t0901317 and Inflammation

The Liver X Receptors (LXRs) are oxysterol-activated transcription factors that upregulate a suite of genes that together promote coordinated mobilization of excess cholesterol from cells and from the body. The LXRs, like other nuclear receptors, are anti-inflammatory, inhibiting signal-dependent induction of pro-inflammatory genes by nuclear factor-κB, activating protein-1, and other transcription factors. Synthetic LXR agonists have been shown to ameliorate atherosclerosis and a wide range of inflammatory disorders in preclinical animal models. Although this has suggested potential for application to human disease, systemic LXR activation is complicated by hepatic steatosis and hypertriglyceridemia, consequences of lipogenic gene induction in the liver by LXRα. The past several years have seen the development of multiple advanced LXR therapeutics aiming to avoid hepatic lipogenesis, including LXRβ-selective agonists, tissue-selective agonists, and transrepression-selective agonists. Although several synthetic LXR agonists have made it to phase I clinical trials, none have progressed due to unforeseen adverse reactions or undisclosed reasons. Nonetheless, several sophisticated pharmacologic strategies, including structure-guided drug design, cell-specific drug targeting, as well as non-systemic drug routes have been initiated and remain to be comprehensively explored. In addition, recent studies have identified potential utility for targeting the LXRs during therapy with other agents, such as glucocorticoids and rexinoids. Despite the pitfalls encountered to date in translation of LXR agonists to human disease, it appears likely that this accelerating field will ultimately yield effective and safe applications for LXR targeting in humans.

Topics: Animals; Atherosclerosis; Benzoates; Benzylamines; Gene Expression Regulation; Humans; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Models, Biological; Molecular Targeted Therapy; Sulfonamides

In the general population, high-density lipoprotein (HDL) cholesterol efflux capacity (HCEC) relates inversely to incident cardiovascular events. Previous studies have suggested that HCEC is decreased in HIV and that antiretroviral therapy (ART) initiation might improve HCEC.. To evaluate HCEC in the context of ART initiation and immune activation in HIV.. Baseline HCEC from 10 ART-naive HIV-infected males and 12 prospectively matched non-HIV-infected males were analyzed. In the HIV cohort, HCEC 6 months after elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF) therapy was evaluated. HCEC served as the primary outcome and was measured by the ability of J774 mouse macrophages to efflux cholesterol. Our ex vivo assay used two cholesterol acceptors [apolipoprotein B (apoB)-depleted sera or purified HDL] and modulation of cellular efflux pathways using a liver X receptor (LXR) agonist.. The median age was 34 years [interquartile range (IQR), 27 to 51], and baseline HDL was 46 mg/dL (IQR, 38 to 61). HCEC was significantly greater in the non-HIV-infected subjects than in the HIV-infected subjects at baseline. HCEC, assessed using apoB-depleted sera, significantly increased after ART (no LXR agonist, baseline: median, 8.1%; IQR, 7.0% to 11.9%; after ART: median, 12.9%; IQR, 10.4% to 21.1%; P = 0.006; LXR agonist, baseline, 1.3% ± 1.3%; after ART, 2.5% ± 1.0%; P = 0.02), although not to the levels in the non-HIV-infected subjects (no LXR agonist: median, 14.9%; IQR, 11.5% to 19.1%; LXR agonist: 5.8% ± 1.3%). HCEC, assessed using purified HDL, did not significantly increase after ART. The change in HCEC with ART related inversely to the change in the percentage of CD14-CD16+ (nonclassical) monocytes (ρ = -0.74, P = 0.04) and directly to the change in the percentage of CD14+CD16- (classical) monocytes (ρ = 0.72, P = 0.045).. Our data suggest improvement of HCEC with E/C/F/TDF and a relationship between the ART-induced decrease in immune activation and ART-induced improvement in HCEC.

Topics: Adult; Animals; Anti-HIV Agents; Anti-Retroviral Agents; Anticholesteremic Agents; Biological Transport; Case-Control Studies; Cells, Cultured; Cholesterol, HDL; HIV; HIV Infections; Humans; Hydrocarbons, Fluorinated; Immunity, Innate; Inflammation; Lipid Metabolism; Liver X Receptors; Male; Mice; Middle Aged; Sulfonamides

