Markers of inflammation and CD8+ T-cell activation, but not monocyte activation are associated with subclinical carotid artery disease in HIV

Abstract

OBJECTIVE

To explore the relationships between lymphocyte and monocyte activation, inflammation, and subclinical vascular disease among HIV-1 infected patients on antiretroviral therapy.

METHODS

Baseline mean common carotid artery (CCA) intima-media thickness (IMT) and carotid plaque (IMT >1.5cm) were evaluated in the first 60 subjects enrolled in the Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN in HIV (SATURN-HIV) trial. All subjects were adults, on stable ART with evidence of heightened T-cell activation (CD8+CD38+HLA-DR+ ≥19%) or increased inflammation (high sensitivity C-reactive protein ≥2mg/L). All had fasting LDL-cholesterol ≤130mg/dL.

RESULTS

78% were men and 65% African-American. Median (IQR) age and CD4+ count were 47(43,52) years and 648(511, 857) cells/µL, respectively. All had HIV-1 RNA<400 cps/mL. Mean CCA-IMT was correlated with log-transformed CD8+CD38+HLA-DR+% (r=0.326, p=0.043), interleukin-6 (r=0.283, p=0.028), soluble vascular cell adhesion molecule (sVCAM, r=0.434, p=0.004), tumor necrosis factor-α receptor-I (TNFR-I, r=0.591, p=<0.0001) and fibrinogen (r=0.257, p=0.047). After adjustment for traditional CVD risk factors, the association with TNFR-I (p=0.007) and fibrinogen (p=0.033) remained significant. Subjects with plaque (n=22, 37%) were older [51(7.7) vs. 43(9.4) years, mean(SD), p=0.002], had higher CD8+CD38+HLA-DR+% [31(24, 41) vs. 23(20,29)%, median(IQR), p=0.046] and higher sVCAM [737(159) vs. 592(160) ng/mL, p=0.008] compared to those without plaque. Pro-inflammatory monocyte subsets and serum markers of monocyte activation (soluble CD163 and soluble CD14) were not associated with CCA-IMT or plaque.

CONCLUSIONS

Participants in SATURN-HIV have a high level of inflammation and immune activation that is associated with subclinical vascular disease despite low serum LDL-C.

Introduction

Chronic HIV infection is associated with an elevated risk of atherosclerotic vascular disease, beginning with the early development of endothelial dysfunction and advancing more rapidly to sub-clinical atherosclerosis and in some cases life-threatening rupture of lipid-laden plaques(1, 2). The important drivers throughout this spectrum of vascular disease in HIV are complex and incompletely understood. Chronic inflammation, including lymphocyte and monocyte activation, may play an important role at each step.

Prior studies in HIV have separately associated sub-clinical atherosclerosis with serum markers of inflammation(3), generalized T-cell activation(4), cytomegalovirus-specific T-cell activation(5), and soluble CD163 (a marker of monocyte activation)(6); although the relative importance of these markers has not previously been compared in a virologically-suppressed population. Additionally, expression of the procoagulant tissue factor (TF) on the surface of monocytes is elevated in HIV-1 infected subjects compared to uninfected controls(7) and is highly prevalent in patients with acute coronary syndrome(8).

The primary aim of this study is to describe the cross-sectional associations of multiple markers of inflammation and immune activation with ultrasound measurements of carotid intima-media thickness and plaque among HIV-1 infected adults on stable antiretroviral therapy (ART).

Methods

Study design

This study is a cross-sectional analysis of the first 60 subjects enrolled in the Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN in HIV (SATURN-HIV) trial. SATURN-HIV is a randomized double-blind placebo-controlled trial designed to measure the effect of rosuvastatin 10mg daily on the progression of subclinical vascular disease. Enrollment began in March 2011.All subjects were ≥18 years of age, without diabetes or known coronary disease, and on stable ART with viral load <400 copies/mL. Additional entry criteria included either evidence of either heightened T-cell activation (CD8+CD38+HLA-DR+ ≥19%) or increased inflammation (high sensitivity C-reactive protein (hs-CRP ≥2mg/L), as well as LDL-cholesterol (LDL-C) ≤130mg/dL. Nineteen percent CD8+CD38+HLA-DR+ is the median level of CD8+ activation among patients successfully treated with ART and the 75th percentile of HIV-uninfected controls in our laboratory(9). Hs-CRP ≥2mg/L was the entry criterion of the JUPITER trial of rosuvastatin in HIV-uninfected adults(10). The study was approved by the Institutional Review Board of University Hospitals Case Medical Center (Cleveland, OH).

