Macrophage inflammatory markers are associated with subclinical carotid artery disease in women with human immunodeficiency virus or hepatitis C virus infection

Abstract

Objective: Infection with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) may be associated with atherosclerosis and vascular disease. Macrophages are a major component of atherosclerotic plaque, and classically activated (M1) macrophages contribute to plaque instability. Our goal was to identify plasma biomarkers that reflect macrophage inflammation and are associated with subclinical atherosclerosis.

Approach and results: We tested whether M1 macrophages produce galectin-3-binding protein in vitro. Then, we measured galectin-3-binding protein and the soluble macrophage biomarkers soluble cluster of differentiation (CD) 163 and soluble CD14 in 264 participants in the Women's Interagency HIV Study. Women were positive for HIV, HCV, both, or neither (66 in each group, matched for age, race/ethnicity, and smoking status). Carotid artery disease was assessed by ultrasound measurement of right distal common carotid artery intima-media thickness, distensibility, and presence of atherosclerotic lesions (intima-media thickness >1.5 mm). Plasma galectin-3-binding protein was higher in HCV+ than HCV- women (P<0.01) but did not differ by HIV status. The 3 inflammatory macrophage markers were significantly correlated with each other and negatively correlated with CD4+ counts in HIV-infected women. We defined a macrophage score as 1, 2, or 3 biomarkers elevated above the median. In models adjusted for traditional risk factors, higher macrophage scores were significantly associated with increased atherosclerotic lesions and lower carotid distensibility. Receiver-operator curve analysis of lesions revealed that the markers added predictive value beyond traditional risk factors and C-reactive protein.

Conclusions: The macrophage inflammatory markers galectin-3-binding protein, soluble CD163, and soluble CD14 are significantly associated with carotid artery disease in the setting of HIV and HCV infection.

Keywords: acquired immunodeficiency syndrome; atherosclerosis; immune system; risk factors; women.

Figure 1. Gal-3BP, CD14 and CD163 expression in human macrophages

Human blood monocytes (all subsets) were incubated with M-CSF (100 ng/ml) for 6 days to produce monocyte-derived macrophages (M0). These macrophages were incubated with interferon-γ for 1 day to produce M1 or with IL-4 to produce M2 macrophages and then challenged with LPS (10 ng/ml). (A) Level of IL-12p70 and IL-10 in the conditioned media were measured by Elisa (B) Gal-3BP mRNA expression was measured by quantitative RT-PCR, expressed relative to H18S. ** P=0.0056 by paired t test, n=3 donors. (C) Gal-3BP protein production was measured using ELISA (P=0.0008). (D) M1 polarized macrophages were treated with cell-permeable antisense (ASO) against LGALS3BP or control sequence (CTL) for 48hr, conditioned media harvested for immunoblotting with anti-Gal-3BP and anti-ERK2 (loading control) antibodies. (E) CD-14 and CD-163 determined by flow cytometry in M0, M1 and M2 polarized macrophages (representative of 3 healthy blood donors). (F) Human postmortem coronary arteries from patients with coronary artery disease were stained with antibodies against Gal-3BP (FITC, green), CD68 (Texas red), and nuclear stain (DAPI, blue) to assess expression of Gal-3BP in plaque macrophages. Lowest panel shows merge of Gal-3BP and CD68; right panels magnify boxed areas. Top: overview (scale bar = 500 μm), other scale bars = 100 μm

Figure 2. Spearman correlations comparing (A) Gal-3BP and sCD14, (B) sCD163 and sCD14, and (C) Gal-3BP and sCD163, across four HIV/HCV strata

White = HIV−/HCV−, red = HIV+/HCV−, blue = HIV−/HCV+, black = HIV+/HCV+. Line represents regression fit to observed data.