Arteriosclerosis, Thrombosis, and Vascular Biology

REVIEW

Impact of Impaired Cholesterol Homeostasis on

Neutrophils in Atherosclerosis
Bradley Tucker ,* James Ephraums,* Thomas W. King, Kaivalya Abburi, Kerry-Anne Rye , Blake J. Cochran  

ABSTRACT: Atherosclerosis is complex chronic disease characterized by intimal cholesterol accumulation and vascular inflammation.
There is a well-established relationship of hypercholesterolemia and inflammation with atherosclerosis. However, the link
between inflammation and cholesterol is not completely understood. Myeloid cells, in particular, monocytes, macrophages, and
neutrophils play essential roles in the pathogenesis of atherosclerotic cardiovascular disease. It is well known that macrophages
accumulate cholesterol, forming foam cells, which drive atherosclerosis-associated inflammation. However, the interaction
between cholesterol and neutrophils remains poorly defined—an important gap in the literature given that neutrophils represent
up to 70% of total circulating leukocytes in humans. Elevated levels of biomarkers of neutrophil activation (myeloperoxidase and
neutrophil extracellular traps) and higher absolute neutrophil counts are both associated with increased rates of cardiovascular
events. Neutrophils contain the necessary machinery to uptake, synthesize, efflux and esterify cholesterol; yet, the functional
consequence of dysregulated cholesterol homeostasis on neutrophil activity remains poorly defined. Preclinical animal data
suggest a direct link between cholesterol metabolism and hematopoiesis, although current evidence in humans has been
unable to corroborate such findings. This review will explore the impact of impaired cholesterol homeostasis neutrophils and
draw focus on the discordant data from animal models and atherosclerotic disease in humans.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: atherosclerosis ◼ biomarker ◼ homeostasis ◼ monocyte ◼ neutrophils

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M
yeloid cells play important roles in immune reg-
ulation and tissue homeostasis. Dysfunctional
monocytes, macrophages, dendritic cells, and
neutrophils have all been demonstrated to contribute to
atherosclerosis.1 Under hypercholesterolemic conditions,
monocytes/macrophages and dendritic cells accumulate
cholesterol, migrate into the vessel wall and accelerate
atherosclerotic lesion progression by secreting proin-
flammatory cytokines—a pathway, which has been exten-
sively reported over the last 3 decades.1,2 However, the
response of neutrophils to hypercholesterolemia has not
been extensively explored. Given that neutrophils are the
most abundant white cell population in human blood, this
gap in knowledge is a limitation in our understanding of
the pathobiology of atherosclerosis and its complications.
There is a well-established relationship between hyper-
cholesterolemia and atherosclerosis. Numerous large-scale
randomized trials have demonstrated that treatment of
hypercholesterolemia with lipid-lowering agents, most nota-
bly statins, dramatically reduces the incidence of cardiovas-
cular events.3 Similarly, the relationship of inflammation with
*B. Tucker and J. Ephraums contributed equally and share first authorship.
For Sources of Funding and Disclosures, see page 625.
© 2023 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb
atherosclerosis has recently been highlighted by several trials
in which anti-inflammatory medications, such as canakinumab
and colchicine, reduce the incidence of cardiovascular events
independent of cholesterol levels.4,5 The current understand-
ing is that inflammation and hypercholesterolemia work syn-
ergistically to promote atherosclerotic plaque development
and progression. However, precisely, how these 2 pathways
are related remains poorly understood.
This review explores the impact of impaired choles-
terol homeostasis on neutrophils in animals and humans.
Moreover, the fundamentals of neutrophil cholesterol
metabolism are discussed, with a particular focus on the
modulation of proinflammatory actions and pathways.
NEUTROPHILS IN CARDIOVASCULAR
DISEASE
Pathobiology
Neutrophils mediate innate immune functions via oxi-
dative burst, phagocytosis and secretion of neutrophil

