Recent studies suggest that patients with HIV are at high risk for cardiovascular events [1–3]. The mechanisms underlying such risk remain unclear. In patients without HIV, inflammation and T-cell activation are important components of the development of atherosclerotic lesions . T cells are present in atherosclerotic lesions and, when activated, produce cytokines which lead to inflammation and promote atherosclerosis . Consistent with these observations, chronic inflammatory infections such as cytomegalovirus (CMV) and Chlamydiae pneumoniae have been implicated in the pathogenesis of atherosclerosis in HIV-uninfected patients [5,6]. This is particularly true in the setting of chronically immunosuppressed heart transplant recipients, where infection with CMV has been associated with cardiac allograft vasculopathy, and where treatment with anti-CMV therapy has been associated with a reduced risk of atherosclerosis-associated complications [7,8].
HIV infection results in chronic inflammation, as measured by the levels of T-cell, B-cell and macrophage/monocyte activation. This increased level of inflammation occurs in the early stages of disease, before the onset of immunodeficiency [9–12]. HIV infection can also have profound effects on T-cell production and turnover. This phenomenon, which appears to be linked to the effect of HIV on immune activation , directly contributes to the progressive CD4 T-cell depletion, perhaps as a consequence of increased rates of T-cell maturation and death and/or decreased rates of de novo T-cell production [14–16].
The mechanisms whereby HIV causes high levels of immune activation remain unknown but are likely multifactorial. Direct activation of T cells through presentation of HIV peptides by antigen presenting cells (APC) may play a role [13,17]. However, over half of CD8 T cells can be activated in untreated HIV infection  and most of these are not HIV-specific . Bystander activation, in which the proinflammatory nature of HIV infection drives polyclonal activation and proliferation of CD4 and CD8 T cells, and homeostatic proliferation may also contribute to the chronic high levels of T-cell activation. Finally, it is likely that some activated CD4 and CD8 T cells are specifically activated upon interactions with microbial agents such as CMV, which may be more common in HIV-infected patients. Still, the increased prevalence of certain infections within the HIV-infected population does not fully account for high levels of inflammation. For example, elevated CMV-specific T-cell responses occurs in the HIV-infected population even in the absence of occult CMV replication and/or CMV-associated disease .
The purpose of this study was to investigate the relationship between measures of chronic T-cell activation and atherosclerosis in HIV-infected subjects. Our primary hypothesis was that carotid intima-media thickness (IMT) would be higher in HIV-infected subjects compared to HIV-uninfected subjects, and that this difference would be independently associated with higher levels of T-cell activation and/or higher CMV-specific T-cell responses. We also investigated the role of high-sensitivity C-reactive protein (hs-CRP), a marker which has previously been associated with atherosclerotic risk in HIV-uninfected individuals .
This is a cross-sectional study of HIV-infected and uninfected adults. HIV-infected subjects were recruited from an ongoing clinic-based cohort based at the University of California (SCOPE). From this cohort, we recruited individuals who were either off antiretroviral therapy for at least 1 year or on a stable antiretroviral regimen for at least 1 year. We excluded individuals who had experienced an acute infection or vaccination within the prior 8 weeks or who were receiving ongoing treatment with an immune-based therapy such as interferon-α. Control subjects were selected mainly from among subjects answering advertisements to participate in clinical studies. All control subjects underwent HIV antibody testing and were documented as HIV antibody negative before enrollment in the study. Twenty-five (26.8%) of the HIV patients and nine (24%) of the controls were involved in a prior study from our group  but all of the measurements of carotid IMT, T-cell activation, CMV-specific T-cell responses, and hs-CRP for all patients were measured at a different time point than the previous study.
Eligible individuals underwent an extensive interview regarding prior cardiovascular risk factors. CMV-specific T-cell immunity, T-cell activation, and hs-CRP were measured at this visit. Carotid IMT measurements were obtained within 4 months of the immunophenotyping analysis.
The University of California, San Francisco Committee on Human Research approved the study and all subjects provided written informed consent.
