FXR and Anti-HIV Therapy Figure 3. FXR gene ablation worsens the severity of dyslipidemia caused by ritonavir. Panel A–F. Plasmatic level of ALT, triacylglycerols, cholesterol, HDL, LDL and glucose in wild type and FXR2/2 mice administered ritonavir, 5 mg/kg/day, for 10 days. In each panel, data are mean6 SE of 6 animal per group. *P,0.05 versus wild type naive; **P,0.05 treatments versus ritonavir wild type. Panel G. Liver weight/liver body weight ratio after 10 days administration of ritonavir. In each panel, data are mean6 SE of 8 animal per group. Panel H. Liver histophatology. H&E staining of liver sections obtained from FXR+/+ and in FXR2/2 mice. Magnification 206. Figure I. RT-PCR analysis of liver expression of genes involved in lipid and cholesterol homeostasis and nuclear receptors. Treatment with ritonavir caused a significant dysregulation of FAS mRNA expression in FXR+/+ and FXR2/2 mice. FXR2/2 mice show a severe reduction of liver expression of SHP and PPARa mRNA that is insensitive to ritonavir. In each panel, data are mean6 SE of 6 animal per group. *P,0.05 versus wild type naive; **P,0.05 treatments versus ritonavir wild type. doi:10.1371/journal.pone.0013238.g003 knockout of SREBP1c abrogated the ability of ritonavir to induce CD36 (Figure 7H versus 7G) highlighting a putative role of SREBP1c in mediating the effect of ritonavir on CD36. To gain further insights on the mechanisms involved in the regulation of CD36 by SREBP1c, we have carried out an analysis of the CD36 promoter in the search for a putative binding site for SREBP1c. Because this analysis revealed the presence of a putative sterol regulatory element (SRE)-binding consensus in the promoter of CD36, we have performed a ChIP assay and PCR amplification using primer pairs that cover the putative CD36-SRE consensus site located at 276 in the CD36 promoter. Results from these experiments, however, were negative, indicating that in RAW264.7 cells induction of CD36 expression caused by ritonavir is not mediated by a direct binding of SREBP1c to the CD36 promoter and is likely mediated by activation of additional mechanisms (Figure S3). Discussion FXR is a nuclear receptor that functions as a bile acid sensor and plays a regulatory role in bile acids, cholesterol and lipid homeostasis [26]. Previous studies have shown that FXR might be an interesting target for preventing development of atheroscle- rosis. This contention results from the aggregate evaluation of the fact that FXR gene ablation results in a pro-atherogenic lipoproteins profile [15] and FXR and ApoE double knockout male mice fed an high fat diet develop an accelerated atherosclerosis in comparison to ApoE2/2 single knock out mice [27]. In addition, we and others have demonstrated that FXR activation protects against athero- sclerosis plaque formation in ApoE2/2 and LDLr2/2 mice [16,17]. In the present study we have provided evidence that FXR activation with CDCA effectively protects against dyslipidemia and atherosclerosis development in rodents fed ritonavir, a widely used HIV PI (Figure 8). Despite the fact that synthetic and semi- synthetic FXR ligands are currently investigated [26], we have decided to use CDCA, the natural FXR ligand, for the following reasons. The prevalence of HIV infection is growing in undeveloped countries [1–3] and the cost of co-medications to prevent HAART’s related side effects add significantly to the already elevated cost for HAART itself. Our aim was therefore to exploit a drug that would have beneficial effects without increasing further the costs of HIV management. CDCA has a low cost and is the only FXR ligand available worldwide that has been PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13238