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 Tucker et al
Nonstandard Abbreviations and Acronyms
ABCG1  TP-binding cassette subfamily G mem-
A • Neutrophils play an important role in the develop-
ber 1
apoA-I apolipoprotein A-I
CANTOS Canakinumab Anti-Inflammatory Throm-
bosis Outcome Study
CCL5 chemokine ligand 5
CXCL1 chemokine (C-X-C motif) ligand 1
CXCL12 chemokine (C-X-C motif) ligand 12
CXCR2 CXC chemokine receptor 2
DNase deoxyribonuclease 1
G-CSF granulocyte colony-stimulating factor
HDL high-density lipoprotein
HMG-CoA 3-hydroxy-3-methyl-glutaryl-coenzyme A
HNP1 human neutrophil peptide 1
ICAM-1 intercellular adhesion molecule-1
IL interleukin
LDL low-density lipoprotein
LDLR low-density lipoprotein receptor
Lp(a) lipoprotein (a)
MCP-1 monocyte chemoattractant protein-1
MI myocardial infarction
MPO myeloperoxidase
NET neutrophil extracellular trap
NLR neutrophil-to-lymphocyte ratio
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oxLDL oxidized low-density lipoprotein
RAP LDL receptor-related protein-associated
protein 1
SOAT2 sterol O-acyltransferase 2
SR-BI scavenger receptor class B type 1
VCAM-1 vascular cellular adhesion molecule-1
extracellular traps (NETs). The role of neutrophils in ath-
erosclerosis has become better understood in recent
years, with elevated numbers of circulating neutrophils
having been shown to be predictive of cardiovascu-
lar events independent of serum cholesterol levels in
humans.6,7 Analogously, heightened neutrophil production
in mice leads to accelerated atherosclerotic lesion devel-
opment,8 and depleting neutrophils with a Ly6G-specific
antibody markedly reduces plaque burden.9 In hyper-
cholesterolemic mice, the circulating neutrophil count is
highly correlated with plaque burden (r=0.95),9 and there
is evidence to suggest that neutrophils preferentially
infiltrate at the shoulders of atherosclerotic lesions.10 In
addition, risk factors for cardiovascular disease, such as
metabolic syndrome, stress, aging, and sleep disorders
have been linked to enhanced myelopoiesis.11
Neutrophils act via multiple mechanisms to promote
plaque progression, destabilization and thrombosis. MPO
(myeloperoxidase), one of the major neutrophil granule
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Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis
Highlights
REVIEW - AL
ment of atherosclerosis.
• The impact of elevated circulating cholesterol and
lipid levels are different between mice and humans.
• Altered cholesterol homeostasis has important
impacts on neutrophil function.
proteins promotes plaque progression by oxidizing LDLs
(low-density lipoproteins), thereby enhancing foam cell
formation.12 In hypercholesterolemic mice, administra-
tion of an MPO inhibitor improves endothelial function,
reduces oxidative stress, and inhibits plaque formation.13
Although neutrophils alone are proatherogenic, this
is enhanced by their ability to recruit proinflammatory
monocytes to the artery wall.14 Activated neutrophils also
release several chemotactic granule proteins including
azurocidin, cathepsin G and HNP1 (human neutrophil
peptide 1, α-defensin).15–17 Azurocidin and cathepsin G
increase endothelial expression of ICAM-1 (intercellular
adhesion molecule-1), VCAM-1 (vascular cell adhesion
molecule-1), and E-selectin,16,17 while HNP1 forms het-
erodimers with platelet-derived CCL5 (chemokine ligand
5) that promote monocyte recruitment.15 Moreover, neu-
trophils damage endothelial cells by increasing produc-
tion of reactive oxygen species, which enhances vascular
permeability.11 Together, these events increase adhesion
of monocytes to the endothelium and their transmigra-
tion into the subendothelial space where they participate
in atherosclerotic plaque development.
Neutrophils contribute to plaque destabilisation by
secreting cytotoxic histone H4-containing NETs, which
lyse vascular smooth muscle cells.18 This cytotoxic effect
of NETs is evidenced by their presence in complicated
plaques with evidence of erosion, rupture, or intraplaque
hemorrhage, but not in intact plaques.19 Inhibition of
NET formation by DNase (deoxyribonuclease 1) treat-
ment attenuates local inflammation and promotes plaque
regression.20 Recent studies have demonstrated that
NET clearance can be regulated by either increasing or
decreasing circulating DNase activity via exercise21 or
