FXR and Anti-HIV Therapy Figure 7. Distinct patterns of gene and protein expression are induced by FXR and PPARa ligands in macrophages challenged with ritonavir. Panel A–D. RT-PCR analysis of gene expression induced by exposure RAW264.7 cells to ritonavir alone or in combination with a PPARa or FXR ligand. Ritonavir slightly increased expression of CD36 mRNA (Figure 7A) while CDCA, but not gemfibrozil, completely abrogated this effect. In addition exposure CDCA, but not to gemfibrozil, reduced SREBP1c mRNA and increased the expression of SHP mRNA (P,0.05 versus ritonavir). In each panel, data are mean6 SE of 5 separate experiments. Panel E. Detection of nuclear translocation of SREBP1c by Western blot analysis. Treatment of RAW264.7 cells with ritonavir increased protein level of SREBP1c and caused its nuclear translocation. Activation of FXR with CDCA reduced total SREBP1c protein and prevented its nuclear translocation. Data shown is representative of three other experiments showing the same pattern. Panel F–H. Transfection of RAW264.7 macrophages with an anti-SREBP1c siRNAs resulted in a robust downregulation of SREBP1c mRNA (<70%, P,0.05 versus naive cells). The knock-down of SREBP1c abrogated the ability of ritonavir to induce CD36 (panel 7H versus panel G). In each panel, data are mean6 SE of 4 separate experiments. doi:10.1371/journal.pone.0013238.g007 including FAS in ApoE2/2 mice, administered a non-fat diet [17]. In the present study we have confirmed that CDCA similarly to its derivative 6-ethyl CDCA, protects against development of a dyslipidemic phenotype in ApoE2/2 mice not administered ritonavir (Figure S1), strongly indicating that feeding ApoE2/2 mice with an HIV PI introduces a further distortion of the metabolic regulation that can not be reverted by FXR and PPARa ligand in this model. The fact that CDCA protects against atherosclerotic plaque formation in ApoE2/2 despite it had no effect on lipid profile, indicates that FXR activation might impact on expression on pro- inflammatory factors [34–40]. Thus, in vivo and in vitro studies, have demonstrated that HIV PIs increase atherosclerotic lesions by a direct action on macrophages [12]. Macrophages play an essential role in atherosclerosis [41]. Gene-deletion and bone marrow–transplantation experiments have provided evidence that macrophages scavenger receptor A and CD36 exert non dispensable effects in mediating the uptake of oxidized LDL by macrophages and promoting the development of atherosclerosis [42,43]. CD36 is the primary mediator of cholesterol accumula- tion in atherosclerosis induced by HIV PIs [12]. One important observation we made is that while administration of ritonavir to ApoE2/2 mice increased the percentage of CD36 positive monocytes, chronic administration of CDCA caused a robust reduction in the percentage of CD14/CD36 positive cells in ApoE2/2 mice treated with ritonavir. A similar pattern was observed when CD36 mRNA expression was measured in the aortic plaques. Similarly to CDCA, gemfibrozil was also effective in attenuating CD36 expression in the aorta and circulating macrophages. These data are consistent with our previous observations indicating that activation of FXR attenuates the expression/function of CD36 in ApoE2/2 naive mice. The above results were confirmed by in vitro studies. Thus, while treating RAW264.7 macrophages with ritonavir and atazanivar resulted in a concentration-dependent induction of CD36 expression, gene and protein, this effect was completely abrogated by treating the cells with CDCA and GW4064 (a synthetic FXR agonist). The concentration of ritonavir used in this experiment is clinically relevant. Indeed, while plasma concentra- tions of ritonavir in HIV infected patients treated with ritonavir PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13238