Statins impair CD1d-mediated antigen presentation through the inhibition of prenylation

Abstract

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.

FIGURE 1

The mevalonate pathway and its targets for prenylation and cholesterol biosynthesis inhibition. Simvastatin and other statins block the biosynthesis of mevalonic acid by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A. The prenylation-specific inhibitors FTI-277 and GGTI-298 inhibit the activities of farnesyl and geranylgeranyl transferases, respectively. The cholesterol biosynthesis inhibitor AY9944 prevents the conversion of 7-dehydrocholesterol into cholesterol by inhibiting 7-dehydrocholesterol reductase.

FIGURE 2

Simvastatin inhibits antigen presentation by CD1d in a cholesterol-independent manner. A, LMTK-CD1d1 cells were treated with the indicated concentrations of simvastatin for 24 h, washed, fixed and co-cultured with the indicated NKT cell hybridomas. *, P<0.05 as compared to vehicle. B, BMDCs were treated with vehicle (DMSO) or simvastatin and co-cultured with NKT cell hybridomas as in A. *, P<0.05 as compared to vehicle. C, LMTK-CD1d1 cells were treated with vehicle or simvastatin (25 and 50 μM) for 24 h. The cells were then washed and treated with the CD1d-specific ligand α-GalCer (500 ng/ml) for 1 h and co-cultured as above. *, P<0.05 as compared to vehicle; **, P<0.05 as compared to simvastatin. D, LMTK-CD1d1 cells were treated with the indicated concentrations of the cholesterol biosynthesis inhibitor AY9944 for 24 h. The cells were washed and co-cultured with the NKT hybridomas as above. *, P<0.05 as compared to vehicle.

FIGURE 3

Mevalonate and GGPP (but not FPP or squalene) reverse simvastatin-induced inhibition of CD1d-mediated antigen presentation. LMTK-CD1d1 cells were treated for 24 h with A, vehicle or simvastatin (50 μM) in the presence or absence of mevalonate (200 and 400 μM): *, P<0.05 as compared to vehicle; **, P<0.05 as compared to simvastatin. B, GGPP (10 and 25 μM): *, P<0.05 as compared to vehicle; **, P<0.05 as compared to simvastatin. C, FPP (10 and 25 μM): *, P<0.05 as compared to vehicle. D, Squalene (SQL; 10 and 25 μM). The cells were then co-cultured with the indicated NKT cell hybridomas as above. *, P<0.05 as compared to vehicle.

FIGURE 4

Inhibition of geranylgeranyl (but not farnesyl) transferase activity substantially impairs CD1d-mediated antigen presentation. LMTK-CD1d1 cells were treated for 24 h with the indicated concentrations of the geranylgeranyl transferase inhibitor GGTI-298 (a) or farnesyl transferase inhibitor FTI-277 (b) and co-cultured with NKT cell hybridomas; *, P<0.05 as compared to vehicle. (c) BMDCs were treated with vehicle or the indicated concentrations of simvastatin, GGTI-298 or FTI-277 for 24 h and co-cultured with the indicated NKT cell hybridomas. *, P<0.05 as compared to vehicle. (d) LMTK-CD1d1 cells transfected with control or GGTase I and II-specific siRNA-expressing vectors were co-cultured with NKT cell hybridomas as above. *, P<0.05 as compared to vehicle.

FIGURE 5

Simvastatin inhibits antigen presentation by MHC class II (but not MHC class I) molecules. (a) L-CD1d-DR4 cells were treated with the indicated concentrations of simvastatin for 24 h in the presence or absence of HSA (10 μM). The cells were then washed, fixed and co-cultured with the 17.9 T cell hybridoma at a T cell:APC ratio of 2:1 for 24 h. (b) Murine TA3 (I-A^k) or (c) C3H/HeJ BMDCs were treated with the indicated concentrations of simvastatin, GGTI-298 or FTI-277 in the presence or absence of HEL (1 mg/ml) for 24 h. The cells were washed, fixed and co-cultured with the 3A9 T cell hybridoma as in (a). The level of IL-2 in the supernatant was measured by ELISA. (d) Effect of prenylation inhibition on MHC class I-mediated antigen presentation. Murine MC57G cells (treated with vehicle or the indicated inhibitors) were mock- or VV-infected for 6 h, fixed and then co-cultured with VV-specific CTL overnight. IFN-γ production was measured by ELISA. Mock-infected (●); lactacystin (■); GGTI-298 (▲); FTI-277 (▼); vehicle (◆); simvastatin (○).

FIGURE 6

Altered intracellular localization of CD1d induced by simvastatin is reversed by mevalonate. LMTK-CD1d1 cells were treated with vehicle (DMSO), simvastatin (50 μM), mevalonate (400 μM), simvastatin + mevalonate, GGTI-298 (10 μM), FTI-277 (μM) or AY9944 (10 μM). The cells were then washed, fixed and stained for CD1d (green) and LAMP-1 (red) and analyzed by confocal microscopy (a). Six random fields were chosen to calculate the percent co-localization (b). *, P<0.05 as compared to vehicle; **, P<0.05 as compared to simvastatin.