Assessment of inflammatory markers and cardiac risk factors
Each study participant underwent an in-depth assessment including a detailed interview and structured questionnaire covering HIV disease history (for the HIV infected subjects), medication exposure, and cardiac risk factors including family history of coronary artery disease, and health-related behaviors. Blood was drawn in the fasting state to measure total and high density lipoprotein (HDL)-cholesterol, triglycerides, and glucose. Low density lipoprotein (LDL)-cholesterol was calculated using the Friedewald formula except in subjects with hypertriglyceridemia, where it was measured directly. hs-CRP was measured using the CardioPhase hs-CRP assay . HIV RNA levels were measured using branched chain DNA method (Quantiplex HIV RNA, Chiron Version 3.0: Chiron Corporation, Emeryville, California, USA). Antibodies (IgG) against CMV were measured using enzyme immunoassay (Quest Diagnostics, Nichols Institute, San Juan Capistrano, California, USA).
Carotid IMT measurements
Carotid B-mode ultrasound recordings were obtained from each subject using the standardized protocol of the Atherosclerosis Risk in Communities Study [22–24]. The right and left carotid arteries were examined with the head in the mid-line position, tilted slightly upward. A 10-MHz linear array probe was used with the GE VividFive Imaging System. The probe was manipulated so that the near and far walls were parallel to it and lumen diameter was maximized in the longitudinal plane. Landmarks were identified and high-resolution, high frame-rate images were recorded digitally in a cineloop format for subsequent measurement without data degradation.
Carotid IMT was measured in 12 predefined segments (6 per side) by one highly experienced vascular technician blinded to the subject's clinical features, including HIV status. No values were imputed for missing segments. One technician did all recordings and all measurements in this study, so as to maximize reproducibility. Measurement reproducibility in our laboratory has been described previously and is greater than 0.9 . A carotid plaque was considered to be present when carotid IMT was > 1.5 mm at any site.
Flow cytometry studies
T cell activation was quantitated as the median number of CD38 antibodies bound per CD4+ and CD8+ T cell using the QuantiBRITE method . Whole blood was stained with PE-conjugated at a ratio of 1:1 with anti-CD38, PerCP-conjugated anti-CD3, APC-conjugated anti-CD4, and FITC-conjugated anti-CD8 (Becton Dickinson Immunocytometry Systems (BDIS), San Jose, California). Following staining, blood was lysed with FACS Lyse (BDIS) and analyzed on a FACSCalibur flow cytometer (BDIS),. The median number of anti-CD38 antibodies bound per CD4+ or CD8+ T cell was then extrapolated by comparing each sample's median relative fluorescence intensity (RFI) to a standard curve of median RFI's from QuantiBRITEPE (BDIS: BD Biosciences, San Jose, California, USA) beads of known PE concentration using the calibration platform in FlowJo software (Tree Star, Oregon).
CMV-specific T cell responses were determined by measuring the proportion of CD4+ and CD8+ T cells that expressed IFN-γ after exposure to CMV pp65 peptides using flow cytometry as previously described [27,28]. The pp65 peptides were a gift from BDIS. A pool of 15 amino acid peptides with 11 amino acid overlaps corresponding to the CMV pp65 protein was used in these studies. Heparin-anticoagulated fresh whole blood was stimulated with this pp65-peptide pool in the presence of brefeldin A. Non-stimulated and superantigen staphylococcal enterotoxin B (SEB)-stimulated blood samples were used as negative and positive controls, respectively. Subsequently, cells were fixed and permeabilized with FACS Lyse and FACS Perm (BDIS) before incubation with FITC-conjugated anti-IFN-γ, PE–conjugated anti-CD69, –PerCPCy5.5 conjugated anti-CD4, and APC conjugated anti-CD3. The fraction of activated, cytokine-secreting (CD69+IFN-γ+) CD4+ and CD8+ T lymphocytes was determined by analysis on a FACSCalibur flow cytometer (BDIS). To reduce the number of false positive responses, only cells that stained brightly for IFN-γ were included for analysis . These “IFN-γ bright” events were defined as CD69+ IFN-γ+ events that fell 3 decades above the IFN-γ-negative population in non-stimulated controls. For each participant, the percentage of CMV-specific CD4+ or CD8+ T cells was defined by the percentage of “IFN-γ-bright” events observed using the pp65-stimulated blood minus the percentage of “IFN-γ-bright” events observed using the participant's non-stimulated blood.
