The distributions of metabolic parameters and pro-inflammatory and anti-inflammatory markers are in Table 1. Overall, a low number of patients had abnormal levels of high-density lipoprotein (HDL) (28.7%, <1.0 mmol/l), LDL (12.0%, >4.1 mmol/l), fasting glucose (1.3%, >6.1 mmol/l), insulin (17.3%, >10 mU/l), or homeostatic model assessment of insulin resistance score (10.7%, >3.0). Both treated and untreated HIV-infected patients had higher proportion with hs-CRP higher than 3.0 mg/l (43.2 and 38.7%, respectively) compared to HIV-negative controls (22.8%), after adjusting for age, diabetes, and hypertension. When further stratifying by median prevalent duration of known HIV infection (Supplementary Table 1, http://links.lww.com/QAD/A368), some significant differences were found in adjusted median triglycerides, LDL, interleukin-6, interleukin-18, HMW adiponectin, and interleukin-10. After adjusting for age, diabetes, and hypertension, waist-to-hip ratio and BMI were significantly correlated with insulin (r = 0.282, r = 0.454); QUICKI (r = −0.285, r = −0.437); total adiponectin (r = −0.316, r = −0.220); and HMW adiponectin (r = −0.257, r = −0.244) as expected.
Relation between carotid intima–media thickness, HIV infection, and antiretroviral therapy
c-IMT values were significantly different in treated HIV-infected patients compared to untreated HIV-infected and HIV-negative individuals after adjusting for age, hypertension, and diabetes (Table 2a). Treated HIV-infected patients were further stratified on protease inhibitor use using the same adjustments, while showing no statistical difference in c-IMT (P = 0.6). As the initial trend could conceivably be explained by the severity of HIV infection, we additionally adjusted for nadir CD4+ cell count and found no significant difference between treated and untreated HIV-infected groups (Table 2a).
However, in a post-hoc analysis, we observed higher c-IMT in patients with longer (0.760 ± 0.008 mm) versus shorter HIV infection (0.731 ± 0.008 mm) in the fully adjusted model (Table 2b), remaining so after additionally adding ART (0.759 ± 0.009 and 0.731 ± 0.009, respectively, P = 0.04). In Supplementary Table 2, http://links.lww.com/QAD/A368, we aimed to further clarify the impact of prevalent duration of known HIV infection: after stratifying HIV-infected groups on both treatment status and median prevalent duration of HIV infection (7.9 years), adjusted mean [±standard error (SE)] c-IMT tended to be thicker with longer duration, irrespective of ART.
We further examined the distribution of prevalent HIV-infection duration, which was then placed in (2–4), (4–8), (8–16), and (16–25) years intervals to ensure more comparable groups with similar HIV-duration. Adjusted mean c-IMT levels increased with prevalent HIV-infection duration in both treated and untreated patients (Fig. 1).
Relation between carotid intima–medial thickness and inflammatory profiles
Adjusting for age, diabetes and hypertension, patients with an anti-inflammatory score below the median had thicker c-IMT (0.754 ± 0.006 mm) than those above the median (0.722 ± 0.006 mm), irrespective of concomitant high or low pro-inflammatory scores (P < 0.001, Fig. 2a and b). This finding held when adding nadir CD4+ cell count to the model (P < 0.001) and while excluding the elite controller patient (P = 0.001). Furthermore, a significantly higher mean c-IMT level among patients with low versus high anti-inflammatory markers was still observed when adding prevalent duration of HIV infection (0.761 ± 0.008 versus 0.725 ± 0.008, respectively, P = 0.002), suggesting that the association between anti-inflammation and c-IMT is independent of HIV-infection duration.
