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1.
Sci Rep ; 6: 31414, 2016 Sep 23.
Article in English | MEDLINE | ID: mdl-27659201

ABSTRACT

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by aortic dilation and rupture leading to sudden death. Currently, no non-surgical treatments are available and novel therapeutic targets are needed to prevent AAA. We investigated whether increasing plasma levels of adiponectin (APN), a pleiotropic adipokine, provides therapeutic benefit to prevent AngII-induced advanced AAA in a well-established preclinical model. In the AngII-infused hyperlipidemic low-density lipoprotein receptor-deficient mouse (LDLR-/-) model, we induced plasma APN levels using a recombinant adenovirus expressing mouse APN (AdAPN) and as control, adenovirus expressing green florescent protein (AdGFP). APN expression produced sustained and significant elevation of total and high-molecular weight APN levels and enhanced APN localization in the artery wall. AngII infusion for 8 weeks induced advanced AAA development in AdGFP mice. Remarkably, APN inhibited the AAA development in AdAPN mice by suppressing aortic inflammatory cell infiltration, medial degeneration and elastin fragmentation. APN inhibited the angiotensin type-1 receptor (AT1R), inflammatory cytokine and mast cell protease expression, and induced lysyl oxidase (LOX) in the aortic wall, improved systemic cytokine profile and attenuated adipose inflammation. These studies strongly support APN therapeutic actions through multiple mechanisms inhibiting AngII-induced AAA and increasing plasma APN levels as a strategy to prevent advanced AAA.

2.
FASEB J ; 29(2): 636-49, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25392268

ABSTRACT

Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40-60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 µg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-α, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by up-regulating liver X receptor-α (LXRα) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.


Subject(s)
Adiponectin/metabolism , Macrophages/cytology , Receptors, Adiponectin/metabolism , Animals , Atherosclerosis , Cell Line , Cytokines/metabolism , Gene Expression Regulation , Humans , Inflammation , Insulin Resistance , Interleukin-10/metabolism , Liver X Receptors , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/cytology , Orphan Nuclear Receptors/metabolism , Phenotype
3.
Circ Res ; 115(4): 442-9, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24935961

ABSTRACT

RATIONALE: The E3 ubiquitin ligase inducible degrader of the low-density lipoprotein receptor (IDOL) triggers lysosomal degradation of the low-density lipoprotein receptor. The tissue-specific effects of the IDOL pathway on plasma cholesterol and atherosclerosis have not been examined. OBJECTIVE: Given that the liver is the primary determinant of plasma cholesterol levels, we sought to examine the consequence of effect of chronic liver-specific expression of a dominant-active form of IDOL in mice. METHODS AND RESULTS: We expressed a degradation-resistant, dominant-active form of IDOL (super IDOL [sIDOL]) in C57Bl/6J mice from the liver-specific albumin promoter (L-sIDOL transgenics). L-sIDOL mice were fed a Western diet for 20 or 30 weeks and then analyzed for plasma lipid levels and atherosclerotic lesion formation. L-sIDOL mice showed dramatic reductions in hepatic low-density lipoprotein receptor protein and increased plasma low-density lipoprotein cholesterol levels on both chow and Western diets. Moreover, L-sIDOL mice developed marked atherosclerotic lesions when fed a Western diet. Lesion formation in L-sIDOL mice was more robust than in apolipoprotein E*3 Leiden mice and did not require the addition of cholate to the diet. Western diet-fed L-sIDOL mice had elevated expression of liver X receptor target genes and proinflammatory genes in their aortas. CONCLUSIONS: Liver-specific expression of dominant-active IDOL is associated with hypercholesterolemia and a marked elevation in atherosclerotic lesions. Our results show that increased activity of the IDOL pathway in the liver can override other low-density lipoprotein receptor regulatory pathways leading to cardiovascular disease. L-sIDOL mice are a robust, dominantly inherited, diet-inducible model for the study of atherosclerosis.