Factors associated with carotid IMT were assessed using unadjusted and adjusted linear regression models. Carotid IMT, hs-CRP, T-cell activation levels, and the percentage of CMV-specific CD8 T cells were log-transformed to meet model assumptions, and effect sizes were expressed on a relative scale for interpretability . Traditional cardiac risk factors (age, gender, diabetes, hypertension, hyperlipidemia, pack-years of cigarette smoking, and family history of coronary artery disease) were considered as potential confounders for all multivariable analyses. For models restricted to HIV-infected participants, duration of HIV infection, years of protease inhibitor use, and CD4 T-cell count nadir were also considered as potential confounders. For final model selection, we removed potential confounding factors from the model; removal of these factors changed the effect size for the primary predictor by less than 10%. Except where noted, comparisons between groups were made with non-parametric tests (i.e., Spearman correlation coefficient; Wilcoxon rank sum tests). A Fisher's exact test was used to compare dichotomous variables between groups.
The clinical features of the 93 HIV-infected subjects and 37 HIV-uninfected controls are listed in Table 1. The mean age of the HIV infected subjects was 48 years; 85 (91%) were male. Most subjects (62%) were Caucasian, 25% were African–American, and 8% were Hispanic. All but 6 of the 93 HIV infected subjects were receiving antiretroviral therapy. The mean duration of HIV infection was 13 ± 4 years and the median CD4 T-cell count was 354 cells/μl. Most (57%) had undetectable HIV RNA levels. Among the subset receiving protease inhibitor therapy (88%), the mean duration of therapy with these drugs was 4 ± 2 years. Compared to controls, HIV-infected subjects were more likely to smoke and have hypertension (P = 0.03 in each case). The HIV-infected subjects also had lower HDL-cholesterol levels (P < 0.01), lower LDL-cholesterol levels (P = 0.01) and higher triglyceride levels (P < 0.01). Ninety-nine percent of the HIV-infected subjects were CMV seropositive as compared to 57% of the controls (P < 0.001). Eight of the 93 HIV patients had a clinical history of CMV disease.
Association between HIV serostatus and carotid IMT
The median carotid IMT in the HIV-infected and uninfected subjects was 0.95 mm and 0.68 mm, respectively (P < 0.001, Fig. 1a). These differences are consistent with prior observations from our group using a different cohort of individuals . Among the HIV-infected subjects, hypertension and age were associated with higher baseline carotid IMT while antiretroviral therapy was not associated with higher carotid IMT. Sixty (65%) of the HIV-infected individuals had plaques compared to 10 (27%) of the HIV-uninfected controls (P < 0.001). The difference between the HIV-infected and uninfected subjects remained significant after adjustment for traditional cardiovascular risk factors (age, gender, diabetes, cigarette smoking, hyperlipidemia, hypertension, and family history of coronary artery disease). After adjusting for these factors, the HIV-infected subjects had a mean 27% greater IMT than HIV-uninfected controls (P < 0.001).
Hs-CRP, T-cell activation, and CMV-specific immunity in HIV-infected versus -uninfected subjects
Hs-CRP values of the HIV-infected subjects ranged from 0.2 to 34 mg/l. The median baseline hs-CRP was 1.1 mg/l [interquartile range (IQR), 0.5–4.6 mg/l), which was higher than that observed in the uninfected controls (0.8 mg/l, P = 0.05) as shown in Fig. 1b. Among the HIV-infected subjects, neither traditional cardiovascular risk factors (cigarette smoking, hypertension, diabetes mellitus, HDL-cholesterol, LDL-and family history of coronary artery disease) nor HIV disease specific factors (viral load, CD4 T-cell nadir, current CD4 T-cell count, and antiretroviral therapy use) were significantly associated with hs-CRP levels.
