With the effectiveness of antiretroviral therapy (ART) at preventing AIDS-related complications, the number of people living with HIV has increased significantly over the past decade. As a result, non-HIV-related comorbidities typically associated with aging, including cardiovascular diseases (CVDs), osteoporosis, cancer, neurocognitive impairment and frailty are of increasing concern among HIV-infected individuals [1–5]. Human HIV infection is suppressed but not eliminated by ART; therefore, HIV-infected individuals successfully treated by ART do not attain normal longevity [2,5–7]. Viral persistence in the face of therapy has been explained by viral latency, lowered effectiveness of drugs in some anatomical sites and cell types, and cell-to-cell spread of the virus [8,9]. As a result, antigen persistence due to HIV infection is a major source of inflammation and substantial immune activation, both of which are linked to ‘inflammaging’ [7,10], a concept that attributes a pro-inflammatory milieu to the aging process.
Notably, inflammation as measured by highly sensitive C-reactive protein (hsCRP), interleukin (IL)-6, D-dimer, cystatin C levels , and increased expression of CD38, HLA-DR, Ki-67 and Bcl-2 by T cells are reported in HIV-infected individuals [12–15]. Mounting evidence supports the role of persistent inflammation and immune activation during suppressive ART as contributing factors not only to an increased CVD risk but also to other HIV-related comorbidities such as non-AIDS-defining malignancies, osteoporosis, cerebral and renal diseases [7,16,17].
Furthermore, a dramatic increase in the incidence of dyslipidemia in ART-treated individuals, especially among those taking protease inhibitors and to a lesser extent, those taking nonnucleotide reverse transcriptase inhibitors has been reported . Therefore, this dyslipidemia, as well as elevated inflammation due to persistent immune activation could contribute to an increased risk of CVD and other non-AIDS-defining comorbidities.
Statins are potent cholesterol lowering medications by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway . Interestingly, statins exert broad spectrum anti-inflammatory and immunomodulatory properties [20,21], which make them a potential choice to determine whether their anti-inflammatory properties could attenuate immune activation/viral replication and as a result reduce the comorbidity risk associated with HIV.
A number of studies have shown anti-HIV activity of statins by disruption of lipid rafts and inhibition of lymphocyte function antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) interactions in vitro[22–26].
In addition, statins have demonstrated delay in cancer cells progression and inhibit cell cycle by upregulating p53 and p21 genes [27–30]. However, the effect of statins on p21 expression in CD4+ T cells in the context of HIV infection has remained unknown.
Here, we examine the immunomodulatory effects of atorvastatin, in HIV pathogenesis in vitro. Atorvastatin as the most prescribed statin,  with the highest safety profiles, was used in this study . We demonstrate a novel role for atorvastatin in reducing HIV-1 infection involving upregulation of p21 in CD4+ T cells through mevalonate pathway. Secondary goals included a comprehensive analysis of the effects of atorvastatin on cellular markers of immune activation. We found that atorvastatin could modify HIV pathogenesis by means of direct antiviral and indirect anti-inflammatory mechanisms.
Materials and methods
Peripheral blood mononuclear cell samples from 12 HIV seronegative individuals were used for this study. The appropriate Institutional Review Boards at the University of Alberta approved the studies. All study participants gave written informed consent to participate in this study.
The CXCR4-utilizing isolate LAI and the CCR5-utilizing HIV-1 strain CSF-Jr were obtained from the AIDS Research and Reagent Programme at the National Institutes of Health. GFP-labeled T-cell tropic single cycle HIV-1SF2 was kindly provided by Dr Noah Sather (Center for Infectious Disease Research).
