need redefinition and deeper characterization. Moreover, monocytes populations are

heterogeneous and have subset specific effector functions3, which are altered as a result of

changes in the local environment and inflammatory response. This Review focuses on

dyslipidaemia and changes in blood lipid profiles that might alter the behaviour of

monocytes, thereby contributing to cardiovascular disease.

[H1] Steady-state production and function

[H2] Origin of tissue-resident macrophages

The identification of fetal-derived, self-maintaining, tissue-resident macrophages has shifted

our understanding of myeloid lineages away from the previously accepted dogma, which

postulated as the core definition of the mononuclear phagocyte system that short-lived,

bone marrow-derived monocytes are required to replenish tissue macrophages 4.

Monocytes have long been known to contribute to the tissue macrophage pool, particularly

during infection and inflammation5. However, the relative contribution of monocytes to

tissue macrophage populations in the steady state has been under scrutiny. Increasingly

sophisticated experiments with fate-mapping technology, adoptive transfer, and parabiosis

models have helped to improve our understanding of the complex contributions of different

cell lineages throughout embryological development to haematopoiesis and the seeding of

tissues with resident macrophage precursors. Data now indicate that monocytes have a

minimal contribution to most tissue-resident macrophage pools in the steady state6,7 or

even under certain inflammatory conditions. Many tissue-resident macrophage populations

have now been identified, but tissue-specific differences cannot yet be fully explained.

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Tissue macrophage ontogeny can be divided into the early ‘primitive’ and subsequent

‘definitive’ haematopoietic waves1, with the bulk of experimental data derived from mouse

models. Primitive haematopoiesis refers to early formation of haematopoietic progenitors,

including macrophage progenitors (or ‘primitive macrophages’) and erythro-myeloid

progenitors, from blood islands of the yolk sac in mice8 and rats9 at embryonic day 8.5–9.0

(E8.5–E9.0), with subsequent colonization of the fetal liver at E10.0–E10.5 via the newly

established circulatory system10,11. Definitive haematopoiesis in the aorta–gonad–

mesonephros (AGM) results in the release of haematopoietic stem cells (HSCs) at E10.5,

which then colonize the fetal liver (E11.0)12. Thus, from E11.0 to E11.5 the fetal liver

becomes the dominant site of haematopoiesis1, before the initiation of bone marrow-

derived haematopoiesis.

Yolk sac-derived primitive macrophages, colonize tissues such as the brain, spleen, pancreas,

kidney, heart and lungs7,13-16. Fetal liver monocytes directly derived from cMyb+ erythro-

myeloid progenitors generated in dorsal aortic endothelial cells17, subsequently adds to the

tissue-resident macrophage population. Exceptions to this fetal monocytic infiltrate includes

the central nervous system, where fetal liver-derived monocytes do not contribute to the

microglial cell population14; the skin, where dual-origin macrophages contribute to the

Langerhans cell pool18; and the gut, which seems to remain dependent on constant renewal

from blood precursors, with data suggesting circulating blood monocytes contribute to the

pool of macrophages and dendritic cells in the intestine19,20.

[H2] Monocyte development

Monocyte development and survival depends on colony-stimulating factor 1 (CSF1; also

known as M-CSF) and its receptor CFS1R (also known as CD115)21-23. Embryonic and adult

haematopoiesis in the steady state results in monocyte development from myeloid

precursor cells found in primary lymphoid organs, including the fetal liver and bone

marrow1. In addition, in mice under some inflammatory conditions the spleen has been

shown also to contribute to monocyte production from precursor cells24.

Following the establishment of bone marrow progenitors, macrophage and dendritic cell

precursors (MDP) — defined as lineage-negative (Lin-), CD117+ (also known as KIT), CD135+

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(also known as FLT3), and CSF1R+ — give rise to monocytes, macrophages, and dendritic

cells25. However, the absolute requirement for MDP as a common precursor for monocyte

and macrophage lineages and resident dendritic cells is debated26.

A potential additional monocyte precursor, termed the common monocyte progenitor

(cMoP), has been identified27. cMoPs derive from, and also differ from, MDPs, and are

characterized by a reduction in the relative expression of CD135 (that is, cMoPs are Lin–

CD117+CD135–CSF1R+)27. cMoPs give rise to mature monocytes in the bone marrow. These

mature monocytes do not subsequently differentiate into either classical dendritic cells or

plasmacytoid dendritic cells 27. Thus in the adult, monocytes can arise from the bone

marrow via myelopoiesis. Myelopoiesis begins from haematopoietic stem and progenitor

cells (HSPCs), which give rise to common myeloid progenitors (CMPs), which in turn give rise

to granulocyte-macrophage progenitors (GMPs) and then cMoPs (FIG. 1). During times of

stress, such as infection, the myeloid branch of haematopoiesis can be expanded to increase

monocyte production through the action of a number of well-characterized cytokines5. This

process is also important in chronic inflammatory disorders, including metabolic disorders

and cardiovascular disease, where monocytosis is present (detailed below). Monocyte

subset production in the bone marrow and the factors influencing blood trafficking and

extravasation into tissue are represented in Figure 1.

[H2] Blood monocytes

Circulation in the blood is a defining characteristic of a monocyte3. Although previously

considered intermediary cells awaiting stimuli towards terminal differentiation into a

macrophage, dendritic cell, or other tissue descendant4,28,29, blood monocytes are now

known to be a heterogeneous population of cells with differing steady-state phenotypes and

as effector cells in response to microbial stimuli3. Furthermore, monocyte gene expression

profiles and functions are conserved across vertebrate species30.

Monocytes can be characterized by the expression levels of surface proteins, RNA

expression profiles and morphology3,31-33. Morphologically, monocytes are spherical cells

with prominent surface ruffles and blebs, a large reniform nucleus with a small nucleolus34.

Differences in size and granularity exist between subsets (detailed below), with non-classical

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cells much smaller and less granular than classical monocytes3. Heterogeneity in monocytes

exists in many species35, including humans, rats, mice, and pigs32,35-42. Two major monocyte

subsets have been identified in mice — ‘classical’ and ‘nonclassical’ — and three subsets

have been identified in humans — classical, intermediate, and nonclassical — with classical

monocytes being the most abundant (~80%) (TABLE 1). In mice, monocytes are commonly

identified in flow cytometry analyses by their characteristic forward and side scatter profiles

and expression of CD11543,44. Monocyte subsets can then be differentiated on the basis of

expression levels of a number of surface markers, including lymphocyte antigen 6C (LY6C),

CD117, CX3C-chemokine ligand 1 (CX3CR1), C-C chemokine receptor type 2 (CCR2), and

CD431,38,43. Classical monocytes are defined as LY6ChighCD117–CX3CR1intCCR2+CD62L+CD43low

(where “int” refers to intermediate). Nonclassical monocytes are

LY6ClowCD117intCX3CR1highCCR2–CD62L–CD43high 38,45-47.

