Introduction
Despite the success of antiretroviral therapy (ART) to inhibit viral replication, people living with HIV (PWH) suffer from accelerated co-morbidities and non-AIDS malignancies [1]. Indeed, in chronic HIV infection, intestinal mucosal dysfunction results in microbial translocation from the gut to the blood which, in turn, mediates generalized immune activation, cellular exhaustion, and chronic inflammation [1]. The resulting chronic inflammation in PWH puts them at risk of premature atherosclerosis and coronary artery disease (CAD) [2,3]. Atherosclerosis is a chronic inflammatory process involving the formation of atherosclerotic plaques within the arterial walls. Their progressive growth leads to narrowing of the lumen of the artery which impairs blood flow [2,3]. Plaques may as well rupture causing myocardial infarction (MI) or ischemic stroke [2,3]. Thus, understanding the immunoregulatory mechanisms involved in early steps of atherosclerosis development in PWH is of particular importance as it may improve cardiovascular disease prevention and intervention strategies.
Regulatory T cells (Tregs) are a subset of CD4+ T cells with various immunosuppressive and anti-inflammatory functions [4,5]. The transcription factor FoxP3 is the master regulator of Treg immunosuppressive functions [4,5]. Tregs can either originate within the thymus from their specific lineage (natural or thymic Tregs) or differentiate from circulating conventional FoxP3− CD4+ T cells into FoxP3+ Tregs during inflammation in the blood or peripheral tissues (induced Tregs) [5]. It is well documented that Tregs exhibit protective effects against cardiovascular disease via the secretion of anti-inflammatory cytokines IL-10 and TGF-β, while a decrease in Treg count is associated with the development and severity of atherosclerosis and MI [6–9]. Accordingly, unstable atherosclerotic plaques contain lower numbers of Tregs resulting in higher inflammation and vulnerability to rupture [8,9]. Furthermore, Tregs play regenerative roles in heart tissue repair, regeneration, and remodeling [10]. More recently, Tregs were shown to promote the regression of the atherosclerotic plaques by stimulation of anti-inflammatory macrophages via IL-10 production [11].
It is also well known that the purinergic metabolic pathway, which involves ectonucleotidases CD39/CD73, is also protective against cardiovascular diseases [12–15]. CD39 hydrolyzes extracellular pools of inflammatory ATP into ADP and AMP and then into immunosuppressive adenosine in concert with CD73 [5,16,17]. Importantly, the expression of CD39 and CD73 on Tregs plays an important role in their immunosuppressive functions [16]. Indeed, adenosine limits endothelial cell activation, monocyte recruitment, and leukocyte adhesion to the vascular endothelium. In addition to its anti-inflammatory properties, adenosine produced by CD39/CD73 can also limit endothelial activation, monocyte recruitment, and leukocyte adhesion to the vascular endothelium [18], while improving cardiac function and preventing vascular remodeling [14].
HIV infection is characterized by increases in frequency and activation of Tregs, that can suppress HIV-specific responses resulting in immune-dysfunction and disease progression [4,5]. Moreover, the expression of CD39 on Tregs and adenosine production is increased during HIV infection and associated with increased immune activation, lower CD4+ T-cell counts, and disease progression [19–21]. Importantly, the atheroprotective roles of Tregs and the CD39/CD73 pathway contrast with their increased expression and activity in HIV infection and the increased risk of atherosclerosis and CAD in PWH. Therefore, we assessed the dynamics of total Tregs and Tregs subsets in PWH with and without CAD compared with HIV-uninfected controls. Herein, we provide evidence of the impairments of Treg differentiation and their distinct migratory potential toward atheroma plaques, which may contribute to the increased prevalence of premature CAD in PWH.