Ex vivo infection assays
CD4+ T cells were isolated, cultured in RPMI supplemented with 10% fetal bovine serum, and made susceptible to HIV-1 infection by in-vitro culture with exogenous recombinant IL-2 (50 U/ml), and phytohemagglutinin (PHA) (2 μg/ml). After 3 days, cells were washed and treated with different concentrations of atorvastatin (Sigma, Oakville, Ontario, Canada) post infection with HIV-1 viruses at multiplicity of infection (MOI) of 0.1 by using magnetofection . Cells were washed extensively and then atorvastatin was added into the wells for 24–48 h. Four to five days post infection p24 analysis by flow cytometry was conducted.
p21 inhibitor (2–10 μmol/l)  was used to block p21 expression in CD4+ T cells 6 h prior to infection. In some assays p53 activity was blocked using Pifithrin-α at 10–20 μmol/l (Sigma). Furthermore, 300 μm L-mevalonate (Sigma) was used to inhibit the effects of atorvastatin on HMG-CoA reductase enzyme.
Cell separation and flow cytometry
CD4+ T cells and monocytes (CD14+ cells) were isolated by negative selection (purity >97%) using a magnetic separation system (STEMCELL Technology, Vancouver, British Columbia, Canada). For phenotypic characterization, CD4+ T cells were stained with appropriate antibodies as indicated for each assay. Viability dye and antibodies directed against CD3, CD4, CD8, CCR5, CD25, CD127, CD38, HLA-DR, Ki67 (BD, Mississauga, Ontario, Canada), FOXP3 (eBiosciences, San Diego, California, USA), TIGIT (Biolegend, San Diego, California, USA), and carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific, Life Technology, Carlsbad, California, USA) were used in these studies. Paraformaldehyde fixed cells were acquired by flow cytometry using a LSRFORTESSA flow cytometer (BD) and analyzed with FlowJo software (Ashland, Oregon, USA).
Reverse transcription-PCR and real-time PCR
Expression of CDKN1A and p53 mRNA was analyzed by reverse transcription (RT)-PCR using standard methods as we described elsewhere [33,35].
Small interfering RNA (siRNA) transduction
RNA interference on CD4+ T cells was performed using a nucleofector device (Lonza) as we described elsewhere . After transfection (48 h), expression of p21 was assessed using RT-PCR and cells were infected with HIV-1. Knockdown of p21 gene expression in CD4+ T cells is shown in Supplementary Figure 1, http://links.lww.com/QAD/A796.
In-vitro T-cells proliferation
CD4+ T-cell proliferation was determined with CFSE assay as we reported elsewhere .
Atorvastatin reduces HIV-1 infection in already infected CD4+ T cells
CD4+ T cells were infected with either X4-tropic (HIV-1LAI; MOI 0.1) or R5-tropic (HIV-1JR-CSF; MOI 0.1) isolates in the presence or absence of atorvastatin (0.25 to 1 μg/ml; 24–48 h post infection). In humans, 80 mg daily of atorvastatin is the highest recommended dose for treatment of hypercholesterolemia , for an individual who weighs 70 kg this dose equals 1.1 mg/kg or 1.1 μg/ml. Therefore, for our experiments we used physiological relevance doses ranging equal from 20 to less that 80 mg/kg. Using these culture conditions, we consistently observed that atorvastatin induced up to at least 85%, and an average of 70%, inhibition of infection in CD4+ T cells with both R5-tropic and/or X4-tropic HIV-1 viral isolates (Fig. 1a). Atorvastatin-induced inhibition of HIV-1 infection in CD4+ T cells was significant for both R5-tropic (P = 0.0079) and X4-tropic isolates (P = 0.0066) (Fig. 1b). Interestingly, atorvastatin exhibited a dose-dependent suppressive effect on HIV-1 infection (HIV-1JR-CSF, Fig. 1c; and HIV-1LAIFig. 1d) and the inhibitory effects were time-dependent, with no effect at 24 h treatment (Fig. 1e and f). However, 48 h treatment provides significant inhibitory effects on HIV-1 replication (Fig. 1a) with no CD4+ T-cell death (Supplementary Figure 2, http://links.lww.com/QAD/A796). Therefore, 48 h atorvastatin treatment was used in all subsequent experiments. Furthermore, to dissect whether the activation status of CD4+ T cells or downstream infection-related changes in cytokine milieu were responsible for the observed effects of atorvastatin, cells were infected by a GFP-labeled single-cycle virus pretreatment with atorvastatin (0.25 and 1 μg/ml for 48 h). Atorvastatin reduced viral infection with this single-cycle virus suggesting that the suppressive effects of atorvastatin are directly due to reducing viral replication in target cells rather than altering downstream infection-related events (Fig. 1g).