Murine monocyte subsets have equivalents in human. Gene expression arrays show that

classical LY6Chigh mouse monocytes resemble human monocytes that express CD14highCD16low

surface markers32. Conversely, nonclassical LY6Clow monocytes are most analogous to human

CD14lowCD16high monocytes32. A third, ‘intermediate’ subset has been reported in humans,

defined by high levels of both CD14 and CD16. This intermediate subset aligns functionally

with murine classical cells3,48, but transcriptionally with nonclassical CD14lowCD16high

monocytes49. The subset of intermediate monocytes expands in a variety of infectious and

inflammatory conditions (for example, cardiovascular disease, rheumatoid arthritis, Crohn’s

disease, and chronic kidney disease)35,50.

Monocyte subsets differ in their response to pathogens. Classical monocytes respond

strongly to bacterial stimuli via Toll-like receptor (TLR) 4, nonclassical monocytes respond to

viral stimuli via the intracellular receptors TLR7 and TLR8, and intermediate monocytes

respond to both bacterial and viral stimuli producing high levels of inflammatory cytokines

such as interleukin (IL)-1. Therefore, intermediate monocytes might represent a subset in

transition between classical and nonclassical cells, displaying characteristics of both

subsets51. Indeed, evidence from murine models indicates that classical monocytes can

transition towards a nonclassical phenotype in bone marrow and blood7 and in the setting of

murine myocardial infarction and healing52, although this transition is yet to be confirmed in

other species and under different environmental conditions. Monocyte ontogeny remains

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enigmatic, particularly in the context of subset origins. For instance, what the half-life of

monocytes is and where monocytes eventually go are still unclear. The current hypothesis is

that both subsets develop from bone marrow progenitors. However, as mentioned

previously, data in mice suggest that in the steady state, LY6Chigh monocytes can give rise to

LY6Clow monocytes7,53, both in the bone marrow and in the blood, although evidence to

conclude confidently a linear relationship between both monocyte subsets is lacking.

Conversion of LY6Chigh to LY6Clow is further supported by stark differences in mouse

monocyte subset survival. Turnover of LY6Chigh monocytes is thought to be 1 day, whereas

LY6Clow monocytes can survive from 2 days to as long as 2 weeks under certain conditions7.

The transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1; also

known as NUR77) has an essential role in the development and survival of LY6Clow

monocytes54 (reviewed in REFS. 52,55-57). LY6Clow monocytes have significantly higher amounts

of NR4A1 than LY6Chigh cells at both the transcriptional52,54 and protein levels54. Nr4a1–/– mice

lack LY6Clow monocytes in the bone marrow, blood, and spleen, and do not have any

patrolling monocytes as assessed by intravital microscopy55. Moreover, MDP precursors that

would usually give rise to blood LY6Clow monocytes do not do so in NR4A1-deficient mice54.

These data suggest that either NR4A1 is involved in monocyte ‘conversion’ or that mature

LY6Chigh monocytes are in fact not the precursors of LY6Clow . One might speculate that three

monocyte subsets exist in mice: bona fide LY6Chigh, LY6Clow matured from LY6Chigh cells, and

LY6Clow patrolling monocytes at the endothelial interface derived from the differentiation of

precursors driven by NR4A1 (FIG. 1). Experiments are underway to examine this hypothesis.

Interestingly, homozygous deletion of Nr4a1 has been shown to accelerate atherosclerosis

in the high-fat diet (HFD)-fed Ldlr-/- mouse model of atherosclerosis in some studies58,59, but

not others60. Finally, definitions of monocyte subsets may be further complicated by recent

reports that LY6Chigh monocytes may be defined by CXCR4 expression in the bone marrow

giving rise to further functional subsets 61, and that a subpopulation of LY6Chigh monocytes

give rise to inflammatory dendritic cells62. Moreover, an ‘atyical’ monocyte with neutrophil

characteristics may be involved with fibrosis 63. More work is now needed to characterise

the ‘plasticity’ of monocytes.

[H2] Monocyte functions

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Although the precise functions of monocyte subsets are yet to be defined, clear evidence

supports subset-specific functions. In mice, classical LY6Chigh monocytes are rapidly recruited

to sites of inflammation, extravasate and differentiate into monocyte-derived

macrophages64. By contrast, mouse nonclassical LY6Clow monocytes, similar to their human

counterparts3, crawl and patrol the luminal side of the vascular endothelium37,55, as shown

by intravital microscopy. Evidence showing the differentiation of LY6Clow monocytes from

LY6Chigh monocytes, the longer half-life of the LY6Clow subset, and its patrolling role have led

some investigators to suggest that these cells are terminally differentiated ‘blood

macrophages’ rather than blood monocytes1. However, we and other researchers have

shown monocytes can migrate out of the blood during dyslipidaemia (detailed below) or

autoimmune inflammation65-67, implying they are not terminally differentiated cells and can

continue to respond to tissue cues. Similarly, the question remains whether the highly

mobile, responsive LY6Chigh monocytes that extravasate into the tissue not always

differentiate into macrophages or dendritic cells, and can uptake antigens as part of a

tissue-surveillance function47.

Nonclassical LY6Clow monocytes, by patrolling the endothelium also have surveillance

functions. Through expression of surface molecules such as CD11a (also known as integrin

αL)55, CX3CR137, intercellular adhesion molecule (ICAM) 1 and ICAM236,68, and protein CYR61

(also known as CCN1)69, LY6Clow monocytes remain in close proximity to the endothelium

and can respond to endothelial damage, perhaps monitoring endothelial integrity and

responding to deposited immune complexes by recruiting inflammatory neutrophils55,70.

Moreover, these surveying monocytes might remove luminal vascular β-amyloid deposits,

as shown in a murine model of Alzheimer disease71. Nonclassical monocytes have also been

shown to scavenge metastasizing tumour cells in a lung tumour mouse model72. More work

is needed to identify the unique functional properties of nonclassical LY6clow monocytes.

Whether these nonclassical monocytes have a direct role in cardiovascular disease is

unclear, although these monocytes have been shown to patrol along the coronary vascular

tree73.

