Myeloid LXR (Liver X Receptor) Deficiency Induces Inflammatory Gene Expression in Foamy Macrophages and Accelerates Atherosclerosis.

BACKGROUND: Cholesterol loaded macrophage foam cells are a prominent feature of atherosclerotic plaques. Single-cell RNA sequencing has identified foam cells as TREM2 (triggering receptor expressed on myeloid cells 2) positive populations with low expression of inflammatory genes, resembling the TREM2 positive microglia of neurodegenerative diseases. Cholesterol loading of macrophages in vitro results in activation of LXR (liver X receptor) transcription factors and suppression of inflammatory genes. METHODS: To test the hypothesis that LXRs mediate anti-inflammatory effects in Trem2 expressing atherosclerotic plaque foam cells, we performed RNA profiling on plaque cells from hypercholesterolemic mice with myeloid LXR deficiency. RESULTS: Myeloid LXR deficiency led to a dramatic increase in atherosclerosis with increased monocyte entry, foam cell formation, and plaque inflammation. Bulk cell-RNA profiling of plaque myeloid cells showed prominent upregulation of inflammatory mediators including oxidative, chemokine, and chemotactic genes. Single-cell RNA sequencing revealed increased numbers of foamy TREM2-expressing macrophages; however, these cells had reduced expression of the Trem2 gene expression module, including phagocytic and cholesterol efflux genes, and had switched to a proinflammatory and proliferative phenotype. Expression of Trem2 was suppressed by inflammatory signals but not directly affected by LXR activation in bone marrow-derived macrophages. CONCLUSIONS: Our current studies reveal the key role of macrophage LXRs in promoting the Trem2 gene expression program and in suppressing inflammation in foam cells of atherosclerotic plaques.

Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency.

BACKGROUND: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. METHODS: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr-/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2V617F). RESULTS: Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk-/- platelets promoted increased NETosis when incubated with Lnk-/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk-/- platelets, as well as increased priming and response of Lnk-/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk-/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ controls. CONCLUSIONS: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

Increased SLAMF7high monocytes in myelofibrosis patients harboring JAK2V617F provide a therapeutic target of elotuzumab.

Monocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.

Liver X Receptors Regulate Cholesterol Metabolism and Immunity in Hepatic Nonparenchymal Cells.

Excess dietary cholesterol intake and the dysregulation of cholesterol metabolism are associated with the pathogenesis and progression of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and fibrosis. Hepatic accumulation of free cholesterol induces activation of nonparenchymal cells, including Kupffer cells, macrophages, and hepatic stellate cells, which leads to persistent inflammation and fibrosis. The nuclear receptors liver X receptor α (LXRα) and LXRß act as negative regulators of cholesterol metabolism through the induction of hepatocyte cholesterol catabolism, excretion, and the reverse cholesterol transport pathway. Additionally, LXRs exert an anti-inflammatory effect in immune cell types, such as macrophages. LXR activation suppresses acute hepatic inflammation that is mediated by Kupffer cells/macrophages. Acute liver injury, diet-induced steatohepatitis, and fibrosis are exacerbated by significant hepatic cholesterol accumulation and inflammation in LXR-deficient mice. Therefore, LXRs regulate hepatic lipid metabolism and immunity and they are potential therapeutic targets in the treatment of hepatic inflammation that is associated with cholesterol accumulation.

Synthesis and In Vitro Evaluation of Novel Liver X Receptor Agonists Based on Naphthoquinone Derivatives.

We aimed to synthesize novel liver X receptor (LXR) agonists with potent agonist activity and subtype selectivity. Our synthetic scheme started with naphthoquinone derivatives, such as menadione and 2,3-dichloro-1,4-naphthoquinone. We introduced different substituents into the naphthoquinone structures, including aniline, piperidine, pyrrolidine, and morpholine, in one or two steps, and thus, we produced 14 target compounds. All 14 synthetic ligands were tested to determine whether they mediated LXR-mediated transcriptional activity. We investigated the transcriptional activity of each compound with two types of receptors, LXRα and LXRß. Among all 14 compounds, two showed weak LXRß-agonist activity, and two others exhibited potent LXRα-agonist activity. We also performed docking studies to obtain a better understanding of the modes of compound binding to LXR at the atomic level. In conclusion, we successfully synthesized naphthoquinone derivatives that act as LXRα/ß agonists and selective LXRα agonists.

