Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS

Comparaison des mécanismes de toxicité redox du cadmium, du cuivre et du zinc : place des métallothionéines et de p53

DNA damage induced by cadmium (Cd) come not only from various antioxidant molecules inactivation, but also from the interference of this metal with other systems involved an alteration of essential metals homeostasis such as zinc (Zn) and copper (Cu). To understand the involvement of oxidative stress in the mechanisms of toxicity induced by metals, we analyzed their impact on antioxidant balance (biomarkers of oxidative stress, the major antioxidant enzymes activities, synthesis of other antioxidants factors such as metallothionein and glutathione) of two cellular lines, a cell line derived from human keratinocytes and immortalized by p53 gene mutation (HaCaT), and a line issue of rat glioblastoma (C6). Our results are in favour of prooxidant effects. The results showed that the 3 metals affect the cells redox status. This is revealed by an increased of oxidized glutathione level (GSSG), MDA level, inducing DNA damage, and not only a decrease in protein thiols groups (SH) levels, glutathione (GSH) level, but also glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GRase) and superoxide dismutases (SODs) activities. The metals impact on the antioxidant enzymes activities is accompanied by an increase in free radicals concentrations such as the hydroxyl radical, which can initiates lipid peroxidation; proteins, GSH oxidation and DNA damage.Unlike other metals, Cd up 50 µM has a relatively low toxicity in HaCaT cells. This resistance is mainly explained by the presence of metallothionein (MTs) in basal state on the one hand and a synthesis of GSH and MTs induced by Cd on the other hand. The MT expression induced by Cd, Cu or Zn and MTs nuclear translocation suggests DNA protection role of these MTs against oxidative damage. The expression of MTs is regulated not only by a synthesis metals-induced but also by the GSH and p53 protein levels whose mutation has an impact on intracellular MTs levels and impact on HaCaT resistance to Cd.These studies indicate that the imbalance between inhibition of antioxidants activities and synthesis of other factors such as GSH and MTs is strongly involved in metals toxicities. This imbalance is the cause of the oxidative stress increasing and explains the C6 cells sensitivity and high mortality induced by Cd. In these C6 cells, the antioxidant enzymes activities, GSH and MTs levels decrease at 20 μM Cd and 475 µM Cu. This decrease is accompanied by an increased cell death. The cell death obtained after incubation with Cd, Cu or Zn is dose-dependent and very different between C6 and HaCaT lines. Indeed, HaCaT cells in the presence of Cd and Cu have a nonapoptotic death while C6 cells, incubated with Cd, die via a p53-dependent apoptosis.Les dommages engendrés par l'exposition au cadmium (Cd) proviennent non seulement de l'inactivation de diverses molécules antioxydantes mais aussi de l'interférence du métal