[H2] Monocytes in atherosclerosis

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Atherosclerosis is a dynamic and chronic inflammatory disease, and thus seems to elicit very

different responses from blood monocytes and tissue-resident macrophages compared with

traditional inflammatory scenarios. Preclinical models of atherosclerosis involve

homozygous deletion of genes encoding proteins associated with lipoprotein clearance,

namely Ldlr and Apoe, in order to generate chronic hyperlipidaemia and the formation of

atherosclerotic plaques. These models have helped to shed light on monocyte production,

migration into the atherosclerotic plaque, and subsequent development of lipid-laden

macrophage foam cells (FIG. 2). In these preclinical models, the hypercholesterolaemic

environment 74-77 or the disruption of cholesterol efflux mediated by ABC transporters78,79

promotes monocytosis, an observation consistent with the monocytosis found in people

with cardiovascular disease80-82. This increase in blood monocytes is a result of an expansion

of the bone marrow progenitors HSPCs, CMPs, and GMPs. Importantly, use of competitive

bone marrow transplantation experiments allowed the tracking of the HSPC progeny into

the atherosclerotic lesion, revealing a preferential accumulation in atherosclerotic lesions of

Apoe–/– monocytes and macrophages compared with wild type cells 74. Interestingly,

monocytosis in the bone marrow and blood compartments might be dependent on

chemokine signalling, specifically mediated by C-C motif chemokine 2 (CCL2), CCR2, CX3CR1,

and C-C chemokine receptor type 5 (CCR5)76,83. Moreover, these chemokines have a pro-

atherogenic role, as shown by the reduction in atherosclerotic plaque size following

combined inhibition of CCL2, CX3CR1, and CCR5 despite higher total cholesterol levels

present in Apoe-knockout mice 83. These studies on atherogenesis subscribe to the dogmatic

model of circulating monocyte extravasation and differentiation into macrophages, followed

by subsequent accumulation in macrophages of modified lipids via scavenger-receptor

uptake to generate foam cells. Bone marrow transplantation models in atherosclerosis-

prone mice indicate that an ’inflammatory’ (LY6Chigh) monocyte subset is readily recruited

into the arterial intimal layer84,85. Atherosclerosis progression depends on the presence and

viability of this monocyte subset, and recruitment from this monocyte pool continues during

lesion growth84-86. Importantly, this inflammatory or classical subset is the predominant

subset to extravasate during atherogenesis. However, reports show nonclassical LY6Clow

monocyte migration to discrete atherosclerotic regions87. This migration might be important

during specific types of dyslipidaemia associated with atherosclerosis, such as

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hypertriglyceridaemia, which has been associated with increased tissue recruitment of

nonclassical monocytes66.

The idea that plaque macrophages arise strictly from continued recruitment of blood

monocytes has been challenged. In 2013, a study demonstrated through a series of elegant

experiments that maintenance and expansion of the lesional pool of macrophages in

established lesions is mainly sustained by local cell proliferation rather than by continuous

recruitment of blood monocytes 2 (FIG. 2a). Interestingly, the study also showed that the

macrophage area in the growing lesions remained unchanged2. These paradoxical results

are difficult to reconcile, but an explanation might be that most of the newly recruited

monocytes undergo rapid cell death, whereas a small pool of macrophages proliferate but

then die after a few divisions 44. However, in contrast to the short-term experiments, long-

term follow-up revealed that almost all lesional macrophages were replaced by newly

recruited cells2 . Collectively, the findings from this study highlight the importance of

monocyte recruitment, macrophage proliferation, and macrophage death during

atherosclerosis progression88.

A population of tissue-resident macrophages, termed adventitial macrophages, has been

identified in the arterial adventitia89. In adulthood, these adventitial macrophages derive

from a pool of adventitial macrophage progenitor cells89. These adventitial macrophages

also derive embryonically from CX3CR1+ erythro-myeloid progenitors (from the yolk sac) and

from fetal liver monocytes 90. Interestingly, the adventitial macrophage population has been

shown to expand in the setting of hyperlipidaemia 89. However, these adventitial

macrophages seem to be a minority within the macrophage pool in the atheroma89, but

might have an important indirect role through the release of cytokines or by inducing

neovascularization.

The above studies focussed on plaque macrophage populations during atherogenesis.

However, recurrent insults, such as those frequently following an acute myocardial

infarction (MI), initiate an additional wave of altered haematopoiesis91. Interestingly, an

increased macrophage burden was observed within atherosclerotic lesions after an acute

MI91. The increase in macrophage burden was independent of local cell proliferation and

was a result of enhanced monocyte recruitment from the spleen91. The acute MI caused

sympathetic overdrive, which mobilises HSPCs from the bone marrow into the spleen,

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where HSPCs undergo extramedullary haematopoiesis and give rise to monocytes involved

in atherosclerosis91. Furthermore, in addition to the role of blood monocyte-derived

macrophages in atherosclerotic lesion progression, monocytes and macrophages with mixed

ontogenies are important in driving an inflammatory response and subsequent repair of the

myocardium after an acute MI92.

[H1] Dyslipidaemia

Dyslipidaemia encompasses blood lipid levels beyond the normal range either when

elevated or low, however is most commonly applied when lipid levels associated with

cardiovascular disease are elevated, such as LDL or when low levels of HDL cholesterol are

found93. Elevations in blood cholesterol levels, in particular are linked with an increased risk

of cardiovascular disease94. The blood lipid pool is made up of lipoproteins that transport

cholesterol, cholesterol esters and fatty acids predominantly as triglycerides to muscle,

adipose tissue, and to the liver for processing and clearance (BOX 1). Free, nonesterified

fatty acids are also thought to be present in the blood, most probably bound to albumin95.

Experimental animal models and human histopathological samples confirm the presence of

cholesterol in atherosclerotic plaques, predominantly in foam cells (thought to be lipid-

laden macrophages)96. Fatty acids have also been shown to accumulate in the

atherosclerotic plaque97. Interestingly, this accumulation is dynamic and the composition of

the fatty acid pool changes over time, which might provide selective stimuli to foam cells

and might represent a dietary influence on plaque composition97.

[H2] Classification

Dyslipidaemias can be classified as primary if the dyslipidaemia has a genetic cause (familial

or inherited dyslipidaemia) or as secondary if the dyslipidaemia results from other medical

conditions (BOX 1). The Frederickson classification98 of familial dyslipidaemia has been

adopted by the WHO99, and remains one of the most frequently used in clinical decision

making, however does not account for HDL nor for gene variants associated with abnormally

elevated lipid levels. For this reason, guidelines produced in by European (ESC/EAS)100 and

American (ACC/AHA)101 associations do not focus on such classifications though they do

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often still retain the names proposed by Frederickson, instead make recommendations for

testing and treatment if lipoprotein parameters and other clinical features are suggestive of

an inherited, familial dyslipidaemia.

Although familial hypercholesterolaemia (FH) is a monogenic disorder and most strongly

correlates with cardiovascular disease, most inherited lipid disorders are polygenic102.

Individuals with FH can have the homozygous or heterozygous form of the disease, which

correlates with both severity of the LDL abnormality and the risk of cardiovascular

disease103,104.

