Catherine Aude-Garcia

Quantitative and Qualitative Changes in V-J α Rearrangements During Mouse Thymocytes Differentiation Implication For a Limited T Cell Receptor α Chain Repertoire

Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR α chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR α repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3′ side than 5′ end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR α repertoire size and suggest a scenario for V usage during differentiation.

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.

Comparative Proteomic Analysis of the Molecular Responses of Mouse Macrophages to Titanium Dioxide and Copper Oxide Nanoparticles Unravels Some Toxic Mechanisms for Copper Oxide Nanoparticles in Macrophages

Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

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

Recent reports have evidenced a role for MEF2C (myocyte enhancer factor 2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show in the present study 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 immunoprecipitations 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 HDAC (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 analysed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is up-regulated 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 in human macrophages, as activators or as repressors of gene transcription.

A combined proteomic and targeted analysis unravels new toxic mechanisms for zinc oxide nanoparticles in macrophages.

UNLABELLED The cellular responses of the J774 macrophage cell line to zinc oxide and zirconium oxide nanoparticles have been studied by a comparative quantitative, protein level based proteomic approach. The most prominent results have been validated by targeted approaches. These approaches have been carried out under culture conditions that stimulate mildly the aryl hydrocarbon receptor, thereby mimicking conditions that can be encountered in vivo in complex environments. The comparative approach with two nanoparticles allows to separate the common responses, which can be attributed to the phagocytosis event per se, from the response specific to each type of nanoparticles. The zinc-specific responses are the most prominent ones and include mitochondrial proteins too, but also signaling molecules such as MyD88, proteins associated with methylglyoxal detoxification (glyoxalase 2, aldose reductase) and deoxyribonucleotide hydrolases. The in cellulo inhibition of GAPDH by zinc was also documented, representing a possible source of methylglyoxal in the cells, leading to an increase in methylglyoxal-modified DNA bases. These observations may be mechanistically associated with the genotoxic effect of zinc and its selective effects on cancer cells. BIOLOGICAL SIGNIFICANCE The responses of the murine J774 macrophage cell lines to two types of metallic oxide nanoparticles (zinc oxide and zirconium dioxide) were studied by a comparative 2D gel based approach. This allows sorting of shared responses from nanoparticle-specific responses. Zinc oxide nanoparticles induced specifically a strong decrease in the mitochondrial function, in phagocytosis and also an increase in the methylglyoxal-associated DNA damage, which may explain the well known genotoxicity of zinc. In conclusion, this study allows highlighting of pathways that may play an important role in the toxicity of the zinc oxide nanoparticles.

Improvements and simplifications in in‐gel fluorescent detection of proteins using ruthenium II tris‐(bathophenanthroline disulfonate): The poor man's fluorescent detection method

Fluorescent detection of proteins is a popular method of detection allying sensitivity, linearity and compatibility with mass spectrometry. Among the numerous methods described in the literature, staining with ruthenium II tris(bathophenanthroline disulfonate) is particularly cost‐effective, but slightly cumbersome owing to difficulties in the preparation of the complex and complexity of staining protocols. We describe here the modifications on both aspects that allow to perform a higher contrast staining and offer a more robust method of complex preparation, thereby maximizing the advantages of the method.

Enhanced susceptibility of T lymphocytes to oxidative stress in the absence of the cellular prion protein

The cellular prion glycoprotein (PrPC) is ubiquitously expressed but its physiologic functions remain enigmatic, particularly in the immune system. Here, we demonstrate in vitro and in vivo that PrPC is involved in T lymphocytes response to oxidative stress. By monitoring the intracellular level of reduced glutathione, we show that PrP−/− thymocytes display a higher susceptibility to H2O2 exposure than PrP+/+ cells. Furthermore, we find that in mice fed with a restricted diet, a regimen known to increase the intracellular level of ROS, PrP−/− thymocytes are more sensitive to oxidative stress. PrPC function appears to be specific for oxidative stress, since no significant differences are observed between PrP−/− and PrP+/+ mice exposed to other kinds of stress. We also show a marked evolution of the redox status of T cells throughout differentiation in the thymus. Taken together, our results clearly ascribe to PrPC a protective function in thymocytes against oxidative stress.

Different in vitro exposure regimens of murine primary macrophages to silver nanoparticles induce different fates of nanoparticles and different toxicological and functional consequences

Abstract Silver nanoparticles (Ag-NPs) are used in a variety of consumers’ goods. Their toxicological impact is currently intensely studied, mostly upon acute exposure, but their intracellular dissolution and fate is rather poorly documented. In this study, murine primary macrophages were exposed to a single high but non-lethal dose of Ag-NPs or to repeated, low doses of Ag-NPs. Cells were either collected immediately after acute exposure or after 72 h of recovery in the NP-free exposure medium. Ag intracellular content and distribution were analyzed by particle-induced X-ray emission, transmission electron microscopy coupled to energy-dispersive spectroscopy analysis and inductively coupled plasma mass spectrometry. In parallel, macrophage functionality as well as inflammatory and thiol-responses were assessed after Ag-NP exposure. We show that Ag accumulation in macrophages is similar upon acute and repeated exposure to Ag-NPs, and that Ag is partly expelled from cells during the 72 h recovery stage. However, acute exposure leads to a strong response of macrophages, characterized by reduced mitochondrial membrane potential, phagocytic capacity and nitric oxide (NO) production upon lipopolysaccharide (LPS) stimulation. Under this condition, we also show an increased release of proinflammatory cytokines as well as a decreased release of anti-inflammatory cytokines. This response is reversible since these biomarkers reach their basal level after the recovery phase; and is much less intense in repeatedly exposed cells. These results suggest that repeated exposure of macrophages to Ag-NPs, which is a more realistic exposure scenario than acute exposure, leads to significant Ag intracellular accumulation but a much less intense toxicological response.

Culture medium associated changes in the core proteome of macrophages and in their responses to copper oxide nanoparticles.

The physiology of cells cultured in vitro depends obviously on the external conditions, including the nutrients present in the culture medium. In order to test the influence of this parameter, J774 macrophages grown either in RPMI or in DMEM were compared by a combination of targeted analyses and a proteomic approach. The two media differ in their glucose, amino acids, and vitamins concentrations, but there were no significant differences in the cell cycle or in the percentage of phagocytic cells in both media, although the phagocytic capacity (i.e. the number of phagocytized particles) was higher in DMEM. Conversely, we found that J774 cells grown in RPMI produced more nitric oxide in response to lipopolysaccharide. The proteomic study highlighted differences affecting the central metabolism and nucleotide metabolism, cytoskeleton, protein degradation, and cell signaling. Furthermore, proteomics showed that J774 cells grown in RPMI or in DMEM and exposed to copper oxide nanoparticles respond rather differently, with only a few proteins similarly modulated between cells grown in both media. Taken together, our results show that the basal state of cells grown in two different media is different, and this may affect the way they respond to an external stimulus or stress.