Oxysterols are oxidized cholesterol derivatives whose systemic levels are found elevated in pregnancy disorders such as gestational diabetes mellitus (GDM). Oxysterols act through various cellular receptors and serve as a key metabolic signal, coordinating inflammation. GDM is a condition of low-grade chronic inflammation accompanied by altered inflammatory profiles in the mother, placenta and fetus. Higher levels of two oxysterols, namely 7-ketocholesterol (7-ketoC) and 7β-hydroxycholesterol (7β-OHC), were observed in fetoplacental endothelial cells (fpEC) and cord blood of GDM offspring. In this study, we tested the effects of 7-ketoC and 7β-OHC on inflammation and investigated the underlying mechanisms involved. Primary fpEC in culture treated with 7-ketoC or 7β-OHC, induced the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling, which resulted in the expression of pro-inflammatory cytokines (IL-6, IL-8) and intercellular cell adhesion molecule-1 (ICAM-1). Liver-X receptor (LXR) activation is known to repress inflammation. Treatment with LXR synthetic agonist T0901317 dampened oxysterol-induced inflammatory responses. Probucol, an inhibitor of LXR target gene ATP-binding cassette transporter A-1 (ABCA-1), antagonized the protective effects of T0901317, suggesting a potential involvement of ABCA-1 in LXR-mediated repression of inflammatory signaling in fpEC. TLR-4 inhibitor Tak-242 attenuated pro-inflammatory signaling induced by oxysterols downstream of the TLR-4 inflammatory signaling cascade. Taken together, our findings suggest that 7-ketoC and 7β-OHC contribute to placental inflammation through the activation of TLR-4. Pharmacologic activation of LXR in fpEC decelerates its shift to a pro-inflammatory phenotype in the presence of oxysterols.

Topics: Diabetes, Gestational; Endothelial Cells; Female; Humans; Inflammation; Liver X Receptors; Oxysterols; Placenta; Pregnancy; Toll-Like Receptor 4

The pathogenesis of chronic obstructive pulmonary disease (COPD) is associated with dyslipidemia, an established co-morbidity. Statins treat hypercholesterolemia, but more recently have been trailed in the setting of COPD for their potential anti-inflammatory benefits. The outcomes of prospective trials however have been inconsistent. Thus, we hypothesize that the variation in results may have been due to statin-induced downregulation of ATP-binding cassette transporter A1 (ABCA1), thereby reducing cholesterol export. This study aims to elucidate whether statin treatment in a cellular model of COPD leads to a decrease in ABCA1 protein expression.. To mimic the inflammatory environment of COPD, two commonly used lung epithelial cell lines (BEAS-2B and A549) were treated with tumor necrosis factor (TNF), and co-treated with cholesterol/25-hydroxycholesterol (25-OH) to mimic dyslipidemia. ABCA1 protein was detected by Western Blotting.. We unexpectedly showed that statins did not affect ABCA1 expression. However, the LXR agonist T0901317 significantly increased ABCA1 expression in both cell lines, while TNF, cholesterol or 25-OH induced ABCA1 protein upregulation in BEAS-2B cells, indicating cell line differences in response. There was also evidence of synergistic impacts of combined treatments on ABCA1 upregulation in BEAS-2B cells.. Statins did not have an impact on ABCA1 expression in lung epithelial cell lines, disproving our original hypothesis. However, we showed for the first time, the effect of the inflammatory cytokine TNF, cholesterol/25-OH, statins and the LXR agonist T0901317 on expression of ABCA1 transporter protein in human lung epithelial cell lines in vitro. We hope that these in vitro studies may prove beneficial for addressing dyslipidemia in COPD in the future.

Topics: A549 Cells; ATP Binding Cassette Transporter 1; Cell Line; Cell Line, Tumor; Cholesterol; Epithelial Cells; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Liver X Receptors; Lung; Sulfonamides; Tumor Necrosis Factor-alpha; Up-Regulation

Liver X Receptors (LXRs) belong to the nuclear receptor superfamily, have been reported that activation of LXRs with synthetic ligands has anti-inflammatory effects in various inflammatory diseases. This study aims at investigating the effects of T0901317 (T0), a synthetic LXRs ligand, on lipopolysaccharide (LPS)-stimulated primary bovine mammary epithelial cells (bMECs). BMECs were stimulated by LPS in the presence or absence of T0. The results showed that treatment with T0 significantly inhibited LPS-induced tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) expression. LPS-induced NF-κB activation was also suppressed by T0. Furthermore, T0 was found to inhibit the translocation of TLR4 to lipid rafts. T0 could activate ATP-binding cassette transporter A1 (ABCA1) dependent pathway which induced cholesterol efflux from cells and disrupted the formation of lipid rafts. Thus, based on those findings we proposed that LXRs agonist might become a novel therapeutic target for inflammation.

Topics: Animals; ATP Binding Cassette Transporter 1; Cattle; Cells, Cultured; Cholesterol; Cytokines; Epithelial Cells; Female; Hydrocarbons, Fluorinated; Inflammation; Lipopolysaccharides; Liver X Receptors; Mammary Glands, Animal; Membrane Microdomains; Signal Transduction; Sulfonamides; Toll-Like Receptor 4

To investigate whether activation of the liver X receptors (LXRs) inhibits amyloid β. Confluent cultures of human primary RPE and ARPE-19 cells pretreated with 5 μΜ of TO901317 (TO90), a synthetic agonist of LXR, or vehicle were incubated with 1 μΜ of Aβ. A negative linear relationship between the Aβ. Activation of the LXRα-ABCA1 axis may alleviate Aβ

