Statins disrupt CCR5 and RANTES expression levels in CD4(+) T lymphocytes in vitro and preferentially decrease infection of R5 versus X4 HIV-1.

Abstract

Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles.
Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus.
The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols.

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Statins Disrupt CCR5 and RANTES Expression Levels in
CD4+T Lymphocytes In Vitro and Preferentially Decrease
Infection of R5 Versus X4 HIV-1
Alexey A. Nabatov1¤a, Georgios Pollakis1, Thomas Linnemann1¤b, William A. Paxton1*, Michel P. de Baar2¤c
1Laboratory of Experimental Virology, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic
Medical Center of the University of Amsterdam, Amsterdam, The Netherlands, 2Primagen, Amsterdam, The Netherlands
Background. Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1
(HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1
incorporation into viral particles. Principle Findings. Here we demonstrate that treatment of an enriched CD4+lymphocyte
population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N),
respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P,0.01 for Sim(A) and Lov). The
lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein
and mRNA expression levels were slightly increased in CD4+enriched lymphocytes treated with statins. Both R5 and X4 HIV-1
were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease
in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. Conclusions. The
results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be
considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in
combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1
activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment
could be considered as a way to modulate immune induction such as during vaccination protocols.
Citation: Nabatov AA, Pollakis G, Linnemann T, Paxton WA, de Baar MP (2007) Statins Disrupt CCR5 and RANTES Expression Levels in CD4+T
Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1. PLoS ONE 2(5): e470. doi:10.1371/journal.pone.0000470
INTRODUCTION
Antiretroviral therapy has expanded the lives of many infected with
HIV-1, however, emerging resistance and the encountered toxicity
indicate that new classes of drugs capable of reducing virus
replication are desired. In clinical trials the cholesterol lowering
class of drugs termed statins have been shown to be beneficial in the
primary and secondary prevention of coronary heart disease [1].
Recently a number of statins have been shown to possess anti-HIV-1
activity through a number of mechanisms and have been proposed
for the treatment of HIV-1 infection. Two main mechanisms have
been proposed to contribute to this inhibitory effect; 1) the down-
modulation of lipid raft formation through modulation of Rho
GTPase activity and 2) the blocking of the interaction between
virion-associated ICAM-1 and cell associated LFA-1 [2,3]. Statins
strongly inhibit the endogenous cholesterol biosynthesis by inhibiting
the rate-limiting enzyme in this biosynthesis process, named [3-
hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase].
Inhibition of small GTP-binding proteins, Rho, Ras, and Rac,
whose proper membrane localization and function are dependent on
isoprenylation, are supposed to be responsible for the pleiotropic
effects of statins [4,5]. Several in vivo studies have suggested
a beneficial effect of statins on HIV-1 viral load measurements,
whicharehighlypredictivefordiseaseprogression,whilstothershave
reported no beneficial effect of statins in controlled trials [2,6,7].
The chemokine receptors CCR5 and CXCR4 have been shown
to serve, in conjunction with the primary CD4 receptor, as HIV-1
coreceptors, which are required for viral entry [8–10]. The
classification of HIV tropism is based on chemokine receptor usage
of either CCR5 (R5 virus), CXCR4 (X4 virus), or both receptors
(R5X4 virus), although the utilization of other chemokine receptors
has been reported [11,12]. These receptors mediate immune cell
responses to a family of soluble chemo-attractant molecules, termed
chemokines. The CC chemokines RANTES, MIP-1a and MIP-1b,
the natural ligands for the CCR5 chemokine receptor, and SDF-1a,
the natural ligand for the CXCR4 coreceptor, have been shown to
successfully block the replication of HIV-1 in vitro [13,14]. These
receptors are therefore likely targets for drug development provided
that no essential cellular functions are affected by the intervention
strategy.Interestingly,ithaspreviouslybeenreportedthatstatinscan
down-regulate thelevelsoftheCCR5chemokine receptor onboth B
and T lymphocytes [15]
CCR5 has been shown to be present in cholesterol rich lipid
rafts, co-localizing at the leading edge of migrating cells [16]. This
receptor, in contrast to CXCR4, has also been shown to be
palmitoylated, which is one of the important modifications in lipid
raft targeting of proteins [17–19]. Importance of the cholesterol
Academic Editor: Douglas Nixon, University of California, San Francisco, United
States of America
Received April 25, 2007; Accepted May 1, 2007; Published May 23, 2007
Copyright: ? 2007 Nabatov et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work was supported by grants from the European Union (QLK2-CT-
1999-01321 and QLK2-CT-2001-01316 ‘‘Eurovac-I and –II’’).