avec d'autres systèmes impliqués dans la régulation de l'homéostasie redox et de l'homéostasie de métaux essentiels comme le zinc (Zn) et le cuivre (Cu). Afin de mieux comprendre l'implication du stress oxydant dans les mécanismes de toxicité induits par ces métaux de transition, nous avons analysé leur impact sur la balance antioxydante (biomarqueurs de stress oxydant, activités des principales enzymes antioxydantes, synthèse d'autres facteurs antioxydants comme les métallothionéines, le glutathion intracellulaire) de deux lignées cellulaires ; une lignée cellulaire dérivée de kératinocytes humains et immortalisée par mutation du gène p53 (HaCaT); ainsi qu'une lignée issue du glioblastome de rat (C6). Nos résultats sont en faveur d'un effet globalement pro-oxydant. Ils montrent que les 3 métaux affectent le statut redox des cellules. Ceci est révélé par une augmentation du taux de glutathion oxydé (GSSG), du taux de MDA, des lésions de l'ADN, et une décroissance non seulement du taux des thiols protéiques (SH), de glutathion (GSH) mais aussi des activités glutathion peroxydase (GPx), catalase (CAT), glutathion réductase (GRase) et des superoxydes dismutases (SODs). L'impact des métaux sur les activités des enzymes antioxydantes s'accompagne d'une augmentation de la quantité de radicaux libres comme le radicale hydroxyle qui peut initier la peroxydation lipidique, ainsi que l'oxydation des protéines, du GSH et de l'ADN.Contrairement aux autres métaux, le Cd présente une toxicité relativement faible dans les cellules HaCaT pour des concentrations inférieures ou égales à 50 µM. Cette résistance s'explique principalement par la présence des métallothionéines (MTs) à l'état basal d'une part et par une synthèse induite par le Cd du GSH et des MTs d'autre part. L'induction des MTs par le Cd, le Cu ou le Zn et leur redistribution nucléaire fait de ces protéines un acteur prépondérant dans la protection du génome contre les dommages oxydatifs. L'expression des MTs est régulée non seulement par une synthèse induite par les métaux mais aussi par le GSH et la protéine p53 dont la mutation a un impact sur le taux intracellulaire de MTs et sur la résistance des HaCaT au Cd. Ces études indiquent que le déséquilibre entre l'inhibition des activités antioxydantes et la synthèse des facteurs comme le GSH et les MTs est très impliqué dans la toxicité des métaux. C'est ce déséquilibre qui est à l'origine de l'augmentation du stress oxydant et qui explique la sensibilité et la forte mortalité des cellules C6 induite par le Cd. En effet dans ces cellules, les activités des enzymes antioxydantes et les taux de GSH et de MTs diminuent dès 20 µM. Cette diminution s'accompagne d'une mort cellulaire accrue. La mort cellulaire obtenue après incubation avec le Cd, le Cu ou le Zn est dose-dépendante et très différente entre les lignées C6 et HaCaT. En effet, les cellules HaCaT en présence de Cd ou de Cu présentent une mort non apoptotique alors que les cellules C6, incubées avec le Cd, meurent par une voie apoptotique p53-dépendante

