Étude des effets des stimulateurs des défenses naturelles des plantes sur l’immunité innée des vertébrés et mise en évidences des acteurs communs par une approche in silico

Prod 2016-283 IPM SPE CNRS DOCTLes pesticides, utilisés pour protéger les cultures, ont souvent des effets délétères pour la santé humaine et l’environnement. Des alternatives telles que les SDN sont actuellement développées pour réduire leur utilisation. Compte tenu de la similarité des motifs moléculaires induisant l'immunité chez les plantes et les animaux, les SDN pourraient interagir avec le système immunitaire inné des vertébrés. Toutefois, lors du processus d'homologation des pesticides, aucun test ne s’intéresse directement à leurs effets pro/anti-inflammatoires bien que des maladies soient favorisées par l’inflammation chronique. En utilisant des cellules mononucléaires du sang périphérique humain, nous avons étudié l’effet cytotoxique et pro/anti-inflammatoire (production d'IL1B) des SDN commercialisés, le but étant de développer un test de sécurité sanitaire applicable en routine. Deux SDN ont eu des effets marqués sur l’activité métabolique et sur la production d'IL1B par les cellules humaines. Pour vérifier si ces produits avaient un effet similaire chez d’autres organismes, nous avons utilisé des larves de poisson zèbre (Danio rerio). Comme chez l'humain, ces deux SDN ont soit un effet pro-inflammatoire ou soit un fort effet toxique. L'immunité innée est une fonction conservée chez tous les eucaryotes et il est probable que certains récepteurs aient été conservés tout au long de l'évolution entre les plantes et les animaux. Les travaux menés ont eu pour but d’identifier par bio-informatique de nouveaux récepteurs chez les plantes à partir des connaissances obtenus chez l'homme. Notre étude a permis de mettre en évidence de nouveaux récepteurs putatifs

Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates

Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03–1 mg mL−1) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1β inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1β. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1β and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection

Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates

International audiencePlant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03-1 mg mL(-1)) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1β inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1β. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1β and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection

Effect of commercially available Plant Defence Stimulators (PDS) on human innate immunity

International audiencePDS (Plant defence stimulators) constitute a recent alternative to pesticides used for crop protection. These compounds called elicitors are of diverse nature, but they all act by stimulating innate immune system of plants. So, plants can better fight pathogens. Furthermore, there are many similarities in pathogen perception systems and cellular signalling in plants and animals. It is well established that many elicitors stimulate both human and plant innate immunity (Zipfel and Felix, 2005). Therefore, it is likely that human innate immunity could be modulated by PDS. The aim of this study is to evaluate pro/anti-inflammatory activity of five different commercially available PDS on human cell models. We studied the pro/anti-inflammatory effect of PDS (Bion® 50WG, Stifenia...) on human peripheral blood mononuclear cells (PBMC). These cells are exposed during twenty hours to various concentrations of PDS or their corresponding active molecules. Pro-inflammatory action is evaluated by measuring the quantity of the inflammatory cytokine IL-1β in the cells supernatants using ELISA test. To study anti-inflammatory effect, we used PBMC treated with LPS to trigger a basal inflammatory response. We then checked if PDS delivered at the same time as LPS modified IL-1β production. In addition, in all the experiments, the viability is evaluated with a XTT test. PDS, which were however used at equal or lower concentrations than in the fields, show different profiles in terms of cytotoxicity and inflammatory modulation. For example, Stifenia was slightly cytotoxic at 1 mg/ml and pro-inflammatory at 0,3 and 1 mg/ml concentrations. Conversely, Bion® 50WG at dosages from 0,3 mg/ml dose-dependently inhibited IL-1β production and proved to be anti-inflammatory. Interestingly, some active molecules have not the same inflammatory profile than their respective formulated PDS from the market. Our results indicate that PDS can differently interact with human innate immunity. We hope to use these particularities to better understand the innate immunity pathways that could be common in plants and animals

Lutter contre les infections bactériennes : le système immunitaire des plantes est aussi très efficace !

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Role of mannitol metabolism in the pathogenicity of the necrotrophic fungus Alternaria brassicicola

In this study, the physiological functions of fungal mannitol metabolism in the pathogenicity and protection against environmental stresses were investigated in the necrotrophic fungus Alternada brassicicola. Mannitol metabolism was examined during infection of Brass/ca oleracea leaves by sequential HPLC quantification of the major soluble carbohydrates and expression analysis of genes encoding two proteins of mannitol metabolism, i.e., a mannitol dehydrogenase (AbMdh), and a mannito1-1-phosphate dehydrogenase (AbMpd). Knockout mutants deficient for AbMdf7 or AbMpd and a double mutant lacking both enzyme activities were constructed. Their capacity to cope with various oxidative and drought stresses and their pathogenic behavior were evaluated. Metabolic and gene expression profiling indicated an increase in mannitol production during plant infection. Depending on the mutants, distinct pathogenic processes, such as leaf and silique colonization, sporulation, survival on seeds, were impaired by comparison to the wild-type. This pathogenic alteration could be partly explained by the differential susceptibilities of mutants to oxidative and drought stresses. These results highlight the importance of mannitol metabolism with respect to the ability of A. brassicrcola to efficiently accomplish key steps of its pathogenic life cycle