Materials and methods
Study population
Peripheral blood mononuclear cells (PBMCs) were collected from 142 study participants from the cardiovascular imaging prospective substudy of the Canadian HIV and Aging Cohort Study (CHACS), including successfully ART-treated PWH with or without subclinical CAD (HIV+CAD+, n = 43; and HIV+CAD−, n = 41) and HIV-negative controls with or without subclinical CAD (HIV−CAD+, n = 31; HIV−CAD−, n = 27). Briefly, CHACS recruits PWH over the age of 40 or having lived with HIV for at least 15 years, as well as HIV-negative controls over the age of 40. Controls are recruited at the same clinics as the PWH participants [22]. Participants from the CHACS cohort who were free of overt cardiovascular disease (had never suffered a MI, coronary revascularization, angina, stroke of peripheral vascular revascularization) and had a 10-year Framingham risk score ranging from 5 to 20% were invited to participate in the cardiovascular imaging substudy. Data on all traditional cardiovascular risk factors was collected prospectively as part of the CHACS visit.
Cardiovascular imaging
In all participants, a 256-slice computed tomography (CT) scanner (Brilliance iCT, Philips Healthcare, Best, The Netherlands) was used to perform non-contrast cardiac CT and contrast-enhanced coronary CT angiography (CCTA). Prospective ECG – gating was used. Coronary artery calcium measurement was performed using Agatston method [23] on non-contrast CT. Coronary atherosclerotic plaque analysis was performed using CCTA images as described previously [24]. The coronary segments were defined as reported in the American College of Cardiology/American Heart Association guidelines for coronary angiography [25]. Plaque volumetric analysis was performed in multi-planar reformat, using the aforementioned semiautomated software. Any presence of plaque was classified as presence of subclinical CAD, while the absence of plaque was classified as absence of subclinical CAD. All imaging studies were performed at the Centre Hospitalier de l’Université de Montréal, QC, Canada, and interpreted by a board-certified cardiothoracic radiologist (CCL). All radiology personnel performing image interpretation and post-processing were blinded to HIV status.
Ethical considerations
The study was conducted in compliance with the principles included in the Declaration of Helsinki and received approval from the Institutional Review Boards of the CHUM-Research Centre (# CE 11.063). All study participants signed written informed consent.
Flow cytometry analysis
Multi-parameter flow cytometry was performed in batches on thawed PBMCs. Predetermined optimal concentrations of fluorochrome-conjugated antibodies were used for staining as detailed in Supplementary Table 1, https://links.lww.com/QAD/C19. Dead cells were excluded for the analysis using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Eugene, Oregon, USA). After extracellular staining, intracellular staining of FoxP3 and Helios was performed following permeabilization with the Transcription Factor Buffer Set (BD Bioscience, Franklin Lakes, New Jersey, USA). Flow cytometry acquisition was performed on a 3-laser BD LSR Fortessa X-20 cytometer and results were analyzed by FlowJo V10.2 (FlowJo, LLC, Ashland, Oregon, USA).
Statistical analysis
GraphPad Prism Software V6.01 (GraphPad Software, San Diego, California, USA) was used for statistical analyses. Results are presented throughout the text as median with interquartile range. One-way analysis of variance (ANOVA) or Kruskal–Wallis tests were performed first to determine whether there were significant differences between the four study groups. Differences among each two study groups were then determined by a t test of unpaired variables, Mann -Whitney U test, or a Fisher's exact test.
Results
Characteristics of the study populations
The demographic and clinical characteristics of the study populations are summarized in Table 1. In our study, all four groups were balanced for age and sex. There were no significant differences in time between HIV diagnosis and ART initiation between the two HIV-positive groups, nor for CD4+ Nadir, CD4+ and CD8+ T-cell counts, CD4+ : CD8+ ratio, and plasma viral load. In addition, no differences have been found in plasma levels of inflammatory soluble markers C-reactive protein (CRP) and d-dimer among the four study groups. As expected, HIV+CAD+ participants had the highest rates of tobacco consumption compared with other study groups and cytomegalovirus (CMV) infection was much more frequent among PWH than uninfected individuals. Importantly, the total volume of plaques and coronary low attenuation plaque volumes [26] were both greater in HIV+CAD+ vs. HIV−CAD+ individuals along with greater calcium scores in PWH, indicating the contribution of HIV infection to the establishment of CAD. In addition, the proportion of individuals treated with statins was also significantly higher in the HIV+CAD+ group. Accordingly, plasma levels of both HDL and LDL were lower in PWH compared with uninfected participants, while no differences were found in LDL : HDL ratio among the four study groups. As statins induce the generation of Tregs [27,28], the possible impact of statin treatment on Tregs and other immunological parameters was further considered and discussed in our study.
