ABSTRACT
Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets.
Subject(s)
Hematopoiesis/immunology , Macrophages/immunology , Orphan Nuclear Receptors/immunology , Spleen/immunology , Animals , Benzoates/pharmacology , Benzylamines/pharmacology , Cell Differentiation/immunology , Flow Cytometry , Immunity, Cellular/immunology , Immunohistochemistry , Liver X Receptors , Macrophages/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , Orphan Nuclear Receptors/agonists , Signal Transduction/immunology , Specific Pathogen-Free Organisms , Spleen/cytologyABSTRACT
Photodynamic therapy (PDT) is a rising and hopeful treatment for solid tumors and others malignancies. PDT uses harmless visible light to activate a tumor-associated photosensitizer (PS). The excited PS generates cytotoxic reactive oxygen species (ROS) that induce damage and death of tumor cells. It is known that certain phytoalexins and phytoanticipins derived from plants often display a PS-like activity due to a phenalenone (PN) moiety-an efficient singlet oxygen photosensitizer-in its skeleton. The aim of this study is to explore the phototoxic properties of PN on the human cell line tumor-derived HL60 (acute promyelocytic leukemia) and to identify the cell-specific targets of ROS involved in the tumor cell death. Our results reveal that PN acts as an excellent PS, showing a potent antitumor cell activity in presence of light. PN-PDT generates intracellular ROS, via oxidation reaction mechanisms type I and II, resulting in an induction of apoptosis. Moreover, both extrinsic (through direct activation of caspase-3) and intrinsic (through mitochondrial depolarization) pathways of apoptosis are induced by PN-PDT. Using pharmacologic inhibitors, we also find that PN-PDT activates caspase-8/tBid and p38-MAPK, triggering the activation of the apoptotic pathways. Although, survival pathways are also promoted through PI3 K/Akt and JNK activation, the net result of PN-PDT is the tumor cell death. The present work identifies to PN, for the first time, as a potent photosensitizer in human tumor cell lines and proposes a mechanism by which ROS induces apoptosis of tumor cell.
Subject(s)
Apoptosis/drug effects , Apoptosis/radiation effects , Caspase 8/metabolism , Phenalenes/pharmacology , Photochemotherapy , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA Fragmentation , Dose-Response Relationship, Drug , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Neoplasms/genetics , Neoplasms/metabolism , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.
Subject(s)
Apoptosis/immunology , Autoimmune Diseases/immunology , DNA-Binding Proteins/agonists , Macrophages/immunology , Receptors, Cytoplasmic and Nuclear/agonists , Spleen/immunology , Animals , Autoimmune Diseases/metabolism , Autoimmune Diseases/pathology , Autoimmunity/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Immune Tolerance/immunology , Liver X Receptors , Macrophages/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Orphan Nuclear Receptors , Phagocytosis/immunology , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/immunology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/immunology , Signal Transduction/immunology , Spleen/cytology , Spleen/metabolism , c-Mer Tyrosine KinaseABSTRACT
The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR-/-) LDLR-/- mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.
Subject(s)
Chemotaxis/physiology , Dendritic Cells/physiology , Liver X Receptors/physiology , ADP-ribosyl Cyclase 1/metabolism , Animals , Cells, Cultured , Dendritic Cells/cytology , Inflammation , Lipid Metabolism , Liver X Receptors/genetics , Macrophages , Mice , Mice, Inbred C57BL , Mice, Knockout , Orphan Nuclear Receptors , Receptors, Cytoplasmic and Nuclear , Signal TransductionABSTRACT
Systemic or intratesticular release of TNF alpha and IL1 beta have been implicated in the reduced testosterone biosynthesis and impaired production of competent spermatozoa found in human patients suffering from sepsis or chronic inflammation. Although in vitro and in vivo studies have demonstrated that TNF alpha and IL1 beta intercept the hypothalamic-pituitary testis axis at different levels, the site(s) of action and relative contribution of each cytokine to the overall testicular failure associated to systemic inflammatory processes remains poorly defined. In this study we show that intratesticular delivery of TNF alpha induced a rapid (4 h) and sustained (up to 24 h) reduction in steroidogenic acute regulatory (StAR) protein expression and testosterone biosynthesis in nonstimulated or human chorionic gonadotropin-treated intact or hypophysectomized rats. Bilateral treatment with cell-permeant short-chain ceramides (C2-cer or C6-cer) reproduced the early (4 h) inhibitory action of TNFalpha on testosterone biosynthesis and testicular StAR expression. The inhibitory action of C2-cer or C6-cer was not observed in animals treated with inactive analogs (dihydroceramide), phosphorylcholine, sphingosine, or sphingosine-1P. In sharp contrast to the previously described ability of IL1 beta to prevent human chorionic gonadotropin-stimulated Leydig cell steroidogenesis in vitro, serum testosterone and testicular StAR protein expression remained unchanged in animals bilaterally injected with this cytokine. These data support the concept that TNF alpha triggers different effector mechanisms to directly inhibit Leydig cell StAR expression and steroidogenesis, which ultimately contribute to the global reproductive failure associated with chronic inflammation and sepsis.