Structural development of tetrachlorophthalimides as liver X receptor ß (LXRß)-selective agonists with improved aqueous solubility.

LXRß-selective agonists are promising candidates to improve atherosclerosis without increasing plasma or hepatic TG levels. We have reported a series of tetrachlorophthalimide analogs as an LXRß-selective agonist. However, they exhibited poor aqueous solubility probably due to its high hydrophobicity and highly rigid and plane structure. In this report, we present further structural development of tetrachloro(styrylphenyl)phthalimides as the LXRß-selective agonists with improved aqueous solubility.

Activation of LXRs using the synthetic agonist GW3965 represses the production of pro-inflammatory cytokines by murine mast cells.

BACKGROUND: The activation of liver X receptor (LXR) α or LXRß negatively regulates the expression of pro-inflammatory genes in mammalian cells. We recently reported that 25-hydroxycholesterol, a representative LXR-activating oxysterol, suppresses IL-6 production in mouse mast cells (MCs) following its engagement of the high-affinity IgE receptor (FcεRI). This finding suggests that murine MCs express functional LXRs; however, the mechanisms underlying the LXR-dependent repression of the MC-mediated production of pro-inflammatory cytokines, including IL-6, are poorly understood. Therefore, we employed the synthetic LXR ligand GW3965 to examine the functions of LXRα and LXRß in the production of pro-inflammatory cytokines by murine bone marrow-derived MCs (BMMCs). METHODS: We prepared BMMCs from wild-type (WT), LXRα(-/-), and LXRα/ß(-/-) mice. Each group of BMMCs was pretreated with GW3965 and then stimulated with IgE+antigen (Ag) or lipopolysaccharide (LPS). Cytokine production was then analyzed using specific ELISA kits. RESULTS: The activation of LXRs by GW3965 significantly attenuated the production of IL-1α and IL-1ß, but not of IL-6, in the WT and LXRα(-/-) BMMCs stimulated with IgE+Ag. However, GW3965 treatment decreased the production of IL-1α, IL-1ß, and IL-6 in WT and LXRα(-/-) BMMCs upon stimulation with LPS, while the GW3965-mediated suppression of cytokine production was nearly absent from the LXRα/ß(-/-) BMMCs. CONCLUSIONS: These findings demonstrate, for the first time, that the activation of LXRs by GW3965 attenuates the antigen- or LPS-induced production of pro-inflammatory cytokines, such as IL-1α and IL-1ß, in murine MCs and that LXRß plays an important role in the LXR-mediated repression of cytokine production.

Protective effect of S-allyl-L-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.

Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca(2+) and leads to activation of calpain, a Ca(2+)-dependent cysteine protease. We have shown previously that S-allyl-L-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, L-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of µ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited µ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 µM), which prevents interaction of Ca(2+) with the Ca(2+)-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 µM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of µ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca(2+)-binding site.

Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1ß secretion.

AIMS: Neutrophil extracellular trap formation (NETosis) increases atherosclerotic plaque vulnerability and athero-thrombosis. However, mechanisms promoting NETosis during atherogenesis are poorly understood. We have shown that cholesterol accumulation due to myeloid cell deficiency of the cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/G1) promotes NLRP3 inflammasome activation in macrophages and neutrophils and induces prominent NETosis in atherosclerotic plaques. We investigated whether NETosis is a cell-intrinsic effect in neutrophils or is mediated indirectly by cellular crosstalk from macrophages to neutrophils involving IL-1ß. METHODS AND RESULTS: We generated mice with neutrophil or macrophage-specific Abca1/g1 deficiency (S100A8CreAbca1fl/flAbcg1fl/fl or CX3CR1CreAbca1fl/flAbcg1fl/fl mice, respectively), and transplanted their bone marrow into low-density lipoprotein receptor knockout mice. We then fed the mice a cholesterol-rich diet. Macrophage, but not neutrophil Abca1/g1 deficiency activated inflammasomes in macrophages and neutrophils, reflected by caspase-1 cleavage, and induced NETosis in plaques. NETosis was suppressed by administering an interleukin (IL)-1ß neutralizing antibody. The extent of NETosis in plaques correlated strongly with the degree of neutrophil accumulation, irrespective of blood neutrophil counts, and neutrophil accumulation was decreased by IL-1ß antagonism. In vitro, IL-1ß or media transferred from Abca1/g1-deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1ß on NETosis was blocked by an NLRP3 inhibitor. CONCLUSION: These studies establish a new link between inflammasome-mediated IL-1ß production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1ß appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation.

