Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.
The endogenous mevalonate pathway utilizes the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA),3 which is responsible for the biosynthesis of cholesterol and isoprenoids, and inhibiting HMG-CoA by statins potently suppresses this pathway (Fig. 1) (1). Statins have been used for the treatment of cardiovascular diseases over the years due to their lipid-lowering effects (2, 3). Their efficacy in the treatment of atherosclerosis has been attributed to both cholesterol-dependent and -independent activities (2, 4, 5). Given the cholesterol-independent effects of statins in inflammation, atherosclerosis, and immunomodulation (2), it is not surprising that the immune response would also be affected independently of their lipid-lowering effects.
In addition to their ability to reduce the level of lipids, statins also inhibit the biosynthesis of isoprenoid intermediates such as geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) (6). Isoprenylation, the attachment of GGPP and FPP, is a posttranslational modification of several proteins including the small GTP-binding proteins Ras, Rho, and Rab (7). Isoprenylation plays some role in the activation and intracellular transport of proteins important for various cellular functions such as differentiation, proliferation, motility, and the maintenance of cell shape (8). Moreover, prenylation enables GTPases to be targeted to the cell membrane and allows their subsequent interaction with downstream signal transduction effector molecules (9). Statins therefore impair the functioning of these small GTPases by preventing their correct membrane targeting (10).
Statins have been shown to affect both innate and acquired immune responses (11). Innate immune functions are inhibited via the targeting of TLR2 and TLR4 expression, preventing the activation of endothelial cells and macrophages (12). In contrast, acquired immune responses are affected by statin-dependent inhibition of Ag presentation by MHC class II molecules or T cell function and by the suppression of costimulatory molecules (e.g., CD40, CD80, and CD86) on macrophages, lymphocytes, and endothelial cells (13, 14). In the adaptive immune response, the inhibition of prenylation plays an important role in the Th1 to Th2 switch and is protective against autoimmune diseases of the CNS, experimental arthritis, autoimmune myocarditis, and systemic lupus erythematosus (15). Statins also inhibit proinflammatory gene expression in a number of leukocytes and vascular cells (15) and also diminish MCP-1 in the neointima (16, 17). NK cell cytotoxicity in patients with cardiovascular disease has also been shown to be impaired by statins (18).
NKT cells are T lymphocytes with innate immune effector functions that are activated by lipid Ags presented by the MHC class I-like CD1d molecule (19, 20, 21). In the lipid-associated disorder atherosclerosis, T cells and NKT cells play crucial roles in the pathology of the disease (22, 23, 24, 25, 26). In advanced plaques, the numbers of dendritic cells, T cells, macrophages, and HLA-DR-expressing cells have been found in higher numbers than initial lesions or stable plaques (27). Furthermore, NKT cells have been shown to contribute to the progression of atherosclerotic lesions (22, 28, 29, 30). Importantly, both CD1d expression and NKT cells have been detected in human atherosclerotic plaques (31, 32, 33). As statins have been found to reduce inflammation and stabilize atherosclerotic plaques by their activities independent of their lipid-lowering effects (34, 35), it is important to understand their ability to regulate Ag presentation. Interestingly, statins have been shown to alter MHC class II-mediated Ag presentation (36, 37), and although CD1d molecules traffic through some of the same endocytic compartments as MHC class II for Ag loading (19), their effects on Ag presentation by CD1d have not been studied. Thus, in the present study, the ability of statins to regulate lipid Ag presentation by CD1d molecules was investigated. It was found that statins significantly impair Ag presentation independent of their lipid-lowering properties, suggesting an important role for prenylation in the control of Ag presentation.
Materials and Methods
Female C3H/HeJ and C57BL/6 mice were purchased from The Jackson Laboratory. All procedures were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine.