CD8 T-cell activation (as measured by CD38 expression) was significantly higher in HIV-infected subjects compared to the uninfected controls (745 versus 282 anti-CD38 antibodies bound per CD8 T cell, P < 0.0001, Fig. 1c). Similar differences were observed with CD4 T-cell activation (1002 versus 640 anti-CD38 antibodies bound per CD4 T cell, P = 0.001, Fig. 1c).
CMV-specific CD8 T-cell responses (as measured by the proportion of cells expressing IFN-γ after stimulation with pp65 peptides) were significantly higher in HIV-infected subjects compared to the uninfected controls (median values of 2.09% and 0.59%, P = 0.002; Fig. 1d). CMV-specific CD4 T-cell responses exhibited the same trends (median values of 0.49% and 0.15% in infected and uninfected subsets, respectively, P = 0.02). This difference in CMV-specific T-cell responses was also observed when we limited our analysis to those subjects who were CMV seropositive (P = 0.02 for CMV-specific CD4 T-cell responses in HIV-infected versus uninfected subjects, and P = 0.003 for CMV-specific CD8 T-cell responses in HIV-infected versus uninfected subjects). Within the HIV group, CD4 nadir was not associated with higher CMV-specific T-cell responses (rho, −0.01, P = 0.88).
Association of hs-CRP, T-cell activation, and CMV-specific immunity with carotid IMT
Having established that measures of inflammation and IMT were higher in the HIV-infected group compared to the uninfected group, we next addressed the question of whether any of these measures were independently associated with IMT (Table 2). T-cell activation was not associated with carotid IMT (rho, 0.009, P = 0.92 for CD8 T-cell activation and rho, −0.05, P = 0.61 for CD4 T-cell activation). In contrast, hs-CRP was associated with carotid IMT; for every twofold increase in hs-CRP, IMT increased by 4% (P = 0.01). However, after adjusting for all traditional risk factors, the association between hs-CRP and IMT was no longer significant (P = 0.09).
There was also a consistent association between the percentage of CMV-specific CD8 T cells and carotid IMT. For every 10-fold increase in the percentage of CMV-specific CD8 T cells, there was a 14% increase in IMT (P < 0.001) (Table 2, Fig. 2). Even after adjustment for age and other traditional cardiac risk factors, each 10-fold increase in the percentage of CMV-specific CD8 T cells was associated with a mean 9% increase in carotid IMT (P < 0.001). Similar trends were observed in the relationship between CMV-specific CD4 T cells and IMT (Spearman's rho, 0.37; P < 0.001). A significant association between CMV-specific CD8 T-cell responses and IMT was found among HIV patients (n = 78, Spearman's rho, 0.31; P = 0.007) and also among uninfected controls (n = 37, Spearman's rho, 0.33; P = 0.047).
Because higher hs-CRP levels and CMV-specific T-cell responses were associated with both HIV infection and IMT, we next determined whether hs-CRP or CMV-specific CD8 T-cell responses might mediate the association between HIV infection and IMT. After adjustment for traditional cardiac risk factors and hs-CRP levels, HIV-infected subjects continued to have a mean 21% greater IMT compared to uninfected controls (P = 0.001) (Table 3). However, after adjusting for the percentage of CD8 CMV-specific T-cell responses in addition to hs-CRP and traditional cardiac risk factors, there was only a non-significant trend between HIV infection and carotid IMT (P = 0.079), suggesting that differences in CD8 CMV-specific T-cell responses might mediate the higher carotid IMT observed in the HIV-infected subjects. CD8 CMV-specific T-cell responses remained independently associated with higher carotid IMT in this model (P = 0.025).
