Protein (SREBP)-1 and 2. SREBPs are master genes and transcription factors that sense liver cholesterol concentrations. In hepatocytes and adipocytes, activation or extended life span of SREBP proteins, resulting from suppression of activated SREBP degradation, causes the nuclear accumulation of activated SREBP-1 and 2 leading to increased lipogenesis by induction of lipogenetic genes such as the fatty acid synthase (FAS) [9,10]. In addition to these effects, PIs act directly on macrophages, another important player in the formation of atherosclerotic plaques. In vitro studies have shown that exposure of macrophages to HIV PIs increases the accumulation of free cholesterol, activates the unfolded protein response (UPR), induces apoptosis, and promotes transition toward a foam cell phenotype [11]. Macro- phage’s recruitment into the vascular wall is one of the earliest events in atherogenesis. This event is mediated by the up- regulation of the scavenger receptor-dependent uptake of lipoprotein sterols by macrophages in the sub-endothelial space, which contributes to the formation of lipid-laden macrophages [13]. The class B scavenger receptor, CD36, mediates the uptake of oxidized (ox) LDL particles by macrophages [13] and is a well identified target of HIV PIs, although the mechanisms that regulate the induction of its expression/activity in response HIV PIs remain elusive [12]. FXR (NR1H4) is an adopted member of the nuclear receptor super-family of ligand-activated regulatory factor highly expressed in entero-hepatic tissues. In the liver, FXR serves as a bile acid sensor and regulates bile acid synthesis and excretion [14]. In target tissues, primary bile acids, cheno-deoxycholic acid (CDCA) and cholic acid (CA), bind and activate the receptor at micromolar concentrations leading to feedback repression of bile acid biosynthesis, an effect that is primarily achieved by repressing the expression/activity of the cholesterol 7a-hydroxylase (CYP7A1), a gene encoding for a protein that regulates the first, and rate limiting, enzymatic step of cholesterol breakdown. Other than bile acid synthesis, FXR intervenes in a variety of physiological processes. In mice, FXR deficiency results in a pro-atherogenic serum lipoprotein profile [15], while FXR agonism reduces triacylglycerols by repressing the expression and activity of SREBP1c in the liver. The phenotypic readouts of these effects is a reduction of the pro- atherogenic lipid profile and an attenuation of the tendency toward development of atherosclerotic plaques in ApoE2/2 and LDLr2/2 mice [16–17], two genetic models of dyslipidemia-driven athero- sclerosis, as well as in rodents strains characterised by insulin resistance and liver steatosis [16–20]. Further on FXR is expressed by cells of innate immunity and both natural and synthetic FXR ligands counter-regulate macrophage activation induced by Toll- like receptor (TLR)-4 ligands [17]. This regulatory effects of FXR on macrophage effector’s function is inhibitory in nature and translates in vivo in an attenuated formation of aortic plaques as well as reduced expression of CD36 on circulating monocytes [17]. Because development of therapeutic interventions to effectively counteract the complications of HIV PIs based therapy is especially urgent, we have investigated whether FXR agonism protects against dyslipidemia and vascular injury induced by ritonavir, a widely used HIV-PIs, in rodent models of dyslipide- mia-driven atherosclerosis. Results FXR Agonism Protects Against Dyslipidemia Induced By Ritonavir HIV-PIs are thought to influence the development of cardiovas- cular disease primarily through increasing plasma triacylglycerols and cholesterol levels [5–8]. Because FXR gene ablations promotes a pro- FXR and Anti-HIV Therapy atherogenic lipid profile and FXR ligands rescue from dyslipidemia in rodents, we have investigated whether FXR agonism attenuates dyslipidemia caused by ritonavir a widely used HIV-PI in mice. The natural FXR ligand CDCA was used because it effectively reduces plasma triacylglycerols levels in dyslipidemic patients [21,22]. We found that 10 days administration of ritonavir to wild type mice increased plasma triacylglycerols, FFA, cholesterol and LDL. This effect was abrogated by co-treating wild type mice with CDCA, and gemfibrozil, a clinically used PPARa, agonist (Figure 1; p,0.05; n = 5–6 per group). Administering mice with ritonavir had no effect on liver weight and liver weight/liver bodyweight ratio (Figure 2A, n = 5,6; P.0.05), but increased significantly the liver content of triacylglycerols (Figure 2B, n = 5,6; P,0.05). Liver accumulation of triacylglycerols translated in the appearance of liver steatosis as demonstrated by the histopathology analysis shown in Figure 2C. Indeed, staining of liver sections with H&E demonstrates that exposure to ritonavir (5 mg/kg) resulted in a microvescicular steatosis and hepatocyte ballooning with minor portal and lobular inflamma- tion. Administering mice with an FXR and a PPARa agonist in combination with ritonavir effectively reduced the severity of the liver microsteatosis, (Figure 2 C). The morphometric analysis of liver sections stained with Oil red-o, a measure of liver triacylglycerols content, confirmed this pattern and corroborated biochemical tissue analyses (Figure 2D). To gain mechanistic insights on the effect exerted by ritonavir we have profiled the liver expression of genes involved in lipid and cholesterol homeostasis and found that treating mice with ritonavir caused a shift in the liver expression of genes involved in lipid homeostasis including SREBP1c and FAS (Figure 2E; n = 6; P,0.05), and genes involved in cholesterol synthesis, such as SREBP2 and HMCoA synthase, although the later was not statistically significant. Co-treating mice with gemfibro- zil and CDCA, attenuated this in pattern. Specifically, CDCA effectively prevented the induction of SREBP1c and FAS caused by ritonavir (Figure 2E, n = 6; p,0.05), thought that no changes were observed in the expression of SREBP2 and HMCoA synthase (Figure 2E). Administering mice with CDCA also increased the expression of SHP mRNA, an nuclear receptor and FXR target gene. Finally, both CDCA and gemfibrozil increased the liver expression of PPARa mRNA (Figure 2E; n = 6; p,0.05 versus ritonavir alone). FXR gene Ablation Exacerbates the Dyslipidemia Caused by Ritonavir Because these data indicate that FXR agonism effectively reduced the severity of dyslipidemia caused by ritonavir, we have investigated in an independent set of experiments whether FXR gene ablation would exacerbate the dyslipidemia caused by the administration of this HIV-PI. As shown in Figure 3, FXR gene ablation exacerbated the severity of dyslipidemia caused by ritonavir (Figure 3A–F, n = 6–8; P,0.05). In addition, mice harbouring a disrupted FXR gene had a more pronounced liver steatosis (Figure 3H) and developed a specific pattern of liver expression of genes involved in lipid and cholesterol metabolism. The main feature of the later being the robust dysregulation of FAS mRNA expression and the marked reduction of the expression of PPARa and SHP mRNAs (Figure 3I; n = 6; p,0.05). FXR Activation Protects Against Atherosclerosis Development In ApoE2/2 Mice Treated With Ritonavir Treating ApoE2/2 mice with the HIV-PI ritonavir alone or in combination with CDCA, and gemfibrozil for three months had no effect on mice body weight and mortality (data not shown). Administration of ritonavir (5 mg/kg/day) to ApoE2/2 however, PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13238