hypercholesterolemia,22 respectively. Additionally, neu-
trophils secrete matrix metalloproteinases that degrade
the extracellular matrix and progressively reduce the
structural integrity of plaques, especially in rupture-prone
shoulder regions.10,23,24
Relationship of Neutrophils With Cardiovascular
Events
Numerous large epidemiological studies have demon-
strated a positive association between neutrophil count
and cardiovascular event risk. In a study by Horne et
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 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis
al,6 which included 3227 participants without an acute
myocardial infarction (MI) but who had been diagnosed
with coronary artery disease on angiography, a neutrophil
REVIEW - AL
count in the fourth quartile was associated with a 2-fold
increased risk of death or MI over a 3.5-year follow-up
period compared with those in the first quartile. These
findings were corroborated by analysis of data from
≈500 000 ostensibly healthy participants from the UK
Biobank25 in which there was a positive linear association
between neutrophil count and cardiovascular events.25
To limit the influence of reverse causality, the authors
excluded participants with comorbid disease at baseline
and those who had an event within the first 2 years of
follow-up. Importantly, the association of neutrophil count
with cardiovascular events remained significant after this
adjustment.25 Similar results were also obtained by Dis-
telmaier et al26 who examined the culprit site to systemic
neutrophil count gradient in 417 patients undergoing
primary percutaneous coronary intervention. The authors
found that those with the greatest increase in culprit site
neutrophils relative to the systemic concentration of neu-
trophils had the highest long-term mortality rate.26 This
effect was later shown to be secondary to localized acti-
vation of the classical complement cascade at the culprit
site.27 In the CANTOS trial (Canakinumab Anti-Inflam-
matory Thrombosis Outcome Study), canakinumab, an
IL (interleukin)-1β neutralizing monoclonal antibody,
reduced circulating neutrophil levels and the incidence
of cardiovascular events.5
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The neutrophil-to-lymphocyte ratio (NLR) is a robust
and inexpensive measure of chronic inflammation that
reflects the dynamic relationship between innate and
adaptive immune responses. The NLR is closely related
to the severity and prognosis of many cardiovascular dis-
eases.28 In a meta-analysis of 10 245 participants with
an acute MI who underwent emergent revascularization,
an elevated NLR at the time of revascularisation was
independently associated with a greater risk of in-hos-
pital and long-term cardiovascular events and mortality.29
This is at least in part due to the fact that the NLR is
proportional to the angiographic severity of acute coro-
nary syndrome, as assessed by the SYNTAX score.30 The
NLR is also predictive of periprocedural complications,31
although this may not be the case in patients undergoing
elective percutaneous coronary intervention.32 In patients
with an acute MI undergoing primary percutaneous coro-
nary intervention, the NLR upon admission is proportional
to the risk of coronary no-reflow.33 More specifically, with
a mean NLR of approximately 3.8, each 1 unit increase
in NLR was associated with 54% increased odds of
coronary no-reflow.33 The prognostic significance of the
NLR was highlighted by a study of 1000 participants
with acute decompensated heart failure and found those
with the highest NLR upon admission had the greatest
risk of both short- and long-term mortality.34 In fact, when
compared with those in the first tertile, those in the third
620   May 2023 Arterioscler Thromb Vasc Biol. 2023;43:618–627. DOI: 10.1161/ATVBAHA.123.316246
tertile of NLR had a 2.2-fold higher mortality rate. The
NLR is also proportional to the risk of a patient having
metabolic syndrome—a well-known risk factor for cardio-
vascular disease.35,36
NETs are the terminal product of neutrophil activation37
and consist of granule proteins, such as neutrophil elas-
tase and MPO, scattered across a backbone of decon-
densed chromatin and histone proteins.38 Under regular
homeostatic conditions, NETs trap and neutralize patho-
gens. However, aberrant NET formation is pathological