Patterns of pro-inflammatory and anti-inflammatory profiles according to prevalent duration of HIV infection
As a group, the prevalence of pro-inflammatory scores above the median remained stable across the reconstructed duration of known HIV infection (Fig. 3a). Conversely, anti-inflammatory markers declined with longer infection duration (P < 0.001, Fig. 3b). Among these inflammatory markers, median-adjusted adiponectin and HMW adiponectin levels carried most of that decline in which respective decreases of 2.67–2.00 μg/ml and 1.29–1.06 μg/ml were observed between patients with 2–4 years of HIV infection versus more than 16 years (P for trend across HIV-duration in four groups < 0.001). Each pro-inflammatory and anti-inflammatory marker is summarized across groups of prevalent duration of HIV infection in Supplemental Figure 1, http://links.lww.com/QAD/A368.
In these carefully selected triads of never-smoking men, we report the persistence of an increased risk of carotid atherosclerosis in HIV-infected patients compared to their age-matched controls. Further, when comparing age-matched HIV-infected ART-naive individuals to individuals receiving ART, increased carotid atherosclerosis was related to prevalent duration of known HIV infection, irrespective of ART. Importantly, the increased carotid atherosclerosis was associated with a low anti-inflammatory profile, irrespective of pro-inflammatory markers values. In the reconstructed HIV history, the values of anti-inflammatory markers declined over time, providing further validation to our findings.
We opted to analyze the inflammatory markers as a whole and were careful to standardize them to allow a comparable contribution to the ratio. This approach is important as it avoids the risk of having one marker with a high numeric value overshadow another one with low, but increased, levels.
We designed this study in never-smokers because smoking remains the most important confounder of the reported increased vascular risk in HIV-infected individuals, among whom smoking is known to be prevalent. Notably, Petoumenos et al. showed that the risk of CVD events in HIV-infected patients decreased with increasing time since smoking cessation, a finding that could put to doubt the existence of a truly increased risk independent of smoking patterns among these patients. Our results determine that the increased risk of carotid atherosclerosis exists independent of smoking. When substituting our adjustment with the Framingham score, the difference remained (P = 0.09).
We carefully selected our two HIV-infected groups with the rationale that, as exposures, both HIV infection and ART were to be given sufficient opportunity to affect carotid atherosclerosis. Nevertheless, dissecting HIV-infection duration and ART duration has been particularly difficult because of their intricacy, aside from the difficulty of finding treatment-naive individuals with a sufficiently long known infection of duration, while not falling in the category of elite controllers.
Two factors are likely to contribute to the discrepancies across studies [19–22] relative to the contribution of infection versus ART toward carotid atherosclerosis in HIV-infected individuals. First, a number of studies did not adjust for nadir CD4+ cell counts . The importance of taking into account nadir CD4+ cell counts is evidenced by Ho et al. who studied 80 HIV-infected men treated with ART and with undetectable plasma RNA levels: after adjustment for cardiovascular risk factors, nadir CD4+ cell counts were independently associated with arterial stiffness. Similarly, Medina et al. report that both nadir and current CD4+ cell counts were significantly elevated with increasing coronary age in HIV-infected patients.
Discrepancies in results across studies are also likely due to the confounding role of smoking. This confounding role is not simply related to the higher prevalence of smoking among HIV-infected versus HIV-negative individuals, but also and significantly to the differential prevalence of smoking between treated and untreated groups among HIV-infected individuals. In a case–control study of 77 HIV-infected men (including 22 ART-naive) and 52 controls, van Vonderen et al. reported that patients exposed to ART had similar c-IMT compared with ART-naive patients. However, because so many of those ART-naive individuals smoked (77%) compared to those receiving ART (56 and 30%, without and with lipodystrophy, respectively, P < 0.05), one could not exclude that ART naive individuals had thicker IMT because of their higher smoking habits, even after statistical adjustment. Similarly, Mangili et al. found a strong association between c-IMT, hs-CRP, and all-cause mortality, which was similar irrespective of exposure to ART. Once again, smoking levels were significantly higher (71%) in those who died compared to those who did not (46%, P < 0.001) raising the possibility of residual bias even after statistical adjustment .
