Clearance of apoptotic cells by macrophages induces regulatory phenotype and involves stimulation of cd36 and platelet-activating factor receptor

Phagocytosis of apoptotic cells (efferocytosis) induces macrophage differentiation towards a regulatory phenotype (IL-10high/IL-12p40low). CD36 is involved in the recognition of apoptotic cells (AC), and we have shown that the platelet-activating factor receptor (PAFR) is also involved. Here, we investigated the contribution of PAFR and CD36 to efferocytosis and to the establishment of a regulatory macrophage phenotype. Mice bone marrow-derived macrophages were cocultured with apoptotic thymocytes, and the phagocytic index was determined. Blockage of PAFR with antagonists or CD36 with specific antibodies inhibited the phagocytosis of AC (~70–80%). Using immunoprecipitation and confocal microscopy, we showed that efferocytosis increased the CD36 and PAFR colocalisation in the macrophage plasma membrane; PAFR and CD36 coimmunoprecipitated with flotillin-1, a constitutive lipid raft protein, and disruption of these membrane microdomains by methyl-β-cyclodextrin reduced AC phagocytosis. Efferocytosis induced a pattern of cytokine production, IL-10high/IL-12p40low, that is, characteristic of a regulatory phenotype. LPS potentiated the efferocytosis-induced production of IL-10, and this was prevented by blocking PAFR or CD36. It can be concluded that phagocytosis of apoptotic cells engages CD36 and PAFR, possibly in lipid rafts, and this is required for optimal efferocytosis and the establishment of the macrophage regulatory phenotype

Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR

OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR) is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV) or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR

Uptake of oxLDL and IL-10 production by macrophages requires PAFR and CD36 recruitment into the same lipid rafts

Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with βCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development

CD36-PAFR complex is present in human carotid plaques.

<p>Frozen sections of human carotid plaques were fixed with acetone and stained with rabbit and with the mouse anti-human CD36 or mouse anti-human CD68. Anti-rabbit IgG DyLight-594 or anti-mouse DyLight-488 were used as a secondary antibody. Colocalization was visualized by confocal microcopy at a 60-fold magnification. In (A), the specificity of anti-PAFR was evaluated by pre-treatment with PAF receptor blocking peptide and after stained for hPAFR. In (B) is shown double staining of PAFR with CD36 or with CD68. Yellow patches signify areas of colocalization. Figures in (A) were used as staining control and were acquired in different magnification. </p

oxLDL induces colocalization of CD36 and PAFR in lipid raft microdomains.

<p>Macrophages were stimulated with oxLDL (30 µg/mL) for 10 minutes. Cells were fixed and stained with CTxB-Alexa 488/anti-CTxB, Alexa-647 anti-PAFR, and phycoerythrin anti-CD36, as described in the methods section. Colocalization was visualized by confocal microcopy at a 60-fold magnification. In A-B, macrophages were stimulated with oxLDL and stained for GM1 lipid raft fraction (green) alone or in combination with PAFR-Alexa-647 (red) or CD36-PE (red). Yellow patches signify areas of colocalization of PAFR or CD36 and GM1. In (C), colocalization images were quantified using the package ImageJ 1.44p and Graph data are presented as mean ± SEM of 10-15 pictures in three independent experiments. Dashed lines signify the non-stimulated cells. In (D), macrophages were triple stained for GM1- Alexa 488 (green), PAFR – Alexa 647(blue) and CD36-PE (red). Colocalization areas of triple stained are visualized in white/gray patches.</p

CD36 and PAFR cooperatively mediate the uptake of oxLDL and IL-10 production.

<p>Macrophages were treated with mAb to CD36 (1:500) alone or in combination with WEB2170 (50 µmol/L) or CV3988 (10 μmol/L) for 30 min and then incubated with FITC-oxLDL for 1 h. In (A-B) The uptake was visualized in confocal microscopy and measured by FACS (B). In (C), after treatment, cells were stimulated with oxLDL (30 µg/mL) and the IL-10 production was evaluated after 24 h by ELISA. Data are presented as mean ± SEM of the mean fluorescence Intensity (MFI). ***p<0.001 comparing with non-stimulated cells. # p<0.05 comparing cells treated with non-treated cells. In (D) HEK 293T cells were transiently transfected with hCD36 and/or hPAFR and stimulated with oxLDL (30 µg/mL). The mRNA expression for IL-10 and IL-12 was evaluated after 5 h. *** p<0.001; * p<0.05 comparing with cDNA3 plasmid control transfected cells. In (E-F), THP-1 monocytes were differentiated into macrophages with PMA, followed by treatment with LDL, moxLDL or oxLDL (30 µg/mL) for 24 h. IL-10 and TGFβ were measured in the supernatant were measured by ELISA. *p<0.05 comparing with non-stimulated cells. </p

Lipid raft integrity is important for oxLDL uptake and IL-10 production.

<p>Macrophages were treated with βCD (2 mmol/L) or with the inactive analog αCD for 5 min. The uptake assay was performed by incubation with FITC-oxLDL (30 µg/mL) for 1 h and visualized by microscopy. Data are presented as representative Figure (A). The fluorescence was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech) (B). IL-10 production was measured in the supernatants after oxLDL stimulation for 24 h (C). Graph Data are presented as mean ± SEM. ***p<0.001 comparing with non-stimulated cells. #p<0.05 comparing βCD treated with non-treated cells.</p

oxLDL induces co-immunoprecipitation of CD36-PAFR-Flotillin-1.

<p>Macrophages were treated with oxLDL (30 μg/mL) or PAF (10^-7 mol/L) for 20 min at 37°C prior addition of the lysis buffer. Cell lysates were subjected to immunoprecipitating and immunoblotting as described in “Methods section” using antibodies to CD36, PAFR (A) and Flotillin-1 (B). Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment and Graph data are presented as mean ± SEM of four experiments. In A, western blot figures were obtained from the same gel. ** p<0.01 comparing oxLDL-stimulated with the non-stimulated cells (dashed lines).</p

Public policies and sports in marginalised communities: the case of Cidade de Deus, Rio de Janeiro, Brazil

Significant economic development has been experienced by Brazilian society in recent years, leading to important changes in its social structures. The country\u27s success in attracting sport mega-events has resulted in the media increasingly portraying the current period as Brazil\u27s “sport decade.” This paper considers the phenomenon of sport participation in Brazil in the context of significant investments in sport mega-events. In particular, it considers how the Brazilian government has been delivering sport and physical activity opportunities for low socio-economic groups and the extent to which people living in a marginalised community have benefited from these developments. This involves an analysis of sport and physical activity projects and programmes supported by the Brazilian federal government in the community of Cidade de Deus, one of the most densely populated favelas in Rio de Janeiro. The area is located close to the Olympic Park, which will host most of the key facilities for the 2016 Olympic Games and so is a community that could be significantly impacted, positively or negatively, by the hosting of the event. The results of the investigation provide insights into the developments that have occurred in the provision of sport and physical activity opportunities to this marginalised community and the role public policies play in facilitating access to sport and physical activity