Furthermore, monocyte-derived macrophages were infected with X4-tropic (HIV-1LAI; MOI 0.1) viral isolate in the presence or absence of atorvastatin (0.5 to 1 μg/ml; 48 h post infection). As shown in Supplementary Figure 3, http://links.lww.com/QAD/A796, atorvastatin inhibited infection of monocytes-derived macrophages in a dose-dependent manner, suggesting that the increased resistance to HIV-1 infection induced by atorvastatin occurs in both activated CD4+ T cells and macrophages. As CD4+ T cells are the primary targets of HIV-1 infection , the remaining studies were performed only on CD4+ T cells. Overall, these results demonstrate that atorvastatin limits viral replication in CD4+ T cells that are already infected in a time- and dose-dependent manner.
Atorvastatin downregulates activation markers on the surface of CD4+ T cells and expands regulatory T cells (Tregs)
CD4+ T cells were cultured in the presence of PHA and IL-2 for 3–4 days to upregulate CCR5 . Atorvastatin downregulated expression of CCR5 approximately 60–80% compared with untreated cultures after 48 h treatment with 0.5 μg/ml (P = 0.0295) and 1 μg/ml (P = 0.0104) respectively (Fig. 2a and b). Interestingly, atorvastatin did not impact expression levels of CXCR4 on CD4+ T cells (data not shown). Thus statins significantly reduce surface expression of CCR5, suggesting that down-regulation of HIV-1 co-receptor could be a mechanism by which statins reduce HIV-1 infection in CD4+ T cells . However, CCR5 is also an activation marker  and thus the effects of atorvastatin on reducing surface expression of CCR5 could simply be the result of its ability to suppress T-cell proliferation and activation . Atorvastatin also decreased expression of other activation markers such as CD38, HLA-DR (Fig. 2c) and T-cell proliferation as measured by Ki-67 and CFSE (Fig. 2d–g). In contrast, atorvastatin upregulated expression of FOXP3 in both CD4+ (Fig. 2h) and CD8+ T cells (Fig. 2i) which is in agreement with its known ability to expand Tregs . Additionally, atorvastatin upregulated expression of TIGIT, an inhibitory receptor, on Tregs (Fig. 2j)  indicating diverse immunoregulatroy properties of atorvastatin.
Atorvastatin reduces HIV-1 infection of resting CD4+ T cells
Some components depending on the activation status of CD4+ T cells can either reduce or enhance CD4+ T-cells susceptibility to HIV-1 infection . To confirm that atorvastatin does not enhance HIV-1 infection in resting CD4+ T cells, we infected nonactivated CD4+ T cells with either the X4-tropic isolate or R5-tropic HIV-1 isolate then treated them with atorvastatin (0.5–1 μg/ml; 48 h post infection) and analyzed for p24 expression. Presence of atorvastatin inhibited HIV-1 infection in resting CD4+ T cells in a dose-dependent (P < 0.0001; Supplementary Figures 4A–C, http://links.lww.com/QAD/A796) and time-dependent manner, with no effect observed at 24 h of stimulation (Supplementary Figure 4D, http://links.lww.com/QAD/A796).