[H2] Dyslipidaemia in cardiovascular disease

[H3] Hypercholesterolaemia. The association between high LDL cholesterol levels in blood

and the development and progression of cardiovascular disease has been known for >100

years105,106. Discovery of the LDL receptor and subsequent development and success of LDL-

lowering drugs, such as statins, have shown a convincing link between LDL-level reduction

and reductions in cardiovascular morbidity and mortality107-111. Beyond the extensively used

statin therapy, which lowers blood cholesterol levels through inhibition of HMG-CoA

reductase, a key and rate-limiting step in its synthesis in the liver. Newer, alternative LDL-

lowering agents, such as ezetimibe (which acts by inhibition of Niemann-Pick C1-like

1 (NPC1L1) protein resulting in reduced absorption of cholesterol in the small intestine, have

also successfully demonstrated in clinical trials improvements in cardiovascular outcome

following heart attack112. These findings suggest that the reduction in the risk of

cardiovascular disease is not unique to statins and probably extends to alternative LDL-

lowering strategies. For example, a potential alternative strategy is to target oxidized forms

of LDL (oxLDL), which have been shown to induce a proinflammatory response in monocytes

in vitro113-115, and whose circulating levels are elevated in cardiovascular disease116-119.

[H3] HDL as a therapeutic strategy. Beyond LDL-levels, interest in HDL cholesterol levels as

a potential therapeutic target has been explored, mainly owing to the role of HDL in reverse

cholesterol transport (RCT), antioxidative and anti-inflammatory properties 120. HDL

promotes cellular cholesterol efflux by interaction with the ATP-binding cassette

transporters ABCA1 and ABCG1121-124. The ratio of total cholesterol to HDL level is used as a

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predictive tool in assessing the risk of cardiovascular disease125. The consistent inverse

relationship between HDL levels and the risk of cardiovascular disease126 prompted

optimism into research aimed at elevating blood HDL levels. However, clinical trials on

therapies aimed to raise HDL levels for reducing the risk of cardiovascular disease have been

largely disappointing 127-132. These negative findings are supported by a Mendelian

randomization trial showing that individuals with a single nucleotide polymorphism (SNPs)

in LIPG that results in elevated HDL levels in blood (approximately a 12% increase) was not

associated with a reduction in the risk of MI 133. Furthermore, other SNPs associated with

lifetime elevations of HDL also did not associate with a reduction in the risk of MI 133.

However, evidence now shows that static measurements of HDL alone are not necessarily

an accurate assessment of the functional role of HDL particles. For instance, HDL particle

size134 and efflux capacity135 might be better predictors for assessing the role of HDL in

altering the risk of cardiovascular disease.

[H3] Hypertriglyceridaemia. Despite some early interest in the role of dietary triglycerides

in the risk of cardiovascular disease136-141, epidemiological data do not support a substantial

link between the levels of triglycerides in blood and the risk of cardiovascular disease 142.

Moreover, pharmacological methods for reducing triglyceride blood levels with the aim of

decreasing the risk of cardiovascular disease in addition to the risk reductions achieved with

statins have been unsuccessful so far143. Nevertheless, lowering triglyceride levels as a

therapeutic strategy is experiencing renewed interest on the basis of findings from

Mendelian randomization analyses144-146, genome-wide association studies147,148 and studies

on SNPs149 suggesting that the residual risk of cardiovascular disease persists despite

achievement of LDL target levels is related to elevated triglyceride levels, rather than low

HDL levels.

[H2] Effect of dyslipidaemia on monocytes

A number of studies have examined the changes in the phenotype of monocytes in response

to lipids, predominantly in the context of atherosclerosis models65,66,150-157. Although

mechanisms associated with atherogenesis remain the focus of much work, the effect of

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dyslipidaemia (that is, variance in particular lipid species) on monocyte phenotype and

function might be more complex. Possible effects of dyslipidaemia on immune function

include human data suggesting an apparent immunoparesis in surgical patients with very

high or low total cholesterol levels are at an increased risk of infection 158. Multiple

experimental data show poor responses of both innate and adaptive immunity to fungal and

bacterial infection in transgenic mice with dyslipidaemia159-161. Indeed, leukocyte interaction

with lipids in hyperlipidaemic environments has been documented for >50 years, when

lipid-filled rat monocytes in response to a HFD were denoted as ‘lipophages’ 162, and

monocyte invasion of the vascular endothelium in HFD-reared pigs was imaged by

transmission and scanning electron microscopy in 1979163 and 1981164, respectively. Some

studies in zebra fish have also shown monocytes can uptake lipids in circulation in a TLR4-

dependent process165.

[H3] Effects of cholesterol. The presence of cholesterol within the atherosclerotic plaque

and the identification of monocyte extravasation into the subendothelial space has focussed

the trajectory of much monocyte research in this pathophysiological context. Elevation in

blood cholesterol, predominantly owing to raised LDL levels, is the most commonly studied

dyslipidaemic condition. As monocytes and lipoprotein particles both circulate in the blood,

monocytes are likely to have physical encounters with cholesterol well before monocytes

migrate into the atherosclerotic lesion and differentiate into tissue macrophages. In most of

the literature in this field, the term ‘monocyte’ is often used interchangeably with

(monocyte-derived) ‘macrophages’, and experimental models are often in vitro scenarios

and/or cell lines that are used to recapitulate potential pathophysiological conditions.

Additionally, these experiments frequently involve transgenic animal models where

cholesterol levels are dramatically elevated to obtain results within an acceptable

timeframe. Therefore, data from experimental studies should be interpreted with some

caution when translating into the human clinical context.

Monocytes undergo morphological, phenotypical, and functional changes in

hypercholesterolaemic environments. Work in patients with familial hypercholesterolaemia

demonstrated intracytoplasmic vacuolation in monocytes and suggested the presence of

monocytosis in these patients166. CD14highCD16low (classical) monocytes from patients with

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familial hypercholesterolaemia preferentially take up oxLDL167.  Exposure of these

monocytes to oxLDL induces an increase in the expression of CD68 (also known as

macrosialin), a macrophage marker and CD11c (also known as integrin αX), an activation

marker, and an improved ability for phagocytosis167. Human monocytes have been shown to

uptake LDL in vitro, but do so more readily with oxLDL via scavenger receptors, including

SRA1, SRA2, SRB1 , CD36, and lectin-like oxidized LDL receptor 1 (LOX1) 168,169 (FIG. 2).

Transgenic animal models of elevated total cholesterol show evidence of neutral lipid

droplets in monocytes150 and, although the most probable explanation for this finding is lipid

uptake, the mechanisms remain unclear, and de novo lipogenesis (described in monocyte-

derived macrophage-like cells170) remains a possibility.

Atherosclerosis has long been considered an inflammatory condition171 and the

identification of cholesterol crystals not just in mature atherosclerotic plaques, but also in

early atherosclerotic plaques172,173, adds weight to the notion that cholesterol might drive

inflammatory atherogenesis (FIG. 2). Cholesterol crystals can induce the release of IL-1

through activation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome

in pre-primed mononuclear cells in vitro172. Activation of the NLRP3 inflammasome and

release of active IL-1 requires two signals. The first (priming) signal is required for the

upregulation of NLRP3 and pro-IL-1β, and can be triggered for example by TLR activation.

