The CCR2+ Monocyte Subsets Increase in Obese Boys but Not Girls with Abnormally High Carotid Intima-Media Thickness: A Pilot Study

Abstract

The differential contribution of monocyte subsets expressing the C-C chemokine receptor 2 (CCR2) to subclinical atherosclerosis in girls and boys is unclear. In this pilot study, we compared classical, intermediate, and nonclassical monocyte subsets expressing CCR2 in 33 obese children of both sexes aged 8 to 16 divided by carotid intima-media thickness (IMT), considering values above the 75th percentile (p75) as abnormally high IMT. Obesity was defined as body mass index above the 95th percentile according to age and sex. Flow cytometry analyses revealed that boys but not girls with IMT ≥ p75 displayed increased CCR2+ cell percentage and CCR2 expression in the three monocyte subsets, compared to boys with IMT < p75. The CCR2+ cell percentage and CCR2 expression in the three monocyte subsets significantly correlated with increased IMT and insulin resistance in boys but not girls, where the CCR2+ nonclassical monocyte percentage had the strongest associations (r = 0.73 and r = 0.72, respectively). The role of CCR2+ monocyte subpopulations in identifying an abnormally high IMT shows a marked sexual dimorphism, where boys seem to be at higher subclinical atherosclerosis risk than girls.

Keywords: CCR2; children; insulin resistance; intima-media thickness; nonclassical monocytes; obesity.

Figure 1

Gating Strategy. We first gated total leukocytes on a time/side scatter density plot and selected the Zombie UV negative cell population for detecting living cells. Next, we gated living cells for singlets on a forward scatter/trigger pulse width density plot. After recognizing cells by size and granularity, we selected monocytes on the HLA-DR gate. Then, we gated monocytes using the rectangular gating strategy on the CD14⁺/CD16⁺ cell population to identify CD14⁺⁺CD16⁻ cells as classical monocytes, CD14⁺⁺CD16⁺ cells as intermediate monocytes, and CD14⁺CD16⁺ cells as nonclassical monocytes (Figure 1). We obtained the median fluorescence intensity (MFI) for CCR2 by considering both positive and negative cell populations. We got the CCR2⁺ cell percentage using fluorescence minus one (FMO) control. For each fluorochrome, we used compensation controls through UltraComp eBeads^TM (Invitrogen^TM, Carlsbad, CA, USA). We analyzed data by the FlowJo 10.0.7 software (TreeStar, Inc., Ashland, OR, USA).

Figure 2

Evaluation of (A) the proportion of CCR2 in classical monocytes and intima media thickness (IMT) and (B) expression of CCR2 in classical monocytes and intima media thickness (IMT) by sex. We considered a high-risk IMT ≥ p75 and compared differences by the student T-test, considering a p value < 0.05 as significant.

Figure 3

Evaluation of (A) the proportion of CCR2 in intermediate monocytes and intima media thickness (IMT) and (B) expression of CCR2 in intermediate monocytes and intima media thickness (IMT) by sex. We considered a high-risk IMT ≥ p75 and compared differences by the student T-test, considering a p value < 0.05 as significant.

Figure 4

Evaluation of (A) the proportion of CCR2 in nonclassical monocytes and intima media thickness (IMT) and (B) expression of CCR2 in nonclassical monocytes and intima media thickness (IMT) by sex. We considered a high-risk IMT ≥ p75 and compared differences by the student T-test, considering a p value < 0.05 as significant.