Subject(s)
Aortic Diseases/enzymology , Aortic Diseases/pathology , Atherosclerosis/enzymology , Diet, High-Fat , Diet, Western , Hypercholesterolemia/enzymology , Liver/enzymology , Ubiquitin-Protein Ligases/metabolism , Albumins/genetics , Animals , Aorta/metabolism , Aorta/pathology , Aortic Diseases/genetics , Apolipoprotein E3/genetics , Apolipoprotein E3/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Cholesterol, LDL/blood , Disease Models, Animal , Humans , Hypercholesterolemia/genetics , Hypercholesterolemia/pathology , Inflammation Mediators/metabolism , Liver X Receptors , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Orphan Nuclear Receptors/metabolism , Plaque, Atherosclerotic , Promoter Regions, Genetic , Receptors, LDL/metabolism , Time Factors , Ubiquitin-Protein Ligases/genetics
4.
PLoS One ; 9(1): e86404, 2014.
Article in English | MEDLINE | ID: mdl-24466075

ABSTRACT

Adiponectin (APN), an adipocytokine produced by adipose tissue, exerts pleiotropic actions regulating inflammation, metabolism and vascular homeostasis. APN levels are inversely correlated with obesity, type-2 diabetes, hypertension and cardiovascular disease. Although renin angiotensin system (RAS) activation in these interrelated metabolic syndrome components increases angiotensin II (AngII) levels leading to vascular damage, it is unknown whether APN under these conditions provides atheroprotection. We investigated whether increasing plasma APN provides atheroprotection in a hypertensive and accelerated atherosclerosis model. Using adenoviral gene transfer, sustained APN expression increased plasma levels of total and high-molecular weight APN, leading to a significant elevation of plasma HDL-cholesterol (HDL-C). Elevated APN levels were strongly atheroprotective, yet had no impact on blood pressure. Notably, gene expression analyses revealed that APN significantly inhibited the expression of pro-inflammatory and atherogenic genes while it increased the expression of the anti-inflammatory cytokine, IL-10 and the cholesterol efflux transporters, ABCA1 and ABCG1 in the artery wall. These findings suggest that increasing APN levels may be an effective therapeutic strategy to inhibit vascular inflammation and accelerated atherosclerosis associated with RAS activation in the metabolic syndrome.


Subject(s)
Adiponectin/genetics , Angiotensin II/metabolism , Atherosclerosis/genetics , Atherosclerosis/metabolism , Gene Expression , Inflammation/genetics , Inflammation/metabolism , Adiponectin/blood , Adiponectin/chemistry , Adiponectin/metabolism , Angiotensin II/adverse effects , Animals , Apolipoproteins/genetics , Apolipoproteins/metabolism , Atherosclerosis/pathology , Blood Pressure/genetics , Cholesterol/metabolism , Disease Models, Animal , Hypertension/chemically induced , Hypertension/genetics , Hypertension/metabolism , Liver/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Knockout , Protein Multimerization , Protein Transport , Receptors, Adiponectin/genetics , Receptors, Adiponectin/metabolism , Receptors, LDL/genetics , Receptors, Scavenger/genetics , Receptors, Scavenger/metabolism
5.
Methods Enzymol ; 480: 117-40, 2010.
Article in English | MEDLINE | ID: mdl-20816207

ABSTRACT

Glycosphingolipids are ubiquitous membrane components that play important roles in signal transduction events thereby affecting many cellular functions, including modulation of the immune response. Whereas many studies focus on the functional roles of glycosphingolipids in mammals, relatively little is known about the structures of glycosphingolipids of pathogenic organisms, and how such pathogen-derived glycosphingolipids influence immune functions of their hosts. Many different glycosphingolipids of the human parasitic helminth Schistosoma mansoni have been structurally characterized. Recent evidence indicates that glycosphingolipids isolated from different life-cycle stages of the parasite have the potential to modulate the function of human dendritic cells, a cell population that is crucial to regulate adaptive immunity in the host. A remarkable finding in this context is that glycosphingolipids derived from adult worms induce maturation of dendritic cells, in contrast to glycosphingolipids of eggs or cercariae. The glycosphingolipid-induced dendritic cell activation requires intact fucose residues on the glycolipids, and is induced via a mechanism that involves both the dendritic cell receptors TLR4 and DC-SIGN. In this chapter, we describe methods to extract glycosphingolipids from the different life-cycle stages of the parasite, techniques to separate them by thin-layer chromatography or high-performance liquid chromatography as well as strategies to structurally characterize the glycan and ceramide moieties of the glycosphingolipids. Moreover, an overview is provided of the structural diversity in the glycosphingolipid-derived glycan moieties found in this helminth. Finally, we discuss methods used to isolate monocyte-derived dendritic cells from human blood and to study the modulation of dendritic cell function by these molecules.