Competing Interests: At time of study MdB was employed by the biotechnology
company Primagen who devised the CCR5 and RANTES mRNA quantification
NASBA assays. All authors declare no conflicts of interest.
* To whom correspondence should be addressed. E-mail: w.a.paxton@amc.uva.
nl
¤a Current address: Department of Molecular Cell Biology and Immunology, Vrije
Universiteit Medical Center, Amsterdam, The Netherlands
¤b Current address: Berlin, Germany
¤c Current address: OctoPlus, Leiden, The Netherlands
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presence in the membrane has recently been demonstrated in
experiments studying the effects of cyclodextrins on CCR5 and
CXCR4 function as both chemokine receptors and as HIV-1 co-
receptors [20,21].
RESULTS AND DISCUSSION
Statins down-modulate expression of CCR5 on CD4+
T lymphocytes
We aimed to analyse whether statins affected CCR5 cell surface
expression on CD4+T lymphocytes isolated from 5 separate
individuals. Isolated CD4+T lymphocytes were activated and
treated with either Sim(N), Sim(A), Lov or Mev and compared for
CCR5 expression on day 3 of treatment. Untreated cells from the
same donors were used as controls (Figure 1A). A final concentration
of 5 mM was utilized for all statins tested with no alterations to either
CD4+T lymphocyte cell counts or cell viabilities identified (data not
shown), indicating lack of toxicity induced by statins. We found that
all statins induced a reduction in the cell surface expression of CCR5
(Figure 1A). There was a statistical significant reduction in CCR5
expression for Sim(A) and Mev treatment (P,0.01) with a trend
towards a reduced expression with Sim(N) and Lov. The lower
reduction in CCR5 expression observed with Sim(N) likely reflects
the lower activity of this drug over its activated form and suggests
that the activity of the compound is responsible for the lowered
expression of CCR5. We tested the expression of CD4 and CXCR4
expression following statin treatment and although a slight reduction
in CD4 expression was observed no decrease in CXCR4 expression
was identified for any of the statins tested (data not shown). This
latter finding indicates that the lowered CCR5 expression is specific
and not due to toxicity.
We proceeded to determine whether the effect of statins on
lowering CCR5 cell surface expression was due to a reduction in
CCR5 mRNA expression or alterations to protein trafficking and
CCR5 association with the cell membrane. We developed and
utilized a Nucelic Acid Sequence Based Amplification (NASBA)
mRNA quantitative assay and utilized it to measure CCR5
mRNA levels in CD4+T lymphocytes. Overall a slight, but not
significant, reduction in CCR5 mRNA expression was detected
when comparing untreated versus statin treated cells (Figure 1B),
with Sim(A) showing the lowest reduction in mRNA synthesis. The
results indicate that the reduced cell surface expression of CCR5
on CD4+T lymphocytes can only in part be explained by down-
regulation of mRNA expression.
Statins up-regulate the expression of RANTES in
CD4+T lymphocytes
Since we observed that statins could marginally down-regulate
CCR5 expression at the cell surface of CD4+T lymphocytes we
wished to identify whether statin treatment had any effect on
modulating RANTES production. We assayed the culture super-
natants of CD4+T lymphocytes treated either with or without
statins for levels of RANTES production. From this we identified
that for the statins tested there is a trend towards an increase in
RANTES secretion (Figure 2A). When we compared mRNA
expression of RANTES from the same cells untreated or treated
with statins utilizing a specifically developed NASBA quantifica-
tion assay we observed a small but insignificant increase in
expression (Figure 2B). This increase in RANTES mRNA
expression also suggests that the concentrations of statins used in
the experiments are not toxic to the cells.
The results on CCR5 expression and RANTES secretion
collectively suggest that statins have the effect of modulating
chemokine and chemokine receptor expression levels. The
lowering of CCR5 cell surface expression is associated with an
increased production of RANTES, possibly reflecting a disruption
to cell signalling or a reduced uptake and degradation of secreted
RANTES by the CD4+
T lymphocyte from the culture
supernatant. An increase in RANTES mRNA expression may
also provide a further block to HIV-1 replication through higher
levels of RANTES competing with virus binding to CCR5.
