FXR and Anti-HIV Therapy Figure 1. FXR and PPAR a ligands attenuate dyslipidemia induced by the HIV protease inhibitor ritonavir in wild type mice. C57Bl/6 mice at the age of 8 weeks were administered 5 m/kg/day ritonavir alone or in combination with the FXR agonist CDCA, 15 mg/kg/day, or the PPARa agonist gemfibrozil, 100 mg/kg/day, for 10 days. Data shown are plasmatic level of ALT, FFA, triacylglycerols, cholesterol, HDL, LDL and glucose measured at the end of the study period. In each panel, data are mean6 SE of 12 animal per group. *P,0.05 ritonavir versus naive; **P,0.05 treatments versus ritonavir alone. doi:10.1371/journal.pone.0013238.g001 resulted in a dramatic increase in the extension of aortic plaques (Figure 4A, n = 10 per group; P,0.05). The development of aortic plaques was significantly attenuated by co-treating ApoE2/2 mice fed ritonavir with CDCA and gemfibrozil (P,0.05 and P,0.01 respectively; Figure 4A). Analysis of Sudan IV staining of whole aortas, to measure neutral lipid content in the vessel, demonstrated that ritonavir exacerbated plaque lipid accumulation in ApoE2/2 mice and that this effect was robustly attenuated by both CDCA and gemfibrozil (Figure 4B; P,0.01). When compared to their wild-type counterparts, ApoE2/2 mice showed a twofold increase in plasma cholesterol and triglycerides levels, a fourfold increase in plasma LDL levels and threefold decrease in plasma HDL levels (Figure 4C–H; n = 10; P,0.01). Twelve-week administration of ritonavir failed to modify this pattern and pharmacological treatments were also unable to attenuate this pro-atherogenic lipid profile (Figure 4; n = 10; P,0.05 versus ritonavir). Because, the later finding was in a striking contrast with previous observations demonstrating that FXR agonism protects against development of pro-atherogenic lipid profile in ApoE2/2, we have investigated whether resistance to the ipolipemic effects of CDCA and gemfibrozil could be ascribed to the administration of ritonavir. For this purpose ApoE2/2 mice were treated with CDCA and gemfibrozil for 12 weeks without ritonavir. Results of these experiments demonstrated that both CDCA and gemfibrozil were effective in reducing plasma levels of triacylglycer- ols and total cholesterol in ApoE2/2 in the absence of ritonavir (Figure S1), strongly supporting the view that the HIV-PI was responsible for the inability of FXR and PPARa agonists to rescue ApoE2/2 mice from development dyslipidemia. Finally, ritonavir treatment had no effect on plasma glucose (Figure 4G). Treating ApoE2/2 mice with ritonavir caused no changes in the pattern of liver expression of genes involved in lipid and cholesterol metabolism, and similarly, no changes in the expression of these gene was detected in mice administered CDCA and gemfibrozil (Figure 5A–G). FXR Agonism Counter-regulates the effect of Ritonavir on CD36 expression In vivo and in vitro As shown in Figure 6, blood monocytes isolated from ApoE2/2 had a significant higher expression of CD36 in comparison to wild type naive mice (44.961.9 versus 29.662.8; n = 6 P,0.05). Treating ApoE2/2 mice with ritonavir for 12 weeks caused a further increase of CD36 expression on circulating monocytes (from 44.961.9 to 58.363.3; n = 6; P,0.05). However, co- treating ApoE2/2 mice with CDCA and gemfibrozil resulted in a robust attenuation of CD36 expression on monocyte’s cell surface and completely reversed the effect exerted by ritonavir (Figure 6A; P,0.05 and P,0.01 versus ApoE2/2 na¨ıve and ApoE2/2 plus ritonavir, respectively). Previous studies have shown that HIV-PIs disrupt cellular lipid homeostasis by increasing the levels of SREBPs and by inducing a SREBP-related increase in CD36 expression and lipid uptake [9,10,11,23]. We have therefore examined whether HIV-PIs directly modulate CD36 expression in cultured macrophages. For this purpose RAW264.7 cells, a macrophage cell line, were directly challenged with HIV-PIs. Results from this experiment (Figure 6B) demonstrate that ritonavir (Figure 6B–D) and atazanavir, another clinically used HIV-PI (Figure S2), increased the cell surface expression of CD36 in a concentration-dependent manner (Figure 6B, n = 5; P,0.05), and that this effect was abrogated by co-treating the RAW264.7 cells with CDCA and gemfibrozil (Figure 6C, n = 5; p,0.05). Modulation of CD36 expression by FXR and PPARa agonism correlated with a reduced uptake ac- LDL by cultured macrophages. Thus, CDCA and gemfibrozil PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13238