Cooperation between Engulfment Receptors: The Case of ABCA1 and MEGF10

The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway

Effects of Deletion of Macrophage ABCA7 on Lipid Metabolism and the Development of Atherosclerosis in the Presence and Absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen

Influence du transporteur ABCA1 sur le microenvironnement lipidique de la membrane plasmique

Le transporteur ABCA1 est impliqué dans le transfert des phospholipides et du cholestérol vers Apo A-I sur la membrane plasmique cellulaire. Comme ABCA1 est un transporteur des lipides, nous avons examiné son effet sur le microenvironnement lipidique de la membrane. Nous avons démontré que l'activité ATP-ase d'ABCA1 modifie sa répartition dans les radeaux lipidiques, ainsi que la répartition d autres protéines de la membrane comme le récepteur de transferrine (TfR), la dynamique cinétique de TfR et réduit l endocytose de TfR. Nous avons montré par des méthodes biophysiques, cationic sensors et FLIM des modifications significatives dans le feuillet membranaire, interne et externe, par l'expression d'ABCA1. Nous avons démontré aussi par FRAP un changement général de la dynamique de la membrane en présence d'ABCA1. Nous avons corrélé la modification des propriétés de la membrane via ABCA1, avec son influence sur l'activation des macrophages en interférant avec la signalisation de l IFNgR.The ABCA1 transporter is involved in the Apo A-I/mediated removal of cellular phospholipids and cholesterol at the cell membrane. Considering that ABCA1 acts as lipid translocator we investigated the effect of the transporter on membrane lipid microenvironment. By biochemical assays, we demonstrated that the ATP-ase activity of ABCA1 modifies the partitioning in lipid rafts of the transporter itself and other membrane proteins such as the transferrin receptor (TfR), TfR dynamic kinetics and down regulates TfR-mediated endocytosis. We then assessed by biophysical methods, cationic sensors and FLIM, significant modifications of membrane attributes at the inner and outer leaflet in the presence of ABCA1. Furthermore, we evidenced overall changes in membrane dynamics in the presence of ABCA1 by FRAP. Finally, we correlate the mechanistic basis of ABCA1-dependent modulation of macrophage phenotype with the influence of ABCA1 on lipid raft dependent signaling downstream of IFNgR.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Influence du transporteur ABCA1 sur le microenvironnement lipidique de la membrane plasmique

Le transporteur ABCA1 est impliqué dans le transfert des phospholipides et du cholestérol vers Apo A-I sur la membrane plasmique cellulaire. Comme ABCA1 est un transporteur des lipides, nous avons examiné son effet sur le microenvironnement lipidique de la membrane. Nous avons démontré que l'activité ATP-ase d'ABCA1 modifie sa répartition dans les radeaux lipidiques, ainsi que la répartition d autres protéines de la membrane comme le récepteur de transferrine (TfR), la dynamique cinétique de TfR et réduit l endocytose de TfR. Nous avons montré par des méthodes biophysiques, cationic sensors et FLIM des modifications significatives dans le feuillet membranaire, interne et externe, par l'expression d'ABCA1. Nous avons démontré aussi par FRAP un changement général de la dynamique de la membrane en présence d'ABCA1. Nous avons corrélé la modification des propriétés de la membrane via ABCA1, avec son influence sur l'activation des macrophages en interférant avec la signalisation de l IFNgR.The ABCA1 transporter is involved in the Apo A-I/mediated removal of cellular phospholipids and cholesterol at the cell membrane. Considering that ABCA1 acts as lipid translocator we investigated the effect of the transporter on membrane lipid microenvironment. By biochemical assays, we demonstrated that the ATP-ase activity of ABCA1 modifies the partitioning in lipid rafts of the transporter itself and other membrane proteins such as the transferrin receptor (TfR), TfR dynamic kinetics and down regulates TfR-mediated endocytosis. We then assessed by biophysical methods, cationic sensors and FLIM, significant modifications of membrane attributes at the inner and outer leaflet in the presence of ABCA1. Furthermore, we evidenced overall changes in membrane dynamics in the presence of ABCA1 by FRAP. Finally, we correlate the mechanistic basis of ABCA1-dependent modulation of macrophage phenotype with the influence of ABCA1 on lipid raft dependent signaling downstream of IFNgR.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

L’élimination des cellules apoptotiques : une phagocytose particulière

Le programme de mort cellulaire par apoptose joue un rôle clé dans le maintien de l’homéostasie cellulaire. La première phase du programme conduit au démantèlement de la cellule. Celle-ci est très précocement reconnue et ingérée, par des phagocytes professionnels ou amateurs, grâce à la mise en œuvre d’une phagocytose spécialisée, l’engulfment ou engloutissement. Contrairement à l’attention portée aux mécanismes régissant la phase effectrice de l’apoptose, les bases moléculaires de l’engloutissement de la proie apoptotique ainsi que les conséquences physiologiques pouvant dériver d’une clairance sub-optimale n’ont été étudiées que récemment. Nous nous proposons de discuter ici les dernières avancées du domaine, ainsi que certains des attributs spécifiques de cette forme de phagocytose

Etude de la sous-classe ABCA de la famille des transporteurs ABC (Analyse génomique et inactivation fonctionnelle du gène ABC-1)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Immunopathological consequences of the loss of engulfment genes: the case of ABCA1

Programmed cell death plays a crucial role in the maintenance of cell homeostasis. An initial, effector phase leads to the generation of apoptotic corpses and is closely followed by a swift clearance by professional or amateur phagocytes. Several aspects distinguish this latter process of engulfment of dying cells from the classical forms of phagocytosis. They concern all aspects of the process from the recognition of the prey to the final outcome, i.e. immunological silence. The engulfment of dead cells is a process highly conserved through evolution and it has been studied in parallel in two systems, mammalian cells and the nematode C. elegans. ABCA1 and its ortholog CED-7 in the nematode are key players of engulfment. Their mode of action somehow original in the panorama of engulfment receptors since they act as lipid transporters. While in the worm the loss of CED-7 has phenotypic consequences exclusively on engulfment, in the mouse the deletion of ABCA1 by homologous recombination has highlighted broad consequences on macrophage biology. Among those we will discuss here the aberrant responses of ABCA1–/– mice to Plasmodium berghei ANKA infection, concerning in particular the development of cerebral malaria (CM), a cytokine-induced immunopathology. This syndrome involves a central role of monocytes and, as shown recently, high levels of circulating microparticles. It was found that ABCA1 loss completely protects against CM and its associated mortality. This observation, together with the demonstration of quantitative and functional modifications of microparticles, suggests that microparticles may be involved in CM pathogenesis. The ABCA1 transporter thus appears to control susceptibility to CM, thereby providing new insights in its pathophysiological mechanisms and potential new therapeutic avenues