Table 1 -
Demographic and clinical characteristics of study groups.
|
Study population, n = 142 |
|
| Characteristics |
HIV+CAD+, n = 43 |
HIV+CAD−, n = 41 |
HIV−CAD+, n = 31 |
HIV−CAD−, n = 27 |
P values |
| Age (years) [Mean ± SD (range)] |
56 ± 6.7 (44–71) |
53 ± 6.8a (41–70) |
58 ± 8.4a (43–75) |
54 ± 6.4 (39–65) |
NS |
| Male, n (%) |
42 (97.7%) |
40 (97.6%) |
28 (90.3%) |
25 (92.6%) |
NS |
| Time since HIV-1 diagnosis (years) [mean ± SD (range)] |
19 ± 7.5b (4–31) |
15 ± 8.5b (2–33) |
NA |
NA |
NA |
| Time since start of ART (years) [mean ± SD (range)] |
15 ± 6.3b (0.2–24) |
11 ± 7.2b (0.2–25) |
NA |
NA |
NA |
| Antiretroviral regimen, n (%) |
|
|
NA |
NA |
NA |
| PI |
21 (48.8%) |
15 (36.6%) |
|
|
|
| INSTI |
8 (18.6%) |
7 (17.1%) |
|
|
|
| NNRTI |
2 (4.7%)b
|
12 (29.3%)b
|
|
|
|
| PI + NNRTI and/or INSTI |
10 (23.3%) |
5 (12.2%) |
|
|
|
| Unknown |
2 (4.7%) |
2 (4.9%) |
|
|
|
| CMV co-infection, n (%) |
35 (81.4%)c
,
d
|
32 (78.0%)a
,
e
|
6 (19.4%)a
,
c
,
f
|
0 (0.0%)d
,
e
,
f
|
P < 0.0001 |
| Statin treatment, n (%) |
24 (55.8%)b
,
c
,
d
|
8 (19.5%)b
|
7 (22.6%)c
|
3 (11.1%)d
|
P < 0.0001 |
| Tobacco consumption (pack-year) [mean ± SD (range)] |
18 ± 19b
,
c
,
d (0–99) |
6.6 ± 12b (0–42) |
7.5 ± 12c (0–50) |
3.6 ± 9.1d (0–38) |
P = 0.0002 |
| BMI (kg/m2) [mean ± SD (range)] |
24.8 ± 4.1d (19.0–34.5) |
26.3 ± 3.7 (21.0–35.5) |
26.9 ± 5.2 (18.6–44.0) |
26.8 ± 3.6d (19.6–35.6) |
NS |
| Framingham score [mean ± SD (range)] |
12 ± 7b (5–30) |
9 ± 4b (4–19) |
11 ± 4 (5–18) |
11 ± 5 (2–18) |
NS |
| Calcium score [mean ± SD (range)] |
380 ± 437b
,
d (0–1979) |
5 ± 18a
,
b (0–82) |
318 ± 547a
,
f (0–2165) |
0.3 ± 2d
,
f (0–9) |
P < 0.0001 |
| Diabetes, n (%) |
6 (14.0%)c
|
3 (7.3%) |
0 (0.0%)c
|
0 (0.0%) |
NS |
| Nadir CD4+ cell count (cells/μl) [mean ± SD (range)] |
215 ± 145 (10–576) |
186 ± 151 (10–750) |
NA |
NA |
NA |
| CD4+ T-cell count (cells/μl) [mean ± SD (range)] |
589 ± 244 (57–1386) |
501 ± 237 (9–980) |
NA |
NA |
NA |
| CD8+ T-cell count (cells/μl) [mean ± SD (range)] |
815 ± 315 (186–1485) |
690 ± 309 (6–1310) |
