Sylvie Marleau

Reduced Inflammation and Tissue Damage in Transgenic Rabbits Overexpressing 15-Lipoxygenase and Endogenous Anti-inflammatory Lipid Mediators

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB4 or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB4. PMN from TG rabbits also exhibited a dramatic reduction in LTB4-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA4) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA4 stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA4 is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.

Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study

The GHRPs (growth hormone-releasing peptides) are a class of small synthetic peptides known to stimulate GH release through binding of a G-protein-coupled receptor (designated GHS-R). We have found that hexarelin, a hexapeptide member of the GHRPs, binds to another protein identified as CD36, a scavenger receptor that is expressed in various tissues, including monocytes/macrophages and the endothelial microvasculature. CD36 is involved in the endocytosis of oxLDL (oxidized low-density lipoprotein) by macrophages, and in the modulation of angiogenesis elicited by thrombospondin-1 through binding to endothelial cells. To define the binding domain for hexarelin on CD36, covalent photolabelling of CD36 followed by enzymic and chemical degradation of the photoligand-receptor complex was performed. A 8 kDa photolabelled fragment corresponding to the CD36-(Asn132-Glu177) sequence has been identified as the hexarelin-binding site. Chemical cleavage of this fragment with CNBr resulted in the release of the free ligand, suggesting that Met169 is the contact point for the ligand within the receptor binding pocket. We conclude that the binding domain for hexarelin on CD36 overlaps with that for oxLDL, which corresponds to residues Gln155-Lys183 of CD36. Hence hexarelin might interfere with the CD36-mediated uptake of modified lipoproteins by macrophages. This may contribute, at least in part, to the anti-atherosclerotic effect of GHRPs in apolipoprotein E-deficient mice.

Histoplasma capsulatum Cell Wall β-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B4 Generation and Role in HIV-1 Infection

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B4 plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B4 and PGE2. Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly β-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that β-glucan plays a signaling role in LB formation. In agreement with this hypothesis, β-glucan-elicited LB formation was inhibited in leukocytes from 5-LO−/−, CD18low and TLR2−/− mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after β-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection.

The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.

AIMS CD36 has been shown to associate with non-receptor Src kinases to activate mitogen-activated protein kinases and trigger cytoskeletal remodelling, important events in foam cell formation and macrophage migration. Yet, its role in regulating circulating mononuclear phagocyte trafficking to atherosclerotic lesions has not been investigated. The aim of the present study was to investigate the role of CD36 in modulating the recruitment of mononuclear phagocytes to the arterial wall and the associated vascular inflammation, using both pharmacological and genetic approaches. METHODS AND RESULTS Apolipoprotein E-deficient (apoE(-/-)) mice fed a high-fat, high-cholesterol diet were treated daily with a CD36 ligand, EP 80317 (300 microg/kg), or 0.9% NaCl for 6 or 12 weeks. Forty-eight hours before sacrifice, mice were injected iv with (111)Indium-labelled macrophages. A 65% (P < 0.001) reduction of labelled macrophage accumulation at aortic lesions was observed in EP 80317-treated mice, mainly at the level of the aortic arch and iliac arteries, correlating with a 43% reduction of atherosclerotic lesion areas. This was associated with reduced phosphorylation of the focal adhesion kinase Pyk2 following stimulation with oxidized phospholipid in a Src kinase- and CD36-dependent manner. At the vascular level, EP 80317 treatment reduced the expression of pro-inflammatory proteins, including NADPH oxidase, inducible nitric oxide synthase, vascular endothelial cell adhesion molecule-1, and CCL2 chemokine. Plasma IL-6 levels were also reduced by 40% (P < 0.05). In contrast, none of these proteins was modulated in EP 80317-treated apoE/CD36 double knockout (apoE(-/-)/CD36(-/-)) mice. CONCLUSION Our results support a role for CD36 signalling in the regulation of mononuclear phagocyte trafficking to atherosclerotic-prone sites and in the associated vascular wall inflammation.