Upregulation of p21 in CD4+ T cells following treatment with atorvastatin reduces HIV-1 replication in CD4+ T cells
We have shown that atorvastatin reduces expression of HIV-1 co-receptor CCR5 and activation markers as a possible inhibitory barrier against HIV-1 infection (Fig. 2). However, in our studies CD4+ T cells were infected with HIV-1 prior to treatment with atorvastatin, indicating that atorvastatin may impact replication of HIV-1 in already infected CD4+ T cells. Recently, p21 has been shown to be strongly upregulated in CD4+ T cells from elite controllers with inverse correlation between CDKN1A (the gene name for p21) mRNA expression levels in CD4+ T cells and their susceptibility to HIV-1 infection . Interestingly, activation of p53/p21 pathway by statins has been reported in different cell types and tumor cells [29,44,45]. To determine whether p53/p21 pathway was involved in the reduced HIV-1 susceptibility of atorvastatin-treated CD4+ T cells, we analyzed CDKN1A mRNA expression in atorvastatin-treated CD4+ T cells by RT-PCR and quantitative RT-PCR. We observed a strong upregulation of CDKN1A mRNA as measured by quantitative PCR (Fig. 3a). Quantitative RT-PCR confirmed that atorvastatin induced a 2–12 fold (an average of >5 fold) increase in mRNA expression of CDKN1A in atorvastatin-treated CD4+ T cells compared with nontreated controls (P < 0.0001; Fig. 3b). In addition, we compared the expression of p21 mRNA in CD4+ T cells in the presence of atorvastatin with the inhibitory effects of atorvastatin on HIV-1 infection in CD4+ T cells. A significant positive correlation between CDKN1A mRNA expression levels in atorvastatin-treated CD4+ T cells and suppression of HIV-1 infection in CD4+ T cells, as determined by p24 expression after infection of ex-vivo atorvastatin-treated CD4+ T cells with HIV-1LAI, was observed (r2 = 0.68, P = 0.0031; Fig. 3c). The expression of p21 is regulated either via p53 or p53 independent pathways . Therefore, we analyzed p53 mRNA expression by RT-PCR following treatment of CD4+ T cells with atorvastatin. As shown in Fig. 3a, upregulation of p53 mRNA expression was evident; however, inhibition of p53 expression did not result in downregulation of p21 (Fig. 3d). Although atorvastatin enhances expression of both p53 and p21 genes, it appears that atorvastatin-mediated upregulation of p21 is p53 independent (Fig. 3d). In agreement, inhibition of p53 did not reverse the p21 dependent effects of atorvastatin on CD4+ T cells to HIV-1 infection in vitro (Fig. 3e). Additionally, it appears that atorvastatin in a time-dependent manner enhances p53/p21 mRNA expression levels and 24 h treatment did not upregulate the expression of these genes (Fig. 3f). Furthermore, we analyzed CDKN1A mRNA expression in atorvastatin-treated monocyte-derived macrophages by RT-PCR. We observed a strong upregulation of CDKN1A mRNA as measured by RT-PCR (Supplementary Figure 3B, http://links.lww.com/QAD/A796). Taken together, these data demonstrated that CD4+ T cells and monocyte-derived macrophages treated with atorvastatin expressed higher levels of p21 than did untreated cells and that levels of p21 expression were inversely correlated to CD4+ T-cell susceptibility to HIV-1 infection.
Atorvastatin via upregulation of p21 in CD4+ T cells suppresses HIV-1 replication
To test whether the elevated p21 levels in atorvastatin-treated CD4+ T cells functionally contribute to their resistance to HIV-1 infection, we performed ex-vivo infection assays of CD4+ T cells using a highly selective small molecule inhibitor of p21 in the presence and absence of atorvastatin. This molecule leads to ubiquitin-mediated proteasomal degradation of p21and pharmacologically inhibits p21 . In the presence of p21 inhibitor, the atorvastatin-induced resistance of activated CD4+ T cells to HIV-1 infection was significantly abrogated for both R5-tropic (P < 0.0001) and X4-tropic (P = 0.0002) viruses in a dose-dependent manner (Fig. 4a–c). Similarly, p21 inhibitor eliminated the preventive effects of atorvastatin on HIV-1 replication in nonactivated CD4+ T cells for both R5-tropic and X4-tropic viruses (Fig. 4d). We reconfirmed the p21 inhibition by performing ex-vivo infection assays of CD4+ T cells transfected with siRNA, inducing effective downregulation of p21 expression, or with control siRNA that does not affect CDKN1A gene expression (Supplementary Figure 1, http://links.lww.com/QAD/A796) in the presence or absence of atorvastatin. In the absence of p21 upregulation, the atorvastatin-induced resistance of activated CD4+ T cells to HIV-1 infection was significantly abrogated (Supplementary Figure 4, http://links.lww.com/QAD/A796) indicating that induction of p21 is critical for atorvastatin-enhanced resistance of CD4+ T cells to HIV-1 infection.
