Myeloid-Derived Suppressive Cells Deficient in Liver X Receptor α Protected From Autoimmune Hepatitis.

Myeloid-derived suppressor cells (MDSCs) emerge as a promising candidate for the immunotherapy of autoimmune hepatitis (AIH). However, targets for modulating MDSC in AIH are still being searched. Liver X receptors (LXRs) are important nuclear receptors linking lipid metabolism and immune responses. Despite the extensive studies of LXR in myeloid compartment, its role in MDSCs is currently less understood. Herein, expression of LXRα was found to be upregulated in AIH patients and colocalized with hepatic MDSCs. In ConA-induced hepatitis, deletion of LXRα led to increased expansion of MDSCs in the liver and alleviated the hepatic injury. MDSCs in LXRα-/- mice exhibited enhanced proliferation and survival comparing with WT mice. T-cell proliferation assay and adoptive cell transfer experiment validated the potent immunoregulatory role of MDSCs in vitro and in vivo. Mechanistically, MDSCs from LXRα-/- mice possessed significantly lower expression of interferon regulatory factor 8 (IRF-8), a key negative regulator of MDSC differentiation. Transcriptional activation of IRF-8 by LXRα was further demonstrated. Conclusion: We reported that abrogation of LXRα facilitated the expansion of MDSCs via downregulating IRF-8, and thereby ameliorated hepatic immune injury profoundly. Our work highlights the therapeutic potential of targeting LXRα in AIH.

Retinal and optic nerve degeneration in liver X receptor ß knockout mice.

The retina is an extension of the brain. Like the brain, neurodegeneration of the retina occurs with age and is the cause of several retinal diseases including optic neuritis, macular degeneration, and glaucoma. Liver X receptors (LXRs) are expressed in the brain where they play a key role in maintenance of cerebrospinal fluid and the health of dopaminergic neurons. Herein, we report that LXRs are expressed in the retina and optic nerve and that loss of LXRß, but not LXRα, leads to loss of ganglion cells in the retina. In the retina of LXRß-/- mice, there is an increase in amyloid A4 and deposition of beta-amyloid (Aß) aggregates but no change in the level of apoptosis or autophagy in the ganglion cells and no activation of microglia or astrocytes. However, in the optic nerve there is a loss of aquaporin 4 (AQP4) in astrocytes and an increase in activation of microglia. Since loss of AQP4 and microglial activation in the optic nerve precedes the loss of ganglion cells, and accumulation of Aß in the retina, the cause of the neuronal loss appears to be optic nerve degeneration. In patients with optic neuritis there are frequently AQP4 autoantibodies which block the function of AQP4. LXRß-/- mouse is another model of optic neuritis in which AQP4 antibodies are not detectable, but AQP4 function is lost because of reduction in its expression.

Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells.

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRß). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.

Liver X Receptors Suppress Activity of Cholesterol and Fatty Acid Synthesis Pathways To Oppose Gammaherpesvirus Replication.

Gammaherpesviruses are oncogenic pathogens that persist in ~95% of the adult population. Cellular metabolic pathways have emerged as important regulators of many viral infections, including infections by gammaherpesviruses that require several lipid synthetic pathways for optimal replication. Liver X receptors (LXRs) are transcription factors that are critical regulators of cellular fatty acid and cholesterol synthesis pathways. Not surprisingly, LXRs are attractive therapeutic targets in cardiovascular disease. Here we describe an antiviral role for LXRs in the context of gammaherpesvirus infection of primary macrophages. We show that type I interferon increased LXR expression following infection. Surprisingly, there was not a corresponding induction of LXR target genes. Rather, LXRs suppressed the expression of target genes, leading to decreased fatty acid and cholesterol synthesis, two metabolic pathways that support gammaherpesvirus replication. This report defines LXR-mediated restriction of cholesterol and lipid synthesis as an intrinsic metabolic mechanism to restrict viral replication in innate immune cells.IMPORTANCE Fatty acid and cholesterol synthesis pathways of the host play important roles in diverse biological systems. Importantly, these two metabolic pathways are also usurped by a number of viruses to facilitate viral replication. In this report, we show that suppression of these pathways by liver X receptors in primary macrophages creates an intrinsic antiviral state that attenuates gammaherpesvirus replication by limiting viral access to the two metabolic pathways.

Loss of the Liver X Receptors Disrupts the Balance of Hematopoietic Populations, With Detrimental Effects on Endothelial Progenitor Cells.