NA |
NA |
NA |
| CD4+ : CD8+ ratio [mean ± SD (range)] |
0.82 ± 0.40 (0.27–1.96) |
0.76 ± 0.38 (0.06–1.51) |
NA |
NA |
NA |
| VL (log10 copies/ml) [mean ± SD (range)] |
1.61 ± 0.07 (1.6–1.96) |
1.60 ± 0.02 (1.59–1.72) |
NA |
NA |
NA |
| CRP (mg/l) [mean ± SD (range)] |
7 ± 12.2 (3.2–82) |
6 ± 5.17 (0.7–29.5) |
5.2 ± 0.8 (5–8) |
8.1 ± 9 (5–32) |
NS |
|
D-Dimer (μg/l) [mean ± SD (range)] |
364 ± 290 (170–1970) |
334 ± 276 (169–1320) |
352 ± 136 (170–580) |
260 ± 57 (170–334) |
NS |
| Fibrinogen (mg/dl) [mean ± SD (range)] |
3.42 ± 1.0b (1.9–6.1) |
2.8 ± 0.6b (2.1–4.6) |
3.1 ± 0.7 (2.1–4.3) |
3 ± 0.7 (2.2–4.2) |
P = 0.0202 |
| Insulin (pmol/l) [mean ± SD (range)] |
96 ± 52 (21–205) |
101 ± 51 (29–212) |
77 ± 41 (14–170) |
73 ± 33 (29–157) |
NS |
| LDL (mmol/l) [mean ± SD (range)] |
2.7 ± 1.2d (0.8–8) |
3 ± 0.7e (1.5–4.6) |
3.1 ± 1.0 (1.7–6.9) |
3.4 ± 0.8d
,
e (1.6–4.8) |
NS |
| HDL (mmol/l) [mean ± SD (range)] |
1.2 ± 0.3d (0.6–2.2) |
1.2 ± 0.3e (0.7–2.3) |
1.3 ± 0.3 (0.9–2.0) |
1.4 ± 0.4d
,
e (0.7–2.9) |
P = 0.0406 |
| LDL : HDL ratio [mean ± SD (range)] |
2.3 ± 1 (0.7–6.6) |
2.5 ± 0.7 (1.0–4.2) |
2.3 ± 0.8 (1.1–5.1) |
2.6 ± 1.0 (0.8–4.6) |
NS |
| Total volume of plaque (mm3) [mean ± SD (range)] |
648 ± 528c (18–1981) |
0 |
391 ± 535c (32–2253) |
0 |
NA |
| Volume of low attenuation plaque (mm3) [mean ± SD (range)] |
200 ± 182 (5–694) |
0 |
120 ± 171 (4–632) |
0 |
NA |
Results are shown as mean ± SD and (range). P values come from the comparison of the four groups by using the ANOVA test. ANOVA, analysis of variance; ART, antiretroviral therapy; CAD, coronary artery disease; CMV, cytomegalovirus; CRP, C-reactive protein; INSTI, integrase strand transfer inhibitor; NA, not applicable; NNRTI, non-nucleoside reverse transcriptase inhibitors; PI, protease inhibitor; VL, viral load.Significant differences (P < 0.05) following t test or Fisher's test are mentioned as follow:
aHIV+CAD− vs. HIV−CAD+.
bHIV+CAD+ vs. HIV+CAD−.
cHIV+CAD+ vs. HIV−CAD+.
dHIV+CAD+ vs. HIV−CAD−.
eHIV+CAD− vs. HIV−CAD−.
fHIV−CAD+ vs. HIV−CAD−.