and been linked to inflammatory and autoimmune dis-
eases.39 NET formation can be triggered by a plethora of
factors, including bacterial proteins, cholesterol crystals
and oxidized LDL.38 Furthermore, maintenance of mice on
a high fat diet has been shown to increase NET forma-
tion in a mouse model of viral pneumonia, suggesting that
the metabolic state can contribute to the proinflammatory
properties of neutrophils.40 NETs also directly contribute to
atherosclerosis by activating macrophages.41
Markers of NET formation have been shown to corre-
late with atherosclerotic disease severity. In a cross-sec-
tional study of 282 participants with suspected coronary
artery disease, Borissoff et al42 found that circulating
levels double-stranded DNA, nucleosomes, citrullinated
histone H4 and MPO-DNA complexes—all markers of
in vivo NET formation—are increased and associated
with more severe coronary artery disease on angiog-
raphy, a prothrombotic state (as evidenced by elevated
thrombin-antithrombin complex and von Willebrand fac-
tor levels) and an increased incidence of cardiovascular
events. Participants in this study with a double-stranded
DNA concentration higher than the median had a 3-fold
higher risk of suffering a major adverse cardiovascular
event.42 This effect is partially explained by the propensity
of NETs to induce plaque destabilization through cyto-
toxic effects on smooth muscle cells.18 In patients with
thrombotic occlusion, the presence and concentration of
intra-thrombus NETs is associated with a worse progno-
sis.43 In patients with an acute MI secondary to an occlu-
sive thrombus, the presence of NETs within the thrombus
was found to be associated with a lower ejection fraction
3 to 8 months after the index event.43 In a similar popula-
tion, the abundance of NETs at the thrombus site was
found to correlate positively with infarct size and nega-
tively with ST-segment resolution.44 Percutaneous coro-
nary intervention of such lesions causes a rapid increase
in the intra-coronary concentration of NETs,45 which pre-
disposes to microvascular obstruction, subsequent peri-
procedural events, and worse long-term outcomes.31,46
RELATIONSHIP OF LIPID LEVELS WITH
NEUTROPHIL COUNT
Numerous preclinical and human studies have investi-
gated the relationship of lipid levels with neutrophil count.
Interestingly, however, these findings are somewhat
 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis
discordant (Table). This suggests that mechanistic links
between lipids and hematopoiesis may vary between
animal and humans, which is a critical consideration in
future studies of cardiovascular disease. Neutrophil
homeostasis is controlled on numerous levels, includ-
ing granulopoiesis, mobilization from the bone marrow,
apoptosis and clearance.65 Changes to any one of these
mechanisms will influence circulating neutrophil levels.
Preclinical Studies
Suzuki et al47 were the first to demonstrate that feeding
rats a lipid-rich diet caused marked leukocytosis. Given
that neutrophils represent the majority of circulating leu-
kocytes, it was reasonable to assume that this reflected
neutrophilia. Similarly, initiation of a high fat diet in New
Zealand White rabbits sharply increased serum choles-
terol levels, as well as leukocyte counts.50 In the same
study, cessation of the high fat diet resulted in a gradual
normalization of neutrophil count. A similar trend has also
been observed in mice.9,48 In fact, when compared with
mice fed a regular chow diet, those fed a high fat diet for 17
weeks had a 2-fold higher circulating neutrophil count—
a finding,which was later corroborated by Drechsler et
al6 who went on to further delinate the mechanisms
involved. Interestingly hypercholesterolemia impacted 2
of the aforementioned levels of neutrophil homeostasis:
granulopoiesis, mobilization from bone marrow and clear-
ance from circulation. More specifically, administration of
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a high fat diet stimulated granulopoiesis by increasing
G-CSF (granulocyte colony-stimulating factor) levels via
TNF and IL-17 dependent mechanisms.9 Mobilization of
neutrophils from the bone marrow was also enhanced
in dyslipidemia, which occurred secondary to increased
CXCL1 (chemokine [C-X-C motif] ligand 1) and CXCR2
(CXC chemokine receptor 2) expression.9 Additionally,
by reducing CXCL12 (Chemokine [C-X-C motif] ligand