Furthermore, our results would indicate that statins could be
utilized as immunomodulatory agents with the capacity to alter
induced immune responses. It has been shown that the Th-1 and
Th-2 cytokine secretory balance can be altered by statin treatment
with the net effect of modulating induced immune responses
against viral antigens [15,22–24]. This could have implications for
modulating the type of cellular immune response mounted in
HIV-1 infected patients, with a preferential skewing towards
a strengthened Th1 response and heightened CTL activity.
Statins preferentially reduce the infection of CD4+T
lymphocytes with an R5 versus X4 virus
Since the CCR5 CC-chemokine receptor and RANTES CC-
chemokine expression levels have been modulated in CD4+T
Figure 1. Statins down-regulate expression of CCR5 on CD4+T
lymphocytes. (A) CCR5 cell surface expression was monitored on
enriched CD4+T lymphocytes treated with Lov, Mev, Sim(A) and Sim(N).
Mean levels of CCR5 expression in the control and statin treated cells
are depict, * indicates P,0.01. (B) Levels of CCR5 mRNA were assayed in
CD4+T lymphocytes treated with Lov, Mev, Sim(A) and Sim(N) on day 4
of treatment. The results are presented as a ratio between untreated
(unt.) cells and cells treated (tr.) with statins.
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Figure 2. Statins upregulate RANTES accumulation in culture supernatant and increase RANTES mRNA expression. (A) RANTES levels were
analyzed in the culture supernatants of cells from five donors treated with Lov, Mev, Sim(A) and Sim(N) on day 4 of treatment and compared to
untreated control cells. (B) Mean levels of RANTES mRNA expression after treatment with the statins in four donors. The results are presented as
a ratio between untreated (unt.) cells and cells treated (tr.) with statins.
doi:10.1371/journal.pone.0000470.g002
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lymphocytes we wished to identify whether HIV-1 infection could
be modulated with statin treatment. Previous studies have shown
that statins have the effect of lowering the in vitro replication of
both R5 and X4 strains of HIV-1 equally [2]. We initially
identified the overall effect of statins on reducing infection with an
R5 and X4 virus on CD4+T lymphocytes. For this purpose we
performed 50% Tissue Culture Infectious Dose per ml (TCID50/
ml) determinations of a X4 and an R5 virus in the continual
presence of the four different statins and compared the results with
non-statin treated cells (Figure 3A). The results demonstrate that
both R5 and X4 viruses have a reduced TCID50/mlwhen cultured
in the presence of statins, with the R5 viruses being inhibited
stronger with Mev, Sim(N) and Sim(A) than the X4 strain by
approximately 2 fold, whilst inhibition with Lov was similar for
both viruses.
We next identified whether pre-treatment of cells with statins
could reduce infection with an R5 or X4 virus when culturing was
performed free from statin treatment. We therefore treated CD4+
T lymphocytes with statins for 3 days before washing and infecting
with an R5 or X4 strain of HIV-1. These were the same cultures
where the CCR5 and RANTES expression levels had been
determined above. After a short period of infection (12 hrs) the
cells were washed and diluted onto fresh non-statin treated CD4+
cells and the resultant viral replication monitored. Pre-treatment
of the cells with each statin separately had no effect on limiting
infection with the X4 virus strain, whereas, statin pre-treatment
had a major effect on limiting infection with an R5 variant at all
cell concentrations tested (Figure 3B and 3C). These results
demonstrate a clear distinction between infection with an R5 and
X4 virus when the culturing is performed under statin free culture
conditions.
