In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes

International audienceBivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/ RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNI production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters

Effet de la qualité lipidique du régime nutritif sur la composition des membanes des hémocytes de C. gigas. Impact sur les capacités de défense

Des études ont déjà montré que la composition lipidique des micro-algues utilisées dans les écloseries et nurseries avait des répercussions sur la croissance, le développement et la survie des bivalves. L'objectif de la présente étude est de déterminer l'influence de l'alimentation sur la composition membranaire des hémocytes et sur les mécanismes de défense des bivalves. Des huîtres, Crassostrea gigas, et des palourdes, Ruditapes philippinarum, ont été nourries avec trois algues: Chaetoceros calcitrans déficiente en 22:6(n-3), lsochrysis galballa déficiente en 20:5(n-3) et 20:4(n-6), Tetraselmis suecica déficiente en 22:6(n-3), 20:5(n- 3) et 20:4(n-6). Dans notre étude, les animaux nourris avec T. suecica présentent le plus faible indice de condition. Les déficiences en acides gras poly-insaturés (AGPI) de l'algue sont probablement à l'origine de cette mauvaise  croissance pour les espèces. Les fortes mortalités observées chez C. gigas pour tous les conditionnements semblent refléter une plus grande sensibilité face au régime mono-spécifique et/ou aux  conditions expérimentales. Au cours de l'expérimentation, les compositions lipidiques des membranes des hémocytes et des branchies ont été fortement influencées par celle de l'alimentation. Néanmoins, une rétention sélective de certains AGPIs a été observée dans les tissus analysés: le 22:6(n-3) pour la palourde et le 20:5(n-3) pour l'huître. Les paramètres immunitaires sont aussi affectés par ces changements de composition dans les membranes des hémocytes. En effet, l'enrichissement de 20:5(n-3) et de 20:4(n-6) semble avoir un effet positif sur la phagocytose et l'activité métabolique des hémocytes de palourdes. Un effet positif du 20:5(n-3) sur l'activité métabolique des hémocytes d'huître a aussi été observé mais il est de moindre importance. Enfin, lorsque les hémocytes d'huître sont placés dans des conditions stressantes,  on observe une corrélation positive entre le 22:6(n-3) contenu dans les hémocytes et le taux de phagocytose. Néanmoins, afin de distinguer plus clairement l'effet de chaque acide gras, de nouvelles expérimentations devront être réalisées en utilisant des aliments artificiels

Modulation des paramètres hémocytaires par la nutrition chez l'huitre creuse Crassostrea Gigas (Implication dans les mortalités estivales)

Cette étude réalisée dans le cadre des mortalités estivales de l'huître C. gigas montre que l'effort de reproduction, associé à une montée thermique, déprime les paramètres énergétiques (glucides, CEA) et hémocytaires (concentration, phagocytose, adhésion). L'augmentation de la ration alimentaire de 4% à 12% du poids sec de l';huître ne compense pas voir aggrave ces dépressions puisque l'excédant énergétique du régime 12% est alloué en priorité à la gamétogénèse. Par contre, après la gamétogénèse, la ration 12% favorise l'acquisition de réserves et corrélativement augmente la concentration en hémocytes. Les paramètres hémocytaires sont aussi sensibles à la qualité de la nourriture. Ainsi, l'apport de 20:4n-6 et/ou de 20:5n-3 par des microalgues ou aliments artificiels tend à augmenter le nombre d'hémocytes. Lorsque ces acides gras sont apportés séparément, il a été établi que le 20:4n-6 stimule la phagocytose et la production d'espèces actives de l'O2 tandis que le 20:5n-3 les inhibe.RENNES1-BU Sciences Philo (352382102) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

The influence of dietary supplementation of arachidonic acid on prostaglandin production and oxidative stress in the Pacific oyster Crassostrea gigas