Subject(s)
Ceramides/administration & dosage , Leydig Cells/metabolism , Phosphoproteins/metabolism , Testis/physiology , Testosterone/antagonists & inhibitors , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Chorionic Gonadotropin/pharmacology , Humans , Hypophysectomy , Injections , Interleukin-1/administration & dosage , Isomerism , Male , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , RNA, Messenger/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Testosterone/biosynthesis , Testosterone/blood , Time FactorsABSTRACT
Despite the good growth performance of several fish species when dietary fish oil is partly replaced by vegetable oils, recent studies have reported several types of intestinal morphological alterations in cultured fish fed high contents of vegetable lipid sources. However, the physiological process implied in these morphological changes have not been clarified yet, since alterations in the physiological mechanisms involved in the different processes of lipid absorption could be responsible for such gut morphological features. The objective of the present study was to investigate the activities of reacylation pathways in fish, the glycerol-3-phosphate and the monoacylglycerol pathways, in order to clarify the intestinal triacylglycerol (TAG) and phospholipid biosynthesis to better understand the morphological alterations observed in the intestine of fish fed vegetable oils. Intestinal microsomes of sea bream fed different lipid sources (fish, soyabean and rapeseed oils) at three different inclusion levels were isolated and incubated with L-[(14)C(U)]glycerol-3-phosphate and [1-(14)C]palmitoyl CoA. The results showed that in this fish species the glycerol-3-phosphate pathway is mainly involved in phospholipid synthesis, whereas TAG synthesis is mainly mediated by the monoacylglycerol pathway. Feeding with rapeseed oil reduced the reacylation activity in both pathways, explaining the high accumulation of lipid droplets in the supranuclear portion of the intestinal epithelium, whereas soyabean oil enhanced phosphatidylcholine synthesis, being associated with the increase in VLDL found in previous studies.
Subject(s)
Dietary Fats/administration & dosage , Intestinal Mucosa/metabolism , Phospholipids/biosynthesis , Plant Oils/administration & dosage , Sea Bream/metabolism , Triglycerides/biosynthesis , Animals , Brassica rapa , Cytidine Diphosphate/administration & dosage , Cytidine Diphosphate/analogs & derivatives , Cytidine Diphosphate Choline/administration & dosage , Ethanolamines/administration & dosage , Fatty Acids, Monounsaturated , Fish Oils/administration & dosage , Glycerophosphates/metabolism , Microsomes/metabolism , Monoglycerides/metabolism , Palmitoyl Coenzyme A/administration & dosage , Rapeseed Oil , Soybean Oil/administration & dosageABSTRACT
We have examined the effects of tumor necrosis factor alpha (TNF alpha) and its second messenger, ceramide, on HMGCoA reductase, the rate-limiting enzyme in the mevalonate pathway. Treatment of human U-937 and HL-60 cells with TNF alpha or C2-ceramide inhibited both expression and activity of HMGCoA reductase in a time-dependent manner. Maturation of p21(ras) was also inhibited in a mevalonate-dependent fashion. The addition of mevalonate to both U-937 and HL-60 cells could also partially prevent TNF alpha and ceramide-induced apoptosis. These results support the hypothesis that the inhibition of HMGCoA reductase expression and the subsequent decrease in prenylation of proteins such as p21(ras) are part of the mechanism by which TNF alpha induces apoptosis in these cells.
Subject(s)
Apoptosis , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Ceramides/antagonists & inhibitors , Ceramides/pharmacology , HL-60 Cells , Humans , Hydroxymethylglutaryl CoA Reductases/biosynthesis , Hydroxymethylglutaryl CoA Reductases/genetics , Mevalonic Acid/pharmacology , Proto-Oncogene Proteins p21(ras)/biosynthesis , RNA, Messenger/biosynthesis , Tumor Necrosis Factor-alpha/antagonists & inhibitors , U937 CellsABSTRACT
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway elicits a survival signal against multiple apoptotic insults. In addition, phase II enzymes such as heme oxygenase-1 (HO-1) protect cells against diverse toxins and oxidative stress. In this work, we describe a link between these defense systems at the level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-derived phenol carnosol induced HO-1 expression at both mRNA and protein levels. Luciferase reporter assays indicated that carnosol targeted the mouse ho1 promoter at two enhancer regions comprising the antioxidant response elements (AREs). Moreover, carnosol increased the nuclear levels of Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays and luciferase reporter assays with a dominant-negative Nrf2 mutant indicated that carnosol increased the binding of Nrf2 to ARE and induced Nrf2-dependent activation of the ho1 promoter. While investigating the signaling pathways responsible for HO-1 induction, we observed that carnosol activated the ERK, p38, and JNK pathways as well as the survival pathway driven by PI3K. Inhibition of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1 promoter. Expression of active PI3K-CAAX (where A is aliphatic amino acid) was sufficient to activate AREs. The use of dominant-negative mutants of protein kinase Czeta and Akt1, two kinases downstream from PI3K, demonstrated a requirement for active Akt1, but not protein kinase Czeta. Moreover, the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-1 inhibitors, further demonstrating that carnosol attenuates oxidative stress through a pathway that involves PI3K and HO-1.