The second signal induces the activation of the inflammasome and caspase 1 to cleave pro-

IL-1. Minimally modified LDL (mmLDL), which provides a chemotactic stimulus to

monocytes174,175 , has also been shown to provide a priming signal for mononuclear cells in

vitro172. Interestingly, the dietary saturated fatty acid palmitic acid induced TLR2-mediated

IL-1 release in a human monocytic cell line in vitro, a process that was abrogated by use of

the polyunsaturated fatty acid docosahexaenoic acid176. Similarly, TLR4 activation induced by

oleic acid and palmitic acid has been reported in macrophage cell lines177. TLR4178,179 and its

downstream signalling adaptors TIR domain-containing adaptor molecule 1 (TICAM-1, also

known as TRIF) and TIR domain-containing adaptor molecule 2 (TICAM-2, also known as

TRAM)180 have been implicated in atherosclerosis. TLR4 stimulation will, on the most part,

result in activation of nuclear factor (NF)-κB, and NF-κB activation can either provide

proinflammatory atherosclerotic stimuli or be atheroprotective depending on the initial

route of activation181,182. A report published in 2016 suggests an alternative pathway of

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NLRP3 inflammasome activation in human monocytes that does not require a second signal

when stimulated through lipopolysaccharide (LPS)–TLR4 signalling183. However,

atherosclerosis might occur independently of NLRP3 signalling184, supporting the notion that

inflammasome activation is not ubiquitous in the plaque. OxLDL is thought to induce

activation of TLR2, TLR4, and TLR6 mediated by CD36 signalling185, which in turn can activate

macrophages186. OxLDL can independently promote the differentiation of human monocytes

into macrophage-like cells in vitro187-189. Moreover, oxLDL might induce monocyte

reprogramming or ‘innate immune memory’ that might alter secondary ligand responses115.

Some data suggest that cholesterol not always represents an inflammatory stimulus.

Intermediates of the cholesterol biosynthetic pathway, such as desmosterol, provide an

anti-inflammatory stimulus via LXR signalling in elicited peritoneal macrophages from Ldlr–/–

mice190 (FIG. 2b). Whether these cholesterol intermediates have anti-inflammatory potential

in monocytes is unknown. Of note, other fatty acids, such as palmitoleic acid, that are

present in neutral lipid-droplets of foamy monocytes have also been shown to have anti-

inflammatory functions191. The cholesterol efflux transporter ABCA1 might also have an anti-

inflammatory role by modulating TLR signalling. In murine macrophages, ABCA1 reduces TLR

trafficking to lipid rafts by reducing lipid raft cholesterol content192. This finding also

supports an anti-inflammatory role of HDL cholesterol. Furthermore, cell cholesterol

removal by cholesterol efflux pathways and administration of reconstituted HDL have been

shown to have direct anti-inflammatory effects on leukocytes120,193,194, and in the production

of classical monocytes by acting on HSPCs79. A study published in 2016 showed the specific

immunomodulatory effects of apolipoprotein A1 (the main HDL apolipoprotein) on

monocyte behaviour195. Acute exposure to apolipoprotein A1 inhibited macrophage

chemotaxis in vitro and monocyte recruitment in vivo195, consistent with previous findings120.

The different aspects of cholesterol, inflammation, and immune interaction have been

reviewed previously196.

[H3] Effects of triglyceride-rich lipoproteins. For the purposes of this section, TGRL is

defined as lipoprotein particles predominantly composed of chylomicrons, chylomicron

remnants, and very low-density lipoprotein (VLDL). Most of the research in blood lipid

abnormalities has focused on the effects of LDL and monocyte/macrophage responses.

15

However, some studies have addressed the effects of TGRL on blood monocytes.

Fluctuations of chylomicron levels and, to a lesser degree, VLDL levels occur after ingesting

lipid-rich or carbohydrate-rich meals197,198 . Importantly, people, especially those in Western

countries, spend much of their day in the postprandial state owing to increased snacking

associated with obesity 199. Allied to this concept, the role of nonfasting triglyceride-levels as

a risk predictor for cardiovascular disease has been shown to be of value, and is endorsed by

the European Atherosclerosis Society (EAS) for clinical assessment200. Furthermore, beyond

their potential role in atherosclerosis, elevated lipid levels are found in individuals with

HIV201 and in conditions characterized by chronic inflammation, such as systemic lupus

erythematosus 202, implying potential alternative interactions between dysregulated

immune cells in these conditions and the presence of elevated blood lipid levels.

Ldlr–/– mice are frequently used to study the effects of dyslipidaemia on innate immunity.

These mice unsurprisingly have elevated LDL levels; however, these mice also have

markedly elevated levels of the chylomicron and VLDL subfractions (which are rich in both

apolipoprotein B-100 and apolipoprotein B-48), suggesting elevations in TGRL203. VLDL is

composed predominantly of triglycerides (70%), but has a small proportion of cholesterol

content (10%)204, some of which can be influenced by dietary intake197. A study published

in 2016 demonstrated the appearance of neutral lipid droplets in monocytes (both subsets)

from Ldlr–/– mice maintained on a HFD compared with monocytes from mice fed a control

chow diet, with evidence of impaired monocyte migration and retention in extravascular

tissue in response to inflammation65. This phenotype of tissue-retained monocytes after lipid

loading might be related to the plaque macrophage retention seen during HFD-induced

atherosclerosis models205,206. Indeed, lipid loading is also seen in monocytes from

hypercholesterolaemic Apoe–/– mice fed a HFD87,157. These lipid-loaded monocytes show an

activated phenotype with increased expression of CD11c in lipid rafts in the cellular

membrane157. These findings are supported by studies in human monocytes — in particular

the classical CD14highCD16low subset — showing an increase in membrane cholesterol content

in response to LDL, but more so following incubation with VLDL in vitro65, echoing the

concept that dyslipidaemic conditions can alter monocyte biology. Whether lipid loading of

monocytes during HFD leads to an exclusive proinflammatory phenotype is a matter of

debate. Nevertheless, the evidence indicates that lipids do alter monocyte behaviour.

16

Indeed, lipoproteins (particularly TGRL) are thought to inhibit monocyte response to

endotoxins by scavenging LPS207.