12) expression, dyslipidemia has been shown to limit the
peripheral clearance of senescent neutrophils.9,66
Triglycerides have been shown to directly activate neu-
trophils.67 A triglyceride-rich meal rapidly increases neutro-
phil numbers in the acute post-randial period in humans.67
Moreover, the expression of markers of neutrophil activa-
tion such as CD11b and CD66b are increased following
triglyceride exposure.67 Conversely, exposure to HDLs
(high-density lipoproteins) and the main HDL apolipopro-
tein component, apoA-I (apolipoprotein A-I), attenuates
neutrophil activation.68 Neutrophils treated with either lipid-
free apoA-I or HDL have a markedly impaired migratory
response, have a limited ability to adhere to the vascular
wall and reduced surface expression of CD11b.68 It is
thought that this anti-inflammatory effect is due to the abil-
ity of HDL and apoA-I to mediate cholesterol efflux.54 While
mechanistically elegant, it remains to be seen whether this
is relevant in humans. There are significant differences
between the hematological systems of mice and humans
Arterioscler Thromb Vasc Biol. 2023;43:618–627. DOI: 10.1161/ATVBAHA.123.316246
at both the levels of hematopoietic stem cells, mature leu-
kocytes and the cross talk between various elements of
the immune system.69 As new murine models of hema-
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topoiesis that more closely emulate humans continue to
emerge,70 hopefully too will our understanding of the rela-
tionship between cholesterol and neutrophils in preclinical
models of atherosclerosis.
Human Studies
As described above, a close association of hypercholes-
terolemia with neutrophilia has been identified in preclini-
cal models of atherosclerosis. However, whether these
mechanistic links exist in humans remains unclear. Bovill
et al60 were among the first to evaluate the relationship of
lipid levels with leukocyte count in humans. In this study
of 5201 participants aged 65 or older, triglycerides were
positively correlated, whereas HDL cholesterol was nega-
tively correlated, with total leukocyte count. Unfortunately,
this study did not specifically determine neutrophil count
nor were any of the reported correlations adjusted for
potential confounding variables. Yet, since this time, several
other cross-sectional and longitudinal studies have sug-
gested a causal relationship of elevated triglycerides and
low HDL cholesterol with neutrophilia, albeit with a very
modest effect size.56,58,59,63,64 Data from the National Health
and Nutrition Examination Survey further indicated that the
association of HDL cholesterol with neutrophil count was
significant in men but not women.59 Although biological dif-
ferences exist that may explain an interaction of sex, such
as hormones levels and body composition, a formal test of
interaction was not performed on these data and similar
findings were not seen in a larger subsequent study.55
Observational data from >300 000 participants from
the UK Biobank was used to investigate the link between
cholesterol levels and leukocytosis directly.55 In contrast
to previous studies, HDL cholesterol was positively asso-
ciated with neutrophil count when data were analyzed
cross-sectionally but failed to remain significant in longi-
tudinal analysis. Cross-sectional data from this study also
revealed inverse correlations of total cholesterol, LDL
cholesterol, apolipoprotein B and lipoprotein(a) with neu-
trophil count, all of which failed to maintain significance
in longitudinal analysis. Only the relationship of triglycer-
ides with neutrophil count was significant over time, ergo
providing further support of a causal relationship.55 How-
ever, a recent Mendelian Randomisation study involving
almost 500 000 participants found no relationship of tri-
glycerides with neutrophil count.71 In the same study,LDL
cholesterol and HDL cholesterol levels were also not
found to be associated with neutrophil count. In contrast
to observational studies, Mendelian Randomization limits
the risk of residual confounding and reverse causation,
thereby providing robust estimates of causality.
Despite strong evidence supporting lipid-induced neutro-
philia in animal studies, and the well described mechanisms
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 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis

Table.  Association of Lipid Levels With Neutrophil Counts

Population Study design Findings Reference

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Animals
 Wistar rats High fat diet •  High fat diet resulted in 2-fold increase in total leukocyte count 47
 C57BL/6J mice High fat diet •  High fat diet resulted in a 2-fold higher circulating neutrophil count 48
 Yorkshire swine High fat diet •  High fat diet caused marked monocytosis as well as a mild neutropenia 49
 New Zealand White rabbits High fat diet •  Total leukocyte levels rose in parallel with serum cholesterol levels after 50
initiation of a high fat diet
•  Withdrawal of the high fat diet led to a reduction in the circulating leuko-
cyte count and serum cholesterol levels
apoE−/− mice
  High fat diet •  High fat diet resulted in marked neutrophilia 9
•  High fat diet increased G-CSF, CXCL1, and CXCR2 expression.
•  High fat diet reduced CXCL12 expression
•  Increased granulopoiesis, mobilization of neutrophils from bone marrow,
decreased neutrophil clearance
apoE−/− mice
  High fat diet •  High fat diet increased granulocyte count 51
apoE−/− mice
  High fat diet •  High fat diet-induced monocytosis, neutrophilia, and expansion of hemato- 52
poietic stem cells in bone marrow
LDLR−/− mice
 
LDLR−/− apoA1+/- mice
  High fat diet •  High fat diet-induced neutrophilia, monocytosis, and expansion of hemato- 53
poietic stem cells in bone marrow relative to LDLR−/− mice
Abca1−/− Abcg1−/− mice
  Chow fed •  Elevated neutrophils in blood and bone marrow of Abca1−/− Abcg1−/− mice 54
over WT and single gene knockout mice
Humans
 417 132 ostensibly healthy participants Cross-sectional •  In cross-sectional analysis, neutrophil count was inversely associated with 55
from the United Kingdom (UK Biobank) and longitudinal total cholesterol, LDL-C, apoB, and Lp(a) levels
•  Also in cross-sectional analysis neutrophil count was positively associated
with triglyceride and HDL-C levels
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•  In longitudinal analysis only the association of triglycerides levels with neu-
trophil count remained significant
 27 566 participants from Korea and the Cross-sectional •  In the cross-sectional analysis, total leukocyte count was positively corre- 56
United States (NHANES, KNHANES, TNT and longitudinal lated with triglycerides and negatively correlated with HDL-C
trial)
•  In the longitudinal analysis, increases in total cholesterol, HDL-C and triglyc-
erides levels were all associated with an increase in total leukocyte count
1992 participants from Japan Longitudinal •  Changes in all cholesterol levels not associated with changes to neutrophil count 57
 10 866 hypertensive participants from Cross-sectional •  Positive correlation of total cholesterol, LDL-C, and triglycerides levels with 58
China (CSPPT) neutrophil count
•  Negative correlation of HDL-C level with neutrophil count
 5647 noninstitutionalized participants from Cross-sectional •  LDL-C was inversely associated with neutrophil count in women but not men 59
the United States (NHANES)
•  HDL-C was inversely associated with neutrophil count in men but not women
•  Triglycerides positively correlated with neutrophil count in both sexes
 5201 participants aged ≥65 years from the Cross-sectional •  Total leukocyte count was positively correlated with triglycerides and nega- 60
United States (Cardiovascular Health Study) tively correlated with HDL-C
 3282 participants from China, approximately Cross-sectional •  In comparison to normolipidemic individuals, those with hypercholesterol- 61
half of which had untreated dyslipidemia emia or hypertriglyceridemia had a significantly higher neutrophil count
 2953 participants with no clinically evident Cross-sectional •  Neutrophil count was positively correlated with triglycerides and negatively 62
CVD from Japan correlated with HDL-C
 2873 participants with no clinically evident Cross-sectional •  Total cholesterol and LDL-C were inversely associated with neutrophil count 63
CVD from the United States (MESA)
•  Triglycerides levels were positively associated with neutrophil count
 652 participants with known CAD risk Cross-sectional •  Serum apoA-I level was negatively correlated with neutrophil count 64
factor(s)