HIV+ coronary artery disease+ individuals are characterized by the highest frequencies of circulating regulatory T cells and thymic output, while their regulatory T cells are less differentiated
We first assessed total Treg frequencies (Fig. 1a) and observed significantly higher frequencies of CD4+CD25highCD127lowFoxP3+ Tregs in HIV+CAD+ individuals among all study groups (Fig. 1b). Moreover, Treg frequencies were higher in PWH compared with HIV-uninfected study participants (Fig. 1b). Significantly, we found heterogeneity in Treg differentiation in HIV+CAD+ individuals compared with the other study groups. Indeed, Tregs in the HIV+CAD+ group were characterized by the highest proportions of naïve and central memory subsets, and the lowest proportions of effector memory and terminally differentiated cells among study groups (Fig. 1c–f). Furthermore, low levels of terminally differentiated Tregs were found in HIV-infected individuals regardless of CAD status (Fig. 1f). Overall, our results showed that despite having higher Treg frequencies, these cells are less differentiated in HIV+CAD+ individuals.
;)
Fig. 1: Frequencies of total regulatory T cells and regulatory T-cell subsets among study groups.
To evaluate the ‘thymic’ vs. ‘induced’ origin of Treg expansion in HIV+CAD+ individuals, we assessed the expression of CD31, as a marker for recently emigrated thymic CD4+ T cells [29], and Helios, as a marker exclusively expressed by thymic Tregs [30,31] (Fig. 2a). The highest frequencies of Tregs recently migrated from the thymus (CD31+ Tregs) as well as thymic Helios+ Tregs and CD31+Helios+ Tregs were observed in HIV+CAD+, while no differences were present among other groups (Fig. 2b–d). In addition, higher frequencies of extra-tymic CD31−Helios− Tregs (induced Tregs) were also found in the HIV+CAD+ group (Fig. 2f). Overall, our results show an increase in Treg generation both within the thymus and extra-tymically. This significant increase in the thymic generation of Tregs could explain the enrichment of less differentiated Treg subsets in the HIV+CAD+ individuals.
Fig. 2: Differential thymic origins of regulatory T cells and expression of ectonucleotidases within study groups.
Lower expression of atheroprotective ectonucleotidases CD39/CD73 by regulatory T cells in HIV+ coronary artery disease+ individuals
Since CD39 and CD73 have atheroprotective [12,13] and cardioprotective functions [14,15], we evaluated their expression on Tregs (Fig. 2b). HIV+CAD+ and HIV−CAD+ individuals had lower frequencies of CD39+ Tregs and lower CD39−CD73− Tregs than HIV+CAD− individuals (Fig. 2g–j), while HIV+CAD+ individuals had the lowest frequencies of CD73+ and CD39+CD73+ among all study groups (Fig. 2h and i). These data suggest a decrease in Treg immunosuppressive/atheroprotective functions in HIV+CAD+ individuals that can contribute to an increase in atheroma plaque size and reduced plaque stability.
Distinct migratory potential of regulatory T cells in HIV+ coronary artery disease+ individuals
To assess whether HIV or CAD status are linked with Treg capacity to migrate towards the atheromatous plaques, we analyzed the expression of CCR6, which has been previously described as a marker of T-cell infiltration to inflammatory tissues [32], as well as CXCR3 and CCR4 which have been shown to promote the development of T-cell infiltration into the cardiac tissue during heart inflammation [33–36] (Fig. 3a). Regardless of CAD status, greater frequencies of memory CCR6+ and CXCR3+ Treg were observed in PWH compared to uninfected individuals (Fig. 3b and c). Significantly, the highest memory CCR4+ Treg frequencies were observed in HIV+CAD+ individuals among all study groups (Fig. 3d), suggesting their greater potential to migrate towards atheroma plaques. However, it is also known that CCR4 interaction with its ligand CCL17 interferes with Treg expansion and maintenance in plaques by promoting their apoptosis and contributing to atherosclerosis [37]. Altogether, Tregs in HIV+CAD+ individuals possess a distinct migratory capacity towards atherosclerotic plaques, which might impede their protective anti-inflammatory role.
Fig. 3: Regulatory T cells migratory capacities among study groups.
