Inés Pineda-Torra

Transcriptional regulation of macrophage arginase 1 expression and its role in atherosclerosis

Atherosclerosis results from a metabolic imbalance and chronic arterial inflammation and macrophages are key during the initiation and progression of atherosclerotic lesions. A number of macrophage subsets have been identified in atherosclerotic plaques. Arginase 1 (Arg1), a marker for the M2 anti-inflammatory subset, hydrolyzes l-arginine into urea and ornithine, a precursor to l-proline and polyamines, which are implicated in tissue repair and wound healing. Additionally, Arg1 inhibits nitric oxide-mediated inflammatory pathways by competing with iNOS for the same substrate, l-arginine. Therefore, changes in Arg1 expression in macrophages may affect the development of atherosclerosis. Here, we present an overview of the transcriptional regulation of macrophage Arg1, focusing on the nuclear receptor family of ligand-activated transcription factors, and the relevance of this regulation to atherosclerosis.

Isolation, Culture, and Polarization of Murine Bone Marrow-Derived and Peritoneal Macrophages

Macrophages are the most specialized phagocytic cells, and acquire specific phenotypes and functions in response to a variety of external triggers. Culture of bone marrow-derived or peritoneal macrophages from mice represents an exceptionally powerful technique to investigate macrophage phenotypes and functions in response to specific stimuli, resembling as much as possible the conditions observed in various pathophysiological settings. This chapter outlines protocols used to isolate and culture murine bone marrow-derived and peritoneal macrophages. Furthermore, we describe how these macrophages can be polarized to obtain specific macrophage subsets with special relevance to atherosclerosis.

Modulation of Macrophage Gene Expression via Liver X Receptor α Serine 198 Phosphorylation

ABSTRACT In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by liver X receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRα serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRα at S198 is nonphosphorylated. In bone marrow-derived macrophages (BMDMs), we also observed induction of CCR7 by ligands that promote nonphosphorylated LXRα S198, and this was lost in LXR-deficient BMDMs. LXRα occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing nonphosphorylated LXRα (RAW-LXRα S198A) compared to RAW264.7 cells expressing wild-type (WT) phosphorylated LXRα (RAW-LXRα WT). Expression profiling of ligand-treated RAW-LXRα S198A cells compared to RAW-LXRα WT cells revealed induction of cell migratory and anti-inflammatory genes and repression of proinflammatory genes. Modeling of LXRα S198 in the nonphosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with the presence of sites for protein interaction. Therefore, gene transcription is regulated by LXRα S198 phosphorylation, including that of antiatherogenic genes such as CCR7.

Liver X receptors in immune cell function in humans

The liver X receptors (LXRs), LXRα and LXRβ, are transcription factors with well-established roles in the regulation of lipid metabolism and cholesterol homeostasis. In addition, LXRs influence innate and adaptive immunity, including responses to inflammatory stimuli, proliferation and differentiation, migration, apoptosis and survival. However, the majority of work describing the role of LXRs in immune cells has been carried out in mouse models, and there are a number of known species-specific differences concerning LXR function. Here we review what is known about the role of LXRs in human immune cells, demonstrating the importance of these receptors in the integration of lipid metabolism and immune function, but also highlighting the need for a better understanding of the species, isoform, and cell-type specific effects of LXR activation.

Post-translational modifications of lipid-activated nuclear receptors: Focus on metabolism.

Posttranslational modifications (PTMs) occur to nearly all proteins, are catalyzed by specific enzymes, and are subjected to tight regulation. They have been shown to be a powerful means by which the function of proteins can be modified, resulting in diverse effects. Technological advances such as the increased sensitivity of mass spectrometry–based techniques and availability of mutant animal models have enhanced our understanding of the complexities of their regulation and the effect they have on protein function. However, the role that PTMs have in a pathological context still remains unknown for the most part. PTMs enable the modulation of nuclear receptor function in a rapid and reversible manner in response to varied stimuli, thereby dramatically altering their activity in some cases. This review focuses on acetylation, phosphorylation, SUMOylation, and O-GlcNAcylation, which are the 4 most studied PTMs affecting lipid-regulated nuclear receptor biology, as well as on the implications of such modifications on metabolic pathways under homeostatic and pathological situations. Moreover, we review recent studies on the modulation of PTMs as therapeutic targets for metabolic diseases.

Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed.

Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity.

Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors.