Cell lines and other reagents
Murine LMTK-CD1d1 and LMTK-CD1d1-DR4 cells were cultured in DMEM (BioWhittaker/Lonza) supplemented with 10% FBS (HyClone), 2 mM glutamine, and 500 μg/ml G418. Simvastatin (sodium salt), farnesyl transferase inhibitor (FTI)-277, and geranylgeranyl transferase inhibitor (GGTI)-298 were purchased from Calbiochem. Mevalonolactone, GGPP, FPP, and squalene were obtained from Sigma-Aldrich. The mouse CD1d-specific NKT cell hybridomas DN32.D3, N37-1A12, N38-2C12, and N38-3C3 were cultured in IMDM (BioWhittaker/Lonza) supplemented with 5% FBS and 2 mM l-glutamine. The HLA-DR4-restricted, human serum albumin (HSA)-specific murine T cell hybridoma, 17.9, was a gift from Dr. Janice Blum (Indiana University School of Medicine, Indianapolis, IN). Mouse B cell lymphoma TA3 cells and the I-Ak-restricted, hen egg lysozyme (HEL)-specific T cell hybridoma 3A9 cells were kindly provided by Dr. Gail Bishop (University of Iowa, Iowa City, IA). HEL was obtained from Sigma-Aldrich. Purified and biotinylated mAbs specific for murine IL-2 were purchased from BD Biosciences. Recombinant mouse IL-2 used as a standard in the ELISA assays was obtained from PeproTech. Abs specific for Rho and Ras were purchased from Cell Signaling Technology; Abs against Rab5a (Santa Cruz Biotechnology), Rab7 (Sigma-Aldrich), and Rab11 (BD Biosciences) were also used in the experiments described below.
Generation of bone marrow-derived dendritic cells (BMDCs)
Bone marrow cells from C3H/HeJ and C57BL/6 mice were cultured in RPMI 1640 supplemented with 2 mM l-glutamine, 50 μM 2-ME, 10% FBS, and antibiotics, as well as 10 ng/ml each of murine GM-CSF and IL-4 as previously described (38). On day 7, the plates were gently flushed (three to four times) to remove the loosely adherent cells, which were subsequently used in analyses as BMDCs.
Small interfering RNA (siRNA) plasmids and stable cell lines
To generate cell lines that stably expressed siRNA targeted against GGTase I, GGTase II, or for the control (nonspecific sequence), we used the pSuppressorNeo vector (Imgenex). Primers targeting the β subunit of GGTase I or GGTase II were designed using Imgenex’s siRNA retriever program. Primers for GGTase I were: forward, 5′-TCGACAGGATAAAGAGGTGGTGCATGGAATTCGATGCACCACCTCTTTCTCCTGTTTTT-3′; reverse, 5′-CTAGAAAAACAGGATAAAGAGGTGGTGCATCGAATTCCATGCACCACCTCTTTATCCTG-3′. Primers for GGTase II were: forward, 5′-TCGACCGAGAAGAAATCCTGGTGTTGGAATTCGAACACCAGGATTTCTTCTCGGTTTTT-3′; reverse, 5′-CTAGAAAAACCGAGAAGAAATCCTGGTGTTCGAATTCCAACACCAGGATTTCTTCTCGG-3′. Primers for each target were annealed, phosphorylated, and ligated into the pSuppressorNeo vector. A portion of the ligation reaction was used to transform chemically competent DH5α Escherichia coli. Drug-resistant clones were selected and propagated, and plasmids were isolated and screened using restriction endonuclease digestions for a unique site found in the siRNA primer sequence. Positive clones were sequenced to ensure the correct insert was present. LMTK cells containing CD1d in the pcDNA-Zeo (Invitrogen) were transfected with either of the GGTase siRNA plasmids or control sequence vector only. Cells containing the siRNA vector were selected for using 500 μg/ml G418, and drug-resistant cells were pooled and maintained as stable cell lines for experiments.