Topics: Amyloid beta-Peptides; Cell Line; Cell Survival; Cells, Cultured; Cellular Senescence; Cytokines; Epithelial Cells; Humans; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Macular Degeneration; NF-kappa B; Peptide Fragments; Retinal Pigment Epithelium; Sulfonamides

Topics: Acute Lung Injury; Animals; Antioxidants; Apoptosis; Disease Models, Animal; Humans; Hydrocarbons, Fluorinated; Inflammation; Interleukin-1beta; Lipid Peroxidation; Liver X Receptors; MAP Kinase Signaling System; Mice; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Paraquat; Reactive Oxygen Species; Sulfonamides; Tumor Necrosis Factor-alpha

Amyloid β (Aβ) is a pathogenic peptide associated with many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The retinal inflammation in response to Aβ is implicated in the pathogenesis of several ocular diseases including age-related macular degeneration, Alzheimer's-related optic neuropathy and glaucoma. In the present study, we found that a single intravitreal injection of oligomeric Aβ1-40 in mouse activated the NLRP3 inflammasome and the NF-κB signaling, induced the production of inflammatory cytokines including TNF-α and IL-6. In addition, Aβ1-40 caused retinal function impairment while no noticeable morphological changes were observed under light microscope. Furthermore, immunohistochemical results showed that Aβ1-40 enhanced the number of Iba1-positive cells in the inner retina. The mRNA expressions of LXRα and LXRβ decreased in the neuroretina of the Aβ1-40-injected mice. No significant difference was found on the protein expressions of LXRs and ABCA1 in both neuroretina and RPE/choroid complex between the Aβ1-40-injected group and the control group. A synthetic LXR ligand, TO901317 (TO90), enhanced the expressions of LXRα and ABCA1 at both mRNA and protein levels in the Aβ1-40-injected mice, while the LXRβ expression was unchanged. TO90 preserved ERG a- and b-wave amplitudes and reduced the number of Iba1-positive cells in the Aβ1-40-treated retina. Furthermore, TO90 down-regulated the mRNA levels of TNF-α and IL-6, as well as the expressions of p-IκBα, NLRP3, caspase-1 and IL-1β in the Aβ1-40-injected animals. We suggest that activation of LXRα and its target gene ABCA1 exerts potent anti-inflammatory effect on the Aβ-treated retina.

Topics: Amyloid beta-Peptides; Animals; Cytokines; Hydrocarbons, Fluorinated; Inflammasomes; Inflammation; Mice, Inbred C57BL; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Retina; Signal Transduction; Sulfonamides

Intracerebral hemorrhage (ICH) induces a series of inflammatory processes that contribute to neuronal damage and neurological deterioration. Liver X receptors (LXRs) are nuclear receptors that negatively regulate transcriptional processes involved in inflammatory responses, but their role in the pathology following ICH remains unclear. The present study investigated the neuroprotective effects and anti-inflammatory actions of TO901317, a synthetic LXR agonist, in a model of collagenase-induced ICH and in microglial cultures.. Mice subjected to collagenase-induced ICH injury were injected with either TO901317 (30 mg/kg) or vehicle 10 min after ICH and subsequently daily for 2 days. Behavioral studies, histology analysis, and assessments of hematoma volumes, brain water content, and blood-brain barrier (BBB) permeability were performed. The protein expression of LXR-α, LXR-β, ATP binding cassette transporter-1 (ABCA-1), and inflammatory molecules was analyzed. The anti-inflammatory mechanism of TO901317 was investigated in cultured microglia that were stimulated with either lipopolysaccharide (LPS) or thrombin.. ICH induced an increase in LXR-α protein levels in the hemorrhagic hemisphere at 6 h whereas LXR-β expression remained unaffected. Both LXR-α and LXR-β were expressed in neurons and microglia in the peri-ICH region and but rarely in astrocytes. TO901317 significantly attenuated functional deficits and brain damage up to 28 days post-ICH. TO901317 also reduced neuronal death, BBB disruption, and brain edema at day 4 post-ICH. These changes were associated with marked reductions in microglial activation, neutrophil infiltration, and expression levels of inflammatory mediators at 4 and 7 days. However, TO901317 had no effect on matrix metalloproteinase-9 activity. In BV2 microglial cultures, TO901317 attenuated LPS- and thrombin-stimulated nitric oxide production and reduced LPS-induced p38, JNK, MAPK, and nuclear factor-kappa B (NF-κB) signaling. Moreover, delaying administration of TO901317 to 3 h post-ICH reduced brain tissue damage and neuronal death.. Our results suggest that enhancing LXR activation may provide a potential therapy for ICH by modulating the cytotoxic functions of microglia.