Atorvastatin upregulates p21 and reduces infection of CD4+ T cells to HIV-1 infection through inhibition of the mevalonate pathway
We hypothesized that the observed effects of atorvastatin on decreasing susceptibility of CD4+ T cells to HIV-1 infection were mediated by inhibiting HMG-CoA reductase. PHA-activated CD4+ T cells were infected with HIV-1 infection prior to treatment with atorvastatin alone or atorvastatin plus L-mevalonate, the product of HMG-CoA reductase. The inhibitory effects of atorvastatin on HIV-1 infection of PHA-activated CD4+ T cells was reversed by L-mevalonate (1 mmol/l) for both R5-tropic (P = 0.028; Fig. 5a–c) and X4-tropic (P < 0.0001; Fig. 5b–d) viruses. Consistent with this, L-mevalonate inhibited the effects of atorvastatin on the infection susceptibility of nonactivated CD4+ T cells in vitro (Supplementary Figure 5, http://links.lww.com/QAD/A796). In addition, CD4+ T cells were treated with atorvastatin (0.5–1 μg/ml) for 48 h in the presence or absence of L-mevalonate (1 mM) and then the expression of p21 mRNA by RT-PCR and real-time PCR was examined. We observed that atorvastatin-mediated upregulation of p21 in CD4+ T cells was abrogated by L-mevalonate (Fig. 5e and f), indicating that these effects were mediated through inhibition of the mevalonate pathway.
Although statins were originally used as hypocholesterolemic drugs in the treatment of CVDs [47,48], their wide spectrum immunomodulatory effects have been highly appreciated in different immunological settings. These drugs have attracted significant interests in the treatment of inflammatory disease, Th1-mediated autoimmune disease and multiple sclerosis due to their diverse impact on immune cells [49–51]. Mechanistically, it has been shown that statins via the mevalonate pathway modify immune responses at different levels, including antigen presenting cells, B cells, T cells, Tregs and endothelial cells [51–56].
Notably, inflammation, as determined by CRP and other inflammatory markers, is increased in HIV-infected individuals even in the presence of ART , which could contribute to an increased CVDs risk and other HIV-related comorbidities [18,57]. Therefore, statins have the potential to regulate dyslipidemia in the setting of HIV-1 infection and reduce hyperimmune activation. In this regard, multiple studies indicated reduction of inflammation and immune activation markers such as CRP, soluble CD14 (sCD14), IL-6, IL-8, TNF-α, HLA-DR and CD38 in HIV-infected/statin-treated individuals [58–61]. However, there are somewhat conflicting data in the field. This may suggest statins affect infection and immune activation via different mechanisms or the discrepancy may be attributed to the specific statin used or the specific HIV target group.
Here, for the first time, we report a novel function for atorvastatin, in regulating CD4+ T-cell susceptibility to HIV-infection. Multiple effects of atorvastatin have been reported in different in-vitro and in-vivo systems and different effects may result from specific interactions with different immune cells [51–56].
Although anti-HIV-1 activities of statins have been attributed to their main mechanism of action, inhibiting HMG-CoA reductase, by reducing viral entry/exit,  and diminishing HIV-1 attachment to target cells by suppressing ICAM-1-LFA-1 interactions , their role on HIV-1 replication in target cells is not well understood.
We report here a novel function for atorvastatin via mevalonate pathway in regulating HIV-1 replication in target cells in vitro.