Differential expression and function of alternative splicing variants of human liver X receptor α.

The liver X receptor α (LXRα) is a nuclear receptor that is involved in regulation of lipid metabolism, cellular proliferation and apoptosis, and immunity. In this report, we characterize three human LXRα isoforms with variation in the ligand-binding domain (LBD). While examining the expression of LXRα3, which lacks 60 amino acids within the LBD, we identified two novel transcripts that encode LXRα-LBD variants (LXRα4 and LXRα5). LXRα4 has an insertion of 64 amino acids in helix 4/5, and LXRα5 lacks the C-terminal helices 7 to 12 due to a termination codon in an additional exon that encodes an intron in the LXRα1 mRNA. LXRα3, LXRα4, and LXRα5 were expressed at lower levels compared with LXRα1 in many human tissues and cell lines. We also observed weak expression of LXRα3 and LXRα4 in several tissues of mice. LXR ligand treatment induced differential regulation of LXRα isoform mRNA expression in a cell type-dependent manner. Whereas LXRα3 had no effect, LXRα4 has weak transactivation, retinoid X receptor (RXR) heterodimerization, and coactivator recruitment activities. LXRα5 interacted with a corepressor in a ligand-independent manner and inhibited LXRα1 transactivation and target gene expression when overexpressed. Combination of LXRα5 cotransfection and LXRα antagonist treatment produced additive effects on the inhibition of ligand-dependent LXRα1 activation. We constructed structural models of the LXRα4-LBD and its complexes with ligand, RXR-LBD, and coactivator peptide. The models showed that the insertion in the LBD can be predicted to disrupt RXR heterodimerization. Regulation of LXRα pre-mRNA splicing may be involved in the pathogenesis of LXRα-related diseases.

Vitamin D receptor activation enhances benzo[a]pyrene metabolism via CYP1A1 expression in macrophages.

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Hairless modulates ligand-dependent activation of the vitamin D receptor-retinoid X receptor heterodimer.

The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds to the vitamin D receptor (VDR) and regulates various physiological and pharmacological processes. Secondary bile acids, such as lithocholic acid (LCA), also act as endogenous VDR ligands. The molecular basis of ligand-selective VDR action remains largely unknown. Hairless (HR) acts as a coregulator of VDR through a direct interaction. HR mutations confer an alopecia phenotype similar to VDR mutations in mice and humans, but the underlying molecular mechanisms have not been elucidated. We examined the effect of HR on VDR activation induced by 1,25(OH)(2)D(3) and LCA. HR repressed VDR transactivation induced by both 1,25(OH)(2)D(3) and LCA. HR also repressed transactivation of VDR E269A and R391A mutants, but less effectively than that of wild-type VDR. These residues are involved in retinoid X receptor (RXR) heterodimer allosteric communication, through which information from ligands is transmitted to dimer and coactivator interfaces. In the presence of HR cotransfection, LCA activated these VDR mutants more effectively than wild-type VDR. In mammalian two-hybrid assays, HR enhanced the association of VDR with a corepressor, nuclear receptor corepressor. These findings indicate that HR affects VDR-RXR heterodimer allosteric communication and corepressor complex formation. Interestingly, HR knockdown in keratinocyte-derived HaCaT cells increased ligand-induced cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) expression but suppressed expression of cathelicidin antimicrobial peptide, indicating that HR acts not only as a corepressor but also as a coactivator. HR may be a VDR modulator that affects the RXR allosteric communication network in order to regulate transcription in a gene-selective manner.

Erythroid lineage Jak2V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis.

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk.

Contrasting functional responses of resident Kupffer cells and recruited liver macrophages to irradiation and liver X receptor stimulation.