Study evaluations

At the initial screening visit, self-reported demographics and medical history were obtained along with a targeted physical exam. Blood was drawn after a 12-hour fast for glucose, lipoproteins, hs-CRP, and %CD8+ T cell activation. If enrollment criteria were met, subjects returned within 30 days for entry evaluations. At entry, a fasting blood draw was obtained for markers of inflammation. HIV-1 RNA level and CD4+ cell count were obtained as part of routine clinical care.

Soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble inter-cellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL6), and soluble tumor necrosis factor-α receptor 1 (sTNFR-I) were determined by quantitative sandwich ELISAs (R&D Systems, Minneapolis, MN). Inter-assay variability ranged from 4.76%–8.77%, 3.43%–7.37%, 2.02%–15.36%, and 3.66%–5.77%, respectively. Hs-CRP and fibrinogen were determined by particle enhanced immunonepholometric assays on a BNII nephelometer (Siemens). Inter-assay variability ranged from 3.01%–6.46% and 3.42%–7.59%, respectively. D-dimer was determined by immuno-turbidometric assay on a STA-R Coagulation Analyzer (Diagnostica Stago). Inter-assay variability ranged from 1.54%–9.03%. Serum levels of soluble CD14 and soluble CD163 were measured using Quantikine ELISA kits (R&D Systems Minneapolis MN).

Flow Cytometry

Monocytes and T-cells were identified by size, granularity, and by expression of CD14 or CD3 and CD8, respectively. Cell surface molecule expression was monitored by staining cells with the following fluorochrome-labeled antibodies: anti-Tissue Factor fluorescein isothiocyanate (FITC) (American Diagnostica, Stamford, CT), anti-CD14 Pacific Blue, anti-CD16 phycoerythrin (PE), (BD Pharmingen, San Diego, CA). In order to assure that monocyte populations were not contaminated by lymphocytes, preliminary experiments using an exclusion-gate that included anti-CD3 FITC, anti-CD20 FITC, anti-CD56 FITC (BD Pharmingen) were performed.

T-cell activation was measured using anti-CD38 PE, anti-HLA-DR FITC, anti-CD3 Peridinin-chlorophyll-protein Complex (PerCP), anti-CD8 PerCP (BD Biosciences), and appropriate isotype control monoclonal antibodies.

Whole blood samples were incubated for 15 minutes on ice with FACS Lyse buffer (BD Biosciences) and then washed in wash buffer (phosphate buffered saline with 1% bovine serum albumin and 0.1% sodium azide). Cells were then stained for 30 minutes in the dark on ice and then washed in wash buffer, fixed in 1% formaldehyde. Monocytes were analyzed using a Miltenyi MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and MACS Quant software (version 2.21031.1, Miltenyi Biotec). T-lymphocytes were analyzed using an LSR II flow cytometer (Becton-Dickinson, San Jose, CA) and FACSDiva software version 6.1.1 (BD Biosciences, San Diego, CA).

Carotid Artery Ultrasound

An ultrasound scan of the carotid arteries was performed using a Philips iU22 ultrasound system with a L9-3 MHz linear array transducer according to American Society of Echocardiography guidelines(11). R-wave gated still frame images of the distal 1cm of the CCA far wall were obtained at 3 separate angles bilaterally (anterior, lateral, and posterior). CCA-IMT was measured offline by a single reader (CTL) using semi-automated edge detection software (Medical Imaging Applications LLC, Coralville, IA). The mean-mean CCA-IMT was used for analysis. Carotid plaque was defined as IMT >1.5cm or > 50% thicker than the adjacent vessel.

Statistical Analysis

Pearson correlation was used to examine the association of CD8+ activation, monocyte subsets, markers of monocyte activation, and serum markers of inflammation with mean CCA-IMT. This study had 80% power to detect a correlation of 0.35. Multivariable linear regression models were then constructed to examine the association of each of these factors with CCA-IMT after adjustment for age, sex, smoking, family history of myocardial infarction (MI), body-mass index (BMI), systolic blood pressure, fasting serum glucose and LDL-C concentrations. In a post-hoc analysis of 3 biomarkers of interest (IL-6, CD8+CD38+HLA-DR+, and sVCAM-1), the relationship with CCA-IMT was examined in models that adjusted for age alone. The t-test was used to compare subjects with and without plaque. For the initial analyses, activated T-cells and monocyte subsets were expressed as a percentage of the total cell population. In a post-hoc analysis, all tests were repeated using absolute numbers of monocytes.