apoA-I indicates apolipoprotein A-I; apoB, apolipoprotein B; CAD, coronary artery disease; CSPPT, China Stroke Primary Prevention Trial; CVD, cardiovascular disease;
CXCL1, chemokine (C-X-C motif) ligand 1; CXCL12, chemokine (C-X-C motif) ligand 12; CXCR2, CXC chemokine receptor 2; G-CSF, granulocyte colony-stimulating
factor; HDL-C, high-density lipoprotein cholesterol; KNHANES, Korea National Health and Nutrition Examination Survey; LDL-C, low-density lipoprotein cholesterol;
LDLR, low-density lipoprotein receptor; Lp(a), lipoprotein (a); MESA, Multi-Ethnic Study of Atherosclerosis; NHANES, National Health and Nutrition Examination Survey;
and TNT, Treating to New Targets Trial.

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 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis

underlying this, it appears that these findings are not reli- Project73 to assess the expression of cholesterol related
ably reproducible in humans. Overall, observational data genes in neutrophils compared with classical monocytes.
in humans is variable, with no clear trends being identified Neutrophils express the required proteins to take up, syn-

across all studies and in comparison, to animal work the cor-
relation between lipid levels and neutrophil count is modest.
This further emphasizes the fact that although numerous sta-
tistically significant relationships have been reported, the bio-
logical relevance of them remains questionable. Given this, it
is unclear whether hyperlipidemia directly influences the cir-
culating neutrophil count in humans. To answer this question,
further research into the underlying mechanisms is required.
IMPACT OF CHOLESTEROL ON
NEUTROPHIL CELLULAR HOMEOSTASIS
The presence of the LDL receptor on the cell surface of
neutrophils has been known for an extensive period of
time.72 We utilized data from the Immunological Genome
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thesize, efflux, and esterify cholesterol (Figure 1). Relative
to classical monocytes, neutrophils express significantly
more of the transcripts that encode SOAT2 (sterol O-acyl-
transferase 2; Soat2), ABCG1 (ATP-binding cassette
subfamily G member 1; Abcg1) and significantly less of
the transcripts that encode LDLR (low-density lipopro-
tein receptor; Ldlr), 3- HMG-CoA (hydroxy-3-methyl-glu-
taryl-coenzyme A reductase; Hmgcr), SR-BI (scavenger
receptor class B type 1; Scarb1), and RAP (LDL receptor-
related protein-associated protein 1, Lrpap1).
Neutrophil function is closely linked to membrane
cholesterol levels (Figure 2). Oh et al74 studied the effect
of alterations in cholesterol membrane content using
methyl-β-cyclodextrin in the neutrophil-like HL-60 cell
line and found that increased cholesterol was associated

Figure 1. Expression of genes involved in the uptake, synthesis, efflux, and esterification of cholesterol in neutrophils and
classical monocytes.
Data represent mean±SD. Data source: Immunological Genome Project.73

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 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis
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Figure 2. Mechanisms by which lipids impact neutrophil function.

A, Cholesterol depletion results in reduced actin polymerization while (B) cholesterol accumulation increases endothelial adhesion via
increased membrane tethering, rolling time and whole cell deformability. C, Treatment of human neutrophils with oxLDL (oxidized low-density
lipoprotein) in vitro results in increased migration, degranulation and NETosis. D, Mice fed a high fat have increased LDL and oxLDL levels, but
neutrophil chemotaxis is impaired.