HIV+ coronary artery disease+ individuals have low levels of T-cell immune activation
We then evaluated the expression of human leukocyte antigen-DR isotype (HLA-DR) and CD38 to determine levels of immune activation, since HIV infection promotes persistent T-cell immune-activation [1] resulting in an increased risk of cardiovascular diseases [38]. The lowest frequencies of activated HLA-DR+ CD4 +and CD8+ T cells as well as CD38+ CD8+ T cells were observed in HIV+CAD+ individuals, while HIV−CAD− individuals showed the highest activated (HLA-DR+) T-cell frequencies among all study groups (Fig. 4a–e). However, using the combination of HLA-DR and CD38 to better determine immune activation status, we detected no differences in HLA-DR+CD38+ expression by T cells among the study groups (Fig. 4d and e). The lack of differences in HLA-DR+CD38+ co-expression is in line with plasma levels of immune activation markers such as CRP and D-dimer (Table 1).
Fig. 4: Immune activation.
Frequencies of regulatory T cells subsets were not affected by statin treatment or antiretroviral therapy regimen
Since statin treatment is known to induce Treg generation [27,28], we assessed if increases in total Treg frequencies are linked to a greater proportion of individuals under statin treatment in the HIV+CAD+ group. We thus excluded all of the statin-treated individuals from the analysis of all study groups. This additional analysis demonstrated that statin treatment did not affect the differences observed among study groups for the frequencies of total Tregs, Treg subsets (naive Tregs, central memory Tregs, effector memory Tregs, terminally differentiated Tregs), Tregs expressing CD39/CD73, Tregs expressing migration markers (CCR4, CXCR3, CCR6), or Tregs expressing origin markers (CD31/Helios) (Supplementary Table 2, https://links.lww.com/QAD/C19). In addition, we observed no changes in our results on the levels of T-cell immune activation after excluding statin-treated participants (Supplementary Table 2, https://links.lww.com/QAD/C19). In addition, since the ART regimens were different in CAD+ and CAD− groups (Table 1), we also assessed if there is a link between ART regimens and Treg subsets. We observed no changes in Treg subsets and immune activation except for the frequencies of CD4+HLA-DR+CD38+ cells (Supplementary Table 3, https://links.lww.com/QAD/C19). Overall, we confirm that neither statin treatment, nor ART regimen affect any of the Treg subsets evaluated in our current study.
Discussion
Tregs and ectonucleotidase expression and activity are known to be atheroprotective, while they are both upregulated during chronic HIV infection. Importantly, these protective functions clearly contrast with increased risk of atherosclerosis and CAD in PWH. Herein, we demonstrated for the first time that PWH with CAD are characterized by a distinctive Treg signature, independent of statins treatment. In these individuals, despite an increase in total Tregs frequencies, Tregs are less differentiated and express lower levels of atheroprotective CD39/CD73. Tregs also display a distinct migratory capacity towards the atherosclerotic plaques to exert their atheroprotective roles.
In line with previous reports [4,5], we also observed higher Tregs frequencies in PWH, but contrasting with the expected lower Tregs levels in patients with CAD, the highest frequency of circulating Tregs were found in PWH with CAD. However, we observed that Tregs of PWH with subclinical CAD were less differentiated than their counterparts in the other study groups, mostly dominated by naive and central memory Tregs. Moreover, the lowest frequencies of effector memory subsets were observed in HIV+CAD+ individuals. This is of particular interest since effector memory T cells migrate into non-lymphoid tissues in response to infection or inflammation [39] suggesting a lower Treg migratory potential toward inflammatory sites such as the atheroma plaque. In addition, the observed differentiation pattern of Tregs, notably higher naïve Treg frequencies in HIV+CAD+ group could indicate increased Treg generation and lower frequencies of antigen-experienced Tregs. In addition, the lowest levels of immune CD4+ and CD8+ T-cell immune activation (HLA-DR and CD38 expression, Fig. 4) as observed in PWH with CAD is in line with the highest total and central memory Treg frequencies in these individuals. Similarly, inverse correlations between Treg frequencies and T-cell immune activation has been previously reported by our team and others [17,19–21]. Of note, in contrast to HLA-DR expression, we observed the lowest CD38 expression by CD8+ T cells in HIV+CAD+ individuals. In PWH, the expression of CD38 by T cells is linked to CD4+ T cells counts and viral load [40,41]. In our study, HIV-infected individuals are long-term ART-treated for an average of 13 years with CD4+ T-cell counts greater than 500 cells/μl, which could explain the normalized CD38 expression by CD4+ T cells in HIV+ individuals. Importantly, it has been previously shown in ART-treated PWH that lower brachial artery flow-mediated dilation was associated with a higher percentage of activated CD8+ T cells but not with activated CD4+ T cells [42].
