The Activation of Liver X Receptors Inhibits Toll-Like Receptor-9-Induced Foam Cell Formation ROSALINDA SORRENTINO,* SILVANA MORELLO, SHUANG CHEN,EDUARDO BONAVITA, AND ALDO PINTO Pharmaceutical Science Department, University of Salerno, 84084 Fisciano, Salerno, Italy Toll-like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of athero activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine ma treated with CpG-ODN, TLR9 agonist, and oxidized particles of LDL (Paz-PC) and FCF was analyzed by means of Oil Red The administration of CpG-ODN plus Paz-PC onto macrophages increased the amount of lipid droplets, correlated to inc tumor necrosis factor (TNF)-a, IFNb, and IP-10. The underlying mechanism by which TLR9 ligation influenced Paz-PC in kB- and IRF7-dependent, as observed by higher levels of phosphorylated IkBa, increased nuclear translocation of the p6 levels of the total IKKa protein and higher release of interferon-dependent cytokines, such as IP-10. Liver X receptors (L lipid cellular transport and negatively modulate TLR-dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG-ODN plus Paz-PC stimulation. In this condition, we observed decreased lev translocation of the p65 subunit, related to the higher presence of LXRa into the nucleus. TNF-a, IP-10, and IFNb levels the administration of GW3965 following CpG-ODN and Paz-PC treatment. In conclusion, the activation of TLR9 facilitate of foam cells in an NF-kB- and IRF7-dependent manner, countered by the activation of LXRs. This study further support anti-atherosclerotic target. J. Cell.Physiol. 223:158–167, 2010. ß 2010 Wiley-Liss, Inc. Atherosclerosis, formerly considered a lipid accumulation vascular disease, is actually classified as a focal and chronic ongoing inflammatory response (Michelsen and Arditi, 2006; Sorrentino and Arditi, 2009). The modification of lipoproteins in the sub-endothelial matrix attracts diverse and multifactorial leukocytes, including monocytes, T cells, B cells, mast cells, dendritic cells (DCs), and neutrophils in the vessel wall (Michelsen and Arditi, 2006; Naiki et al., 2008; Sorrentino and Arditi, 2009). The activation of monocytes in the subendothelial matrix leads to the differentiation into macrophages that ingest modified lipids and become foam cells (Naiki et al., 2008). This phenomenon promotes the overcoming release of a variety of inflammatory mediators, with the resultant of a chronic inflammation. Several outstanding studies suggested the relevance of Toll- like receptors (TLRs) in the atherosclerosis pathology, either using human or animal models (Sorrentino and Arditi, 2009). TLRs recognize a wide range of bacterial molecular patterns, instructing the innate immune system to a specific adaptive response (Michelsen and Arditi, 2006; Sorrentino and Arditi, 2009). TLR4 and TLR2 participate to the induction and progression of the atheroma (Michelsen et al., 2004; Michelsen and Arditi, 2006; Mullick et al., 2008; Sorrentino and Arditi, 2009). Foam cell formation (FCF) is a key event for the accumulation of lipids and it can be enhanced by the presence of bacteria or pathogen-associated molecular patterns (PAMPs) (Naiki et al., 2008; Sorrentino and Arditi, 2009). Indeed, C.pneumoniae, Gram-negative bacteria, can promote FCF in the presence of oxidized low-density lipoprotein (LDL) (Chen et al., 2008). The authors related this effect to the recognition of the bacterium by TLR2 and TLR4 (Naiki et al., 2008), that exacerbated the atheroma in a mouse model of atherosclerosis (Naiki et al., 2008). The progression/exacerbation of the atherosclerotic plaques was also observed when LPS (5), TLR4 ligand, Pam3CySK4 (Mullick et al., 2008), TLR2 ligand, or other pathogens, such as H. influenzae, P. gingivalis, were administered into atherosclerotic-prone mice (Michelsen and Arditi, 2006). Hence, bacteria or bacteria-derived molecules, such as nucle acids, could facilitate the formation of the atherosclerotic plaques. The recognition of PAMPs by antigen presenting ce (APCs) can promote the activation of cytoplasmic receptors, such as TLR9, other than the membrane TLR2 and TLR4. Thu the main goal of our study was to evaluate the implication of TLR9 in FCF in the presence of oxidized LDL particles. Liver X receptors (LXRs), nuclear receptors, play a pivotal role for lipid metabolism and are master-regulators of cholesterol metabolism (Bensinger and Tontonoz, 2008). LX controlreverse cholesterol metabolism and also counterbalance the pro-inflammatory effects of TLRs (Castri et al., 2003). Of therapeutic interest, LXRs and TLRs exert a negative cross-talk onto each others’ signaling pathway (Castrillo et al., 2003; Chen et al., 2008; Naiki et al., 2008), a example, in the case of TLR4 activation that is negatively Abbreviations: LXRs,liver X receptors; TLR9,toll-like receptor 9; FCF,foam cell formation. This article was originally published on 4 Jan 2010. An error was subsequently identified. Shuang Chen has been added as the third author in the corrected version published 25 January 2010. Summary Sentence: The activation of TLR9 facilitates the Formation of Foam Cells in an NF-kB- and IRF7-dependent manner, contrasted by the activation of LXRs. Contract grant sponsor: University of Salerno (FARB 2007–2008). *Correspondence to: Rosalinda Sorrentino, Pharmaceutical Science Department, University of Salerno, 84084 Fisciano, Salerno, Italy.E-mail: rosalinda.sorrentino@googlemail.com Received 9 October 2009; Accepted 12 November 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 4 January 2010. DOI: 10.1002/jcp.22022 ORIGINAL ARTICLE 158 J o u r n a l o f J o u r n a l o f Cellular Physiology Cellular Physiology ß 2 0 1 0 W I L E Y - L I S S , I N C .