Hsue et al. have shown that increased atherosclerosis can occur in the absence of ART in HIV-infected patients. In their interesting study, c-IMT in controllers was not different than in HIV-infected untreated patients and higher than in seronegative controls , suggesting a role for chronic inflammation. However, 85% of the controllers in Hsue's study were former or current smokers compared to 53% of the controls. We provide the first evidence in never-smokers, thereby bolstering the validity of an independent association, rather than one mediated by smoking. Naturally, although the exclusion of smokers in our study limits generalizability, it provides validity.
Studies looking at the role of inflammation with regard to CVD in HIV-infected patients have largely focused on specific inflammatory markers, with, sometimes, conflicting results. Among pro-inflammatory markers, a report comprising the Strategies for Management of Anti-Retroviral Therapy (SMART), International Network for Strategic Initiatives in Global HIV Trials, Coronary Artery Development in Young Adults, and Multi-Ethnic Study of Atherosclerosis studies showed that hs-CRP, D-dimer, interleukin-6, and cystatin C levels are elevated in HIV-infected persons even with HIV-RNA suppressed . Kuller et al. found that interleukin-6 and D-dimer levels increased when ART was discontinuous and were related to all-cause mortality, raising the possibility that interrupting ART may further increase the risk of death by elevating these inflammatory makers levels [29,30]. However, Baker et al. subsequently reported that interleukin-6 and hs-CRP did not decline significantly after the introduction of ART. Further, it has been reported that both interleukin-6 and interleukin-18 levels are associated with an increased CVD risk in the general population, and that adipose tissue, including adipose tissue macrophages, produces resistin but also SAA associated with insulin resistance and systemic inflammation in the general population and in HIV-infected patients [32,33]. However, in the SMART study, SAA levels at study entry were not associated with an increased mortality rate, contrary to interleukin-6 and CRP . Moreover, these two markers increased in patients with CVD as compared to those without cardiovascular events.
Among anti-inflammatory markers, activation of the innate and acquired immune system also results in the release of immunosuppressive anti-inflammatory cytokines as interleukin-10 and interleukin-27 [34,35], which were reported to be inversely related to HIV-viral load but a relationship with cardiovascular risk was not previously investigated [36,37]. Adipose tissue also releases adiponectin with insulin-sensitizing and anti-inflammatory properties, whose levels are inversely related to viral load in HIV-infected patients . A relationship with subclinical cardiac damage and hypoadiponectinemia has been observed in HIV-infected patients .
Building on these previous works to guide our choice of markers, and conscious of the limitations of single marker studies and analyses, we surmised a priori that immune/inflammatory system imbalances might be a better barometer than isolated measures of systemic inflammation. Our finding of the increased risk of carotid atherosclerosis among individuals with low anti-inflammatory profile seems to validate that view and is consistent with reports that hypoadiponectinemia is a marker of vascular damage  and that individuals with lower adiponectin also had significantly lower HDL and higher triglycerides, for example . We can only speculate about the reasons why anti-inflammatory mediators decline with prevalent duration of HIV infection and the association with higher risk as determined by c-IMT. In response to injury/infection, a first wave of pro-inflammatory cytokine production is initiated and followed by a second wave of anti-inflammatory mediators such as interleukin-10, which play an important role in resolution of inflammation/injury and the maintenance of homeostasis. It is plausible that a small defect in anti-inflammatory response during the first years of HIV infection would sustain a pro-inflammatory state and be self-perpetuating with time, leading to progressive and sustained decline in anti-inflammatory response. The latter has been linked to alterations in vascular function, increased atherosclerosis and prevalence of high-risk lesions, particularly in association with ageing [40–42].