BACKGROUND: The liver X receptors (LXRs; α/ß) are nuclear receptors known to regulate cholesterol homeostasis and the production of select hematopoietic populations. The objective of this study was to determine the importance of LXRs and a high-fat high-cholesterol diet on global hematopoiesis, with special emphasis on endothelial progenitor cells (EPCs), a vasoreparative cell type that is derived from bone marrow hematopoietic stem cells. METHODS AND RESULTS: Wild-type and LXR double-knockout (Lxrαß -/- ) mice were fed a Western diet (WD) to increase plasma cholesterol levels. In WD-fed Lxrαß -/- mice, flow cytometry and complete blood cell counts revealed that hematopoietic stem cells, a myeloid progenitor, and mature circulating myeloid cells were increased; EPC numbers were significantly decreased. Hematopoietic stem cells from WD-fed Lxrαß -/- mice showed increased cholesterol content, along with increased myeloid colony formation compared with chow-fed mice. In contrast, EPCs from WD-fed Lxrαß -/- mice also demonstrated increased cellular cholesterol content that was associated with greater expression of the endothelial lineage markers Cd144 and Vegfr2, suggesting accelerated differentiation of the EPCs. Treatment of human umbilical vein endothelial cells with conditioned medium collected from these EPCs increased THP-1 monocyte adhesion. Increased monocyte adhesion to conditioned medium-treated endothelial cells was recapitulated with conditioned medium from Lxrαß -/- EPCs treated with cholesterol ex vivo, suggesting cholesterol is the main component of the WD inducing EPC dysfunction. CONCLUSIONS: LXRs are crucial for maintaining the balance of hematopoietic cells in a hypercholesterolemic environment and for mitigating the negative effects of cholesterol on EPC differentiation/secretome changes that promote monocyte-endothelial adhesion.

Dysregulation of Kupffer Cells/Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα Knockout Male Mice.

Liver X receptor (LXR) α expression is mainly localized to metabolic tissues, such as the liver, whereas LXRß is ubiquitously expressed. LXRα is activated by oxysterols and plays an important role in the regulation of lipid metabolism in metabolic tissues. In macrophages, LXRs stimulate reverse cholesterol transport and regulate immune responses. Although a high-cholesterol diet induces severe steatohepatitis in LXRα-knockout (KO) mice, the underlying mechanisms linking lipid metabolism and immune responses remain largely unknown. In this study, we investigated the role of LXRα in the pathogenesis of steatohepatitis by assessing the effects of a high-fat and high-cholesterol diet (HFCD) on hepatic immune cell proportion and function as well as lipid metabolism in wild-type (WT) and LXRα-KO mice. HFCD feeding induced severe steatohepatitis in LXRα-KO mice compared with WT mice. These mice had higher cholesterol levels in the plasma and the liver and dysregulated expression of LXR target and proinflammatory genes in both whole liver samples and isolated hepatic mononuclear cells. Flow cytometry showed an increase in CD68+CD11b+ Kupffer cells/macrophages and a decrease in invariant natural killer T cells in the liver of HFCD-fed LXRα-KO mice. These mice were more susceptible to lipopolysaccharide-induced liver injury and resistant to inflammatory responses against α-galactosylceramide or concanavalin-A treatment. The findings provide evidence for activation of bone marrow-derived Kupffer cells/macrophages and dysfunction of invariant natural killer T cells in LXRα-KO mouse liver. These findings indicate that LXRα regulates hepatic immune function along with lipid metabolism and protects against the pathogenesis of nonalcoholic steatohepatitis.

Liver X receptors constrain tumor development and metastasis dissemination in PTEN-deficient prostate cancer.

Advanced prostate cancer (PCa) is a clinical challenge as no curative therapeutic is available. In this context, a better understanding of metastasis and resistance mechanisms in PCa is an important issue. As phosphatase and tensin homolog (PTEN) loss is the most common genetic lesion in such cancer, we investigate human data sets for mechanisms that can constrain cancer evolution in this setting. Here we report a liver X receptor (LXR) signature, which tightly correlates with PTEN loss, in PCa. Accordingly, the LXR pathway is deregulated in prostate carcinomas in Pten-null mice. Genetic ablation of LXRs in Pten-null mice, exacerbates PCa invasiveness and metastatic dissemination, which involves mesenchymal transition and accumulation of matrix metalloproteinases. Mechanistically, PTEN deletion governed LXR transcriptional activity through deregulation of cholesterol de novo synthesis, resulting in accumulation of endogenous LXR ligands. Our study therefore reveals a functional circuit linking PTEN and LXR, and highlights LXRs as metabolic gatekeepers that are able to constrain PCa progression.Treatment of prostate cancer, especially in its advanced stage, is still challenging; therefore, strategies to prevent metastatic dissemination are of great interest. Here the authors reveal a crucial role for liver X receptors in suppressing prostate carcinogenesis and metastatic progression in PTEN-null tumors.

