T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Insight into the physiologic roles of the LXRs has been greatly facilitated by the discovery of potent synthetic agonists. Here we show that one of these compounds, T0901317, is also a high-affinity ligand for the xenobiotic receptor pregnane X receptor (PXR). T0901317 binds and activates PXR with the same nanomolar potency with which it stimulates LXR activity. T0901317 induces expression not only of LXR target genes, but also of PXR target genes in cells and animals, including the scavenger receptor CD36, a property not shared by more specific LXR ligands, such as GW3965. Activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect. These results suggest that many of the biological activities heretofore associated with LXR activation may be mediated by PXR, not LXR. Since T0901317 has been widely used in animals to study LXR function, the in vivo effects of this compound ascribed to LXR activation should be re-examined.     

Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse

Actions of thromboxane (TXA(2)) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA(2) is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA(2)-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA(2) on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA(2)-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease.     

The role of the liver X receptor in chronic obstructive pulmonary disease

Background

There is a need for novel anti-inflammatory therapies to treat COPD. The liver X receptor (LXR) is a nuclear hormone receptor with anti-inflammatory properties.

Methods

We investigated LXR gene and protein expression levels in alveolar macrophages and whole lung tissue from COPD patients and controls, the effect of LXR activation on the suppression of inflammatory mediators from LPS stimulated COPD alveolar macrophages, and the effect of LXR activation on the induction of genes associated with alternative macrophage polarisation.

Results

The levels of LXR mRNA were significantly increased in whole lung tissue extracts in COPD patients and smokers compared to non-smokers. The expression of LXR protein was significantly increased in small airway epithelium and alveolar epithelium in COPD patients compared to controls. No differences in LXR mRNA and protein levels were observed in alveolar macrophages between patient groups. The LXR agonist GW3965 significantly induced the expression of the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. In LPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10 and CCL5, whilst stimulating IL-10 production.

Conclusions

GW3965 did not significantly suppress the production of TNFα, IL-1β, or CXCL8. Our major finding is that LXR activation has anti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolar macrophages.     

Liver X receptor stimulates cholesterol efflux and inhibits expression of proinflammatory mediators in human airway smooth muscle cells

Human (h) airway smooth muscle (ASM) cells are important mediators of the inflammatory process observed in asthma and other respiratory diseases. We show here that primary hASM cells express liver X receptor (LXR; alpha and beta subtypes), an oxysterol-activated nuclear receptor that controls expression of genes involved in lipid and cholesterol homeostasis, and inflammation. LXR was functional as determined by transient assays using LXR-responsive reporter genes and by analysis of mRNA and protein expression of endogenous LXR target genes in cells exposed to LXR agonists. LXR activation induced expression of the ATP-binding cassette transporters ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein AI and high-density lipoprotein acceptors, pointing to a role for hASM cells in modulating cholesterol homeostasis in the airway. Under inflammatory conditions, hASM cells release a variety of chemokines and cytokines that contribute to inflammatory airway diseases. Activation of LXR inhibited the expression of multiple cytokines in response to proinflammatory mediators and blocked the release of both granulocyte macrophage colony-stimulating factor and granulocyte colony stimulating factor. LXR activation also inhibited proliferation of hASM cells and migration toward platelet-derived growth factor chemoattractant, two important processes that contribute to airway remodeling. Our findings reveal biological roles for LXR in ASM cells and suggest that modulation of LXR activity offers prospects for new therapeutic approaches in the treatment of asthma and other inflammatory respiratory diseases.     

Oxidant stress modulates murine allergic airway responses

The allergic inflammation occurring in asthma is believed to be accompanied by the production of free radicals. To investigate the role of free radicals and the cells affected we turned to a murine model of allergic inflammation produced by sensitization to ovalbumin with subsequent aerosol challenge. We examined oxidant stress by measuring and localizing the sensitive and specific marker of lipid peroxidation, the F2-isoprostanes. F2-isoprostanes in whole lung increased from 0.30 +/- 0.08 ng/lung at baseline to a peak of 0.061 +/- 0.09 ng/lung on the ninth day of daily aerosol allergen challenge. Increased immunoreactivity to 15-F2t-IsoP (8-iso-PGF2alpha) or to isoketal protein adducts was found in epithelial cells 24 h after the first aerosol challenge and at 5 days in macrophages. Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased immunoreactivity after ovalbumin challenge. Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes while supplemental vitamin E suppressed their formation. Similar changes in immunoreactivity to F2-isoprostanes were seen. Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant. Our findings indicate that allergic airway inflammation in mice is associated with an increase in oxidant stress, which is most striking in airway epithelial cells and macrophages. Oxidant stress plays a role in the production of airway responsiveness.     