In this study of HIV-infected and uninfected subjects who were not selected based on any prior history of cardiovascular disease, we observed that HIV-infected subjects had significantly higher carotid IMT measurements than HIV-uninfected controls. This effect remained highly significant after controlling for all known cardiovascular risk factors, confirming prior observations from our group  and others [30–34]. In addition, HIV-infected subjects had significantly higher levels of hs-CRP, T-cell activation, and CMV-specific CD4 and CD8 T-cell responses. After controlling for all other traditional risk factors, CMV-specific T-cell responses were strongly and consistently associated with IMT. Importantly, after controlling for CMV-specific immune responses, carotid IMT measurements were no longer higher in HIV subjects compared to controls. Taken together, these findings suggest that the accelerated atherosclerosis in HIV disease may be mediated by an increased inflammatory response that is directed against CMV.
There is a growing body of literature indicating that HIV-infected subjects are at increased risk for developing accelerated atherosclerosis. For example, we have previously reported that HIV-infected subjects have higher carotid IMT measurements compared to age-matched HIV-negative controls, and that these carotid IMT measurements progress rapidly during follow-up . Similar observations have been made by other groups [30–34], although at least one cross-sectional study failed to confirm these findings . Consistent with these observations, HIV-infected subjects appear to have a higher risk of coronary events than HIV-uninfected adults . To what degree this is related to antiretroviral treatment directly or to the disease itself has not been fully resolved [2,3,36]. However, a large randomized clinical study of continuous antiretroviral therapy versus intermittent antiretroviral therapy was discontinued early due to an excessive number of clinical events in the intermittent arm. Preliminary data suggests that interruption of therapy was associated with a significant increase in the risk of cardiovascular events . Whether this increase in events is due to HIV-related factors or treatment-related factors remains unknown and will require additional investigation.
HIV may have an independent role in accelerating atherosclerosis, which is supported by both clinical and pathologic observations. HIV-infected subjects with acute coronary syndromes are generally younger than HIV-uninfected individuals and their post-angioplasty restenosis rates are higher [38,39]. Also, the pathology of HIV-associated coronary lesions is often distinct from that observed in uninfected individuals .
The mechanism for this increased risk of atherosclerosis in setting of HIV infection has not been fully elucidated. Since HIV infection is associated with early and marked increases in measures of T-cell activation, and since chronic inflammation is associated with accelerated atherosclerosis , we reasoned that inflammation may account for the higher IMT levels observed in these subjects. In our study, we found that hs-CRP levels (a measure of inflammation) were higher in HIV-infected individuals compared to uninfected controls. However, these levels were not consistently associated with IMT after adjusting for other factors. Whether hs-CRP in HIV subjects will be predictive of future cardiac events is not known and would need to be addressed in a larger prospective study. In contrast to our initial hypothesis, we found no association between the level of activated CD4 or CD8 T cells and carotid IMT.
Role of CMV infection in atherosclerosis
The presence of chronic infections has been postulated as a risk factor for both chronic high-level inflammation and for accelerated atherosclerosis. This is particularly true for CMV infection, which has been associated with atherosclerosis in immunosuppressed patients. CMV-seropositive cardiac transplant recipients have higher rates of atherosclerosis and worse survival rates than transplant recipients who are CMV seronegative . Evidence linking CMV infection and transplant-associated atherosclerosis has also been described both epidemiologically  and in animal models . Treatment of cardiac transplant patients with the anti-CMV drug, ganciclovir, prevents the development of transplant-associated atherosclerosis [7,8]. Finally, in uninfected patients, CMV seropositivity in combination with an elevated hs-CRP is an independent predictor of mortality in patients with coronary artery disease . These data, in aggregate, suggest that CMV infection may directly or indirectly result in accelerated atherosclerosis.
