FXR and Anti-HIV Therapy Figure 2. FXR and PPAR a ligands attenuate liver steatosis induced by the HIV protease inhibitor ritonavir in wild type mice. Panel A. Liver weight/liver bodyweight ratio after 10 days of administration of ritonavir alone or combination with CDCA or gemfibrozil. Panel B. Liver triacylglycerols (mg/mg protein) content. Treating mice with CDCA and gemfibrozil protected against increased liver triacylglycerols accumulation caused by ritonavir. In both panel A and B, data are mean6 SE of 12 animal per group. *P,0.05 ritonavir versus naive; **P,0.05 treatments versus ritonavir alone. Panel C and D. Histophatologic analysis of livers obtained from wild type mice administered with ritonavir alone or in combination with CDCA or gemfibrozil. Panel C, H&E staining, and panel D, Oil-Red-O staining, showing liver accumulation of triacylglycerols and moderate steatosis. Treatment of mice with ritonavir (5 mg/kg) caused the appearance of microvescicular steatosis and hepatocyte ballooning with minor portal and lobular inflammation. Administering mice with an FXR and PPARa agonist in combination with ritonavir effectively reduced the severity of the liver microsteatosis. E&O, magnification 206; Oil red O, magnification 1006. Panel E. RT-PCR analysis of liver expression of genes involved in lipid and cholesterol homeostasis and nuclear receptors. Treatment with ritonavir induced the expression of genes involved in triacylglycerols synthesis (SREBP1c and FAS), as well as genes involved in cholesterol synthesis (HMCoA synthase). CDCA effectively reduced SREBP1c and FAS accumulation caused by ritonavir while no changes are were observed in the expression of SREBP2 and HMCoA synthase. Administering mice with CDCA also increased the expression of SHP mRNA. Finally, both CDCA and gemfibrozil increased the liver expression of PPARa mRNA. In each panel, data are mean6 SE of 6 animal per group. *P,0.05 ritonavir versus naive; **P,0.05 treatments versus ritonavir alone. doi:10.1371/journal.pone.0013238.g002 effectively attenuated ac-HDL uptake caused by ritonavir (Figure 6D, n = 5; P,0.05 versus ritonavir alone). In addition, exposure of RAW264.7 cells to GW4064, a non steroidal FXR ligand, attenuated CD36 induction caused by ritonavir and atazanavir (P,0.05; n = 4; Figure S2). We have then examined the mechanism involved in CD36 induction caused by ritonavir. As illustrated in Figure 7A exposure of RAW264.7 to ritonavir resulted in robust increase in the expression of CD36 mRNAs (P,0.05, n = 4). This pattern was significantly shifted by treating the cells with CDCA. Thus exposure of RAW 264.7 cells to the FXR ligand, but not to gemfibrozil, attenuated induction of CD36 caused by ritonavir and reduced the expression of SREBP1c mRNA by approximately 50%, while caused a 4–5 fold increase in SHP mRNA (Figure 7 B– C; P,0.05, versus ritonavir). Importantly, both gemfibrozil and CDCA increased the expression of ABCA1, a gene involved in cholesterol secretion by macrophages (Figure 7D; P,0.05 versus ritonavir alone). In addition to these genomic effects, ritonavir interfered with cell handling of SREBP1c. Thus exposure of RAW264.7 to ritonavir leads to its translocation to the nucleus, an event that is associated with binding of this regulatory factor to its target genes (Figure 7E), This effect was robustly attenuated by CDCA but not by gemfibrozil, indicating that FAXR and PPARa agonists act on different steps of post-translation regulation of CD36. The expression of the CD36 gene should be properly regulated to ensure its functions [24,25]. Because an involvement of SREBPs in the activation of CD36 is an interesting possibility of coordination of a homeostatic balance between fatty acids and sterols, we have examined whether SREBP1c mediates CD36 mRNA upregulation caused by HIV-PIs. For this purpose expression of SREBP1c in RAW264.7 macrophages was knocked down by transfecting the cells with a validated anti-SREBP1c siRNAs. As shown in Figure 7F, transfecting the cells with anti- SREBP1c siRNAs resulted in robust downregulation of SREBP1c mRNA (approximately 70%, P,0.05 versus naive cells). The PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13238