Vitamin D is known for its essential role in the binding of calcium by the body and in bone health. Severe vitamin D deficiency, usually defined as a serum level of 25-hydroxyvitamin D3 [25(OH)VD3] below 12 ng/ml , has also been associated with a variety of conditions such as cardiovascular risk, insulin resistance and diabetes, various cancers, and overall mortality in vulnerable populations (e.g. elderly or individuals at high risk of coronary disease). Vitamin D deficiency is also very common in patients infected with HIV and may play a deleterious role during HIV infection [2,3]. More specifically, there seems to be a link between severe vitamin D deficiency and the clinical course of patients with HIV. Vitamin D levels have been shown to be positively associated with quantitative immune reconstitution under therapy, and antiretroviral therapy (ART) treated patients with vitamin D deficiency have an increased risk of death from any cause and AIDS-defining events .
Increasing evidence supports a link between vitamin D deficiency and a proinflammatory state characterized by elevated blood markers of inflammation in the general population  and in HIV-infected individuals [6,7]. However, the consequence of vitamin D deficiency on immune parameters remains poorly characterized. Several studies suggest that vitamin D has an immunomodulatory role, influencing diverse subpopulations of hematopoietic cells such as monocytes and macrophoges, dendritic cells, and B and T cells . Vitamin D deficiency could therefore affect the status of the immune system in HIV-infected patients. Our objective here was to study the potential impact of vitamin D deficiency on phenotypic alterations of lymphocytes in HIV-infected patients with severe vitamin D deficiency, and to investigate the effect of vitamin D supplementation on these markers. In particular, we focused our study on the expression of CD38 on memory CD8+ T cells, which is a robust and commonly used marker of systemic immune activation in HIV infection, and a well established correlate of disease progression .
Materials and methods
Patients were recruited from three hospitals in Paris (Hôtel-Dieu, Cochin and Pitié-Salpêtrière). Inclusion criteria were HIV-1 infection, being on ART for at least 3 years, with plasma HIV RNA below 20 copies/ml for at least 1 year, with CD4+ T-cell counts at least 350 cells/μl, and aged between 40 and 55 years old. Efavirenz-treated individuals were not included, as treatment with this molecule has been associated with lower levels of vitamin D [3,10]. Moreover, only individuals with phototype I to IV (i.e. pale white to light brown skins) were included to avoid a potential bias on vitamin D levels because of dark brown and black skins. Patients presenting evidence of hepatitis C virus (HCV) coinfection were also excluded to avoid a confounding effect of HCV replication on immune activation levels . Patients were selected to enter two groups: deficit in or normal levels of vitamin D (i.e. 25(OH)VD3 < 12 ng/ml or ≥30 ng/ml, respectively). Serum 25(OH)VD3 levels were measured using the chemiluminescent Liaison XL 25(OH)VD3 assay (Diasorin, Saluggia, Italy). Although preidentification of potential study individuals was based on serum 25(OH)VD3 levels evaluated during previous routine visits, an assessment of vitamin D levels was always performed at the same time as blood cell collection, and the final selection of patients studied was based on a posthoc analysis. Peripheral blood mononuclear cells (PBMCs) were purified on Ficoll density gradient (Eurobio, Courtaboeuf, France) and frozen before used for analysis. The study was approved by the local institutional ethics committee and written informed consents were obtained from the patients.
Lymphocyte subset analysis by flow cytometry
Immunophenotyping of PBMCs was performed using directly conjugated monoclonal antibodies, obtained from commercial sources as follows: anti-CD4-peridinin-chlorophyll-cyanin 5.5 (PerCP5.5), anti-CCR7-phycoerythrin-cyanin 7 (PeCy7), anti-CD38-allophycocyanin (APC), anti-Ki67-FITC, anti-CD27-phycoerythrin, anti-CD19-V450, anti-CD21-APC (BD Biosciences, San Diego, California, USA), anti-CD27-Alexa700, anti-CD57-pacific-blue (Ozyme, Saint-Quentin-en-Yvelines, France), anti-CD8-APC-eFluor780 (eBiosciences, San Diego, California, USA), anti-CD45RA-ECD, anti-IgD-FITC, anti-CD38-PeCy7 (Beckman Coulter, Miami, Florida, USA) and anti-IgM-Alexa700 (Clinisciences, Nanterre, France). CD4+ or CD8+ naive, central memory, or senescent T lymphocytes were defined as CD45RA+ CCR7+ CD27+, CD45RA− CCR7+ CD27+, or CD57+ cells, respectively. Naive or memory B cells were defined as IgM+ IgD+ CD38+ CD21+ CD27−, or CD38− CD21+ CD27+, respectively. Plasmoblasts were defined as IgM− IgD− CD38++ CD21− CD27++.
Differences between groups were compared using the non-parametric unpaired Mann–Whitney U or paired Wilcoxon tests. The correlations between variables were analyzed by Spearman's rank test using Prism 5 software (GraphPad, La Jolla, California, USA). P values of less than 0.05 were considered statistically significant.
Patient follow-up and analysis
To test the existence of an association between vitamin D deficiency and immune parameters, we implemented both a cross-sectional study and a longitudinal study. For the cross-sectional study, we compared cellular immunological markers of patients presenting a deficit in vitamin D versus those with normal levels. Fifty-three patients fulfilling the inclusion criteria were included (38 men and 15 women), with 23 patients presenting a deficit in vitamin D (serum 25(OH)VD3 <12 ng/ml) and 30 displaying normal levels (serum 25(OH)VD3 ≥30 ng/ml). Vitamin D low and high patient groups were matched for CD4+ T-cell counts and nadir, age and sex (Table 1). Supplementation with vitamin D was proposed to the patients with severe 25(OH)VD3 deficiency and consisted in the administration of 100.000 IU every 14 days for 3 months (without calcium supplementation), followed by 100.000 IU monthly for an additional 9 months. We therefore took the opportunity to follow longitudinally the effect of vitamin D supplementation on immunological markers as patients returned for their medical checkup between 6 and 12 months after the initiation of vitamin D supplementation. Sixteen out of the 17 patients enrolled in this follow-up study presented significantly increased vitamin D levels after the initiation of vitamin D supplementation (Fig. 1a).