CD36-mediated cholesterol efflux is associated with PPARγ activation via a MAPK-dependent COX-2 pathway in macrophages

AIMS Growth hormone-releasing peptides (GHRPs) as CD36 selective ligands feature potent anti-atherosclerotic activity that is associated with an upregulation of the peroxisome proliferator-activated receptor gamma (PPARgamma)-liver X receptor alpha (LXRalpha)-ATP-binding cassette (ABC) transporter pathway. However, the mechanism involved in PPARgamma activation in response to CD36 signalling has yet to be determined. Therefore, the present study aims to elucidate the upstream molecular mechanisms through which EP 80317, a selective CD36 ligand, promotes lipid efflux from macrophages through PPARgamma activation. METHODS AND RESULTS [3H]-Cholesterol- and [3H]-methylcholine chloride-labelled murine macrophages treated with EP 80317 showed a significant increase in cholesterol and phospholipid efflux to both apolipoprotein A-I and high-density lipoprotein in a CD36-dependent manner. Lipid efflux was associated with enhanced activation of PPARgamma. The signalling pathway by which this CD36 ligand promoted lipid efflux involved an increase in intracellular 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) levels induced by extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent cyclooxygenase-2 (COX-2) expression, leading to PPARgamma activation. In agreement, EP 80317-mediated cholesterol efflux was abrogated by inhibitors of PPARgamma, ERK1/2, and COX-2 as well as ABC transporter inhibitors, whereas a p38 mitogen-activated protein kinase inhibitor had no effect. CONCLUSION These findings suggest a central role for the prostanoid 15d-PGJ2 in PPARgamma activation and the upregulation of the ABC transporter pathway in response to CD36 activation by synthetic GHRPs analogues. The resulting enhanced cholesterol efflux might explain, at least in part, the atheroprotective effect of selective CD36 ligands.

Toll-like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet-activating factor

Activation of toll‐like receptors (TLRs) and polymorphonuclear leukocyte (PMN) accumulation at infection sites are critical events of host defense. The involvement of leukotriene (LT) B4 and platelet‐activating factor (PAF) in TLR ligand‐induced activation of inflammatory cell functions is essentially unknown. Using an in vitro model of human PMN migration through human endothelial cell monolayers’ we demonstrate that prototypic ligands of TLR1/2, 2/6, 3, 4, 5, and 7/8 promote PMN migration, an effect markedly inhibited by 3 LTB4 receptor antagonists (70–80% inhibition at 100 nM compared to vehicle‐treated cells), 3 PAF receptor antagonists (20–50% inhibition at 10 nM), 3 LT biosynthesis inhibitors (75–85% inhibition at 100 nM), and 1 cytosolic phospholipase A2α (cPLA2α) inhibitor (90% inhibition at 1 μM). Accordingly, selected TLR ligands caused Ser505‐phosphorylation of cPLA2α and measurable LTB4 and PAF biosynthesis in the transmigration assay. As negative controls, interleukin‐8‐ and formyl‐methionyl‐leucyl‐phenylalanine‐elicited migration in vitro was not inhibited either by an LTB4 receptor antagonist or by the cPLA2α inhibitor. Finally, LTB4 and PAF receptor antagonists inhibited (up to ~65% at optimal doses) TLR ligand‐induced PMN infiltration in the mouse air‐pouch model. These studies unravel the critical involvement of de novo LTB4 and PAF biosynthesis in PMN migration elicited by TLR ligands.—Lefebvre, J. S., Marleau, S., Milot, V., Lévesque, T., Picard, S., Flamand, N., Borgeat, P. Toll‐like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet‐activating factor. FASEB J. 24, 637–647 (2010). www.fasebj.org

GPR103b functions in the peripheral regulation of adipogenesis.

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and -26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and -26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC(50)s of 2.3 +/- 1.2 and 1.1 +/- 1.0 nm, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR-gamma, and C/EBP-alpha), was increased by 2- to 3-fold after treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis.