International audienceIn a previous study, dietary supplementation with arachidonic acid (ARA) to oysters Crassostrea gigas increased haemocyte numbers, phagocytosis, and production of reactive oxygen species level (ROS) by haemocytes (Delaporte et al., 2006). To assess if the observed stimulation of these cellular responses resulted from changes of ARA-related prostaglandin (PG) production, we analysed prostaglandin E2 metabolite (PGEM) content on the same oysters fed three levels of ARA. Dietary supply of polyunsaturated fatty acids (PUFA) could also induce an oxidative stress that could similarly increase cellular responses; therefore, two indicators of oxidative stress were analysed: peroxidation level and antioxidant defence status. Together the observed positive correlation between ARA and PGEM levels and the absence of lipid peroxidation and antioxidant activity changes supports the hypothesis of an immune stimulation via PG synthesis. Although ARA proportion in oyster tissues increased by up to 7-fold in response to ARA dietary supplementation, peroxidation index did not change because of a compensatory decrease in n-3 fatty acid proportion, mainly 22:6n-3. To further confirm the involvement of PG in the changes of haemocyte count, phagocytosis and ROS production upon ARA supplementation, it would be interesting to test cyclooxygenase and lipooxygenase inhibitors in similar experiments

Impact of food availability on energy storage and defense related hemocyte parameters of the Pacific oyster Crassostrea gigas during an experimental reproductive cycle

Le texte intégral est accessible via Archimer: http://archimer.ifremer.fr/doc/2006/publication-1549.pdfInternational audienceThe aim of this study was to test the effect of food quantity on energy storage and defense capacities of oysters during a reproductive cycle. One-year-old Crassostrea gigas oysters were fed two different dietary rations (4% and 12% of oyster dry weight in algal dry weight per day) in controlled experimental conditions over an annual cycle. Oyster dry weights, carbohydrate and lipid contents, energetic adenylate charge, and hemocyte parameters of oysters were significantly affected by reproductive processes related to seasonal temperature variation and, to a lesser extent, by the dietary rations. Energy parameters decreased during gametogenesis as gonads developed then increased during the gonad resorption phase. The additional energy provided to oysters fed the 12% diet compared to oysters fed the 4% diet was allocated mainly to the development of more gonad tissue. Regardless of diet, hemocyte concentrations were also seasonally affected. Hemocyte concentrations were low during gametogenesis and significantly increased during the gonadal resorption and tissue restoration phase. Phagocytic activity and adhesive capacity of hemocytes were temporarily inhibited during gametogenesis and were at their lowest levels in June. Oysters fed the 12% diet had significantly higher hemocyte concentrations and lower phagocytosis activity and reactive oxygen species production compared to those fed the 4% diet

Impact of 20 : 4n-6 supplementation on the fatty acid composition and hemocyte parameters of the Pacific oyster Crassostrea gigas

International audienceArachidonic acid (20:4n-6, ArA) and its eicosanoid metabolites have been demonstrated to be implicated in immune functions of vertebrates, fish, and insects. Thus, the aim of this study was to assess the impact of ArA supplementation on the FA composition and hemocyte parameters of oysters Crassostrea gigas. Oyster dietary conditioning consisted of direct addition of ArA solutions at a dose of 0, 0.25, or 0.41 mu g ArA per mL of seawater into tanks in the presence or absence of T-Iso algae. Results showed significant incorporation of ArA into gill polar lipids when administered with algae (up to 19.7%) or without algae (up to 12.1%). ArA supplementation led to an increase in hemocyte numbers, phagocytosis, and production of reactive oxygen species by hemocytes from ArA-supplemented oysters. Moreover, the inhibitory effect of Vibrio aestuarianus extracellular products on the adhesive proprieties of hemocytes was lessened in oysters fed ArA-supplemented T-Iso. All changes in oyster hemocyte parameters reported in the present study suggest that ArA and/or eicosanoid metabolites affect oyster hemocyte functions

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

International audienceThe aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect

Stimulation of HIV-1 Replication in Immature Dendritic Cells in Contact with Primary CD4 T or B Lymphocytes ▿

Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence

Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites

International audienceHigh variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, halfsibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Rivière d'Auray (Brittany) and Ronce (Marennes-oléron Basin, Poitou Charentes). Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with Vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production. Among the six families analyzed, three have shown high survival during summer (named as "resistant", mean mortality 5.2%) and three experienced high mortality during summer (named as "susceptible", 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/ hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events