Morin, a novel liver X receptor α/ß dual antagonist, has potent therapeutic efficacy for nonalcoholic fatty liver diseases.

BACKGROUND AND PURPOSE: Morin is a natural occurring flavonoid in many dietary plants and has a wide range of beneficial effects on metabolism; however, the mechanism underlying its action remains elusive. EXPERIMENTAL APPROACH: A reporter assay and the time-resolved FRET assay were used to identify morin as a dual antagonist of liver X receptor (LXR)-α and -ß. Morin (100 mg. 100 g-1 diet) was administered to high-fat diet-induced obese or LXRß-/- mice. The pharmacological effects and mechanism of action of morin were evaluated by Western blot and RT-PCR analyses. KEY RESULTS: From the in vitro assays, morin was shown to be a dual antagonist of LXRα and LXRß. In vivo, morin blunted the development of liver hepatic steatosis, reduced body weight gains, lowered triglyceride levels and improved glucose and insulin tolerance in mice fed a high-fat diet. Mechanistically, morin inhibited 3T3-L1 adipocyte differentiation and lipid formation in human hepatic HepG2 cells and suppressed the mRNA expression of genes downstream of LXR. Consistently, the effects of morin on metabolic disorders were attenuated in LXRß-/- mice. CONCLUSION AND IMPLICATIONS: Our data reveal that morin is a dual antagonist of LXRα and LXRß and suggest that morin may alleviate hepatic steatosis and other associated metabolic disorders via the suppression of LXR signalling and, therefore, shows promise as a novel therapy or nutraceutical for nonalcoholic fatty liver disease.

Deficiency of liver-X-receptor-α reduces glucose uptake and worsens post-myocardial infarction remodeling.

Liver X receptor α (LXRα) is an endogenous protective receptor against ischemic heart diseases. However, whether LXRα regulated glucose metabolism in ischemic heart diseases has not been investigated. In this study we investigated the involvement of LXRα on glucose metabolism in cardiac remodeling after myocardial infarction (MI). MI was induced in mice by permanent ligation of the left anterior descending coronary artery (LCA). Genetic LXRα deletion significantly worsened cardiac remodeling and impaired cardiac function at 4 weeks after MI. Cardiac 18F-fluorodeoxyglucose (FDG) uptake by positron emission tomography (PET) demonstrated that the FDG standardized uptake value (SUV) was significantly lower in LXRα-/- mice as compared to WT mice. Mechanistically, GLUT1/4 and AMPK phosphorylation were significantly downregulated while CD36 expression was markedly upregulated in LXRα-/- mice. This study demonstrated that deficiency of LXRα decreased glucose uptake after MI, resulting in a metabolic shift that suppressed glucose metabolism, which was in association with adverse cardiac remodeling.

EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux.

OBJECTIVE: The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and ß (LXRß, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. APPROACH AND RESULTS: We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. CONCLUSIONS: In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1.

Liver X receptor ß increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts.

Liver X receptors (LXRs) including LXRα and LXRß are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRß but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRß regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRß-/- mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRß-/- mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRß increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRß represents a key regulator of body water homeostasis.

Liver X Receptor ß Is Involved in Formalin-Induced Spontaneous Pain.

Increasing evidence indicates that the liver X receptor(LXR) ß modulates inflammatory pain. However, the molecular mechanisms through which LXRß modulates pain are unclear. Here, we found that LXRß-null mice responded more strongly to acute noxious stimuli than wild-type (WT) littermates (in the hot plate and Hargreaves tests) and had augmented tonic inflammatory pain (in the formalin test). This increased reactivity to inflammatory pain was accompanied by enhanced formalin-evoked Fos and pERK staining of second-order nociceptive neurons. Immunohistochemistry showed that the expression of CGRP, SP, and IB4 was increased in the lamina I-II of the lumbar dorsal horns in formalin-injected LXRß knockout (KO) mice compared with the WT controls. In addition, LXRß deletion in the mice enhanced the formalin-induced inflammation with more activated microglia and astrocytes in the spinal cord. Furthermore, the levels of pro-inflammatory cytokines (IL-1ß ,TNF-α) as well as NFκB in the formalin-injected paw were elevated by the loss of LXRß. Taken together, these data indicate that LXRß is involved in acute as well as inflammatory pain, and thus, it may be considered as a new target for the development of analgesics.