Subject(s)
Chemistry Techniques, Analytical/methods , Glycosphingolipids/analysis , Glycosphingolipids/chemistry , Schistosoma mansoni/chemistry , Adult , Animals , Antigens, Helminth/analysis , Antigens, Helminth/chemistry , Antigens, Helminth/metabolism , Carbohydrate Sequence , Dendritic Cells/metabolism , Glycosphingolipids/isolation & purification , Glycosphingolipids/metabolism , Humans , Immunochemistry/methods , Molecular Sequence Data , Receptors, Immunologic/metabolism , Schistosoma mansoni/metabolism , Structure-Activity Relationship
6.
Mol Immunol ; 47(7-8): 1544-52, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20170964

ABSTRACT

In schistosomiasis, a major human parasitic disease caused by helminths, different life-stages of the parasite contribute to the developing host immune response. To increase our understanding of the mechanisms that play a role in shaping the host immune responses, we have investigated the effects of schistosome glycoconjugates on the phenotype of dendritic cells (DCs), which form a crucial link between the innate and the adaptive immunity. We show here that Schistosoma mansoni worm glycolipids induce DC activation as indicated by upregulation of the maturation markers CD80, CD86 and MHC-II, as well as the production of the cytokines interleukin-12 p40 (IL-12 p40), IL-10, IL-1beta, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha). Co-culture of glycolipid-primed DCs with naïve T cells results in skewing of the T cell response towards a Th1 profile. Remarkably, the DC activation is dependent on fucosylated glycan moieties of the glycolipids. On the DCs, the C-type lectin DC-SIGN and TLR4 are both critically involved in the induced activation, as was demonstrated by using monoclonal antibodies that block interaction of these receptors with the glycolipids. Furthermore, whereas the worm glycolipids were not able to activate HEK 293 cells expressing TLR4, they did show TLR4 activation after introduction of DC-SIGN in the HEK 293-TLR4 cells. Our data provide evidence for a novel function of DC-SIGN as an essential co-receptor for TLR4-induced activation of human DCs. This mechanism of TLR4 activation by worm glycolipids may contribute to eliciting Th1 immune responses in schistosome infection.


Subject(s)
Cell Adhesion Molecules/immunology , Dendritic Cells/immunology , Glycolipids/immunology , Lectins, C-Type/immunology , Receptors, Cell Surface/immunology , Schistosoma mansoni/immunology , Toll-Like Receptor 4/immunology , Animals , Cell Differentiation , Cell Line , Coculture Techniques , Dendritic Cells/cytology , Dendritic Cells/metabolism , Fucose/metabolism , Humans , Inflammation/immunology , Phenotype
7.
Int J Parasitol ; 40(2): 215-22, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19695255

ABSTRACT

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galalpha1-3GalNAc-R and GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galalpha1-3Gal-R as well as significant levels of Galalpha1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.


Subject(s)
Disaccharides/immunology , Haemonchiasis/veterinary , Haemonchus/immunology , Sheep Diseases/immunology , Sheep Diseases/prevention & control , Animals , Antibodies, Helminth/blood , Glycoproteins/administration & dosage , Glycoproteins/immunology , Haemonchiasis/immunology , Haemonchiasis/parasitology , Haemonchiasis/prevention & control , Helminth Proteins/administration & dosage , Helminth Proteins/immunology , Immunoglobulin G/blood , Sheep , Sheep Diseases/parasitology , Vaccination , Vaccines/administration & dosage , Vaccines/immunology
8.
Mol Immunol ; 46(16): 3292-9, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19699526

ABSTRACT

Galectin-3 (Gal-3) is a beta-galactoside binding lectin displaying both intracellular and extracellular immune functions. In Schistosoma mansoni infection, Gal-3 has been associated with the induction of a T helper 2 response. Whereas dendritic cells (DCs) play a pivotal role in the regulation of T cell differentiation, little is known about the regulation of Gal-3 expression in DCs. In this study we determined Gal-3 mRNA and protein levels during in vitro differentiation of human monocytes into immature DCs (iDCs), by culturing monocytes in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Gal-3 mRNA levels show a moderate, transient increase during iDC generation, accompanied by elevated cell-associated Gal-3 protein. Our data show that culturing monocytes with IL-4 alone strongly increases Gal-3 mRNA levels, whereas GM-CSF induces a low increase in Gal-3 mRNA. The combined data indicate that GM-CSF reduces IL-4 induced Gal-3 mRNA levels during the generation of iDC. Remarkably, stimulation of monocytes with GM-CSF results in secretion of significant amounts of Gal-3 in the medium, whereas iDCs do not release detectable amounts of Gal-3, indicating a suppressive role of IL-4 on GM-CSF induced Gal-3 secretion. Finally, our data demonstrate that all differentiated cell types tested show a significantly lower capacity to bind Gal-3 on the cell surface than monocytes. In conclusion, Gal-3 expression in iDCs is restricted, and Gal-3 protein is localized mainly intracellular, due to the opposite actions of IL-4 and GM-CSF. By these properties, the DCs may be protected against Gal-3 induced phosphatidylserine (PS) exposure and/or apoptosis.