Similar to mouse monocytes in vivo, human monocytes of both subsets accumulate neutral

lipid in vitro in response to incubation with VLDL (and peculiarly LDL)65,151,152,154,156. The

accumulation of neutral lipid is associated with an impairment in chemotaxis and

chemokinesis in in vitro assays, potentially explained by alterations in cytoskeletal

rearrangement modulated by the small GTPase RAS homologue gene family member A

(RHOA)65 (FIG. 2b) . However in mouse models, in vivo monocyte lipid accumulation did not

result in altered patrolling behaviour65, which suggests that defects in migration after lipid

loading could be restricted to the extravascular tissue compartment. In vivo work in mouse

models where chronic TGRL elevation is induced pharmacologically support a more complex

role for nonclassical LY6Clow monocytes. Administration of poloxamer-407 elevates

circulating triglyceride levels in wild type mice by inhibiting lipoprotein lipase (LPL) — which

catalyses the conversion of VLDL to intermediate-density lipoprotein (IDL) — and cholesterol

7α-hydoxylase (CYP7A1) — involved in the transformation of cholesterol into bile acids —

without causing apparent hypercholesterolaemia66,208. This model of hypertriglyceridaemia

results in a noticeable reduction in peripheral counts of nonclassical LY6Clow monocytes

owing to the increase in their crawling behaviour and endothelial retention (without

alteration of bone marrow cMoP, total monocyte, or subset numbers)66. This increase in

crawling and retention led to mass monocyte extravasation into all organs examined (heart,

liver, and kidney) and accumulation of F4/80+CD68+ macrophages66. The exact mechanisms

are unknown, but did not produce an overt inflammatory response, although the model did

involve the overexpression in tissue macrophages of the chemokine CCL4 and a G protein-

coupled receptor-dependent mechanism66. Fascinatingly, in this model of poloxamer-407-

induced hypertriglyceridaemia monocytes did not have intracellular lipid accumulation,

which might indicate a role for LPL in lipid uptake or lipophage formation. Indeed, lipid

droplet incorporation into monocytes might be mediated by LPL. LPL generates free,

nonesterified fatty acid from TGRL that might promote an inflammatory phenotype, as

shown in some studies152,209.

Taken together, these findings indicate that cholesterol and TGRL can influence monocyte

subset effector functions (FIG. 2b). Further work is required to understand the potential

17

effect of dyslipidaemic environments on immune functions and cardiovascular disease.

Speculating that monocytes can deliver a lipid load during atherogenesis, rather than the

restricted theory of only tissue macrophages responding to retaining modified cholesterols,

would be intriguing. The contribution of monocytes as effective lipid carriers, and indeed

the role of monocytes in systemic lipid metabolism, warrants further study.

[H2] Effect of dietary lipids on monocytes

Contributions of dietary lipids to cardiovascular disease remain enigmatic, and any potential

role of dietary lipids in modulating monocyte and macrophage biology has not been studied

in detail. Dietary studies are inherently difficult to perform, as the majority require

substitution of one diet component while maintaining similar calorific content. Analyses in

dietary studies usually involve an element of observation, and are reliant on questionnaires

and participant reporting; therefore, these studies are subject to numerous sources of bias.

Despite these limitations, long-term and short-term dietary studies have helped to inform

us of potential deleterious effects of dietary lipids. The effects of so-called ‘healthy eating’

on disease continues to be of interest, particularly its role in cardiovascular disease210. Diets

rich in saturated fatty acids have been implicated in cardiovascular disease211-219, whereas

monounsaturated fatty acids (MUFA)217,220,221 and polyunsaturated fatty acids (PUFA) 222-224

are suggested as cardioprotective. Whether dietary saturated fat is indeed associated with

cardiovascular disease remains controversial, particularly in light of updated guidelines

continuing to support a diet low in saturated fat225, even after several retrospective meta-

analyses have found no link between the incidence of cardiovascular disease and saturated

fat intake224,226,227. However, these studies support the notion that diets rich in trans-fat are

deleterious to health and that MUFA-rich diets and omega-3 PUFA-rich diets are

beneficial224,226,227. Although these data help to establish epidemiological trends, these

studies do not help to clarify the mechanisms by which dietary lipid might influence the risk

of cardiovascular disease. Short-term and acute feeding studies have been used to elucidate

these mechanisms. Diets rich in saturated fat can activate proinflammatory pathways in

peripheral blood mononuclear cells228 and in monocytes152,209,229, whereas MUFA-rich diets

seem to induce anti-inflammatory signalling pathways228. Possible mechanisms through

which saturated fat might drive an inflammatory response are varied; for instance,

18

saturated fat might promote trafficking of gut endotoxin into the systemic circulation229-232,

and although early in vitro data177,233-236 and limited evidence in vivo237 suggested that the

mechanism might be mediated through direct stimulation of TLR by saturated fat , other

studies indicate that this mechanism is unlikely and show that the inflammatory response is

a result of endotoxinaemia238. Immune cells are altered within hours of meal consumption. A

meal rich in saturated fat can activate monocytes, as assessed by increased expression of

CD11c, accumulation of cytosolic intracellular lipid analysed ex vivo, and increased

adherence to vascular cell adhesion molecule 1 (VCAM1) substrate in a shear stress

model152. In flow cytometry analyses, human leukocytes239 and monocytes152 isolated from

blood in the postprandial phase show an increase in side scatter profile, implying an

increase in granularity resulting from an increase in lipid internalization. This increase in side

scatter is also found in monocytes from Apoe–/– mouse raised on a chronic HFD87,157.

Therefore, similar to dyslipidaemic environments, the postprandial response of consuming a

diet high in saturated fat might lead to formation of lipophages or ‘foamy’

monocytes152,157,240. A study published in 2016 showed that the postprandial response

mediates monocyte activation in individuals with metabolic syndrome156. The detailed

mechanisms of when and how monocyte subsets respond to dietary lipid loading remain to

be determined.

[H1] Conclusions

In this Review, we have briefly discussed the role of fetal macrophages and embryonic

monocytes in maintaining the tissue-resident macrophage pool (FIG. 1). The role of these

fetal tissue-resident macrophages in cardiovascular disease and response to dyslipidaemia

remains to be fully explored. We have elaborated on the heterogeneity of bone marrow

derived mouse and human monocytes and some essential differences in subset effector

functions during steady state and dyslipidaemia (FIG. 1, 2). The modulation of effector

functions of blood monocytes during dyslipidaemia might increase the risk of

atherosclerosis and accelerate the progression of the disease. In summary, dyslipidaemia —

with differing effects of hypercholesterolaemia and hypertriglyceridaemia — can alter bone

marrow production of monocyte precursors and mature monocyte subsets (particularly

classical monocytes); promotes the lipid endocytosis and accumulation of neutral lipid

19

droplets in monocyte subsets; induces an increased endothelial accumulation and

extravasation of nonclassical monocytes; increases tissue retention of monocytes and

accumulation of tissue-resident macrophages; and promotes a proinflammatory phenotype

and modulates chemotaxis in monocytes (FIG. 2b). Together, these responses could

aggravate tissue damage during atherogenesis and, we hypothesize, this increased tissue

damage might increase the risk of plaque rupture. This hypothesis requires more

investigation, but does raise the possibility that lipid-lowering therapies can modulate

monocyte behaviour towards a response that might help to reduce cardiovascular events.