Here, we found that treatment of HIV-1-infected CD4+ T cells with atorvastatin significantly inhibited HIV-1 infection by both X4 and R5-tropic isolates. These suppressive effects of atorvastatin were mediated through mevalonate pathway interactions as blocking mevalonate pathway using L-mevalonate abolished the atorvastatin-mediated resistance of CD4+ T cells to HIV-1 infection. We also found that atorvastatin in a time-dependent manner significantly reduces surface expression of CCR5; T cells activation markers such as CD38, HLA-DR, Ki67; and inhibits cell proliferation. Alternatively, atorvastatin expands Tregs, providing a potential mechanism of dampening immune activation and rendering CD4+ T cells less prone to HIV-1 infection. Although, it has been reported that statins inhibit HIV-1 entry  and diminish HIV-1 attachment to target cells , their role on cell cycle inhibitor has not been reported. Here we show that the cell-cycle inhibitor, p21, which is directly involved in reducing the susceptibility of CD4+ T cells to HIV-1 by inhibiting viral reverse transcription and mRNA transcription in elite controllers , was strongly upregulated in atorvastatin-treated CD4+ T cells. Furthermore, we demonstrated that atorvastatin-mediated upregulation of p21 occurs via mevalonate pathway as blocking this pathway using L-mevalonate abolished the effects of atorvastatin-mediated upregulation of p21. Despite the fact that atorvastatin treatment enhanced p53 mRNA expression in CD4+ T cells, blockade of p53 expression did not impact the outcome of viral infection in CD4+ T cells, demonstrating that atorvastatin upregulates p21 through a p53 independent pathway.
To best of our knowledge, the data presented here are the first demonstration that atorvastatin is directly involved in reducing replication of HIV-1 in CD4+ T cells via upregulation of p21 in vitro. The observation that inhibition of p21 expression using p21 inhibitor or siRNA abrogates the effects of atorvastatin on HIV-1 infection in CD4+ T cells provides direct proof that the mechanism of action by atorvastatin in already infected CD4+ T cells is mediated by specifically targeting p21. However, more studies are needed to determine whether p21 upregulation occurs in patients on statins. There have been several studies demonstrating reduced T-cells activation, sCD14, patrolling monocytes, lipoprotein-associated phospholipase A2 concentrations in subjects on ART [60,62–64] which decreases the risk of non-AIDS-defining events in an AIDS clinical trials group . Therefore, it appears that statin therapy in HIV-1-infected individuals could be beneficial by reducing persistent immune activation and subsequently decreasing the risk of CVD and other non-AIDS-defining comorbidities. However, despite our observation and other reported studies showing promising in-vitro HIV-1 inhibition [22–25], this has not been confirmed in HIV-1-infected patients on statins [61,66,67]. In agreement with others  we have shown that atorvastatin expands Tregs in a dose-dependent manner which likely contributes to indirect immunomodulatory effects of statins, as Tregs play a crucial role in counteracting inflammation and autoimmunity. Interestingly, we also observed that atorvastatin upregulates expression of TIGIT on CD4+ T cells in a dose-dependent manner. TIGIT binding to CD155 on dendritic cells leads to reduce IL-12p40 and concomitantly increases IL-10 production . Importantly, TIGIT attenuates antitumor cytotoxic T lymphocyte (CTL) responses , and it was recently reported as a mechanism by which Tregs exert their suppressive capabilities . Although our data provides another potential mechanism by which statins may mediate their immunomodulatory effects, more studies need to be undertaken to identify potential adverse effects. For instance, it has been reported that statin therapy decreases the Th1/Th2 ratio and attenuates CTL response which is necessary in the maintenance of adequate anti-HIV CTL response .
In conclusion, we show for the first time that at cellular level, atorvastatin, via mevalonate pathway, upregulates p21, which subsequently prevents HIV-1 replication. We also provide evidence that atorvastatin directly reduces immune activation and expands Tregs. Considering the ability of atorvastatin to lower viral load and suppress immune activation, it may be a potential drug for reducing the burden of excessive immune activation and preventing accelerated again in HIV-1-infected individuals. However, probably not all patients will benefit from a statin, due to the enormous heterogeneity in HIV-1-infected individuals. Thus, further studies, particularly long-term randomized placebo-controlled trials, are required to fully determine whether statins are beneficial and have the clinical potential to prevent non-AIDS-defining comorbidities.
We thank our study volunteers for providing samples and supporting this work. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR).
Author contributions: S.E. designed, supervised all of the research, performed most of the research, analyzed the data and wrote the manuscript. S.M. performed some of the research and helped in writing part of the manuscript. R.W. provided new reagents and advised on the experiments. All authors read and edited the manuscript.
Conflicts of interest
We declare no competing financial interests.
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