Subject(s)
Cell Differentiation/physiology , Dendritic Cells/metabolism , Galectin 3/biosynthesis , Gene Expression Regulation/physiology , Monocytes/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Differentiation/drug effects , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Galectin 3/immunology , Gene Expression Regulation/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-4/immunology , Interleukin-4/metabolism , Interleukin-4/pharmacology , Monocytes/cytology , Monocytes/immunology , Phosphatidylserines/genetics , Phosphatidylserines/immunology , Phosphatidylserines/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Schistosomiasis mansoni/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism
9.
Carbohydr Res ; 344(12): 1501-7, 2009 Aug 17.
Article in English | MEDLINE | ID: mdl-19541294

ABSTRACT

Several parasitic helminthes, such as the human parasite Schistosoma mansoni, express glycoconjugates that contain terminal GalNAc beta1-4(Fuc alpha1-3)GlcNAc beta-R (LDNF) moieties. These LDNF glycans are dominant antigens of the parasite and are recognized by human dendritic cells via the C-type lectin DC-SIGN. To study the functional role of the LDNF antigen in interaction with the immune system, we have developed an easy chemoenzymatic method to synthesize multivalent neoglycoconjugates carrying defined amounts of LDNF antigens. An acceptor substrate providing a terminal N-acetylglucosamine was prepared by coupling a fluorescent hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N'-diacetylchitobiose. By the subsequent action of recombinant Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase and human alpha1,3-fucosyltransferase VI (FucT-VI), this substrate was converted to the LDNF antigen. We showed that human FucT-VI has a relatively high affinity for the unusual substrate GalNAc beta1-4GlcNAc (LDN), and this enzyme was used to produce micromolar amounts of LDNF-DAP. The synthesized LDNF-DAP was coupled to carrier protein via activation of the DAP moiety by diethyl squarate. By varying the molar glycan:protein ratio, neoglycoconjugates were constructed with defined amounts of LDNF, as was determined by MALDI-TOF analysis and ELISA using an anti-LDNF antibody.


Subject(s)
Antigens, Helminth/chemistry , Glycoconjugates/chemical synthesis , Lactose/analogs & derivatives , Animals , Chromatography, High Pressure Liquid , Disaccharides/chemistry , Enzyme-Linked Immunosorbent Assay , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Glycoconjugates/chemistry , Glycoconjugates/metabolism , Humans , Lactose/chemistry , Models, Theoretical , Molecular Structure , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Pyridines/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization
10.
Infect Immun ; 75(11): 5148-57, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17785480

ABSTRACT

Galectin-3 (Gal-3) is a multifunctional beta-galactoside-binding lectin that senses self-derived and microbial glycoconjugates. Although Gal-3 is important in immune reactions and host defense in some experimental models, the function of Gal-3 during helminthic diseases (e.g., schistosomiasis) is still elusive. We show that, compared to wild-type Schistosoma mansoni-infected mice, infected Gal-3-/- mice have a reduced number of T and B lymphocytes in the spleen, develop reduced liver granulomas at 7 weeks (acute phase) and 14 weeks (chronic phase) postinfection, and mount a biased cellular and humoral Th1 response. In an attempt to understand this latter phenomenon, we studied the role of endogenous Gal-3 in dendritic cells (DCs), the most potent antigen-presenting cells, both in vitro and in vivo. Although Gal-3 deficiency in DCs does not impact their differentiation and maturation processes, it greatly influences the strength (but not the nature) of the adaptive immune response that they trigger, suggesting that Gal-3 deficiency in some other cell types may be important during murine schistosomiasis. As a whole, this study implies that Gal-3 is a modulator of the immune/inflammatory responses during helminthic infection and reveals for the first time that Gal-3 expression in DCs is pivotal to control the magnitude of T-lymphocyte priming.


Subject(s)
Dendritic Cells/immunology , Galectin 3/deficiency , Galectin 3/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Animals , Antibodies, Protozoan/blood , B-Lymphocytes/immunology , Cytokines/biosynthesis , Female , Granuloma/pathology , Immunoglobulin G/blood , Liver/pathology , Mice , Mice, Knockout , Spleen/immunology , T-Lymphocytes/immunology
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