Total RNA was isolated from 1 × 106 cells using the High Pure RNA Isolation kit (Roche). One microgram of this RNA was used to generate cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche), using random hexamers. The mixture of primers and template was denatured (10 min at 65°C) before the reverse transcription reaction. This cDNA served as the template in real-time PCR reactions. The Universal Probe method (Roche) was used to determine expression levels. To the FastStart Universal Probe Master Mix (ROX), we added primers for the gene of interest or β-actin (purchased from Integrated DNA Technologies), cDNA, and one of the Universal Probes. Primers and probes were designed using ProbeFinder version 2.43 for mice. To analyze GGTase I expression, we used Universal Probe no. 11 with forward (5′-TGCTTAGCAGGCTTGAGAGC-3′) and reverse (5′-TTCA GGAACCGCACAGAAG-3′) primers. To analyze GGTase II expression, Universal Probe no. 67 was used with forward (5′-GCCTATGTTCAGAGCCTACA-3′) and reverse (5′-CACCGCACAAAATGAGAATC-3′) primers. For analysis of β-actin expression, Universal Probe no. 11 was used with forward (5′-ACTGCTCTGGCTCCTAGCAC-3′) and reverse (5′-CCTGCTTGCTGATCCACAT-3′) primers. Real-time PCR was performed using an Mx3000P (Stratagene) instrument with MxPro software (version 4.01). The following parameters were used: 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 s, then 60°C for 1 min, during which time the analysis was done. Results were analyzed using the standard defaults except that the Mx4000 v1.00 to v3.00 algorithm was used for the adaptive baseline. The comparative cycle threshold (Ct) of each transcript in the different cell lines was obtained. The ΔΔCt method was used with β-actin as the control to calculate changes in expression.
NKT cell coculture assays
LMTK-CD1d1 cells were treated with various concentrations (0–50 μM) of simvastatin, GGTI-298 (0–10 μM), or FTI-277 (0–10 μM) for 24 h. Cells treated with vehicle only (DMSO) served as the negative control. In a separate set of experiments, cells were treated with simvastatin (50 μM) in the presence or absence of mevalonate (200 and 400 μM) for 24 h or α-galactosylceramide (α-GalCer; 500 ng/ml for 1 h). The cells were then washed with PBS, fixed in 0.05% paraformaldehyde, and cocultured with the indicated NKT cell hybridomas as described previously (39). In parallel, BMDCs were treated with the indicated concentrations of simvastatin for 24 h, washed, fixed, and used in NKT cell assays as above. LMTK-CD1d1 cells transfected with empty vector or GGTase-specific siRNA were also cocultured with NKT cells as above. LMTK-CD1d1 cells were treated with various doses of β-methyl cyclodextrin (β-MCD; 0–20 mM) and nystatin (0–20 μg/ml) for 2 h. The cells were washed, fixed, and cocultured with the indicated NKT cell hybridomas as above.
Determination of cellular cholesterol concentration
LMTK-CD1d1 cells, treated with vehicle, simvastatin (25 and 50 μM), or AY9944 (10 and 20 μM) overnight, were scraped from 6-well plates. The cells (5 × 106) were washed twice with cold PBS and centrifuged at 2000 rpm. The pellet was resuspended in 0.5 ml of isopropyl alcohol, sonicated for 3 min, and cleared by centrifugation at 10,000 rpm for 10 min. Supernatants were decanted and isopropyl alcohol was evaporated. A volume of 50 μl of isopropyl alcohol was added to each tube to resuspend the material, and aliquots were used to measure the cholesterol level using the EnzyChrom assay kit (BioAssay Systems) with cholesterol standards used for calibration according to the manufacturer’s instructions.
MHC class II Ag presentation assay
L-CD1d1-DR4 cells (40) were treated with 10 μM HSA in the presence or absence of different concentrations of simvastatin for 24 h. The cells were then washed with PBS, fixed in paraformaldehyde, and cocultured with the 17.9 T cell hybridoma, with IL-2 production determined as above. Because these L cells also express CD1d1 molecules, an NKT cell coculture was performed in parallel as a positive control. In other experiments, the murine B cell lymphoma TA3 cell line was treated with vehicle, simvastatin, FTI-277, or GGTI-298 in the presence or absence of 1 mg/ml HEL and cocultured with the HEL-specific, I-Ak-restricted 3A9 T cell hybridoma, and IL-2 production was measured as above. BMDCs generated from C3H/HeJ mice were treated with LPS (1 μg/ml) overnight. The cells were washed and treated with the indicated concentrations of simvastatin, FTI-277, and GGTI-298 in the presence or absence of HEL. After two washes in PBS, the BMDCs were cocultured with the 3A9 T cell hybridoma as above.
Cytotoxic T lymphocyte assay
To determine the effect of prenylation inhibition on Ag presentation by MHC class I molecules, MC57G cells were treated with vehicle (DMSO), simvastatin (50 μM), FTI-277 (10 μM), or GGTI-298 (10 μM) overnight and then mock-infected or infected with vaccinia virus (VV; multiplicity of infection of 5) for 6 h in the presence or absence of inhibitors. The cells were washed, fixed, and cocultured for 24 h with splenocytes isolated from C57BL/6 mice on day 6 after a VV infection (1 × 106 PFU/mouse, i.p.). Supernatants were collected to measure the amount of IFN-γ in the cocultures by ELISA.