Phosphorylation of LXRα impacts atherosclerosis regression by modulating monocyte/macrophage trafficking

LXRα activation in macrophages enhances regression of atherosclerotic plaques in mice by regulating genes crucial for cholesterol efflux, cell motility and inflammation. Diabetes, however, impairs plaque regression in mice. LXRα is phosphorylated at serine 198 (pS198), which affects the expression of genes controlling inflammation, lipid metabolism and cell movement. We hypothesize that LXRα function is affected by hyperglycemia through changes in LXRα pS198. Indeed, macrophages cultured in diabetes relevant high glucose versus normal glucose display alterations in LXR-dependent gene expression and increased LXRα pS198. We therefore examined the consequence of disrupting LXRα phosphorylation (S196A in mouse LXRα) during regression of atherosclerosis in normal and diabetic mice. We find that phosphorylation deficient LXRα S196A reduces macrophage retention in plaques in diabetes, which is predicted to be anti-atherogenic and enhance plaque regression. However, this favorable effect on regression is masked by increased monocyte infiltration in the plaque attributed to leukocytosis in LXRα S196A mice. RNA-seq of plaque macrophages from diabetic S196A mice shows increased expression of chemotaxis and decreased expression of cell adhesion genes, consistent with reduced macrophage retention by LXRα S196A. Thus, the non-phosphorylated form of LXRα precludes macrophage retention in the plaque. Our study provides the first evidence for a physiological role of LXRα phosphorylation in modulating atherosclerosis regression. Compounds that prevent LXRα phosphorylation or ligands that induce the conformation of non-phosphorylated LXRα may selectively enhance macrophage emigration from atherosclerotic plaques.

PS5:87 Juvenile-onset sle immunopathogenesis could be associated with altered immune cell plasma membrane lipids and lipoprotein metabolism

Background Juvenile-onset systemic lupus erythematosus (JSLE) is an autoimmune disorder characterised by immune dysregulation, chronic inflammation and increased cardiovascular risk. Our findings in adult-onset SLE link immune cell dysregulation with defects in plasma membrane signalling platforms (lipid rafts). In JSLE little is known about the immune profile or whether abnormal lipid metabolism contributes to pathogenesis. Methods Flow cytometry was used to measure metabolic marker expression on immune cell subsets from 39 healthy donors (HCs) and 35 JSLE patients. Metabolic biomarker analysis including lipoprotein composition was performed on matching serum. Results JSLE patients had significantly elevated membrane lipid rafts in T-cells, B-cells and plasmacytoid dendritic cells compared to HCs suggesting dysregulated membrane receptor signalling. Furthermore, lipid raft expression correlated positively with cell activation markers, disease activity, erythrocyte sedimentation rate and dsDNA titre and negatively with complement protein C3 supporting the hypothesis that altered metabolism is associated with JSLE pathogenesis. Importantly, ROC curve analysis showed that lipid raft expression on these cell types is an excellent diagnostic of high disease activity in JSLE. Metabolomic analysis of matching serum revealed that high disease activity patients had significantly decreased atheroprotective high density lipoproteins (HDL) and increased atherogenic low density lipoproteins (LDL) suggesting altered transport of lipids. In addition, lipids associated with membrane rafts such as sphingomyelin, phosphatidylcholine, phosphoglycerides and cholesterol correlated negatively with HDL in high disease activity patients but positively in low disease activity patients. Immune cell lipid rafts correlated positively with LDL and negatively with HDL together suggesting altered lipid uptake/efflux from these cells; this may alter immune cell signalling in JSLE patients. Stratification of patients based on their lipid profile by hierarchical clustering revealed 3 groups that were unique in both immnophenotype and clinical presentation. Conclusions Differences in the metabolic profiles of immune cell subsets and lipoprotein lipid transport in JSLE contribute to disease pathogenesis and severity. Regulation of lipid metabolism may therefore have therapeutic benefit for JSLE patients providing a dual effect of reducing inflammation and atherosclerotic risk. These therapeutics may perform better in patients that present specific clinical and phenotypic features.

Changes In LXRα Phosphorylation Promote A Novel Diet-Induced Transcriptome That Alters The Transition From Fatty Liver To Steatohepatitis

Understanding the transition from fatty liver or steatosis to more advanced inflammatory and fibrotic stages of non-alcoholic fatty liver disease (steatohepatitis), is key to define strategies that alter or even reverse the progression of this pathology. The Liver X Receptor alpha (LXRα) controls hepatic lipid homeostasis and inflammation. Here we show that mice carrying a mutation that abolishes phosphorylation at Ser196 (S196A) in LXRα exhibit reduced hepatic inflammation and fibrosis when challenged with a high fat-high cholesterol diet, despite displaying enhanced hepatic lipid accumulation. This protective effect is associated with reduced cholesterol accumulation, a key promoter of lipid-mediated hepatic damage. Reduced steatohepatitis in S196A mice involves the reprogramming of the liver transcriptome by promoting diet-induced changes in the expression of genes involved in endoplasmic reticulum stress, extracellular matrix remodelling, inflammation and lipid metabolism. Unexpectedly, changes in LXRα phosphorylation uncover novel diet-specific target genes, whose regulation does not simply mirror ligand-induced LXR activation. These unique LXRα phosphorylation-sensitive, diet-responsive target genes are revealed by promoting LXR occupancy and cofactor recruitment in the context of a cholesterol-rich diet. Therefore, LXRα phosphorylation at Ser196 critically acts as a novel nutritional sensor that promotes a unique diet-induced transcriptome thereby modulating metabolic, inflammatory and fibrotic responses important in the transition to steatohepatitis.