Topics: Animals; Anti-Inflammatory Agents; Behavior, Animal; Blood-Brain Barrier; Body Water; Cerebral Hemorrhage; Collagenases; Gene Expression Regulation; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Sulfonamides

Liver X receptors (LXRs) in the nucleus play important roles in lipid metabolism and inflammation. The mechanism of LXR regulation of the LPS-induced Toll-like receptor 4 (TLR4) inflammatory signaling pathway remains to be elucidated. C57/BL6 mice were randomly divided into four groups: control, T0901317 (a LXRs agonist), LPS and T0901317+LPS. Additionally, Kupffer cells isolated from male C57/BL6 mice were divided into the same four groups. A decreased amount of inflammatory cells infiltrated the portal areas and the hepatic sinusoids in the livers of mice in the T0901317+LPS group than in those of mice in the LPS group. In the T0901317+LPS group, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor alpha (TNF-α) were lower, while the serum level of interleukin-10 (IL-10) was higher. In vitro, Kupffer cells pretreated with T0901317 for 24h presented reduced TNF-α, interferon-beta (IFN-β) and interleukin-1 beta (IL-1β) levels, while the IL-10 level increased; however, the mRNA and protein expression levels of interferon regulatory factor 3 (IRF3) and glucocorticoid receptor-interacting protein 1 (GRIP1) were not significantly reduced. The co-IP data illustrated that LXRα bound to GRIP1 specifically in the T0901317+LPS group, while less IRF3 was bound to GRIP1 in the T0901317+LPS group than in the LPS group. Furthermore, the DNA-binding activity of NF-κB was decreased by pretreating Kupffer cells with T0901317 for 24h. These results suggest that activated LXRα competes with IRF3 for GRIP1 binding, thus repressing IRF3 and NF-κB transcriptional activity and inhibiting the inflammatory response initiated by LPS in Kupffer cells.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cytokines; Hydrocarbons, Fluorinated; Inflammation; Inflammation Mediators; Interferon Regulatory Factor-3; Kupffer Cells; Lipid Metabolism; Lipopolysaccharides; Liver X Receptors; Male; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; NF-kappa B; Signal Transduction; Sulfonamides; Toll-Like Receptor 4; Transcriptional Activation

Macrophages play a central role in atherosclerosis development and progression, hence, targeting macrophage activity is considered an attractive therapeutic. Recently, we documented nanomedicinal delivery of the anti-inflammatory compound prednisolone to atherosclerotic plaque macrophages in patients, which did however not translate into therapeutic efficacy. This unanticipated finding calls for in-depth screening of drugs intended for targeting plaque macrophages.. We evaluated the effect of several candidate drugs on macrophage activity, rating overall performance with respect to changes in cytokine release, oxidative stress, lipid handling, endoplasmic reticulum (ER) stress, and proliferation of macrophages. Using this in vitro approach, we observed that the anti-inflammatory effect of prednisolone was counterbalanced by multiple adverse effects on other key pathways. Conversely, pterostilbene, T0901317 and simvastatin had an overall anti-atherogenic effect on multiple pathways, suggesting their potential for liposomal delivery.. This dedicated assay setup provides a framework for high-throughput assessment. Further in vivo studies are warranted to determine the predictive value of this macrophage-based screening approach and its potential value in nanomedicinal drug development for cardiovascular patients.

Topics: Animals; Anti-Inflammatory Agents; Atherosclerosis; Cell Proliferation; Cytokines; Endoplasmic Reticulum Stress; High-Throughput Screening Assays; Humans; Hydrocarbons, Fluorinated; Inflammation; Inflammation Mediators; Lipid Metabolism; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; Plaque, Atherosclerotic; Prednisolone; RAW 264.7 Cells; Reactive Oxygen Species; Signal Transduction; Simvastatin; Stilbenes; Sulfonamides; Transfection

The liver X receptor (LXR) isoform LXR‑α has a significant role in lipid metabolism and innate immunity. Overexpression of neuron‑derived orphan nuclear receptor‑1 (NOR‑1) in macrophages reduces the synthesis of inflammatory cytokines and chemokines. However, to date, the mechanisms via which NOR‑1 inhibits lipopolysaccharide (LPS)‑induced inflammation in Kupffer cells (KCs) via LXR‑α have not been elucidated. T0901317 is the most potent LXR‑α ligand, leading to its activation. In the present study, KCs were isolated from C57BL/6 mice and randomly divided into five groups: Control, T0901317, LPS, LPS + T0901317 and LPS + T0901317 + NOR‑1 small hairpin (sh)RNA groups. In order to investigate the role of NOR‑1 in inflammation, shRNA targeting NOR‑1 was used to specifically knock down NOR‑1 mRNA in KCs. The expression levels of LXR‑α and NOR‑1 in KCs were determined by reverse transcription quantitative polymerase chain reaction and western blot analyses. The protein levels of tumor necrosis factor (TNF)‑α and interleukin (IL)‑10 in the supernatant of KCs were evaluated by ELISA. The results revealed that LXR‑α expression in the T0901317 group was higher than that in the control group; furthermore, LXR‑α expression was higher in KCs treated with LPS + T0901317 compared with that in KCs treated with LPS only. The expression levels of NOR‑1 in each group showed a similar trend. shRNA targeting of NOR‑1 suppressed the mRNA expression of NOR‑1, but had no influence on LXR‑α mRNA expression. NOR‑1 protein expression was augmented in the LPS + T0901317 group compared with that in the LPS + T09 + shRNA group. In the supernatant of KCs, the TNF‑α levels in the LPS + T0901317 group were lower than those in the LPS group, whereas the IL‑10 levels were higher in the LPS + T0901317 group compared with those in the LPS group. The results of the present study suggested that ligand T0901317 promotes LXR‑α expression, which consequently suppresses LPS‑induced inflammation by elevating NOR‑1 expression in KCs.