LMTK-CD1d1 cells were treated with the indicated concentrations of simvastatin in the presence or absence of mevalonate. The cells were lysed in lysis buffer (25 mM HEPES buffer (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 25 mM sodium fluoride, 10 mM MgCl2, 1 mM EDTA, 10 mM sodium orthovanadate, 25 mM β-glycerophosphate), containing Complete protease inhibitor tablets (Roche Diagnostics). The amount of protein in cell lysates was estimated using Bio-Rad protein assay reagents. Equal amounts of protein were loaded into each well and resolved on a 10% SDS-PAGE gel, and subsequently transferred to a polyvinylidene difluoride membrane (Millipore). The blot was processed with Abs specific for the indicated proteins and bands developed using chemiluminescence before exposure on film (41).
For assessing the relative distribution of GTPases in statin-treated cells, LMTK-CD1d1 cells were treated with simvastatin in the presence or absence of mevalonate. Cells were lysed in sucrose-containing HEPES buffer (200 mM sucrose, 20 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, and Complete protease inhibitor tablets) on ice for 15 min. The lysates were clarified by centrifugation at 1000 rpm for 5 min at 4°C. The resulting supernatant was centrifuged at 30,000 rpm at 4°C for 30 min. The resulting supernatant was then removed (cytosolic fraction) and the membrane pellet was washed in sucrose-containing HEPES buffer by centrifugation at 30,000 rpm for 30 min. Equal amounts of protein were loaded into each well and were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The blots were probed with Abs specific for the individual GTPases, as well as markers for the cytosolic (GAPDH) and membrane (flotillin) fractions, and developed as above.
LMTK-CD1d1 cells were plated in sterile glass-bottom 35-mm dishes coated with collagen (MatTek) at a density of 1 × 106 cells per dish. Cells were treated with the indicated concentrations of GGTI-298, FTI-277, AY9944, or simvastatin with or without mevalonate for 24 h at 37°C. The cells were stained for CD1d and lysosome-associated membrane protein 1 (LAMP-1) followed by Texas Red- and FITC-conjugated secondary Abs, and then analyzed by confocal microscopy as previously described (39, 40). The percentage colocalization of CD1d and LAMP-1 was determined using MetaMorph software (version 5; Molecular Devices), in which six random fields were chosen from each picture.
The data were analyzed by a one-way ANOVA and unpaired two-tailed Student’s t test using GraphPad Prism software (version 5.0 for Windows; GraphPad Software). A p value of <0.05 was considered significant. The error bars in the bar graphs show the SD from the mean of triplicate samples.
Statin-induced inhibition of CD1d-mediated Ag presentation is prenylation-dependent
To determine whether prenylation is important for CD1d-mediated Ag presentation, murine LMTK-CD1d1 fibroblasts were treated with various concentrations of the statin simvastatin for 24 h and cocultured with NKT cells. LMTK-CD1d1 cells treated with simvastatin showed a dose-dependent reduction in the stimulation of NKT cells, with an almost 50–60% decrease in Ag presentation at the highest concentration used, as compared with vehicle-treated cells (Fig. 2,A). To ensure that the effect of prenylation inhibition on CD1d-mediated Ag presentation could be observed in primary APCs, BMDCs were treated with different concentrations of simvastatin as above. As found with LMTK-CD1d1 cells, simvastatin treatment of BMDCs also caused a substantial reduction in Ag presentation by CD1d (Fig. 2,B). Simvastatin did not alter CD1d cell surface expression on either LMTK-CD1d1 or BMDCs (supplementarly Fig. 1, A and B).4 Nonetheless, inhibiting prenylation could have caused some qualitative changes in the functional expression of CD1d molecules. To test this possibility, simvastatin-pretreated LMTK-CD1d1 cells were incubated with the CD1d-specific ligand α-GalCer to determine whether there was an effect on exogenous Ag presentation. As expected, α-GalCer substantially enhanced the stimulation of Vα14+ (e.g., DN32.D3), but not Vα14− (e.g., N37–1A12) NKT cells. Interestingly, simvastatin-treated LMTK-CD1d1 cells could not stimulate NKT cells to the same extent as vehicle-treated cells in the presence of α-GalCer (Fig. 2 C). Therefore, these results suggest that simvastatin treatment impairs both endogenous and exogenous lipid Ag presentation.