In the murine liver, there are two major macrophage populations, namely resident Kupffer cells (resKCs) with phagocytic activity and recruited macrophages (recMφs) with cytokine-producing capacity. This study was performed to clarify the functional differences between these two populations, focusing on their susceptibility to radiation and response to stimulation via liver X receptors (LXRs), which are implicated in cholesterol metabolism and immune regulation. Liver mononuclear cells (MNCs) were obtained from C57BL/6 (WT) mice with or without 2 Gy irradiation, and the phagocytic activity against Escherichia coli (E. coli) as well as TNF-α production were compared between the two macrophage populations. To assess LXR functions, phagocytosis, TNF-α production, and endocytosis of acetylated low-density lipoprotein (LDL) were compared after synthetic LXR ligand stimulation. Furthermore, LXRα/ß knockout (KO) mice and LXRα KO mice were compared with WT mice. Irradiation decreased intracellular TNF-α production by recMφs but did not affect the phagocytic activity of resKCs. In vitro LXR stimulation enhanced E. coli phagocytosis by resKCs but decreased E. coli-stimulated TNF-α production by recMφs. Phagocytic activity and acetylated LDL endocytosis were decreased in both LXRα/ß KO mice and LXRα KO mice, with serum TNF-α levels after E. coli injection in the former being higher than those in WT mice. In conclusion, resKCs and recMφs exhibited different functional features in response to radiation and LXR stimulation, highlighting their distinct roles liver immunity and lipid metabolism.

Liver X receptors regulate natural killer T cell population and antitumor activity in the liver of mice.

The nuclear receptors liver X receptor α (LXRα) and LXRß are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/ß in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRß in NKT cells using mice deficient for LXRα and/or LXRß, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/ß-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/ß-KO hepatic mononuclear cells and in LXRα/ß-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/ß-KO mice. LXRα/ß-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/ß-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRß are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

Frontline Science: Concanavalin A-induced acute hepatitis is attenuated in vitamin D receptor knockout mice with decreased immune cell function.

The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D, 1α,25-dihydroxyvitamin D3 , and regulates various physiologic processes, such as bone and calcium metabolism, cellular proliferation and differentiation, and immunity. VDR is highly expressed in the intestine, kidney, bone, and macrophages, but is expressed at a low level in the liver. The liver is a major metabolic organ and also acts as an immune gateway for dietary nutrients and xenobiotics. In this study, we investigated the function of VDR in hepatic immune cells, such as Kupffer cells/macrophages, utilizing VDR knockout (KO) mice. We showed that VDR is functionally expressed in hepatic mononuclear cells, specifically resident Kupffer cells. We examined the role of VDR in acute hepatitis induced by concanavalin A (Con-A) and found that Con-A-induced hepatitis is attenuated in VDR-KO mice compared to wild-type (WT) mice. Con-A-induced hepatitis is known to be mediated by NKT cell activation, cytokine production, and reactive oxygen species (ROS) production in Kupffer cells/macrophages. However, the proportions of Kupffer cells/macrophages and the NKT cell activation were similar in the liver of WT and VDR-KO mice and inflammatory cytokine gene expression was increased in VDR-KO mice. On the other hand, plasma and hepatic ROS levels were decreased in the liver of VDR-KO mice compared to WT mice. The phagocytic activity of resident Kupffer cells and hepatic neutrophils were also decreased in VDR-KO mice. Therefore, VDR is necessary for Con-A-induced acute hepatitis and plays an important role in hepatic immune cell functions.

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 regulate hepatic F4/80 + CD11b+ Kupffer cells/macrophages and innate immune responses in mice.

The liver X receptors (LXRs), LXRα and LXRß, are nuclear receptors that regulate lipid homeostasis. LXRs also regulate inflammatory responses in cultured macrophages. However, the role of LXRs in hepatic immune cells remains poorly characterized. We investigated the role of LXRs in regulation of inflammatory responses of hepatic mononuclear cells (MNCs) in mice. Both LXRα and LXRß were expressed in mouse hepatic MNCs and F4/80+ Kupffer cells/macrophages. LXRα/ß-knockout (KO) mice had an increased number of hepatic MNCs and elevated expression of macrophage surface markers and inflammatory cytokines compared to wild-type (WT) mice. Among MNCs, F4/80+CD11b+ cells, not F4/80+CD11b- or F4/80+CD68+ cells, were increased in LXRα/ß-KO mice more than WT mice. Isolated hepatic MNCs and F4/80+CD11b+ cells of LXRα/ß-KO mice showed enhanced production of inflammatory cytokines after stimulation by lipopolysaccharide or CpG-DNA compared to WT cells, and LXR ligand treatment suppressed lipopolysaccharide-induced cytokine expression in hepatic MNCs. Lipopolysaccharide administration also stimulated inflammatory cytokine production in LXRα/ß-KO mice more effectively than WT mice. Thus, LXR deletion enhances recruitment of F4/80+CD11b+ Kupffer cells/macrophages and acute immune responses in the liver. LXRs regulate the Kupffer cell/macrophage population and innate immune and inflammatory responses in mouse liver.