To better evaluate the origin of Treg expansion in PWH with CAD, we assessed the expression of CD31 as a marker of Tregs recent migration from the thymus, and Helios, a specific marker of thymic Tregs [29–31]. We showed that HIV+CAD+ individuals have the highest frequencies of CD31+ Tregs, Helios+ Tregs, and recently migrated thymic Tregs CD31+Helios+ Tregs. These results, along with increased naïve Tregs frequencies demonstrate highly active Treg generation and output from the thymus. Particularly, our results are in contrast with previous reports on thymic output and CD31+ Tregs frequencies in HIV− uninfected individuals with CAD. Indeed, in patients with both CAD and heart failure, higher susceptibility of recent thymic emigrant CD31+ Tregs to apoptosis has been associated with significant decreases in Treg frequencies and their thymic output (CD31 expression), as well as impairment in Treg immunoregulatory functions [43,44]. Higher susceptibility of CD31+ Treg to apoptosis could partially explain the lower atheroprotective functions of Tregs in CAD+ PWH in our study. In addition, lower expression of FoxP3 and Helios is also observed in patients with hypertension [45]. Our data showing an increase in both recent thymic emigrants and thymic Tregs in PWH with CAD suggests that HIV infection contributes to a higher thymic output of these cells. Accordingly, increased generation of thymic Tregs could explain the higher Treg frequencies observed and their less differentiated stage in PWH with CAD. In addition to an increase in thymic Treg generation, we also observed higher frequencies of extra-thymic CD31−Helios− Tregs in the HIV+CAD+ individuals among study groups. However, the lack of Helios expression has been linked to lower stability and immunoregulatory functions of Tregs [46].
The discordance between higher frequencies of circulating Tregs and the presence of CAD suggest that Tregs in HIV+CAD+ individuals might be either dysfunctional and/or have less migratory potential to the atheroma plaques to exert their immunosuppressive functions. We thus evaluated the expression of atheroprotective ectonucleotidases CD39 and CD73. Our group and others have previously reported that there is an increase in immunosuppressive CD39+ Tregs during HIV infection which is associated with immune dysfunction and HIV disease progression [17,19–21]. Moreover, by limiting tissue inflammation, platelet activation, and recruitment [47,48], in addition to the inhibition of endothelial activation and leukocyte recruitment [49], CD39 and CD73 have shown important cardioprotective and atheroprotective functions. Here, we demonstrated that HIV+CAD+ individuals have lower frequencies of both CD39+ and CD73+ Tregs. In addition, CD39 is also a well known marker of T-cell activation [50]. Thus, lower frequencies of CD39+ Tregs in individuals with CAD+, regardless of their HIV status, indicates lower Treg activation and immunosuppressive functions in CAD. Overall, our results suggest that despite higher Tregs frequencies in CAD+ PWH, at least part of the immunoregulatory functions of Tregs is altered in these individuals, which potentially contributes to the increase in atheroma plaque burden in our cohort (Table 1) and potentially, to decreased plaque stability. Accordingly, impairments in cytokine secretion and immunosuppressive functions of circulating Tregs were already reported in individuals with acute coronary syndrome [51].
