Monocyte Subsets in Cardiovascular Disease: A Biomarker Perspective

Abstract

Endothelial dysfunctions together with a dysregulated immune response and lipid accumulation are important confounding factors in the onset and chronic development of atherosclerosis. Recently, a large body of data has emerged on the sequential involvement of different immune cell types, including monocytes, in the pathology of this disease. In this condensed review, we aim to highlight some of the recent basic research and clinical findings on monocyte subsets published since our joint European Society of Cardiology consensus document, and re-evaluate their potential relevance as surrogate biomarkers in coronary artery disease.

Keywords

monocytes - atherosclerosis - leukocytes - activation

Monocyte Biology

Monocytes are the largest blood leukocytes in adults and have a rapid turnover with relatively short lifespan of only a few days in the bloodstream.[1] [2] As cells of innate immunity, they are released from the bone marrow and serve as precursors to tissue macrophages and dendritic cells. Monocytes can secrete inflammatory cytokines and have distinct surface receptors such as integrins, G-protein-coupled and toll-like receptors (TLRs) that allow adhesion, crawling, and migration, as well as direct immune response and phagocytosis.[3] [4] [5] [6] In addition to resolving inflammation, monocytes are involved in angiogenesis, tissue remodeling after injury or scavenging apoptotic cells, and toxic debris at homeostatic conditions.[3] [4] [5] [6]

Monocyte Heterogeneity: Phenotype and Function

According to the expression intensity of the lipopolysaccharide (LPS) receptor CD14 and the FcγIII receptor CD16, human monocytes have typically been divided into three major subsets: classical CD14²⁺CD16⁻, intermediate CD14²⁺CD16⁺, and nonclassical CD14⁺CD16²⁺.[7] These subsets differ significantly in phenotype and function.

The CD14²⁺CD16⁻ subset (∼85% of total monocytes) highly expresses Ccr2, Cxcr4, FcγRI, L-selectin, and scavenger receptor class A.[5] [8] [9] [10] In contrast, CD14⁺CD16²⁺ monocytes (∼10%) are smaller and less dense showing higher levels of SLAN, CD31, CD11c, and Cx3cr1 but much lower levels of Ccr2 compared with the classical subset.[5] [8] [9] [10] The CD14²⁺CD16⁺ subset (∼5%) can be clearly distinguished from the nonclassical by the expression of Ccr2.[10] This lowest subset is strong pro-inflammatory with some unique features that distinguish it from the other two. So far, CD14²⁺CD16⁺ monocytes are enriched in bone marrow and have the highest expression of angiogenic markers, CD163, MHC class II, HLA-DR, and miR-6087of any subset.[5] [8] [9] [10] [11] Recent comprehensive data obtained by modern technology like mass cytometry with clustering algorithm and RNA sequencing suggest even higher heterogeneity among each subset with three newly identified nonclassical and four classical monocytes.[12]

Functionally, classical monocytes are professional phagocytes that generate reactive oxygen species (ROS) and secrete cytokines upon LPS stimulation.[5] In contrast, nonclassical monocytes do not generate ROS and are weak phagocytes that preferentially take up oxidized LDL (ox-LDL), but secrete substantial amounts of inflammatory cytokines after TLR-dependent activation by viruses and nucleic acids.[5] [6] Thus, nonclassical monocytes may serve as patrolling immune cells that selectively remove virally infected or injured cells and detoxify ox-LDL. The lower phagocytic activity of CD14⁺CD16²⁺ compared with classical monocytes has been efficiently exploited to enumerate the number and ratio of both subsets by their differential uptake of magnetic nanoparticles.[13] Finally, the intermediate subset does not produce ROS, but shows the highest secretion of tumor necrosis factor-α and interleukin (IL)-1β in response to LPS.[6]

Monocyte subsets undergo linear maturation kinetics in the periphery that is accompanied by a gradual increase in the expression of specific miRNAs.[11] Isotope labeling has revealed that they enter the bloodstream as classical, leave it as intermediate, and re-enter as nonclassical.[14] In contrast to the circulation, we found that the intermediate subset was highly enriched in the bone marrow.[15] This medullar monocyte pool seems to require Ccr2, Cxcr4, and possibly CD44 for mobilization and may give rise to more specialized peripheral Ccr2⁺ classical monocytes with implication during inflammation and infection, whereas nonclassical monocytes are less abundant in the bone marrow.

Monocyte Subsets in CAD: Risk Prediction and Treatment

Epidemiological data 20 years ago have described leukocytosis as an independent risk factor and predictor of future cardiovascular events.[16] Monocyte counts were also positively correlated with in-stent restenosis.[17] Sequential analysis of monocytes by flow cytometry over the last decade has further refined this knowledge and allowed a more detailed assessment of the prognostic value of each subset ([Table 1]).

