Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations

Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents

High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.Comment: cpyright: American Society of Biochemistry and Molecular Biolog

Flt3(+) macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia

BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFκB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-α (TNFα), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia

ETUDE D'UNE BANQUE D'ADNC DIFFERENTIELLE DE CELLULES DENDRITIQUES. CARACTERISATION DE LA MOLECULE CD1E

AFIN D'ANALYSER LE PROGRAMME GENETIQUE PLUS SPECIFIQUE AUX CELLULES DENDRITIQUES (CDS), NOUS AVONS UTILISE UNE APPROCHE PAR LA BIOLOGIE MOLECULAIRE EN CONSTRUISANT UNE BANQUE D'ADNC DIFFERENTIELLE DE CDS HUMAINES DIFFERENCIEES A PARTIR DES MONOCYTES DU SANG PERIPHERIQUE. NOUS AVONS CIBLE NOS TRAVAUX SUR L'ETUDE PLUS APPROFONDIE DE DEUX TRANSCRITS. LE PREMIER CORRESPONDAIT A LA MRP4, MOLECULE DE LA FAMILLE DES MULTIDRUG RESISTANCE ASSOCIATED PROTEIN, DONT LA SEQUENCE COMPLETE DEMEURAIT MECONNUE AU MOMENT OU J'AI ENTREPRIS CE TRAVAIL. LE SECOND TRANSCRIT CORRESPONDAIT AU CD1E. LES GENES CD1 FORMENT UNE FAMILLE REGROUPANT CINQ GENES CHEZ L'HOMME. SEULES CEPENDANT QUATRE GLYCOPROTEINES CD1A, CD1B, CD1C ET CD1D AVAIENT ETE IDENTIFIEES. EXPRIMEES A LA SURFACE DES CPAGS ELLES SONT IMPLIQUEES DANS LA PRESENTATION D'ANTIGENES GLYCOLIPIDIQUES A DES LYMPHOCYTES T ET NKT. CONCERNANT LE CD1E, SEULS QUELQUES TRANSCRITS AVAIENT ETE MIS EN EVIDENCE DANS DES LIGNEES LEUCEMIQUES T ET L'EXISTENCE D'UNE MOLECULE CD1E RESTAIT A DEMONTRER. LA PLUS GRANDE PARTIE DE CE TRAVAIL DESTINEE A CARACTERISER LES MOLECULES CD1E PERMET D'AFFIRMER AUJOURD'HUI QUE LES CINQ GENES CD1 HUMAINS SONT EXPRIMES DANS LES CDS. CD1E SE DISTINGUE DES AUTRES PAR SA LOCALISATION EXCLUSIVEMENT INTRACELLULAIRE. SON EXPRESSION ET SA LOCALISATION TRES PARTICULIERE ONT EGALEMENT ETE RETROUVEES DANS D'AUTRES TYPES DE CDS, INCLUANT LES CELLULES DE LANGERHANS DE L'EPIDERME ET LES CDS THYMIQUES. LE ROLE DE CETTE MOLECULE DANS LA PRESENTATION ANTIGENIQUE EST SUGGERE PAR SA RELOCALISATION, DANS LES CDS EN COURS DE MATURATION, VERS DES ENDOSOMAUX TARDIFS DE TYPE MIIC, CONNUS POUR ETRE IMPLIQUES DANS LE CHARGEMENT EN ANTIGENES DES MOLECULES CD1 ET DES MOLECULES DU CMH DE CLASSE II. UNE ETUDE PLUS APPROFONDIE DE LA FONCTIONNALITE DES MOLECULES CD1E, A PARTIR DES CONNAISSANCES DE BASE ACQUISES A CE JOUR, PERMETTRA TRES PROBABLEMENT DE MIEUX COMPRENDRE LA BIOLOGIE DE LA PRESENTATION ANTIGENIQUE DES MOLECULES CD1.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Interactions de la protéine de stress hsp70 avec les cellules dendritiques humaines (Etude du trafic de certains antigènes de surface des cellules de Langerhans humaines)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Remodelage hépatique et cellule b pancréatique (Apport d'un modèle d'hépatectomie partielle chez le rat diabétique)

Le foie est le site receveur de la greffe d îlots pancréatiques. Des données suggèrent le rôle d un remodelage hépatique, et notamment d une régénération hépatique, dans la survie des îlots greffés dans le foie d animaux diabétiques. Peu de travaux se sont intéressés aux influences précoces et réciproques des modèles expérimentaux que sont l hépatectomie partielle et le diabète post streptozotocine. Des modifications pancréatiques post hépatectomie, décrites alors que la régénération hépatique est achevée, sont rapportées. Les effets b cellulaires précoces de la régénération hépatique, imputables aux modifications systémiques observées après hépatectomie, sont mal caractérisés. Nos résultats apportent des informations sur le modèle d hépatectomie partielle dans un contexte de diabète expérimental et des données physiopathologiques sur les conséquences pancréatiques de la régénération hépatique. En ayant caractérisé conjointement l influence hépatocytaire et métabolique du diabète post streptozotocine sur le processus de régénération hépatique chez le rat Lewis, nous mettons en avant les conséquences hépatiques de ce diabète expérimental. Nous montrons un effet rapide et précoce de la régénération hépatique sur l îlot pancréatique. Nos résultats suggèrent que les modifications systémiques induites par la régénération hépatique seraient impliquées, mais avec des effets atténués en cas de diabète post streptozotocine. Ce travail suggère un impact hépatique de ce diabète expérimental et des effets b cellulaires synchrones d une régénération hépatique. Ces effets réciproques peuvent aider à comprendre le rôle de l environnement hépatique dans l efficacité de la greffe d îlots.The liver is the implantation site for islet transplantation. Some data suggest role of hepatic remodeling process, and in particular liver regeneration, in the subsistence of the islets transplanted in liver of diabetic animals. Few works were interested in early and reciprocal influences of experimental models represented by partial hepatectomy and streptozotocin diabetes. Some pancreatic consequences following partial hepatectomy, even though the liver regeneration is achieved, are reported. The early b cell effects of the liver regeneration, attributed to the systemic modifications observed after a partial hepatectomy are not well characterized. Our results provide informations on the animal models of partial hepatectomy and physiopathological data on the pancreatic consequences of the liver regeneration. Having characterized both the hepatocytic and metabolic influence of streptozotocin diabetes on the liver regeneration process on the Lewis rat, we highlight the liver consequences of this experimental diabetes. We show a fast and early effect of the liver regeneration on pancreatic islet. Our results suggest that the systemic modifications induced by a liver regeneration are involved, but with limited effects in case of streptozotocin diabetes. This work suggests that this experimental diabetes has a hepatic impact and that there are early b cell effects of a liver regeneration. These reciprocal effects can help in understanding the role of the liver tissue in the efficiency of islet transplantation.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Dual roles for MEF2A and MEF2D during human macrophage terminal differentiation and c-Jun expression.: MEF2A and MEF2D in c-Jun expression during macrophage differentiation

International audienceRecent reports have evidenced a role for the Myocyte Enhancer Factor 2C (MEF2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show here that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A/MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunopoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of Trichostatin A, a histone deacetylase inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D:HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D:HDAC1 complexes, we analyzed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is upregulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D:p300 activator complex. The targets of MEF2A/D:HDAC1:HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D human macrophages, as activator or as repressor of gene transcription