This result indicate that statins can have a direct effect on
limiting virus replication of both R5 and X4 viruses but also that
R5 viruses may be preferentially inhibited further through
a disruption in the CC-chemokine receptor and CC-chemokine
expression patterns. This anti-viral effect would be in addition to
the previously described mechanisms of statin inhibition induced
through modulation of Rho GTPase activity as well as disruption
to incorporation of LFA-1 into the virus membrane [3]. The
preferential inhibition of R5 viruses and the disruption of CC-
chemokine and CC-chemokine expression patterns that can
modulate Th1, hence CTL responses, would advocate for the
use of statins early in the infection history of a patient. Whether
statins alone or in combination with other antiretroviral drugs
could confer a clinical advantage needs to be addressed. The
lowering of CCR5 cell surface expression has known clinical
advantages since individuals heterozygous for the 32 bp deletion in
their CCR5 gene are statistically more likely to harbor lower viral
loads and progress slower in their disease course [25]. In-
terestingly, it has been shown that CD4+T lymphocytes isolated
from individuals heterozygous for the 32 bp deletion have lowered
in vitro R5 infection profiles which correspond to lower CCR5 cell
surface expression and increased RANTES production [26,27].
The early intervention of HIV-1 infection with statins may
therefore have the beneficial effect of providing a time-delay
before having to prescribe the more conventional antiretroviral
drug regimes. Alternatively, statins could be considered for
prophylactic use in treating individuals at high risk of HIV-1
infection or those most recently exposed.
We demonstrate here that statin treatment of CD4+T
lymphocytes in vitro has the effect of reducing CCR5 cell surface
expression and providing for the heightened secretion of RANTES
in culture supernatant. Both CCR5 and RANTES mRNA
expression levels show a trend towards a decrease and an increase,
respectively. This altered regulation of chemokine and chemokine
receptor expression correlates with the preferential reduction in
infection of CD4+T lymphocytes with an R5 versus X4 virus
isolate. These results warrant for the further consideration of
statins as anti-HIV-1 agents, especially in the early phases of
infection or in a prophylactic capacity. Additionally, statins could
be considered for use as immunomodulatory compounds such as
in heightening or broadening immune responses during vaccina-
tion protocols.
MATERIAL AND METHODS
Statins
The following statins were utilized in this study: lovastatin,
mevastatin and simvastatin. The latter was used both in active and
non-active forms, meaning the activity to inhibit HMG-CoA
reductase. Activation occurred by hydroxylation of the statin using
NaOH applying standard protocols. The non-active form
nevertheless can get active as a result of normal intracellular
metabolic processes.
Cells and statin treatment
Peripheral Blood Mononuclear Cells (PBMC) were isolated from
human donors and treated with PHA (2 mg/ml) and recombinant
IL-2 (100 units/ml) as described earlier [28]. The following day
the activated PBMCs underwent CD8+lymphocyte depletion
according to the manufacturers instructions and the enriched
CD4+T lymphocytes were treated with filter sterilized stock
solutions of various statins (5 mM), reaching final concentrations
of 5 mM. Following incubation with statins for 3 days the cells
were utilized in the various assays described below. Supernatants
from the cells after 3 days of incubation with statins were harvested
as after 20 hrs of culturing in the presence of the viruses and stored
at 280uC.
Viruses
We utilized virus SF162, a CCR5 using virus and an envelope
modified LAI virus which had been altered in the V1V2 and V3
region as previously described (+6X.10DgV3) and that was of the
X4 phenotype [28].
Flow cytometry
The cells were stained using anti-CD4-PerCP, anti-CCR5-FITC,
anti-CD4- PE (all from BD PharMingen) using standard labelling
protocols. Signals were acquired on a FACS Calibur flow
cytometer (BD Biosciences) with CellQuest software (BD Bios-
ciences).
Enzyme immunoassay
Levels of RANTES in cell supernatants were measured with
specific ELISA kits from R&D Systems according to the
manufacturer’s instructions.
HIV infection assay
Viruses were monitored for their TCID50/mlvalues in the absence
and presence of statins (all at 5 mM final concentration). Briefly,
PHA activated CD4+-enriched lymphocytes (as obtained above)
were plated at 26105cells/well in 96-well plates with 5 fold serial
dilutions of the virus (8 replicas). On day 7 the medium was
replenished and on day 14 the p24 levels were determined utilizing
a standard p24 ELISA assay, with the TCID50/mlcalculated. Virus
replication assays were performed on statin treated or untreated
CD4+T lymphocytes. Cells were washed with PBS, resuspended
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Figure 3. Stain treatment preferentially inhibits the replication of R5 versus X4 HIV-1 (A) TCID50/mlinfectious profiles of an R5 (black bars) and X4
virus (white bars) were monitored on CD4+T lymphocytes in the presence of Lov, Mev, Sim(N) and Sim(A) and compared to non-statin treated cells.