TLR-7 agonist attenuates airway reactivity and inflammation through Nrf2-mediated antioxidant protection in a murine model of allergic asthma

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant–antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.     

Glucocorticoid and estrogen receptors are reduced in mitochondria of lung epithelial cells in asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GRα and ERβ in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERβ, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GRα and ERβ in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.     

Alcohol reduces airway hyperresponsiveness (AHR) and allergic airway inflammation in mice

There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.     

Flt-3 ligand reverses late allergic response and airway hyper-responsiveness in a mouse model of allergic inflammation

Flt3 ligand (Flt3-L) is a growth factor for dendritic cells and induces type 1 T cell responses. We recently reported that Flt3-L prevented OVA-induced allergic airway inflammation and suppressed late allergic response and airway hyper-responsiveness (AHR). In the present study we examined whether Flt3-L reversed allergic airway inflammation in an established model of asthma. BALB/c mice were sensitized and challenged with OVA, and AHR to methacholine was established. Then mice with AHR were randomized and treated with PBS or 6 microg of Flt3-L i.p. for 10 days. Pulmonary functions and AHR to methacholine were examined after rechallenge with OVA. Treatment with Flt3-L of presensitized mice significantly suppressed (p < 0.001) the late allergic response, AHR, bronchoalveolar lavage fluid total cellularity, absolute eosinophil counts, and inflammation in the lung tissue. There was a significant decrease in proinflammatory cytokines (TNF-alpha, IL-4, and IL-5) in bronchoalveolar lavage fluid, with a significant increase in serum IL-12 and a decrease in serum IL-5 levels. There was no significant effect of Flt3-L treatment on serum IL-4 and serum total IgE levels. Sensitization with OVA significantly increased CD11b(+)CD11c(+) cells in the lung, and this phenomenon was not significantly affected by Flt3-L treatment. These data suggest that Flt3-L can reverse allergic airway inflammation and associated changes in pulmonary functions in murine asthma model.     

Mycoplasma pneumoniae infection increases airway collagen deposition in a murine model of allergic airway inflammation

Mycoplasma pneumoniae (Mp) has been linked to chronic asthma. Airway remodeling (e.g., airway collagen deposition or fibrosis) is one of the pathological features of chronic asthma. However, the effects of respiratory Mp infection on airway fibrosis in asthma remain unclear. In the present study, we hypothesized that respiratory Mp infection may increase the airway collagen deposition in a murine model of allergic airway inflammation in part through upregulation of transforming growth factor (TGF)-beta1. Double (2 wk apart) inoculations of Mp or saline (control) were given to mice with or without previous allergen (ovalbumin) challenges. On days 14 and 42 after the last Mp or saline, lung tissue and bronchoalveolar lavage (BAL) fluid were collected for analyses of collagen and TGF-beta1 at protein and mRNA levels. In allergen-na?ve mice, Mp did not alter airway wall collagen. In allergen-challenged mice, Mp infections did not change airway wall collagen deposition on day 14 but increased the airway collagen on day 42; this increase was accompanied by increased TGF-beta1 protein in the airway wall and reduced TGF-beta1 protein release from the lung tissue into BAL fluid. Our results suggest that Mp infections could modulate airway collagen deposition in a murine model of allergic airway inflammation with TGF-beta1 involved in the collagen deposition process.     

Ligand specificity and evolution of liver X receptors

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.     