Topics: Animals; DNA-Binding Proteins; Hydrocarbons, Fluorinated; Inflammation; Interleukin-10; Kupffer Cells; Lipopolysaccharides; Liver X Receptors; Male; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Orphan Nuclear Receptors; Receptors, Steroid; Receptors, Thyroid Hormone; RNA, Messenger; Sulfonamides; Tumor Necrosis Factor-alpha; Up-Regulation

The liver-X-receptors have shown anti-inflammatory ability in several animal models of respiratory disease. Our purpose is to investigate the effect of LXR ligand in allergen-induced airway remodeling in mice. Ovalbumin-sensitized mice were chronically challenged with aerosolized ovalbumin for 8 weeks. Some mice were administered a LXR agonist, T0901317 (12.5, 25, 50 mg/kg bodyweight) before challenge. Then mice were evaluated for airway inflammation, airway hyperresponsiveness and airway remodeling. T0901317 failed to attenuate the inflammatory cells and Th2 cytokines in bronchoalveolar lavage fluid. But the application of T0901317 reduced the thickness of airway smooth muscle and the collagen deposition. Meanwhile, T0901317 treatment evidently abolished the high level of OVA-specific IgE, TGF-β1 and MMP-9 in lung. So LXRs may attenuate the progressing of airway remodeling, providing a potential treatment of asthma.

Topics: Airway Remodeling; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Collagen; Female; Hydrocarbons, Fluorinated; Immunoglobulin E; Inflammation; Ligands; Liver X Receptors; Lung; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Muscle, Smooth; Orphan Nuclear Receptors; Ovalbumin; Sulfonamides; Transforming Growth Factor beta1

To investigate whether a synthetic LXR agonist TO901317 (TO90) ameliorates ocular inflammation in a mouse model of experimental autoimmune uveitis (EAU) and to explore its underlying mechanism.. EAU was induced with subcutaneous injection of IRBP161-180 peptide (SGIPYIISYLHPGNTILHVD) in B10.RIII mice. TO90 (50 mg/kg/d) or vehicle was administrated orally for successive 16 days or 8 days as prevention or effector phase, respectively. The severity of EAU was evaluated with clinical and histological scores. The levels of LXRs, NF-κB subunit p65, and an LXR target gene ABCA1 in the retina were detected with real-time PCR and Western blotting. The expressions of proinflammatory genes, including TNF-α, IL-1β, IL-6, MCP-1, IFN-γ, and IL-17, were detected by real-time PCR. IRBP-specific lymphocyte proliferation was detected by MTT. Intracellular IFN-γ and IL-17 in CD4(+) T cells were measured by flow cytometry.. We found both LXRα and LXRβ were expressed in mouse retina. After administering TO90 orally to B10.RIII mice, the expression of LXRα but not LXRβ was upregulated in the naïve mice. Compared with naïve mice, LXRα expression was increased in vehicle and TO90-treated EAU mice, but the LXRβ expression was unchanged. The protein level of ABCA1 was enhanced in TO90-treated naïve and EAU mice but was unchanged in vehicle-treated EAU mice, suggesting activation of LXRα by TO90 is ligand dependent. TO90-mediated activation of LXRα improved the clinical and morphological scores in EAU mice. Meanwhile, activation of LXRα decreased the expressions of proinflammatory cytokines, including TNF-α, IL-1β, IL-6, MCP-1, IFN-γ, and IL-17 in the retina. TO90 treatment inhibited IRBP-specific immune responses. The proportions of Th1 and Th17 expressing IFN-γ and IL-17 were reduced in TO90-treated EAU mice in both prevention and effector phases. Furthermore, TO90 significantly downregulated the expressions of an NF-κB subunit p65 at the protein and mRNA levels.. TO90 activates LXRα and potently attenuates ocular inflammation in EAU. Alleviation of ocular inflammation could partially result from inhibition of the NF-κB signaling pathway. TO90 reduces IFN-γ and IL-17 expression in both prevention and treatment scenarios. Our data suggest that the LXR agonist may become a novel class of therapeutic agent for autoimmune uveitis.