(B and C) CD4+enriched lymphocytes were treated with Lov, Mev, Sim(A) and Sim(N) for 4 days then infected with an X4 (B) or R5 strain (C) of HIV-1
and cultured in the absence of statins as well as compared to non-statin treated cells. Non-statin treated cells are depict with closed squares and
statin treated cells are depict with open circles.
doi:10.1371/journal.pone.0000470.g003
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in RPMI culture medium (containing 10% FCS and pen/strep) at
a concentration of 46106cells/ml in the absence or presence of
statins (5 mM). On day 4 of culture the cells were washed, counted
and then incubated with HIV-1 viral stocks at 100 TCID50per
well in 96 well culture plates. The virus was incubated for 12 hrs
with the cells in 200 ml (26105cells/well), washed twice with PBS,
resuspended in 200 ml of RPMI (containing 10% FCS and pen/
strep) and cultured at 37oC for 10 days with all infections
performed in triplicate. The infected cells were co-cultured in
different ratios of infected to non-infected cells (fixed amount at
16105cells). Cultures were fed with fresh media on days 4 and 7
and HIV CA-p24 was determined in culture supernatant on day
10 using a standard CA-p24 ELISA assay.
NASBA
RNA was isolated from 36105cells using a silica-based total
nucleic acid isolation protocol. One tenth of the total nucleic acids
were used for three different reactions to quantify the number of
nuclear genomic molecules, the number of CCR5 mRNA
molecules and the number of RANTES mRNA molecules. The
NASBA reactions contained the following reagents: Tris-HCl
(40 mM, pH=8.3), MgCl2 (12 mM), KCl (90 mM), DTT
(5 mM), dNTPs (1 mM each), rNTPs (2 mM each), DMSO
(15% v/v), oligonucleotides: target-P1 and target-P2 (0.2 mM
each) and the target molecular beacons (0.04 mM each) to detect
the amplicons in real-time (Table 1). If the number of genomic
DNA copies was determined, the samples were incubated for
12 minutes at 37uC with 2 units MspI per reaction to allow the
restriction enzyme digestion, subsequently heated to 95uC for
3 min. to inactivate the MspI enzyme and to denature the DNA
allowing efficient annealing of the P1-primers after cooling to
41uC. For the mRNA assays, the samples were incubated 5 min.
at 65uC, before cooling to 41uC. After 1 min. at 41uC, the NASBA
enzyme mixtureswere added
[0.08 units],T7 RNA polymerase
[9.6 units] and sorbitol [0.375 M] per reaction) after which the
amplification was followed for 60 min. at 41uC in a fluorescence
reader with a thermostat, measuring every 30 sec. the fluorescein
(extinction: 485 nm/emission: 518 nm) signals. All samples were
assayed in duplicate.
(BSA[2.1 mg],
[64 units],
RNase
AMV-RT
H
Statistical analyses
Students T-test in the case of normal distributed data was used to
analyse significant differences between sets of data. Non-para-
metric tests as the Wilcoxon Rank test were used to analyse
significance between data sets in case of not-normally distributed
data. Data are presented as mean+/2SD with P values ,0.05
(two-sided) considered as statistically significant.
ACKNOWLEDGMENTS
Author Contributions
Conceived and designed the experiments: WP GP AN TL Md. Performed
the experiments: AN TL. Analyzed the data: AN. Wrote the paper: WP
GP AN Md.
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Table 1. Probes and primers for CCR5 and RANTES NASBA assay’s
..................................................................................................................................................
CCR5
Primer 59 (59R39):AAT TCT AAT ACG ACT CAC TAT AGG GAG CAG CGG CAG GAC CAG CCC CA
Primer 39 (59R39):TTT GGG GTG GTG ACA AGT GTG ATC A
Probe (59R39):FAM – CGT ACG TCC ATA CAG TCA GTA TCA ATT CGT ACG
RANTES
Primer 59 (59R39):AAT TCT AAT ACG ACT CAC TAT AGG GAA ACA GGC AAA TTT GTG TAA GTT CA
Primer 39 (59R39):TGC CCC GTG CCC ACA TCA AGG A
Probe (59R39):FAM – GCA TGC TCA CCC GAA AGA AGC GCC AAG CAT GC
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PLoS ONE | www.plosone.org7May 2007 | Issue 5 | e470