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Acknowledgments

K.J.W laboratory is funded by BHF CRTF to S.R. (FS/14/50/30856), MRC (MR/M003159/1),

Kidney Research UK (RP_019_20160303) and Imperial Biomedical Research Centre. A.J.M is

supported by a Career Development Fellowship from the NHMRC, a Future Leader

Fellowship from the NHF, and Project Grants from the NHMRC and Diabetes Australia. We

would like to thank members of the Woollard lab for helpful discussion and lab work

underpinning data for this Review.

Author contributions

S.R. and K.J.W. researched data for the article and wrote the manuscript. A.J.M. and K.J.W.

provided substantial contribution to the discussion of content, and reviewed and edited the

manuscript before submission.

34

Competing interests statement

The authors declare no competing interests.

Key points

The mononuclear phagocyte system comprises heterogeneous cells derived from

fetal and bone marrow precursors

Seeding of fetal tissue macrophages is distinct from monocyte-derived macrophages,

highlighting the independent effector functions of monocyte subsets

Dyslipidaemia, characterized by hypercholesterolaemia and/or

hypertriglyceridaemia, increases the risk of cardiovascular disease

Dyslipidaemia increases the production of monocytes through myelopoiesis

Blood monocyte subsets under dyslipidaemic conditions change their behaviour and

activation, and increase their extravasation in response to triglyceride-rich

lipoproteins and cholesterol

Box 1 | Definitions of hyperlipidaemia

[b1] Hyperlipidaemia

Hyperlipidaemia is used to describe elevated levels of one or more of total

cholesterol, low-density lipoprotein cholesterol and triglycerides (TG).

[b1] Hypertriglyceridaemia

Hypertriglyceridaemia is an aberrantly elevated level of TG in the blood (normal <1.7,

high 1.7-9.9, severe >10 mmol/L). TG represent the bulk form in which fatty acids (fat)

are transported in the blood. Triglycerides are packaged into lipoproteins,

predominantly in chylomicrons and very-low density lipoproteins (VLDL). Chylomicrons

transport the bulk of dietary fat, whereas VLDL transport fatty acids coming from the

diet (exogenous) and produced in the body (endogenous).

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Elevated levels of nonfasting TG are correlated with increased risk of cardiovascular

disease144,145,147,148.

[b1] Hypercholesterolaemia

Hypertriglyceridaemia can be found in isolation or in addition to elevated levels of

cholesterol (hypercholesterolemia). Of note, elevated cholesterol levels refer to levels of

cholesterol-carrying lipoproteins, LDL and HDL.

High LDL levels strongly correlate with increased risk of cardiovascular disease103,104,107-111.

Conversely, high HDL levels might be correlated with reduced a reduced risk of

cardiovascular disease126.

[b1] Classification

Hyperlipidaemia can be caused by primary (genetic) factors or by secondary

(nongenetic) factors such as lifestyle habits or medical conditions

[b2] Genetic (primary) hyperlipidaemia.

Familial combined hyperlipidaemia. Polygenetic cause. Elevation of LDL, TG or

both.

Familial hypercholesterolemia (heterozygous or homozygous). Mutation in LDL

receptor leads to elevated LDL cholesterol.

Familial dysbetalipoproteinaemia. Autosomal recessive. Majority homozygous

mutation in ApoE. Elevated total cholesterol and TG. Associated with metabolic

syndrome.

Hypertriglyceridemia. Elevated TG. Common and rare genetic variants. Moderate

severity (2-10 mmol/L) caused by polygenetic effect influencing VLDL production

and removal. Monogenic causes: LPL, ApoC2, ApoA5, LMF1, GPIHBP1, GPD1. A

gain of function mutation in ApoC3 leads to high ApoC3/TG levels.

Other genetic disorders of lipoprotein metabolism, e.g. Tangiers disease (very

low HDL).

[b2] Secondary hyperlipidaemia. Several factors can cause hyperlipidaemia, such as

lifestyle habits (high fat diet, physical inactivity, alcohol consumption)241,242, medical

conditions including diabetes mellitus243, hypothyroidism244, and some forms of

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kidney disease245,246, and the use of certain drugs (e.g. anti-hypertensive agents247,

hormone replacement therapy248, contraceptives249 and some antipsychotics250).

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Figure 1 | Monocyte and macrophage ontogeny. Monocytes derive from haematopoietic

precursors in bone marrow or spleen. Monocytes give rise to classical subsets — defined in

mice as lymphocyte antigen 6C (LY6C)high and in humans as CD14highCD16low — and

nonclassical subsets — defined in mice as LY6Clow and in humans as CD14lowCD16+.

Production and emigration of monocyte subsets depends on defined signals and chemokine

receptors, such as C-C motif chemokine 2 (CCL2)36,83, C-C chemokine receptor type 2

(CCR2)16,36,38,76,251,252, and C-X-C chemokine receptor type 4 (CXCR4)61 for LY6Chigh monocytes,

and the microRNA miR-14631, nuclear receptor subfamily 4 group A member 1 (NR4A1)52,

transcription factor RelB31, and sphingosine 1-phosphate receptor 5 (S1PR5)253 for LY6Clow

monocytes. Some monocytes remain attached to the endothelium and patrol the blood

vessel lumen; other LY6Clow monocytes can derive from LY6Chigh monocytes. Inflammatory

cues induce monocyte extravasation (particularly of LY6Chigh monocytes) and polarization

into macrophages. These monocyte-derived macrophages mix with tissue-resident

macrophages derived from yolk sac and fetal liver precursors. The macrophage pool

receives tissue specific signals to define phenotypes and effector functions and, along with

other cues such as damage-associated molecular patterns (DAMPs) and pathogen-

associated molecular patterns (PAMPs), define the spectrum of tissue macrophages found in

vivo. Dotted lines represent hypothesized or unknown pathways. AGTR1A, type 1A

angiotensin II receptor; Ang II, angiotensin II; cMoP, common monocyte progenitor; CMP,

common myeloid progenitor; CX3CL1, CX3C-chemokine ligand 1; CX3CR1, CX3C-chemokine

receptor 1; GMP, granulocyte-macrophage progenitor; HSC, haematopoietic stem cell,

ICAM, intercellular adhesion molecule; Int, intermediate; JAMs, junctional adhesion

molecules; LFA1, lymphocyte function-associated antigen 1; MDP, macrophage and

dendritic cell precursor; PECAM1, platelet endothelial cell adhesion molecule; PSGL1, P-

selectin glycoprotein ligand 1; VLA4, very late antigen 4; VCAM1, vascular cell adhesion

molecule 1.