It has been demonstrated that atheromatous plaques are more vulnerable to rupture when they contain low numbers of Tregs [8] and, more recently, that Tregs can induce regression of atherosclerotic plaques [11]. We thus assessed if there is an impaired migration capacity within our study groups via their expression of CCR6 as a homing marker to inflammatory tissues [32] as well as CCR4 and CXCR3 as markers of migration towards atherosclerotic lesions and heart inflammation [33–36]. Increased frequencies of both memory CXCR3+ and memory CCR6+ Tregs were observed in PWH vs. HIV-uninfected individuals regardless of CAD. Memory CXCR3+ Tregs are important since this population is known to be protective in inflammation [52]. Significantly, we observed significantly higher frequencies of memory CCR4+ Tregs in HIV+CAD+ individuals, which could increase Treg migration to atheroma plaques, where these cells could lose their stability and suppressive functions upon interaction with the chemokine CCL17 and ultimately decrease in their numbers within the plaques [37]. Conversely, blockage of CCL17 with a specific antibody has resulted in Tregs expansion and reduced atheroprogression [37]. Accordingly, CCR4-deficient mice are prone to have prolonged graft survival in a chronic cardiac transplant rejection model [35]. Overall, our data suggest that in PWH with CAD, Tregs may not only have impaired atheroprotective functions, but they might also have a distinctive migratory capacity within atherosclerotic plaques to exert their protective anti-inflammatory properties.
Statins are the most common drugs used as prevention against atherosclerosis. Importantly, statin treatment reduces inflammation, immune activation [53] and increases the frequencies, stability and functional capacity of Tregs [27,28]. Therefore, we assessed whether higher levels of circulating Tregs and the low levels of immune activation in PWH with CAD are associated with higher statin use within this group. After excluding statin-treated individuals from all analyses, we detected no change in our results, indicating an insignificant effect of statin in all our analyzed markers. Currently, guidelines about statin use for HIV-infected individuals remain flexible in regards to the necessity to initiate statin treatment among individuals at low or medium cardiovascular risk [54], and no conclusive studies on the effect of statins in the HIV-infected population have been published. Moreover, in HIV-infected individuals, the efficacy/effects of statin treatment could vary according to ART regimens and type of statin used [55]. The ongoing REPRIEVE trial is currently assessing the efficacy of statins for cardiovascular primary prevention in PWH [56].
Overall, we demonstrated here a peculiar and distinctive dynamics of Tregs subsets in PWH with or without subclinical CAD compared with HIV-uninfected controls. Our results contribute to a better understanding of Tregs dynamics in PWH and their contribution to accelerated CAD in these individuals. These findings pointed to Tregs as potential targets for immunotherapeutic approaches to decrease the incidence of CAD in PWH.
Acknowledgements
First and foremost, the authors thank all study participants for their time and significant dedication to this study. The authors also thank Stephanie Matte for nursing services, Annie Chamberland for coordination, and Mohamed Sylla for specimen banking.
Author contributions: M.-A.J. designed the study; C.R., A.Y., T.S., O.F. performed the experiments. C.H.-L., Ch.T., C.T., M.D. provided access to specimens and clinical data from the Canadian HIV and Aging Cohort Study; C.R., A.Y., M.-A.J., C.C.-L., M.E.-F., C.T.C., M.D. analyzed, discussed, and interpreted results throughout the study. A.Y. and M.-A.J. wrote the article. All authors contributed to the refinement of the study protocol and reviewed and approved the final article.
The current study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S) to M.-A.J. This study was also supported by CIHR-funded Team Grant # HAL-157985 to and HIV Clinical Trial Network (CTN 272 study) to C.C.-L., M.E.-F., Ch.T., C.T., M.D. and M.-A.J. A.Y. is supported by a FRQ-S doctoral scholarship. C.T.C. and M.D. hold FRQ-S Junior 1 and Junior 2 Clinician-researcher salary awards, respectively. M.A.-J. holds the CIHR Canada Research Chair tier 2 in Immuno-virology.
Conflicts of interest
The authors declare that there is no conflict of interest regarding the publication of this article.
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