Additionally, HIV-specific immune response decreases during HIV infection . This phenomenon has been largely explained by exhausted HIV-specific T cells, which generally lose their polyfunctional capacity and are associated with decreased production of the pro-inflammatory marker interferon-γ and decreased responsiveness to interleukin-12, interleukin-18, and interleukin-21 [44,45]. The low pro-inflammatory and low anti-inflammatory profile, which had the highest c-IMT, could be the result of increased frequency of exhausted CD8 T cells in these patients. As data were not collected on these specific cellular functions, this hypothesis remains speculative. Nevertheless, our results suggest that the anti-inflammatory profile was relevant for c-IMT whereas the pro-inflammatory was not. This was unexpected and needs to be further explored prospectively; likewise, although the inflammatory markers were chosen a priori, we cannot naturally exclude that other pro-inflammatory/anti-inflammatory ratios might reveal different associations with c-IMT.
The magnitude of c-IMT differences reported here is in keeping with values considered clinically relevant in prior studies. A cross-sectional difference of 0.03 mm in c-IMT was associated with a 15-mmHg increase in SBP ; likewise, a cross-sectional difference of 0.04 mm in c-IMT was equivalent to a 10-year age difference in individuals without bulbar plaque . Thus, notwithstanding obvious design differences, our finding of 0.03–0.04 mm mean difference between both individuals with longer (versus shorter) HIV infection duration and individuals with low anti-inflammatory profile (versus high) seems to meet the threshold of clinically relevant differences.
This study shares with others the limitations of cross-sectional data; therefore, although we were able to reconstruct a historical cohort through the known duration of HIV infection and treatment, inferences on causality cannot be made. In addition, the duration of HIV infection was based on first positive serology and not initial infection. Treated patients may have presented with later stages of HIV diagnoses and thus longer cumulative duration of inflammation prior to treatment. We did adjust our models by nadir CD4+ cell count as a means to correct for differences in HIV-infection severity. Finally, some caution should be heeded when interpreting results stratified by HIV duration, as these were not the originally intended groupings and resulted from post-hoc analyses.
In summary, and to our knowledge, this is the first study reporting the preponderent association of HIV infection (rather than ART) to increased atherosclerosis in never-smokers, thus determining the validity of the relationship independent of this important confounder. We also report that a low level of anti-inflammatory/insulin-sensitizing profile (irrespective of the pro-inflammatory profile) seems associated with increased carotid atherosclerosis. Larger studies should incorporate and validate the import of inflammatory imbalance in the occurrence of atherosclerosis.
We are grateful to the patients. We acknowledge Ludivine Laurans, Lydie Houssou, and Marie-Christine Rincon for their respective technical assistance for quantifying inflammatory and metabolic markers; Manuela Sebire, Alina Ciuchete, Loïc Desquilbet, Nelly Desplanque, and Catherine Lupin for participant recruitment, management, and data collection; Dr Jürgen Tredup, Dr Pauline Campa, Dr Diane Bollens, Dr Bénédicte Lefebvre, Dr Marie-Caroline Meyohas, Dr Jérôme Pacanowski, Dr Zineb Ouazene, Dr Gilles Raguin, Dr Phillippe Tangre, Dr Laurent Fonquernie, and Dr Karine Lacombe for stellar patient recruitment; Dr David Jacobs for statistical discussions; Sandrine Couffin-Cadiergues and Maria Trumeau for administrative assistance.
This work was supported by the Agence Nationale de Recherche sur le Sida et les Hépatites (ANRS grant number 2007/303); and Sidaction (grant number AI 20). Moïse Desvarieux is also the recipient of a Chair in Chronic Disease from the École des Hautes Etudes en Santé Publique, France. This collaboration was rendered possible by a Contrat d’Interface (to M.D.) between Inserm and Hôpital Saint-Antoine. A.B. was awarded a postdoctoral fellowship from the ANRS.
Conflicts of interest
No authors reported conflicts of interest with the present study.
F.B. reports receiving research grants from Boehringer-Ingelheim and Gilead Sciences.
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antiretroviral; carotid intima–media thickness; HIV; infection duration; inflammatory imbalance; smoking
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