Topics: Animals; Autoimmune Diseases; Blotting, Western; Cytokines; Disease Models, Animal; Gene Expression Regulation; Hydrocarbons, Fluorinated; Immunity, Cellular; Inflammation; Liver X Receptors; Mice; Mice, Inbred Strains; Orphan Nuclear Receptors; Real-Time Polymerase Chain Reaction; Retina; Sulfonamides; T-Lymphocytes; Uveitis

Liver X receptors (LXRs) has been emerged as negative regulators of cardiomyocytic inflammation. The cellular process of autophagy is believed to play a protective role in myocardium during the inflammatory status. In this study, we investigated the role of LXRs agonist TO901317 (TO) on lipopolysaccharides (LPS)-induced myocardial inflammation and autophagy. The results showed that TO pretreatment significantly reduced the LPS-induced infiltration of inflammatory cells, elevation of NF-κB protein, TNF-α, and IL-6 mRNA levels in the myocardium. Moreover, LPS stimulated autophagy in neonatal mice heart, and this effect was further enhanced by TO pretreatment as evidenced by increased LC3-II/GAPDH ratio increment. Furthermore, TUNEL assay revealed LPS stimulation also increased the number of apoptotic cells in the myocardium, and the increment was inhibited by TO pretreatment. Our findings suggested that attenuation of inflammation and apoptosis, and enhancement of autophagy by TO may contribute to the protection of myocardium under inflammatory condition.

Topics: Animals; Animals, Newborn; Autophagy; Hydrocarbons, Fluorinated; Inflammation; Lipopolysaccharides; Liver X Receptors; Mice; Myocardium; Orphan Nuclear Receptors; Protective Agents; Sulfonamides

Alzheimer's disease (AD) is characterized by extracellular deposit of β-amyloid (Aβ) and accumulation of intracellular neurofibrillary tangles in the brain. Prostaglandin F2α (PGF2α) is one of the major metabolites of arachidonic acid (AA), and plays essential roles in a series of key physiological processes like luteolysis and parturition. Additionally, PGF2α is also involved in the regulation of chronic and acute inflammation processes. Recent clinical studies have revealed the high content of PGF2α metabolite, 15-keto-dihydro-PGF2α in AD patients, implying the activation of in vivo PGF2α biosynthesis. However, the mechanism underlying the involvement of PGF2α in the progression of AD still remains unclear. Here we discovered that PGF2α selectively antagonized LXR (liver X receptors)/RXR (retinoid X receptor α) and RXR/RXR dimers. Cell based assays indicated that PGF2α effectively antagonized the activation of LXR agonist (t0901317) on Aβ clearance via inhibiting apolipoprotein E (apoE) expression, and cell apoptosis alleviation by accelerating inflammatory response to Aβ or Lipopolysaccharide (LPS) in microglia. Therefore, our current findings have addressed the potential association of PGF2α with AD progression, and highlighted that inhibition of PGF2α biosynthesis might be a useful therapeutic strategy against AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Line; Dinoprost; Gene Expression Regulation; Humans; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Microglia; Orphan Nuclear Receptors; Peptide Fragments; Protein Multimerization; Protein Structure, Quaternary; Retinoid X Receptors; Sulfonamides

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

LXR is another member of the superfamily of nuclear hormone receptors that heterodimerizes with RXR and regulates the intracellular levels of cholesterol through gene induction of enzymes and proteins involved in the cholesterol metabolism and transport. LXR ligands inhibit the gene expression of proinflammatory mediators in immunostimulated macrophages; in vivo studies have shown that activation of LXR reduces the inflammatory response in a murine model of contact dermatitis and atherosclerosis. No reports have addressed a role for LXRs in pathophysiology of intestinal ischemia. The aim of this study was to investigate the effects of T0901317, a potent LXR ligand, in a mouse model of SAO shock, which was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. Mice were killed at 60 min after reperfusion. This study provides the evidence that T0901317, LXR agonist, modulates: the development of SAO shock; the infiltration of the tissue with PMNs; the expression of TNF-alpha and IL-1beta; the nitration of tyrosine residues; NF-kappaB expression; the MAPK phosphorylation (ERK, JNK, and p38); FasL; apoptosis; Bax and Bcl-2 expression; and the degree of tissue injury caused by SAO shock. Our results imply that LXR agonists may be useful in the therapy of inflammation.

Topics: Animals; bcl-2-Associated X Protein; Cholesterol; Hydrocarbons, Fluorinated; Inflammation; Inflammation Mediators; Interleukin-1beta; Intestine, Small; Liver X Receptors; Macrophages; Male; MAP Kinase Signaling System; Mice; NF-kappa B; Orphan Nuclear Receptors; Reperfusion Injury; Retinoid X Receptors; Splanchnic Circulation; Sulfonamides; Tumor Necrosis Factor-alpha

Atherogenic lipids and chronic inflammation drive the development of cardiovascular disorders such as atherosclerosis. Many cardiovascular drugs target the liver which is involved in the formation of lipid and inflammatory risk factors. With robust systems biology tools and comprehensive bioinformatical packages becoming available and affordable, the effect of novel treatment strategies can be analyzed more comprehensively and with higher sensitivity. For example, beneficial as well as adverse effects of drugs can already be detected on the gene and metabolite level, and prior to their macroscopic manifestation. This chapter describes a systems approach for a prototype CV drug with established beneficial and adverse effects. All relevant steps, for example, experimental design, tissue collection and high quality RNA preparation, bioinformatical analysis of functional processes, and pathways (targeted and untargeted) are addressed.