with increased membrane tethering, whole cell deform-
ability, and increased rolling time, resulting in increased
endothelial adhesion time.75 Cholesterol depletion has
also been linked to reduced actin polymerization, sug-
gesting that abnormalities in lipid rafts may prevent actin-
myosin-mediated cell motility.74,75 In vitro studies with
isolated human neutrophils have shown that oxLDL (oxi-
dized LDL) activates migration and degranulation76 and
increases NET formation in a dose-dependent manner.77
In contrast, a mouse model of diet-induced dyslipidemia,
which results in LDL and oxLDL levels similar to those
seen in obesity, has shown impaired neutrophil chemo-
taxis.78 In this inflammatory state, the chemoattractant
protein MCP-1 (monocyte chemoattractant protein-1) is
elevated, but oxLDL fails to induce CD11b transcription
and F-actin polymerization, which are necessary for neu-
trophil chemotaxis and function.78 A more recent study
confirmed these findings, showing that neutrophil migra-
tory ability depends on both the integrity of the lipid raft
structure and the phosphorylation of the cortical and cel-
lular skeleton assembly protein β-adducin.79
Lipid Droplets
Lipid droplets are lipid-rich organelles, which accumu-
late in the cytoplasm of inflammatory cells in response
to endoplasmic reticulum stress.80 Ordinarily, lipid drop-
lets in neutrophils sequester fatty acids, diacylglycerols,
and ceramides, which inhibits lipid peroxidation. How-
ever, increased lipid droplet accumulation in neutrophils
may induce inflammation by increasing eicosanoid syn-
thesis.81 In a recent study of metastatic breast cancer,
it was reported that lung mesenchymal cells drive lipid
accumulation in neutrophils and that these lipids serve
as an energy reservoir, which can be transferred to meta-
static tumour cells.82 Autophagy of lipid droplets is also
a source of free fatty acids as neutrophils shift from gly-
colysis to fatty acid oxidation during differentiation.83

624   May 2023 Arterioscler Thromb Vasc Biol. 2023;43:618–627. DOI: 10.1161/ATVBAHA.123.316246
 Tucker et al Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis
FUTURE DIRECTIONS
The role of lipoproteins in atherosclerotic cardiovascu-
lar disease is well documented. However, despite the
widespread use of statin-based therapies, cardiovas-
cular disease remains the leading cause of morbidity
and mortality worldwide.84 This has created an oppor-
tunity to identify new therapeutic targets that reduce
inflammation and improve clinical outcomes. Therapies
that target neutrophils offer an attractive addition or
alternative to conventional therapies for treatment
of atherosclerotic cardiovascular disease as they are
implicated in the initiation, propagation, and complica-
tions of the disease.11 The fact that the function of
neutrophils is impacted under conditions of hypercho-
lesterolemia and impaired lipid homeostasis presents
opportunities for the development of novel systemic
and more importantly, targeted interventions. Future
therapies that specifically target neutrophil activation
may yield an improved risk profile.
While MPO is generally regarded as the most important
NET-associated protein in the context of inflammation and
cardiovascular disease, the impact of targeting constitu-
ent elements of NETs and the impact of this in inhibiting
atherosclerosis remains unclear. NETs contain a wide
range of proteomic and genomic constituent elements85
that show considerable heterogeneity based on disease
state,86–88 inducing factor86,89 and the action of exogenous
proteases.90 This has yet to be explored in neutrophils
Downloaded from http://ahajournals.org by on April 27, 2023
isolated from subjects with hypercholesterolemia. Future
studies should examine the impact of altered lipid homeo-
stasis on NET composition and the specific targeting of
proinflammatory NET-associated factors.
ARTICLE INFORMATION
Received January 12, 2023; accepted March 7, 2023.
Affiliations
Sydney Medical School, Faculty of Medicine and Health, University of Sydney,
NSW, Australia (B.T., J.E.). Royal Prince Alfred Hospital, Camperdown, NSW, Aus-
tralia (B.T., J.E.). School of Biomedical Sciences, Faculty of Medicine & Health,
UNSW Sydney, NSW, Australia (T.W.K., K.A., K.-A.R., B.J.C.).
Sources of Funding
This work was supported by an Idea Grant from the National Health and Medical
Research Council of Australia (APPP2004064) to K.-A. Rye and B.J. Cochran.
Disclosures
None.
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