HIV infection appears to have an important impact on the magnitude of the immune response against CMV. In particular, as shown here and as previously observed in other studies, circulating CMV-specific T-cell responses are markedly elevated in CMV-seropositive HIV-infected subjects compared to CMV-seropositive HIV-uninfected controls [13,45–48]. Since both inflammation and CMV have been implicated in atherosclerosis in immunosuppressed subjects, and since HIV-infected subjects are commonly infected with CMV, we hypothesized that enhanced T-cell responses against CMV may contribute to high IMT in the setting of HIV disease. In our current study, we observed that IFN-γ production in response to CMV peptides was indeed strongly and consistently associated with high IMT. Notably, this association was stronger than that observed for hs-CRP and remained significant after controlling for all other risk factors. The association between CMV-specific inflammation and IMT found in our study may be secondary to immune reconstitution as opposed to a direct etiologic role of CMV.
Several other observations provide indirect support for our finding that CMV-associated inflammation contributes to accelerated atherosclerosis in HIV disease. The diseased coronary arteries of HIV-infected subjects show pathologic similarities to transplant vasculopathy lesions; both develop rapidly and are circumferential as opposed to sectorial . Also, CMV has been independently associated with an increased risk of restenosis after rotational atherectomy  and our group has reported very rapid rates of restenosis in the setting of HIV infection . The role of CMV in the development of restenosis after angioplasty in HIV infection deserves further investigation (as well as the role of drug eluting stents in this process).
The primary limitation of our study is its cross-sectional design, with the measurements of inflammation and IMT taken at a single point in time. Carotid IMT reflects the cumulative effects of risk factors acting over many years, whereas hs-CRP, T-cell activation, and CMV-specific T-cell responses were measured only once and may not reflect prior levels. However, our group and others have reported that levels of both T-cell activation and CMV-specific T-cell responses tend to remain in a ‘steady-state’ when measured longitudinally [11,47]. If so, then single measurements may be reflective of the level of inflammation during prolonged periods of time in our patients. Still, variations in hs-CRP, T-cell activation and CMV-specific immunity over time might be expected to weaken the relationship of these measurements to carotid IMT, and would likely bias this study toward the null hypothesis. The association between CMV-specific inflammation and IMT found in our study may be secondary to a generalized immune reconstitution-associated inflammatory syndrome as opposed to a direct etiologic role of CMV, although the absence of an association between T-cell activation and IMT argues against this finding being due to a generalized process. Nonetheless, future studies will be needed to investigate more thoroughly the longitudinal relationship between CMV-associated inflammation and subsequent risk of coronary disease. Such studies are ongoing at our center, but will likely take several years to complete. This study may have lacked the power to detect smaller correlations between IMT and T cell activation, CMV-specific T-cell responses, and hs-CRP.
Additional studies are also needed to determine whether CMV-associated T-cell responses and hs-CRP are associated with a higher risk of coronary events in HIV-infected subjects. If confirmed, our study would support the earlier use of antiretroviral treatment, given the consistent observation that therapy in general reduces HIV-associated inflammation. It would also prompt studies aimed at determining if anti-CMV treatment might be a useful adjunct for HIV-infected subjects at high risk for atherosclerosis-related complications. Of note, similar approaches have been considered in the setting of transplant vasculopathy [7,8]. Although antibiotic treatment of bacterial infections does not appear to alter the cardiac event rate among individuals with coronary artery disease , treatment of CMV infection in HIV-infected subjects may still be beneficial.
P.Y. Hsue is a recipient of a Clinical Scientist Development Award from the Doris Duke Charitable Foundation, a grant from the NIH (K23 AI066885), and a Beginning Grant-in-Aid from the American Heart Association. The work was also supported in part by grants from the NIH (AI47062 to JMM, AI052745 to SGD), the California AIDS Research Center (CC99-SF-001 to SGD), and the UCSF/Gladstone Institute of Virology & Immunology Center for AIDS Research (P30 MH 59037). The study was performed in the General Clinical Research Center (GCRC) at San Francisco General Hospital (5-MOI-RR00083). JMM is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and the NIH Director's Pioneer Award Program, part of the NIH Roadmap for Medical Research, through grant number DPI OD00329.
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