Identification of biological markers of liver X receptor (LXR) activation at the cell surface of human monocytes.

BACKGROUND: Liver X receptor (LXR) α and LXR ß (NR1H3 and NR1H2) are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and ß and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD) markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping. METHODOLOGY/PRINCIPAL FINDINGS: By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment. CONCLUSIONS/SIGNIFICANCE: We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers.

Liver X receptor-mediated induction of cholesteryl ester transfer protein expression is selectively impaired in inflammatory macrophages.

OBJECTIVE: Cholesteryl ester transfer protein (CETP) is a target gene for the liver X receptor (LXR). The aim of this study was to further explore this regulation in the monocyte-macrophage lineage and its modulation by lipid loading and inflammation, which are key steps in the process of atherogenesis. METHODS AND RESULTS: Exposure of bone marrow-derived macrophages from human CETP transgenic mice to the T0901317 LXR agonist increased CETP, PLTP, and ABCA1 mRNA levels. T0901317 also markedly increased CETP mRNA levels and CETP production in human differentiated macrophages, whereas it had no effect on CETP expression in human peripheral blood monocytes. In inflammatory mouse and human macrophages, LXR-mediated CETP gene upregulation was inhibited, even though ABCA1, ABCG1, and SREBP1c inductions were maintained. The inhibition of CETP gene response to LXR agonists in inflammatory cells was independent of lipid loading (ie, oxidized LDL increased CETP production in noninflammatory macrophages with a synergistic effect of synthetic LXR agonists). CONCLUSIONS: LXR-mediated induction of human CETP expression is switched on during monocyte-to-macrophage differentiation, is magnified by lipid loading, and is selectively lost in inflammatory macrophages, which suggests that inflammatory cells may not increase the circulating CETP pool on LXR agonist treatment.

Induction of transglutaminase 2 by a liver X receptor/retinoic acid receptor alpha pathway increases the clearance of apoptotic cells by human macrophages.

RATIONALE: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that are involved in the control of cholesterol homeostasis and inflammatory response. Human monocytes and macrophages express high levels of these receptors and are appropriate cells to study the response to LXR agonists. OBJECTIVE: The purpose of this study was to identify new LXR targets in human primary monocytes and macrophages and the consequences of their activation. METHODS AND RESULTS: We show that LXR agonists significantly increase the mRNA and protein levels of the retinoic acid receptor (RAR)alpha in primary monocytes and macrophages. LXR agonists promote RARalpha gene transcription through binding to a specific LXR response element on RARalpha gene promoter. Preincubation of monocytes or macrophages with LXR agonists before RARalpha agonist treatment enhances synergistically the expression of several RARalpha target genes. One of these genes encodes transglutaminase (TGM)2, a key factor required for macrophage phagocytosis. Accordingly, the combination of LXR and RARalpha agonists at concentrations found in human atherosclerotic plaques markedly enhances the capabilities of macrophages to engulf apoptotic cells in a TGM2-dependent manner. CONCLUSIONS: These results indicate an important role for LXRs in the control of phagocytosis through an RARalpha-TGM2-dependent mechanism. A combination of LXR/RARalpha agonists that may operate in atherosclerosis could also constitute a promising strategy to improve the clearance of apoptotic cells by macrophages in other pathological situations.

7beta-Hydroxycholesterol and 25-hydroxycholesterol-induced interleukin-8 secretion involves a calcium-dependent activation of c-fos via the ERK1/2 signaling pathway in THP-1 cells: oxysterols-induced IL-8 secretion is calcium-dependent.

Oxysterols found in oxidized low-density lipoproteins are probably involved in the appearance of atheroma; some are cytotoxic and some able to induce cytokine secretion. An oxysterol-induced interleukin-8 (IL-8) secretion in human monocytes/macrophages has been previously noticed, but the mechanisms remained unclear. In this paper, we investigated the signaling pathways leading to the induction of IL-8 secretion in monocytic THP-1 cells treated with 7beta-hydroxycholesterol, a cytototoxic oxysterol, or with 25-hydroxycholesterol, an oxysterol non-cytotoxic toward this cell line. The oxysterol-induced IL-8 secretion appears to be a calcium-dependent phenomenon as shown by the use of calcium channel blockers, which strongly decreased IL-8 secretion and IL-8 messenger RNA (mRNA) levels. Fluo-3 staining used in flow cytometry and video microscopy revealed an oxysterol-induced Ca(2+) influx, varying according to the oxysterol studied, leading to the activation of the MEK/ERK1/2 pathway as demonstrated by Western blot analysis. ERK activation led to an increase of c-fos mRNA and/or an activation of c-fos. Luciferase reporter gene assay using constructs of the human IL-8 gene promoter and Transam assay revealed the involvement of the AP-1 transcription factor in oxysterol-dependent IL-8 secretion. These results demonstrate that oxysterol-induced IL-8 secretion is a calcium-dependent phenomenon involving the MEK/ERK1/2 pathway leading to the activation of IL-8 gene via AP-1 (c-fos).