To determine whether simvastatin affected CD1d-mediated Ag presentation through its effects on cholesterol biosynthesis, LMTK-CD1d1 cells were treated with a highly specific inhibitor of cholesterol biosynthesis, AY9944. Notably, whereas AY9944 did not alter Ag presentation by CD1d molecules even at the highest concentration used (Fig. 2 D), it did substantially inhibit cholesterol biosynthesis (supplemental Fig. 2A). Moreover, treatment of LMTK-CD1d1 cells with cholesterol-depleting agents such as nystatin and β-MCD had only a modest effect on CD1d-mediated Ag presentation (supplemental Fig. 2, B and C). The latter results of β-MCD are in line with those recently reported (42). However, it is important to note that β-MCD concentrations >10 mM are toxic (data not shown).
Mevalonate reverses simvastatin-induced inhibition of CD1d-mediated Ag presentation
Mevalonate, the precursor for the biosynthesis of cholesterol and isoprenoids, is considered a therapeutic target for autoimmune diseases (15, 43). Simvastatin inhibits the biosynthesis of mevalonate by inhibiting the enzyme HMG-CoA reductase. Thus, to determine whether the simvastatin-induced inhibition of CD1d-mediated Ag presentation was mevalonate pathway-dependent, exogenous mevalonate was added to simvastatin-treated CD1d+ cells. Although exogenous mevalonate was able to substantially reverse the effect of simvastatin on CD1d-mediated Ag presentation (Fig. 3,A), this approach could not distinguish between whether simvastatin impaired Ag presentation by inhibiting lipid biosynthesis or by isoprenylation (or both). To address this question, LMTK-CD1d1 cells were treated with simvastatin in the presence or absence of the geranylgeranylation precursor GGPP, the farnesylation precursor FPP, or the cholesterol biosynthesis precursor squalene. The addition of GGPP, but not FPP, significantly reversed the inhibitory effects of simvastatin on CD1d-mediated Ag presentation (Fig. 3, B and C). In contrast, squalene had no effect (Fig. 3 D). These results strongly suggest that the simvastatin-induced inhibition of Ag presentation by CD1d is solely prenylation-dependent.
The inhibition of geranylgeranylation alters CD1d-mediated Ag presentation
To further investigate the role of prenylation in the simvastatin-induced inhibition of CD1d-mediated Ag presentation, BMDCs were treated with simvastatin, the farnesylation-specific inhibitor FTI-277, or geranylgeranylation-specific inhibitor GGTI-298 for 24 h. CD1d-mediated Ag presentation was significantly reduced in cells treated with simvastatin or GGTI-298, whereas FTI-277 treatment had only a modest effect (Fig. 4,A). Neither of the inhibitors altered the cell surface expression of CD1d molecules (data not shown). Geranylgeranylation is controlled by two enzymes: GGTase I and GGTase II (44). To determine whether GGTase I and/or GGTase II can regulate CD1d-mediated Ag presentation, the activity of these enzymes was reduced using an RNAi system. Consistent with the GGTI-298 treatment above (Fig. 4,A), LMTK-CD1d1 cells transfected with RNAi for either GGTase I, or GGTase II in particular, were impaired in their Ag presentation ability (Fig. 4 B).