Soluble guanylyl cyclase expression is reduced in allergic asthma

Soluble guanylyl cyclase (sGC) is an enzyme highly expressed in the lung that generates cGMP contributing to airway smooth muscle relaxation. To determine whether the bronchoconstriction observed in asthma is accompanied by changes in sGC expression, we used a well-established murine model of allergic asthma. Histological and biochemical analyses confirmed the presence of inflammation in the lungs of mice sensitized and challenged with ovalbumin (OVA). Moreover, mice sensitized and challenged with OVA exhibited airway hyperreactivity to methacholine inhalation. Steady-state mRNA levels for all sGC subunits (alpha1, alpha2, and beta1) were reduced in the lungs of mice with allergic asthma by 60-80%, as estimated by real-time PCR. These changes in mRNA were paralleled by changes at the protein level: alpha1, alpha2, and beta1 expression was reduced by 50-80% as determined by Western blotting. Reduced alpha1 and beta1 expression in bronchial smooth muscle cells was demonstrated by immunohistochemistry. To study if sGC inhibition mimics the airway hyperreactivity seen in asthma, we treated na?ve mice with a selective sGC inhibitor. Indeed, in mice receiving ODQ the methacholine dose response was shifted to the left. We conclude that sGC expression is reduced in experimental asthma contributing to the observed airway hyperreactivity.     

CCR8 is not essential for the development of inflammation in a mouse model of allergic airway disease

Chemokine receptors play an important role in the trafficking of various immune cell types to sites of inflammation. Several chemokine receptors are differentially expressed in Th1 and Th2 effector populations. Th2 cells selectively express CCR3, CCR4, and CCR8, which could direct their trafficking to sites of allergic inflammation. Additionally, increased expression of the CCR8 ligand, TCA-3, has been detected in affected lungs in a mouse model of asthma. In this study, CCR8-deficient mice were generated to address the biological role of CCR8 in a model of allergic airway disease. Using two different protocols of allergen challenge, we demonstrate that absence of CCR8 does not affect the development of pulmonary eosinophilia and Th2 cytokine responses. In addition, administration of anti-TCA-3-neutralizing Ab during allergen sensitization and rechallenge failed to inhibit airway allergic inflammation. These results suggest that CCR8 does not play an essential role in the pathogenesis of inflammation in this mouse model of allergic airway disease.     

Baseline airway hyperreactivity in A/J mice is not mediated by cells of the adaptive immune system

Human asthma is characterized by increased airway hyperreactivity to a variety of bronchoconstricting agents. Aberrant type 2 immune responses in the lung have been associated with airway hyperreactivity in both human asthma and in murine models of allergic airways disease. Despite their intrinsically elevated basal airway reactivity to smooth muscle constricting agents, A/J mice demonstrated no inherent inflammatory cell infiltration nor elevation of type 2 cytokines in the lung. Crossed bone marrow reconstitution experiments between A/J and MHC congenic B10.A mice revealed enhanced airway reactivity only in A/J recipients, irrespective of whether they had been reconstituted with A/J or B10. A hemopoietic cells. Further, A/J-derived bone marrow cells did not affect the reactivity of B10.A recipients. Although mice on RAG-deficient and IL-4-deficient backgrounds demonstrate substantial abrogation of allergen-induced airway hyperreactivity, these gene deletions had no impact on the elevated baseline reactivity when backcrossed onto A/J mice. Thus, in these mice, basal airway hyperreactivity is maintained independently of type 2 immunity induced by allergens.     

Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells

Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Paeonol attenuates airway inflammation and hyperresponsiveness in a murine model of ovalbumin-induced asthma

Paeonol, the main active component isolated from Moutan Cortex, possesses extensive pharmacological activities such as anti-inflammatory, anti-allergic, and immunoregulatory effects. In the present study, we examined the effects of paeonol on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice sensitized and challenged with ovalbumin were administered paeonol intragastrically at a dose of 100?mg/kg daily. Paeonol significantly suppressed ovalbumin-induced airway hyperresponsiveness to acetylcholine chloride. Paeonol administration significantly inhibited the total inflammatory cell and eosinophil count in bronchoalveolar lavage fluid. Treatment with paeonol significantly enhanced IFN-γ levels and decreased interleukin-4 and interleukin-13 levels in bronchoalveolar lavage fluid and total immunoglobulin E levels in serum. Histological examination of lung tissue demonstrated that paeonol significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. These data suggest that paeonol exhibits anti-inflammatory activity in allergic mice and may possess new therapeutic potential for the treatment of allergic bronchial asthma.