Table 1 Correlation of different monocyte subsets to CAD and outcomes

Investigators	Subjects (n)	Condition	Target measure	Main outcome
Shantsila et al	245	STEMI	IMD	↑MACE
Abo-Aly et al	100	STEMI	IMD	↑MACE
Boidin et al	245	STEMI	IMD	↑MACE, ↓LVEF
Foster et al	18	STEMI	CD11c+ IMD	↑Troponin, ↑CK, ↑plaque necrosis
Yamamoto et al	50	Stable CAD	IMD	↓Fibrous cap thickness
Arnold et al	40	Stable CAD	IMD, NCM	↑CAD severity
Merinopoulos et al	30	Elective PCI	IMD	↑Chronic post-PCI
Rogacev et al	951	Elective PCI	IMD	↑MACE
Lo et al	588	Asymptomatic	IMD	↑Plaque score, ↑coronary stenosis
Markofski et al	45	Older adults	TLR+ CM, CD16+	↑Cardiometabolic risk

Abbreviations: CAD, coronary artery disease; CK, creatine kinase; CM, classical monocytes; IMD, intermediate monocytes; LVEF, left ventricular ejection fraction; MACE, major adverse cardiovascular events; NCM, nonclassical monocytes; PCI, percutaneous coronary intervention; STEMI, ST-elevation myocardial infarction; TLR, toll-like receptor.

Recently published data have shown that intermediate and nonclassical monocytes predict major adverse cardiovascular events and poor outcome in patients with ST-elevation myocardial infarction (STEMI).[18] [19] [20] Intermediate monocyte activation, as assessed by CD11c expression, was also positively correlated with peak levels of troponin and creatin kinase in patients with myocardial infarction (MI).[21] Beyond MI, CD14²⁺CD16⁺ monocyte counts were associated with coronary plaque vulnerability in a prospective study of patients with coronary artery disease (CAD).[22] The intermediate subset further predicts severe coronary stenosis in asymptomatic individuals as shown in another retrospective study.[23] Another study revealed a persistent increase in this subset after elective percutaneous coronary intervention as part of the residual inflammation.[24] Collectively, these data are consistent with the first prospective, large cohort study showing that intermediate monocytes are independent predictors of cardiovascular events in risk patients undergoing elective coronary angiography.[25] Furthermore, intermediate and nonclassical monocytes were positively correlated to CAD severity and older participants without manifested chronic disease but with high cardiometabolic risk had elevated percentages of only nonclassical monocytes.[26] [27]

In patients with stable CAD on statin treatment, high levels of PCSK9 associated with increased classical but decreased nonclassical monocytes while the intermediate subset did not differ.[28] The effects of statins on monocyte subsets are elusive and a prospective randomized trial is still missing. In diseases other than CAD, treatment with prednisolone in patients with immune thrombocytopenia reduced the number of intermediate monocytes and attenuated their pro-inflammatory phenotype.[29] Beta blockers also blunted the mobilization of nonclassical monocytes after acute exercise in healthy cyclist.[30] Further data in healthy donors have shown that classical monocytes generate more angiotensin and express higher levels of both angiotensin-converting enzyme type 1 (ACE1) and ACE2 than the other subsets.[31]

Finally, in healthy human volunteers under LPS-induced low-grade inflammation, intermediate and nonclassical monocytes showed the highest inflammatory response by their expression of IL-6 and IL-8.[32] Given the role of IL-6 and IL-8 in the pathogenesis of cardiovascular disease, this may have implications for risk prediction and potential new treatment options ([Table 2]).

Table 2 The magnitude of the effects of different treatments on monocyte subsets

Investigators	Subjects (n)	Condition	Treatment	Main outcome
Krychtiuk et al	69	Stable CAD	Statin	↑PCSK9, ↑CM, ↓NCM
Williams et al	11	Immune thrombocytopenia	Prednisolone	↓IMD
Graff et al	14	Exercise	Nadolol	↓NCM
Rutkowska-Zapała et al	4–12	Healthy donors	No	↑Ang II, ↑ACE1/2 in CM
Thaler et al	12	Low-grade inflammation	LPS	↑IL-6/8 in IMD and NCM

Abbreviations: ACE, angiotensin-converting enzyme; Ang, angiotensin; CAD, coronary artery disease; CM, classical monocytes; IL, interleukin; IMD, intermediate monocytes; LPS, lipopolysaccharide; NCM, nonclassical monocytes.

Concluding Remarks

Differential monitoring, especially of intermediate monocyte counts, appears to predict cardiovascular events and may also be of interest in identifying individuals with subclinical atherosclerosis. In addition, subset-tailored drug targeting could lead to the development of novel and more specific therapeutics. The effects of approved drugs such as glucocorticoids, statins, PCSK9- or ACE-inhibitors, and β blockers could also be re-evaluated in relation to monocyte subsets.

It is still essential to define reference values for monocyte subsets in the general population by means of a standardized flow cytometry protocol. Meanwhile, the common markers such as CD14, CD16, HLA-DR, and Ccr2 are available as dried, pre-aliquoted multicolor antibody cocktail and could be used routinely on simple flow cytometers equipped with two lasers. There is also consensus on the gating strategy and the enumeration of absolute counts for each subset.[2] However, the above reagents are currently licensed for research use only. It is therefore important to work toward having them certified for clinical diagnostic use. While standard instrument equipment and easy-to-use protocols are more useful for clinical routine, expensive, high-performance technology with enhanced resolution, such as multiparameter flow cytometry or even mass cytometry, seems essential and more appropriate for basic research.

Taken together, a comprehensive approach could integrate monocyte subsets, alone or in multiple panels, as novel reliable predictive and diagnostic biomarkers, but also as selective therapeutic targets in cardiovascular precision medicine.

Conflict of Interest

None declared.

The review process for this paper was fully handled by Gregory Y. H. Lip, Editor in Chief.

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Publication History

Received: 30 April 2024

Accepted: 18 June 2024

Accepted Manuscript online:
19 June 2024

Article published online:
06 July 2024