Inhibition of prenylation impairs MHC class II (but not MHC class I)-mediated Ag presentation
As with CD1d molecules, MHC class II molecules traffic through late endocytic compartments (19), and statins have been shown to also affect Ag presentation by this pathway (36, 37), although the role of prenylation was not studied. Thus, to determine whether the inhibition of prenylation alters MHC class II-mediated Ag presentation under the same conditions as observed with CD1d, HLA-DR4-transfected LMTK-CD1d1 (L-CD1d-DR4) cells (40) were treated with HSA in the presence or absence of simvastatin and cocultured with the HLA-DR-restricted, HSA-specific T cell hybridoma 17.9. These same cells were cocultured with NKT cell hybridomas as a control side-by-side. MHC class II-mediated Ag presentation was reduced in a concentration-dependent manner without a change in cell surface MHC class II molecule expression (Fig. 5,A and data not shown), similar to that observed with CD1d. Statins have been shown to reduce the cell surface expression of induced (e.g., by CD40 ligation or IFN-γ) MHC class II molecules, whereas the constitutive expression of MHC class II was found to be unaltered (36, 37, 45). For the present study, the effect of prenylation inhibitors on murine MHC class II (I-Ak)-mediated Ag presentation was also determined by treating the mouse B cell lymphoma TA3 or BMDCs from C3H/HeJ (H-2k) mice in the presence or absence of HEL with different concentrations of simvastatin, FTI-277, or GGTI-298. Simvastatin and GGTI-298 caused a significant reduction in MHC class II-mediated Ag presentation, whereas FTI-277 had only a modest effect in TA3 cells (Fig. 5,B) and BMDCs (Fig. 5 C). Therefore, these results suggest that under similar conditions that reduce Ag presentation by CD1d, statins also alter MHC class II-mediated Ag presentation by targeting the geranylgeranylation pathway.
CD1d molecules are structurally similar to MHC class I molecules (19). Therefore, it was also important to know whether prenylation inhibition alters Ag presentation by MHC class I. To address this question, murine MC57G fibroblasts were treated with vehicle, simvastatin, GGTI-298, or FTI-277 overnight. The cells were mock-infected or infected with VV for 6 h in the presence or absence of prenylation inhibitors. The cells were then washed, fixed, and cocultured with splenocytes from C57BL/6 mice infected 6 days previously with VV as a source of VV-specific CTLs. The production of IFN-γ was used as a measure of VV-specific T cell recognition. Although inhibiting prenylation had no effect on Ag presentation by MHC class I molecules, as expected, the proteosomal inhibitor lactacystin caused a significant reduction (Fig. 5 D).
Intracellular distribution of CD1d is altered in cells following prenylation inhibition
As the inhibition of prenylation caused a reduction in CD1d-mediated Ag presentation without altering its cell surface level, this effect might have been due to intracellular changes caused by preventing prenylation. Thus, LMTK-CD1d1 cells were treated with simvastatin, FTI-277, GGTI-298, or the cholesterol biosynthesis inhibitor AY9944, and colocalization of CD1d with the late endosome/lysosome marker LAMP-1 was analyzed by confocal microscopy. A significant decrease in the colocalization of CD1d and LAMP-1 was observed in cells treated with either simvastatin or GGTI-298 (Fig. 6), whereas exposure to FTI-277 or AY9944 had no effect. In agreement with our NKT cell assay results, when cells were treated with simvastatin in the presence of mevalonate, there was a recovery in CD1d/LAMP-1 colocalization (Fig. 6). These data suggest that alterations in CD1d intracellular trafficking following inhibition of prenylation substantially impair Ag presentation by CD1d molecules.
The widespread clinical use of statins has decreased the rate of morbidity and mortality of persons suffering from cardiovascular diseases. Besides their lipid-lowering effects, statins also improve or restore endothelial function by decreasing inflammation and enhancing the stability of atherosclerotic plaques (45). Additionally, statins also regulate prenylation and are immunomodulatory (6). For example, statins promote Th2 polarization and inhibit Th1 development, resulting in reduced inflammation in various model systems (46). Thus, these drugs have an advantage over other cholesterol-lowering agents in the treatment of cardiovascular disease, where inflammation plays a critical role in its progression. In the present study, inhibiting cholesterol did not reduce Ag presentation by CD1d. Further, squalene, a cholesterol precursor, was not able to reverse the inhibition of CD1d-mediated Ag presentation by simvastatin. This suggests that the ability of simvastatin and other statins to regulate Ag presentation by CD1d is independent of their effects on cholesterol biosynthesis. Besides cholesterol, other pathways targeted by statins are those that regulate the supply of isoprenoids, particularly FPP and GGPP (7). In the context of Ag presentation by CD1d, we found that inhibition of geranylgeranylation, but not farnesylation, significantly impaired CD1d (and MHC class II)-mediated Ag presentation. Statins alter the prenylation of specific GTPases (47) (supplemental Fig. 3), important for the trafficking of molecules through the endocytic pathway. Prior studies have shown that Rho and Rab GTPases can regulate intracellular trafficking of a variety of proteins and the efficiency of Ag processing and presentation within various endocytic compartments in dendritic cells and B cells (49, 50). Thus, inhibiting the prenylation of these GTPases may have contributed to the altered trafficking of Ag presenting molecules (i.e., both CD1d and MHC class II) through endocytic compartments and consequent effects on Ag presentation (19). Our observation that either simvastatin or GGTI-298 can affect the intracellular localization of CD1d is consistent with this idea.