Topics: Animals; Atherosclerosis; Cardiovascular Agents; Female; Gene Regulatory Networks; Humans; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Mice; Orphan Nuclear Receptors; RNA; Software; Statistics as Topic; Sulfonamides; Systems Biology

Ovarian hyperstimulation syndrome is a frequent complication occurring during in vitro fertilization cycles. It is characterized by a massive ovarian enlargement associated with an accumulation of extra vascular fluid. Here we show that liver X receptor (LXR)-alpha and LXR-beta deficient mice present many clinical and biological signs of ovarian hyperstimulation syndrome: ovarian enlargement, hemorrhagic corpora lutea, increased ovarian vascular permeability, and elevated estradiol. Ovulation stimulation resulted in excessive ovarian response to exogenous gonadotropins because follicle number and estradiol production were higher in transgenic mice. LXR deficiency also leads to perturbations in general inflammatory status, associated with ovarian il-6 deregulation. Upon treatment with the synthetic LXR agonist T09101317, serum estradiol and expression of star and cyp11a1 genes were markedly increased in wild-type mice, showing that LXRs are key regulators of ovarian steroidogenesis. These results suggest that LXRs control the ovulation by regulating endocrine and vascular processes.

Topics: Animals; Chorionic Gonadotropin; DNA-Binding Proteins; Estradiol; Female; Hydrocarbons, Fluorinated; Inflammation; Liver X Receptors; Mice; Mice, Knockout; Orphan Nuclear Receptors; Ovarian Hyperstimulation Syndrome; Ovulation; Ovulation Induction; Receptors, Cytoplasmic and Nuclear; Sulfonamides

To explore the possible mechanism of the inhibitory effect of liver X receptor alpha (LXRalpha) on lipopolysaccharide (LPS)-induced inflammation in mouse Kupffer cells (KCs).. The KCs isolated from the liver of male KM mice and cultured in RPMI 1640 containing 20% FBS for 24 h were divided into control, LPS, T0901317, and LPS+T0901317 groups with corresponding treatments. The expressions of LXRalpha, interferon regulatory factor 3 (IRF3) and glucocorticoid receptor interacting protein 1 (GRIP1) in the KCs were detected by Western blotting. The levels of interferon beta (IFNbeta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA).. The level of LXRalpha protein was highest in T0901317 group and lowest in LPS group, and was significantly higher in LPS+T0901317 group than in LPS group but lower than in T0901317 group (P<0.05). The levels of IRF3 and GRIP1 protein were the highest in LPS group, and significantly lowered by T0901317 treatment (P<0.05). The expression of IRF3 and GRIP1 proteins in LPS group and LPS+ T0901317 group were significantly higher than those in the control and T0901317 groups (P<0.05). The concentration of IFN-beta was significantly higher in LPS group than in the control and T0901317 group (P<0.05), and decreased in LPS+T0901317 group in comparison with that in LPS group (P<0.05). IFN-beta was the lowest in T0901317 group. The levels of TNF-alpha and IL-1beta were the highest in LPS group (P<0.05), and comparable between the other 3 groups (P>0.05).. Pre-treatment with T0901317 before LPS stimulation can suppress the expressions of IRF3 and GRIP1 to inhibit the inflammation and hence Kupffer cell activation.

Topics: Animals; Cells, Cultured; Hydrocarbons, Fluorinated; Inflammation; Interferon Regulatory Factor-3; Kupffer Cells; Lipopolysaccharides; Liver X Receptors; Male; Mice; Nuclear Receptor Coactivator 2; Orphan Nuclear Receptors; Sulfonamides

Nuclear factor-kappaB (NF-kappaB) plays a critical role in cell growth and inflammation during the progression of cardiac hypertrophy and heart failure. Several members of nuclear receptor superfamily, including liver X receptors (LXRalpha and LXRbeta), have been shown to suppress inflammatory responses, but little is known about their effects in cardiomyocytes.. We investigated LXR expression patterns in pressure overload-induced hypertrophic hearts and the hypertrophic growth of the LXRalpha-deficient hearts from mice (C57/B6) in response to pressure overload. The underlying mechanisms were also explored using cultured myocytes. We found that cardiac expression of LXRalpha was upregulated in pressure overload-induced left ventricular hypertrophy in mice. Transverse aorta coarctation-induced left ventricular hypertrophy was exacerbated in LXRalpha-null mice relative to control mice. A synthetic LXR ligand, T1317, suppressed cardiomyocyte hypertrophy in response to angiotensin II and lipopolysaccharide treatments. In addition, LXR activation suppressed NF-kappaB signalling and the expression of associated inflammatory factors. Overexpression of constitutively active LXRalpha and beta in cultured myocytes suppressed NF-kappaB activity.. LXRs are negative regulators of cardiac growth and inflammation via suppressing NF-kappaB signalling in cardiomyocytes. This should provide new insights into novel therapeutic targets for treating cardiac hypertrophy and heart failure.