No impact of vitamin D levels on lymphocyte subset distribution
Evidence indicates that vitamin D can directly affect both T and B lymphocytes, in particular the proliferative capacity and differentiation of naive and memory T cells [8,12]. Moreover, vitamin D levels have been inversely associated with leukocyte telomere length [13,14]. We therefore investigated the potential influence of vitamin D levels on the changes in lymphocyte subset distribution and counts that usually characterize treated HIV-1-infected patients (e.g. decrease in naive or central memory T cells, and increase in senescent memory T cells) . The percentages and absolute counts of CD4+ and CD8+ T-cell and B-cell subsets (see Materials and Methods section) were analyzed. No differences between both vitamin D high and low groups with respect to these parameters were observed (Table 1). Moreover, vitamin D supplementation had no influence on the differentiation phenotype of either T-cell or B-cell subsets (data not shown). These data rule out a primary role of vitamin D in the altered lymphocyte distribution that characterizes HIV-1-infected patients.
Decreased immune activation upon vitamin D supplementation
Of note, we observed that patients with low vitamin D levels displayed a reduced CD4/CD8 ratio (P = 0.03, Table 1), which is usually a sign of increased immune activation in HIV-infected patients receiving effective ART . We also analyzed CD38 expression on the surface of memory CD8+ T cells as a robust marker of immune activation. Although there was substantial interindividual variability, patients with severe vitamin D deficiency displayed modest but statistically significant higher levels of CD38 expression, compared with individuals with normal vitamin D (P = 0.04, Table 1). We thus monitored CD4/CD8 ratio, as well as cellular activation (i.e. CD38 expression) and cell cycling (i.e. Ki67 expression) of memory CD8+ T cells in vitamin D supplemented individuals. These patients presented a higher CD4/CD8 ratio post-compared with pre-supplementation (Fig. 1b). Moreover, vitamin D increase upon supplementation was associated with a reduction in cellular immune activation levels (Fig. 1c). We also found that serum levels of vitamin D and the expression of the cellular immune activation markers were inversely correlated in this longitudinal setting (Fig. 1d). Vitamin D status appears therefore to impact on cellular immune activation levels.
HIV-1-infected patients on long-term suppressive ART still retain significantly higher immune activation levels compared with uninfected controls. This represents an important issue as persistent immune activation has been associated with increased mortality and both AIDS and non-AIDS-defining illnesses in these patients . Reducing persistent immune activation levels in treated patients is thus a priority. Several approaches, including antiretroviral intensification (e.g. with raltegravir or maraviroc), treatment of coinfections (e.g. cytomegalovirus or HCV) and use of pharmacological (e.g. chloroquine or statins) or biological (e.g. probiotics) agents are currently investigated to this end . In the present study, we show that increasing vitamin D levels in deficient patients owing to supplementation results in a reduction of immune activation levels even in patients with a sustain control of plasma HIV-RNA on ART. In a recent study, no effect of vitamin D supplementation on T-cell activation was observed, thus contrasting with our data . However, patients included in that work had a significantly higher vitamin D at baseline (close to 30 ng/ml on average) compared with our study (less than 12 ng/ml for inclusion), and vitamin D supplementation in these individuals resulted in a markedly lower increase in 25(OH)VD3 levels (mean increase of 5 ng/ml, compared with 30 ng/ml in our study). Our findings support an important role for vitamin D in HIV pathogenesis, and provide a rational for the benefits of vitamin D supplementation to reduce immune activation levels in vitamin D-deficient patients. However, further evaluations in larger randomized clinical trials are needed to assess the potential of vitamin D supplementation to lessen the rate of associated comorbidities in HIV-1-infected patients.
As vitamin D can affect the functional attributes and gene expression of hematopoietic cells , supplementation in vitamin D might restore some of the cellular functional abnormalities in HIV-infected patients. The basis for the present benefit of vitamin D may also be found in its capacity to promote FOXP3 expression in CD4+ T cells , and induce regulatory T-cell populations . Induced regulatory T cells able to suppress immune activity may eventually influence immune activation levels. Although further investigation is needed, vitamin D could also regulate immune activation levels through its ability to induce autophagy , which has a central role for cell survival and the regulation of inflammatory mechanisms. Vitamin D is capable of inducing autophagy in monocytes/macrophages and in cancer cells leading to the elimination of mycobateria and cell death, respectively. Recently, vitamin D-mediated autophagic response has even been shown to inhibit HIV replication . Last, 1,25(OH)2VD3 seems to confer a protective effect against nucleoside reverse transcriptase inhibitor -induced mitochondrial toxicity . These different mechanisms involving vitamin D may play a role in the reduction of immune activation levels associated with vitamin D supplementation, reported in the present study.
The authors are very grateful to the patients and staff of the infectious diseases and Internal Medicine departments of the Hôpital Pitié Salpêtrière, Hôpital Dieu and Hôpital Cochin in Paris.
Source of funding: This work was supported by Sidaction and the French Agence Nationale de la Recherche sur le SIDA et les Hépatites (ANRS) and the ANR (Project ANR-09-JCJC-0114-01).
Conflicts of interest
There are no conflicts of interest.
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