Figure 2 | Effect of dyslipidaemia on monocyte recruitment, migration, and cell behaviour.

a | Hypercholesterolaemia promotes production of classical monocytes, defined in mice as

lymphocyte antigen 6C (LY6C)high, by inducing the proliferation of bone marrow precursors,

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which in turn leads to blood monocytosis, endothelial dysfunction, and increased

recruitment mediated by chemokine receptor signals such as C-C chemokine receptor type 5

(CCR5)83,254, and ultimately leads to increased tissue monocyte-derived macrophages.

Hypertriglyceridaemia increases accumulation of patrolling nonclassical monocytes (LY6C low)

and extravasation, possibly mediated by C-C motif chemokine 4 (CCL4)66. Dyslipidaemia

might lead to increased proliferation of tissue-resident macrophages. Monocytes can

endocytose circulating lipids, leading to formation of ‘foamy’ monocytes, which might then

deliver lipids from the blood to peripheral tissues, contributing to increased lipid retention

in tissues (via heparin sulphate proteoglycans; HSPGs) and to increased lipid endocytosis by

tissue macrophages, promoting foam cell formation. Red lines indicate pathways

upregulated in dyslipidaemia. b | Lipids can enter the cell via regulated uptake, mediated

for example by the LDL receptor (LDLR) and the LDLR-related protein 1 (LRP1), or via

unregulated uptake, mediated among others by scavenger receptors such as CD36 and

SRA1, and by Toll-like receptors (TLRs). Neutral lipids and cholesterol can then accumulate

inside the cell, leading to proinflammatory or anti-inflammatory responses depending on

the lipid species and lipid modification. Lipid entry will also lead to activation of intracellular

cholesterol storage and efflux pathways (reverse cholesterol transport). Increased lipid

loading modulates the cytoskeleton by blocking signalling components such as the GTPase

RAS homologue gene family member A (RHOA), leading to defects in migration and possible

tissue retention of macrophages65. ABCA1, ATP-binding cassette transporter subfamily A

member 1, ABCG1, ATP-binding cassette transporter subfamily G member 1; CXCL16, C-X-C

motif chemokine 16; LOX1, lectin-type oxidized LDL receptor 1; LPL, lipoprotein lipase;

mmLDL, minimally modified LDL; oxLDL, oxidized LDL; NEFA, non-esterified fatty acid; TGRL,

triglyceride-rich lipoprotein; VLDL, very low-density lipoprotein.

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Table 1 | Monocyte subsets in blood

Monocyte subset

Common surface proteins used as lab markers to differentiate subsets

Mouse

Classical LY6Chigh CX3CR1int CD62L+ CCR2+ CD43low

Nonclassical LY6Clow CX3CR1high CD62L- CCR2- CD43high

Human

Classical CD14high CD16low CD62L+ CD43low HLA-DRlow

Nonclassical CD14low CD16high CD62L- CD43high HLA-DRhigh

Intermediate CD14high CD16high CD62Lint CD43int HLA-DRint

Glossary

Mononuclear phagocyte system: part of the immune system that consists of phagocytic cells, namely monocytes and macrophages but also tissue specific phagocytes such as Kupffer cells, microglia, osteoclasts and Langerhans cells Adoptive transfer: a technique for introducing labelled cells of interest into a host animal for the purposes of tracking and/or imaging, or to ascertain effects of the donor cells on a host

Parabiosis model: a technique used in order to study the effects of one biological system on another through the surgical conjoining of circulatory and physiological systems. Mice and rats are commonly use in these models.

Aorta–gonad–mesonephros: region of the embryonic mesoderm that includes the dorsal aorta and is implicated in definitive haematopoietic stem cell myelopoeiesis.

Common monocyte progenitor: a monocyte progenitor cell in the bone marrow distinct from macrophage and dendritic cell precursor (MDP) cells

Plasmacytoid dendritic cell: although their appearance resembles that of plasma cells, their function resembles that of conventional myeloid dendritic cells (DCs)

Lipoprotein: a carrier molecule that contains and transports lipid (namely cholesterol and triglyceride) within the circulation, and consists of apolipoprotein molecules and a single layer phospholipid membrane. Examples include LDL, VLDL and HDL as well as chylomicrons.

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ABC transporters: ATP-binding cassette transporters: actively transport molecules in or out of cells. An example is the ABCA1 transporter that plays a role in cholesterol export to HDL.

Oxidized LDL: an LDL molecule where the lipoprotein envelope and/or cholesterol content has been oxidised by free radicals. Oxidised forms of LDL are thought to play an important role in atherosclerosis and can stimulate macrophages accumulate lipid and accelerate conversion to macrophage foam cells.

Reverse cholesterol transport: the transport of cholesterol from tissues or cells out and back to the liver for clearance. Cholesterol can be transported via ABCA1 to HDL that acts as an acceptor molecule. This process is thought to be cardioprotective.

Neutral lipid droplets: lipids without a polar charge that can be found within cells, such as adipocytes and commonly refers to cholesterol esters and triglycerides.

NLRP3 inflammasome: an important oligomer molecule that forms part the cellular inflammatory response, particularly in innate immune cells resulting in the release of cytokines such as interleukin-1 beta.

Minimally modified LDL: a unique form of oxidised LDL enriched with aldehyde containing phosphatidylcholines

Reconstituted HDL: a synthetic form of HDL used in order to improve cholesterol transport out of the cell (efflux) with potential therapeutic value against cardiovascular disease.

Chylomicrons: also known as "ultra-low density lipoprotein", these large lipoprotein particles are secreted by the gut endothelial cells and predominantly transport dietary lipid (triglycerides and cholesterol) in the circulation. Chylomicron levels rise following dietary intake of fat or carbohydrate and fall following fasting.

Very low-density lipoprotein: these lipoproteins predominantly transport triglyceride produced by the body (muscle, liver) but can also be altered by dietary intake.

Author biographies

Dr Shamim Rahman is a Cardiology Specialist Registrar in London, specialising in Coronary Intervention. He is currently reading towards a PhD studying the role of monocyte subset responses to dietary lipid in the context of atherosclerosis, infection and immunity.

Associate Professor Andrew Murphy is the head of the Haematopoiesis and Leukocyte Biology Laboratory at Baker Heart and Diabetes Institute in Melbourne, Australia. His main

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interest is how the haematopoietic system is altered in the setting of cardiovascular disease and comorbidities. His group also studies fundamental biological process of haematopoiesis.

Dr Kevin Woollard is a group leader within the Division of Immunology and Inflammation at Imperial College London. His lab studies the ontogeny and effector functions of monocyte subpopulations and their role in vascular inflammation. Their main focus is the biology of monocytes and macrophages in autoimmune vasculopathies and atherosclerosis.

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