Topics: Adenoviridae; Angiotensin II; Animals; Cardiomegaly; Cells, Cultured; DNA-Binding Proteins; Hydrocarbons, Fluorinated; Inflammation; Lipopolysaccharides; Liver X Receptors; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Orphan Nuclear Receptors; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sulfonamides; Toll-Like Receptor 4

Liver X receptors (LXRs) have previously been implicated in the regulation of inflammation and have, in general, been ascribed an antiinflammatory role. This study was therefore undertaken to explore the biologic mechanisms of LXRs in vivo and in vitro in an experimental inflammatory arthritis model.. Male DBA/1 mice were immunized with type II collagen and treated from an early or established stage of arthritis with 2 different concentrations of the LXR agonists T1317 and GW3965 or vehicle control. The mice were monitored for articular inflammation and cartilage degradation by scoring for clinical signs of arthritis, histologic examination of the joints, and analysis of serum cytokine and antibody levels. In vitro, primary human monocytes and T cells were cultured in the presence of GW3965 or T1317, and the concentrations of proinflammatory cytokines were measured by multiplex assay.. Contrary to expectations, LXR agonism with the use of 2 discrete, specific molecular entities led to substantial exacerbation of articular inflammation and cartilage destruction in this murine collagen-induced arthritis model. This was associated ex vivo with elevated cytokine expression, with enhanced Th1 and Th17 cellular responses, and with elevated collagen-specific autoantibody production. In vitro, LXR agonists, in concert with lipopolysaccharide, promoted cytokine and chemokine release from human monocytes, and similar effects were observed in a T cell-macrophage coculture model that closely recapitulates the pathways that drive synovial cytokine release.. Since LXRs are present in rheumatoid arthritis (RA) synovium, these results suggest that LXR-mediated pathways could exacerbate the chronic inflammatory response typical of RA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cartilage, Articular; Cells, Cultured; Disease Models, Animal; DNA-Binding Proteins; Humans; Hydrocarbons, Fluorinated; Inflammation; Interleukin-17; Interleukin-1alpha; Interleukin-6; Lipopolysaccharides; Liver X Receptors; Male; Mice; Mice, Inbred DBA; Monocytes; Orphan Nuclear Receptors; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sulfonamides

Topics: Cell Division; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Inflammation; Interleukin-2; Interleukin-7; Leukemia, Lymphocytic, Chronic, B-Cell; Liver X Receptors; Orphan Nuclear Receptors; Sulfonamides; T-Lymphocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) and liver X receptor-alpha (LXRalpha) are nuclear ligand-activated transcription factors, which regulate lipid metabolism and inflammation. Murine J774.2 macrophages were stimulated with Escherichia coli lipopolysaccharide (concentration, 10 microg/mL) with or without the PPARgamma ligand, 15-deoxy-Delta prostaglandin J2 (15d-PGJ2), or the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317 (concentration range, 0.01-10 micromol/L), alone or in combination. Nitric oxide (NO) metabolites and tumor necrosis factor alpha production, inducible NO synthase expression, and mitochondrial respiration were measured. When added to the cells as single agents, 15d-PGJ2, 22(R)-hydroxycholesterol, or T0901317 reduced the lipopolysaccharide-induced NO and tumor necrosis factor alpha production and the inducible NO synthase expression, and partially maintained mitochondrial respiration in a concentration-dependent manner. When added to the cells in combination at suboptimal concentrations, 15d-PGJ2 with 22(R)-hydroxycholesterol, or 15d-PGJ2 with T0901317, exerted anti-inflammatory effects similar to much higher concentrations (10,000-fold to 100,000-fold) of each ligand alone. The anti-inflammatory effects of these ligands, alone or in combination, were associated with reduction of nuclear factor-kappaB activation and with enhancement of PPARgamma DNA binding. LXRalpha expression was upregulated in response to 15d-PGJ2 and to the LXRalpha ligands when added alone or in combination. Immunoprecipitation experiments revealed that PPARgamma interacted with LXRalpha. Our data demonstrate that the PPARgamma ligand, 15d-PGJ2, and the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317, although binding to different nuclear receptors (i.e., PPARgamma and LXRalpha, respectively), affect mediator production through common cell signaling events and exert a synergistic potentiation in a combined treatment at suboptimal concentrations. Thus, our data suggest that PPARgamma and LXRalpha may interact in controlling the inflammatory response in macrophages.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; DNA-Binding Proteins; Drug Synergism; Hydrocarbons, Fluorinated; Hydroxycholesterols; Inflammation; Ligands; Lipopolysaccharides; Liver X Receptors; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Orphan Nuclear Receptors; PPAR gamma; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sulfonamides; Tumor Necrosis Factor-alpha