Comparaison des mécanismes de toxicité redox du cadmium, du cuivre et du zinc : place des métallothionéines et de p53

DNA damage induced by cadmium (Cd) come not only from various antioxidant molecules inactivation, but also from the interference of this metal with other systems involved an alteration of essential metals homeostasis such as zinc (Zn) and copper (Cu). To understand the involvement of oxidative stress in the mechanisms of toxicity induced by metals, we analyzed their impact on antioxidant balance (biomarkers of oxidative stress, the major antioxidant enzymes activities, synthesis of other antioxidants factors such as metallothionein and glutathione) of two cellular lines, a cell line derived from human keratinocytes and immortalized by p53 gene mutation (HaCaT), and a line issue of rat glioblastoma (C6). Our results are in favour of prooxidant effects. The results showed that the 3 metals affect the cells redox status. This is revealed by an increased of oxidized glutathione level (GSSG), MDA level, inducing DNA damage, and not only a decrease in protein thiols groups (SH) levels, glutathione (GSH) level, but also glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GRase) and superoxide dismutases (SODs) activities. The metals impact on the antioxidant enzymes activities is accompanied by an increase in free radicals concentrations such as the hydroxyl radical, which can initiates lipid peroxidation; proteins, GSH oxidation and DNA damage.Unlike other metals, Cd up 50 µM has a relatively low toxicity in HaCaT cells. This resistance is mainly explained by the presence of metallothionein (MTs) in basal state on the one hand and a synthesis of GSH and MTs induced by Cd on the other hand. The MT expression induced by Cd, Cu or Zn and MTs nuclear translocation suggests DNA protection role of these MTs against oxidative damage. The expression of MTs is regulated not only by a synthesis metals-induced but also by the GSH and p53 protein levels whose mutation has an impact on intracellular MTs levels and impact on HaCaT resistance to Cd.These studies indicate that the imbalance between inhibition of antioxidants activities and synthesis of other factors such as GSH and MTs is strongly involved in metals toxicities. This imbalance is the cause of the oxidative stress increasing and explains the C6 cells sensitivity and high mortality induced by Cd. In these C6 cells, the antioxidant enzymes activities, GSH and MTs levels decrease at 20 μM Cd and 475 µM Cu. This decrease is accompanied by an increased cell death. The cell death obtained after incubation with Cd, Cu or Zn is dose-dependent and very different between C6 and HaCaT lines. Indeed, HaCaT cells in the presence of Cd and Cu have a nonapoptotic death while C6 cells, incubated with Cd, die via a p53-dependent apoptosis.Les dommages engendrés par l'exposition au cadmium (Cd) proviennent non seulement de l'inactivation de diverses molécules antioxydantes mais aussi de l'interférence du métal avec d'autres systèmes impliqués dans la régulation de l'homéostasie redox et de l'homéostasie de métaux essentiels comme le zinc (Zn) et le cuivre (Cu). Afin de mieux comprendre l'implication du stress oxydant dans les mécanismes de toxicité induits par ces métaux de transition, nous avons analysé leur impact sur la balance antioxydante (biomarqueurs de stress oxydant, activités des principales enzymes antioxydantes, synthèse d'autres facteurs antioxydants comme les métallothionéines, le glutathion intracellulaire) de deux lignées cellulaires ; une lignée cellulaire dérivée de kératinocytes humains et immortalisée par mutation du gène p53 (HaCaT); ainsi qu'une lignée issue du glioblastome de rat (C6). Nos résultats sont en faveur d'un effet globalement pro-oxydant. Ils montrent que les 3 métaux affectent le statut redox des cellules. Ceci est révélé par une augmentation du taux de glutathion oxydé (GSSG), du taux de MDA, des lésions de l'ADN, et une décroissance non seulement du taux des thiols protéiques (SH), de glutathion (GSH) mais aussi des activités glutathion peroxydase (GPx), catalase (CAT), glutathion réductase (GRase) et des superoxydes dismutases (SODs). L'impact des métaux sur les activités des enzymes antioxydantes s'accompagne d'une augmentation de la quantité de radicaux libres comme le radicale hydroxyle qui peut initier la peroxydation lipidique, ainsi que l'oxydation des protéines, du GSH et de l'ADN.Contrairement aux autres métaux, le Cd présente une toxicité relativement faible dans les cellules HaCaT pour des concentrations inférieures ou égales à 50 µM. Cette résistance s'explique principalement par la présence des métallothionéines (MTs) à l'état basal d'une part et par une synthèse induite par le Cd du GSH et des MTs d'autre part. L'induction des MTs par le Cd, le Cu ou le Zn et leur redistribution nucléaire fait de ces protéines un acteur prépondérant dans la protection du génome contre les dommages oxydatifs. L'expression des MTs est régulée non seulement par une synthèse induite par les métaux mais aussi par le GSH et la protéine p53 dont la mutation a un impact sur le taux intracellulaire de MTs et sur la résistance des HaCaT au Cd. Ces études indiquent que le déséquilibre entre l'inhibition des activités antioxydantes et la synthèse des facteurs comme le GSH et les MTs est très impliqué dans la toxicité des métaux. C'est ce déséquilibre qui est à l'origine de l'augmentation du stress oxydant et qui explique la sensibilité et la forte mortalité des cellules C6 induite par le Cd. En effet dans ces cellules, les activités des enzymes antioxydantes et les taux de GSH et de MTs diminuent dès 20 µM. Cette diminution s'accompagne d'une mort cellulaire accrue. La mort cellulaire obtenue après incubation avec le Cd, le Cu ou le Zn est dose-dépendante et très différente entre les lignées C6 et HaCaT. En effet, les cellules HaCaT en présence de Cd ou de Cu présentent une mort non apoptotique alors que les cellules C6, incubées avec le Cd, meurent par une voie apoptotique p53-dépendante