All statistical tests were two-sided with a 0.05 significance level. Analyses were performed with SAS version 9.2 (SAS Institute, Cary, NC).

Results

Median (IQR) age was 47(43,52) years. 78% were men and 65% African-American. Median BMI was 27(24,31) kg/m²LDL-C was 102(86,114) mg/dL, CD4+ count was 648(511, 857) cells/µL, and ART duration was 104(50, 149) months. 58% were current smokers. 35% had a family history of MI, 45% were on protease inhibitors. Twenty-nine (48%) participants screened in with an hs-CRP ≥2mg/L; 31(52%) participants screened in with CD8+ activation ≥19%. Twelve participants (20%) had both hs-CRP ≥2mg/L and CD8+ activation ≥19%. Median hs-CRP of all participants was 2.45(0.78,4.55) mg/L. Among the 39 total participants who were tested for T-cell activation, median CD8+CD38+HLA-DR+ was 27(21,35)%. Median CCA-IMT among all participants was 647(628, 732) µm.

The associations of traditional risk factors, HIV-related factors, markers of inflammation and immune activation with mean CCA-IMT are displayed in Table 1. A statistically significant correlation was observed between CD8+ T-cell activation and CCA-IMT; although, the association did not remain statistically significant after adjustment for traditional risk factors. Significant correlations were also observed in the expected direction with age, sex and family history of MI as well as several serum markers of inflammation (IL-6, sVCAM-1, sTNFR-I, and fibrinogen). Age, family history of MI, sTNFR-I, and fibrinogen remained independently associated with CCA-IMT after adjustment for traditional risk factors. In a post-hoc analysis of CD8+CD38+HLA-DR+, IL-6 and sVCAM-1, adjustment for age alone resulted in significant attenuation of the relationship between the 3 biomarkers and CCA-IMT (all p>0.15). Soluble CD14, soluble CD163, monocyte subsets and TF expression, and HIV-related factors were not associated with CCA-IMT in these analyses.

Table 1.

Associations of traditional risk factors, HIV-related factors, inflammation, and immune activation with mean common carotid artery intima-media thickness.

Traditional risk factors
	Pearson Correlation*		Multivariable linear regression§
	r	P-value	Adjusted P-value
Age	0.444	<0.001	0.003
Male sex	0.292	0.023	0.616
Current smoking	0.022	0.868	0.885
Body mass index	−0.284	0.028	0.302
Systolic blood pressure	0.044	0.738	0.651
Fasting glucose	−0.107	0.415	0.333
Low-density lipoprotein	−0.019	0.887	0.328
Family history of MI	0.260	0.049	0.013
HIV-related factors
Years since HIV diagnosis	0.299	0.061	0.851
Current CD4+ count	−0.164	0.214	0.826
Nadir CD4+ count	−0.038	0.785	0.795
Current PI use	0.130	0.323	0.781
Duration of PI use	0.061	0.749	0.645
Inflammation markers
Interleukin-6	0.283	0.028	0.411
sICAM-1	−0.027	0.836	0.904
D-Dimer	0.087	0.509	0.218
sVCAM-1	0.434	0.004	0.677
sTNFR-I	0.591	<0.001	0.007
Fibrinogen	0.257	0.047	0.033
hs-CRP	0.170	0.195	0.624
Lymphocyte activation
log CD8+CD38+HLA-DR+	0.326	0.043	0.657
Monocyte activation
log soluble CD14	0.163	0.214	0.631
log soluble CD163	0.132	0.315	0.485
CD14+CD16−	−0.063	0.647	0.829
CD14+CD16+	0.080	0.564	0.876
CD14dimCD16+	−0.025	0.858	0.813
CD14+CD16−TF+	−0.037	0.786	0.529
CD14+CD16+TF+	0.053	0.699	0.454
CD14dimCD16+TF+	0.050	0.716	0.833

^*Pearson correlation was used to test for univariable associations

^§Multivariable linear regression was used to test for associations after adjustment for all traditional risk factors listed in the table

r, correlation coefficient; MI, myocardial infarction; PI, protease inhibitor; sICAM-1, soluble intercellular adhesion molecule; sVCAM-1, soluble vascular cell adhesion molecule; sTNFR-I, tumor necrosis factor-á receptor I; hs-CRP, high-sensitivity C-reactive protein; TF, tissue factor

More than a third of the participants (22 of 60, 37%) had evidence of atherosclerotic plaque formation in one or more carotid vessel. Consistent with the CCA-IMT findings, participants with plaque had higher CD8+ activation but similar proportions of monocyte subsets and monocyte tissue factor expression (Figure 1). Those with plaque were also older [51(7.7) vs. 43(9.4) years, mean (SD), p=0.002] and had higher serum concentration of sVCAM [737(159) vs. 592(160) ng/mL, p=0.008] compared to those without plaque. No statistically significant differences in other serum markers of inflammation were observed. A repeated analysis using absolute numbers of each of the monocyte subsets confirmed a lack of association with either CCA-IMT or carotid plaque (data not shown).