Atherosclerosis is an immunoinflammatory disease of arterial walls where both innate and adaptive immune responses contribute to disease development and progression (22). Oxidized low-density lipoprotein is a major cause of injury in atherosclerosis, and adaptive immune responses to oxidized low-density lipoprotein contribute to the development of the disease. Lipid and peptide Ags derived from oxidized low-density lipoprotein bind to CD1d and MHC class II molecules to activate proinflammatory T cells (48). A reduction in Ag presentation by either of these pathways results in diminished atherosclerosis. NKT cells have been implicated as proatherogenic in a mouse model of atherosclerosis (49). Furthermore, CD1d-expressing vascular dendritic cells have been found to be in association with NKT cells in atherosclerotic plaques (31). Some dendritic cells interact with T cells within atherosclerotic lesions, whereas others appear to migrate to regional lymph nodes and activate mainstream T cells (50). Such T cell activation requires a transition of dendritic cells from immature to mature forms. In healthy arterial walls, dendritic cells are in their immature form, whereas in atherosclerotic lesions, dendritic cells express high levels of HSP70 that contribute to their activation and maturation (51). NKT cells appear to play a greater role in the progression of atherosclerosis due to their activation by both immature and mature dendritic cells (20). The results of the present study show that the inhibition of prenylation reduces both CD1d- and MHC class II-mediated Ag presentation. Thus, one mode of the anti-atherosclerotic action of statins, in addition to their cholesterol-lowering activity, may be through their inhibitory effect on proinflammatory Ag presentation pathways mediated by both CD1d and MHC class II molecules. In line with this, it is notable that the adoptive transfer of NKT cells or their activation by the CD1d-specific ligand α-GalCer aggravates disease in mouse models of atherosclerosis (22). The present study shows that inhibition of prenylation modulates the effector activity of NKT cells by impairing lipid Ag presentation by CD1d.
How might this translate in vivo? In addition to its well known low-density lipoprotein-lowering properties, simvastatin is not very bioavailable upon oral administration due to its poor absorption (52), despite showing beneficial effects through immune-mediated mechanisms (53, 54). Furthermore, altering Ag presentation by this class of cholesterol-lowering drugs may lead to somewhat increased susceptibility of statin-treated patients to various pathogens, particularly virus infections (3). Therefore, under some conditions, the long-term use of statins may contribute to the impairment of both innate and adaptive immune responses, with consequent effects; such treated patients would simply need to be monitored more closely.
We thank Gail Bishop for the 3A9 and TA3 cells, Janice Blum for the 17.9 T cell hybridoma, Wen Tao and Janardhan Sampath for advice and help with the RT-PCR, and Keith March for helpful discussions.
The authors have no financial conflicts of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health grants RO1 AI46455 and PO1 AI056097 (to R.R.B.), and by NSF CHE-0194682 from the National Science Foundation (to J.G.H.). G.J.R. and R.M.G. were supported by National Institutes of Health Training Grants T32DK007519 and T32HLO7910, respectively.
Abbreviations used in this paper: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; BMDC, bone marrow-derived dendritic cell; α-GalCer, α-galactosylceramide; β-MCD, β-methyl cyclodextrin; FPP, farnesyl pyrophosphate; FTI, farnesyl transferase inhibitor; GGPP, geranylgeranyl pyrophosphate; GGTI, geranylgeranyl transferase inhibitor; HEL, hen egg lysozyme; HSA, human serum albumin; LAMP-1, lysosome-associated membrane protein 1; siRNA, small interfering RNA; VV, vaccinia virus.
The online version of this article contains supplemental material.