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

International audienceCell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells

Oxidative Stress Induced by Cadmium in the C6 Cell Line: Role of Copper and Zinc

International audienceIn this report, we have investigated the role of copper (Cu) and zinc (Zn) in oxidative stress induced by cadmium (Cd) in C6 cells. Cells were exposed to 20 μM Cd, 500 μM Cu, and 450 μM Zn for 24 h. Then, toxic effects, cellular metals levels, oxidative stress parameters, cell death, as well as DNA damage were evaluated. Cd induced an increase in cellular Cd, Cu, and Zn levels. This results not only in the inhibition of GSH-Px, GRase, CAT, and SOD activities but also in ROS overproduction, oxidative damage, and apoptotic cell death not related to Cu and Zn mechanisms. The thiol groups and GSH levels decreased, whereas the lipid peroxidation and DNA damage increased. The toxicity of Zn results from the imbalance between the inhibition of antioxidant activities and the induction of MT synthesis. The increase in Cu and Zn levels could be explained by the disruption of specific transporter activities, Cd interference with signaling pathways, and metal displacement. Our results suggest that the alteration of Cu and Zn homeostasis is involved in the oxidative stress induced by Cd

The toxicity redox mechanisms of cadmium alone or together with copper and zinc homeostasis alteration: its redox biomarkers.

International audienceCadmium (Cd) is a toxic metal and can induce and/or promote diseases in humans (cancer, aging diseases, kidney and bone diseases, etc.). Its toxicity involves many mechanisms including the alteration of copper (Cu) and zinc (Zn) homeostasis leading to reactive oxygen species (ROS) production, either directly or through the inhibition of antioxidant activities. Importantly, ROS can induce oxidative damages in cells. Cadmium, Cu and Zn are also able to induce glutathione (GSH) and metallothioneins (MT) synthesis in a cell-type-dependent manner. As a consequence, the effects induced by these three metals result simultaneously from the inhibition of antioxidant activities and the induction of other factors such as GSH and MT synthesis. MT levels are regulated not only by the p53 protein in a cell-type-dependent manner, or by transcription factors such as metal-responsive transcription factor 1 (MTF-1) and cellular Zn levels but also by cellular GSH level. As described in the literature, DNA damage, GSH and MT levels are sensitive biomarkers used to identify Cd-induced toxicity alone or together with Cu and Zn homeostasis alteration

Metallothionein expression in HaCaT and C6 cell lines exposed to cadmium

International audienceMetallothioneins (MT) are low-molecular weight, cysteine-rich metal-binding proteins. MT play a role in the homeostasis of essential metals such as zinc (Zn) and copper (Cu), detoxification of toxic metals such as cadmium (Cd) and protection against oxidative stress. In this study, we examined the expression of MT in HaCaT and C6 cells as a strategy to enhance protection against Cd-mediated toxicity. At basal level, HaCaT cells showed higher MT level than C6 cells which could explain the resistance of HaCaT cells. Western blot showed that C6 cells treated with 20 μmol/L Cd for 24 h did not express any MT. MT were initially expressed in the cytoplasmic or periplasmic compartment and were then translocated in the nucleus after 24 h treatment by Cd both in HaCaT and C6 cells. In addition, the cell treatment with Cd was followed by an increase in the cellular zinc level but the electrophoretic mobility shift assay (EMSA) experiment did not show any translocation of metal-responsive transcription factor-1 (MTF-1) to the nucleus of HaCaT cells. These absence of translocation could be due to the presence of MT in these cells at the basal state. The translocation study in HaCaT cells suggested that the MT translocation in the nucleus was greater than observed in C6 cells. The latter observation could explain HaCaT cells resistance to Cd concentrations up to 50 μmol/L. Our results suggested that the C6 cell sensitivity was correlated with the decrease in MT level at 20 μmol/L Cd occurring after the transcription of MT gene