Figure 1.

Immune activation in participants with plaque compared to those without plaque. A Proportions of T-cell activation as measured by %CD8+CD38+DR+. B Proportions of two pro-inflammatory monocyte subsets, CD14+CD16+ and CD14^dimCD16+. C Proportions of these two monocyte subsets that express tissue factor on the cell surface. Similar results were seen for a third monocyte subset, CD14+CD16-. T-tests were used to test for statistical significance.

Discussion

CD8+ T-cell activation and inflammation, but not monocyte activation or TF expression, appear to be associated with subclinical vascular disease in virologically suppressed HIV-1 infected patients with high levels of baseline inflammation and immune activation. The moderate associations with some biomarkers appear to be independent of traditional risk factors; although, this study may not have adequate power to show independent relationships with other markers of inflammation. Our findings confirm a previous study linking carotid IMT with inflammation(3) and are consistent with data from the Women’s Interagency HIV Study (WIHS) that associated CD8+ activation with carotid plaque in a mixed population of ART treated and untreated participants(4). In contrast to the WIHS cohort, we additionally describe an association between CD8+ activation and CCA-IMT and extend these findings to a mostly male population.

Although both carotid plaque and IMT provide independent predictive value for cardiovascular events(12, 13), each reflects a different pathophysiology and distinct stage of atherogenesis. IMT thickening may occur with vascular remodeling in the absence of atherosclerosis(14); however, there is evidence that IMT above a certain level more likely represents early atherosclerotic plaque(15). Our findings suggest that CD8+ activation and inflammation are linked to both vascular remodeling and plaque. This association is partly related to aging, although the relationship with sTNFR-I and fibrinogen appears to be independent of several traditional CVD risk factors including age.

The role of T-cells and the adaptive immune response in atherosclerosis has long been recognized. Although mouse models suggest CD4+ cells may be more atherogenic than CD8+ cells within plaques, circulating CD8+ cells may also promote atherosclerosis in response to activation by intracellular infections such as cytomegalovirus or Chlamydia pneumoniae(16). Kaplan et al have demonstrated that in HIV, CD8+ activation may be more predictive of carotid plaque(4) while CD4+ activation is more closely associated with carotid stiffness, another measure of subclinical vascular disease(17).

Blood monocytes and tissue macrophages are also well-described mediators of atherosclerosis. In humans, pro-inflammatory CD14+CD16+ monocytes produce unique cytokine profiles in response to Toll-like receptor ligands such as bacterial lipopolysaccharide (LPS), whereasCD14^dimCD16+ cells typically serve an anti-inflammatory function as they “patrol” the endothelium, but become pro-inflammatory in response to viral infections(18). Soluble markers of monocyte activation such as sCD14 and sCD163 have been linked to mortality(19) and coronary disease(6) in HIV. Surprisingly, neither proportions of inflammatory monocytes nor sCD14 or sCD163 were associated with CCA-IMT or plaque in our study. Monocyte TF expression also did not correlate with subclinical atherosclerosis, but may still be an important driver of cardiovascular risk and thrombotic plaque rupture. Our group has demonstrated that the profile of TF expression on monocyte subsets in HIV is similar to uninfected patients with acute coronary syndrome(20).

The small sample size is a notable limitation of our study. Specifically, in regards to monocyte activation, our study may not have adequate power to detect a small correlation of CIMT with sCD14 and sCD163; however, the nearly absent observed correlations between CIMT and the monocyte subsets are unlikely to significantly change with a larger sample size. As in all cross-sectional studies, we cannot prove causality or rule out residual confounding. These observations were made in a select population of virologically controlled patients on ART with favorable lipid profiles and may not be generalized to all patients with HIV infection. Finally, because of the number of statistical tests performed, there is a risk of type I error.

In conclusion, participants in SATURN-HIV have a high level of baseline inflammation and immune activation that is associated with subclinical vascular disease despite low serum LDL-C. We anticipate that longitudinal follow-up of this trial will help clarify the detrimental effects of chronic immune-activation and inflammation on vascular disease in this population.