EP 80317, a selective CD36 ligand, shows cardioprotective effects against post-ischaemic myocardial damage in mice

AIMS The CD36 receptor plays an important role in facilitating fatty acid transport to the heart. The present study aimed to assess whether EP 80317, a selective synthetic peptide ligand of CD36, is cardioprotective in a murine model of myocardial ischaemia and reperfusion (MI/R) injury. METHODS AND RESULTS Mice were pretreated with daily subcutaneous injections of EP 80317 for 14 days before being subjected to a 30 min ligation of the left anterior descending coronary artery. The treatment reduced the infarct area and improved myocardial haemodynamics and function, as shown by an increase in cardiac output, ejection fraction and stroke work, and a reduced total peripheral resistance. In contrast, administration of EP 51389, a tripeptide analogue devoid of binding affinity to CD36, did not protect against myocardial injury. Six hours after myocardial reperfusion, EP 80317-treated mice showed reduced myocardial fatty acid uptake, as assessed by micro-positron emission tomography, in agreement with reduced levels of circulating non-esterified fatty acids. Studies using [(14)C]-palmitate infusion revealed reduced lipolysis, although no significant change in insulin or catecholamine plasma levels were observed. Increased expression levels of adipogenic and anti-lipolytic genes further supported an effect of EP 80317 in preventing fatty acid mobilization from adipose tissue. No effect of the treatment was observed in CD36(-/-) mice. CONCLUSION Our results show that pretreatment with EP 80317 protected the heart against damage and dysfunction elicited by MI/R, along with a transient reduction in peripheral lipolysis. Our findings support CD36 as a novel target for the treatment of ischaemic cardiopathy.

Induction of Anti-Anti-Idiotype Antibodies Against Sulfated Glycosaminoglycans Reduces Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—The pathogenesis of atherosclerosis is associated with the early retention of low-density lipoproteins that are trapped in the extracellular matrix of the arterial intima by interaction with glycosaminoglycan side chains of proteoglycans. Mutant mouse/human chimeric antibodies of the murine monoclonal antibody P3, which react with N-glycolyl–containing gangliosides and sulfated glycosaminoglycans, were tested for their potentially antiatherogenic properties through the induction of an idiotypic antibody network that may specifically interfere with the binding of low-density lipoproteins to proteoglycan side chains, low-density lipoprotein modification, and foam cell formation. Methods and Results—Apolipoprotein E–deficient mice fed a high-fat, high-cholesterol diet received 5 to 6 doses of chP3R99 or chP3S98 mutant antibodies, showing high and low reactivity, respectively, against their respective antigens. Both chimeric antibodies elicited an immunodominant anti-idiotypic response in the absence of adjuvant. A striking (40%–43%) reduction (P<0.01) in total lesion areas was observed in 18-week-old mice immunized with chP3R99, but not chP3S98, compared with PBS-treated mice. The antiatherosclerotic effect was associated with increased mice sera reactivity against heparin and sulfated glycosaminoglycans, including chondroitin and dermatan sulfate. In addition, purified IgG from chP3R99-immunized mice blocked the retention of apolipoprotein B–containing lipoproteins within the arterial wall of apolipoprotein E−/− mice. Conclusion—The present study supports use of active immunization and the mounting of an idiotypic antibody network response against glycosaminoglycans as a novel approach to target atherosclerosis.

Analysis of albuterol in human plasma based on immunoaffinity chromatograhic clean-up combined with high-performance liquid chromatography with fluorimetric detection

A method combining immunoaffinity chromatography with high-performance liquid chromatography was developed for the determination of albuterol in human plasma. The immunoaffinity chromatography, based on the specific interaction of albuterol with the immobilized antibody raised against it, was used as a clean-up step. Albuterol eluted from this immunochemical solid-phase clean-up step was analysed by reversed-phase high-performance liquid chromatography with fluorimetric detection. The performance of the assay was validated on six normal volunteers after a 4-mg oral dose of albuterol, which gave a peak plasma concentration in the range 6.67-15.31 ng/ml at 3-4 h after the dose. Plasma levels (0.79-1.56 ng/ml) of albuterol could be detected up to 24 h after the dose.