Shift of monocyte subsets along their continuum predicts cardiovascular outcomes

Highlights

• Some discrete monocyte subsets predict cardiovascular outcomes, but monocytes have now been redefined as a continuum.

• We tested whether shift of monocytes along their continuum provides incremental prognostic information.

• We found that shift of monocyte subsets along their continuum predicted adverse cardiovascular outcomes more than their frequency.

• These data illustrate how the concept of monocyte continuum can be used to model cardiovascular risk.

Abstract

Background and aims

Distribution of monocyte subsets has been shown to predict cardiovascular outcomes. However, monocytes form a continuum and categorization into discrete subsets may be an oversimplification. We herein aimed at establishing whether distribution of monocytes based on CD14 and CD16 fluorescence intensity provides incremental and complementary information on cardiovascular outcomes beyond enumeration of traditional subsets.

Methods

A cohort of 227 patients at high cardiovascular risk was characterized at baseline and followed for a median of 4 years. We quantified monocytes subsets by flow cytometry based on CD14 and CD16 expression and evaluated the continuous distribution of CD14 and CD16 fluorescence within each subset.

Results

A consistent shift toward higher CD16 fluorescence intensity within each monocyte subset was observed in patients with type 2 diabetes, despite no change in their frequencies. Patients with coronary artery disease (CAD) at baseline showed a doubling of CD14⁺⁺CD16⁺ intermediate monocytes and a shift of non-classical and classical monocytes towards intermediates ones. During follow-up, cardiovascular death or cardiovascular events occurred in 26 patients, who showed monocyte skewing similar to those of patients with baseline CAD. In fully adjusted Cox proportional hazard regression models, higher CD16 expression on classical monocytes, but not the level of intermediate monocytes or other subsets, independently predicted adverse cardiovascular outcomes.

Conclusions

Shift of monocyte subsets along the CD14/CD16 continuum, more than their frequencies, predicted adverse cardiovascular outcomes. This finding illustrates how the concept of monocyte continuum can be used to model the cardiovascular risk.

Introduction

Chronic inflammation is a hallmark feature of atherosclerosis and cardiovascular diseases. Traditional cardiovascular risk factors are associated with various changes in the activation state of innate immunity [1]. Monocyte-macrophages are key components of the innate immune system, and are involved in several pathologic processes linked to cardiovascular diseases, such as adipose tissue expansion [2], atherosclerotic plaque growth [3], and myocardial remodelling [4]. Notably, monocytes and macrophages display considerable heterogeneity, and can exert either protective or harmful effects in the cardiovascular system [5]. This is paradigmatically represented by the Yin/Yang distinction of macrophages into pro-inflammatory pro-atherosclerotic M1 versus anti-inflammatory anti-atherosclerotic M2 [6]. It is now clear that the M1/M2 polarization dichotomy is an oversimplification and that the spectrum of macrophage diversity is a continuum [7]. Although monocytes generate macrophages during inflammation, in steady-state conditions, tissue resident macrophages originate from local proliferation rather than differentiation of monocytes recruited from the circulation [8]. For this reason, monocytes and macrophages are today conceived as distinct entities.

Monocytes are traditionally distinguished in 3 different subsets based on expression of the LPS co-receptor CD14 and the scavenger receptor CD16 (FcγRIII), which are important for cellular function [9]. While the majority of monocytes in basal conditions express high levels of CD14 and low levels of CD16 (classical CD14⁺⁺CD16^neg/dim monocytes), inflammation triggers the appearance of monocytes expressing low levels of CD14 and high levels of CD16 (non-classical CD14⁺CD16⁺⁺ monocytes), possibly through an intermediate state (CD14⁺⁺CD16⁺) [10]. A kinetic study in humans has recently clarified that monocytes undergo sequential transition from the classical to intermediate and non-classical form in steady state and during inflammation [11].

Alterations in the frequency of monocyte subsets have been associated with vascular disease, such as unstable plaque features [12] and restenosis [13]. Remarkably, an expansion of intermediate monocytes has been associated with adverse cardiovascular outcomes [14]. Although intermediate monocytes display a distinct gene expression signature from classical and non-classical ones [15], new data on sequential transition now clearly suggest that monocytes form a continuum [12]. To model this concept, flow cytometry approaches have evolved to divide the monocyte population within the CD14/CD16 plot in a far higher number of subsets than traditionally done, confirming the existence of a continuous transition, rather than distinct subsets [16].

Our previous studies on pre-diabetes, diabetes, and hypercholesterolemia detected no change in traditional monocyte subsets defined by CD14/CD16 expression [17], [18], [19]. We herein hypothesize that identification of monocyte subsets based on discrete gating and fluorescence thresholds may be relatively insensitive to disease states. Single cell transcriptomic analyses support the idea that monocyte phenotypes represent a continuum, revealing significant heterogeneity within individual monocyte subsets [20], To account for this continuum, in addition to determining the frequency of monocyte subsets by traditional CD14 and CD16 discrete gates, in the present study, we also analysed the continuous distribution of CD14 and CD16 fluorescence intensity in the whole monocyte population and within each subset. Such analysis has been carried out in a cohort of patients with high cardiovascular risk to verify whether distribution of monocyte subsets along the CD14/CD16 continuum provides complementary information with respect to their frequencies, and is differentially associated with prevalent and incident cardiovascular disease. Intriguingly, we found that significant displacement of CD14/CD16 expression can occur even in the absence of changes in the frequency of monocyte subsets, and that distribution of CD16 expression predicts adverse cardiovascular outcomes more than the levels of intermediate monocytes.