1α-Hydroxy derivatives of 7-dehydrocholesterol are selective liver X receptor modulators.

The nuclear receptors liver X receptor (LXR) α and LXRß are involved in the regulation of lipid metabolism, inflammation, immunity, cellular proliferation, and apoptosis. Oxysterols are endogenous LXR ligands, and also interact with other nuclear and membrane receptors. We previously reported that a phytosterol derivative with a 1α-hydroxy group acts as a potent LXR agonist with intestine-selective action and that 25-hydroxy and 26/27-hydroxy metabolites of 7-dehydrocholesterol (7-DHC) exhibit partial LXR agonism. In this study, we report that 1α-hydroxy derivatives of 7-DHC, 1α-OH-7-DHC and 1,25-(OH)2-7-DHC, act as LXR modulators. Luciferase reporter gene assays showed that 1α-OH-7-DHC activates LXRα and LXRß and that 1,25-(OH)2-7-DHC activates both LXRs and vitamin D receptor. Examination of cofactor peptide association showed that the 1α-hydroxy derivatives, specifically 1,25-(OH)2-7-DHC, induce association of coactivator/corepressor peptide in a different manner from the agonist T0901317. Docking modeling and alanine mutational analysis of LXRα demonstrated that 1,25-(OH)2-7-DHC interacts with LXRα residues in a manner distinct from potent agonists, such as T0901317 and 24(S),25-epoxycholesterol. 1α-OH-7-DHC and 1,25-(OH)2-7-DHC induced expression of LXR target genes in a cell type- and gene-selective manner. 1,25-(OH)2-7-DHC effectively suppressed lipopolysaccharide-stimulated proinflammatory gene expression in an LXR-dependent manner. Therefore, 1α-hydroxy derivatives, such as 1,25-(OH)2-7-DHC, are unique LXR modulators with selective agonistic activity and potent transrepression function. These oxysterols have potential as LXR-targeted therapeutics for inflammatory disease.

Development of Tetrachlorophthalimides as Liver†X Receptorâ€ ß (LXRß)-Selective Agonists.

Liver X receptor (LXR) agonists are candidates for the treatment of atherosclerosis via induction of ABCA1 (ATP-binding cassette A1) gene expression, which contributes to reverse cholesterol transport (RCT) and to cholesterol efflux from the liver and intestine. However, LXR agonists also induce genes involved in lipogenesis, such as SREBP-1c (sterol regulatory binding element protein 1c) and FAS (fatty acid synthase), thereby causing an undesirable increase in plasma and hepatic triglyceride (TG) levels. Recent studies indicate that LXRα contributes to lipogenesis in liver, and selective LXRß activation improves RCT in mice. Therefore, LXRß-selective agonists are promising candidates to improve atherosclerosis without increasing plasma or hepatic TG levels. However, the ligand-binding domains in the two LXR isoforms α/ß share high sequence identity, and few LXR ligands show subtype selectivity. In this study we identified a tetrachlorophthalimide analogue as an LXRß-selective agonist. Structural development led to (E)-4,5,6,7-tetrachloro-2-(2-styrylphenyl)isoindoline-1,3-dione (24 a), which shows potent and selective LXRß agonistic activity in reporter gene assays. In binding assays, compound 24 a bound to LXRß preferentially over LXRα. It also induced the expression of ABCA1 mRNA but not SREBP-1c mRNA in cells. Compound 24 a appears to be a promising lead compound for therapeutic agents to treat atherosclerosis without the side effects induced by LXRα/ß dual agonists.