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International audience; Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 μM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination. © Copyright © 2020 Vatzia, Pierron, Hoog, Saalmüller, Mayer and Gerner.

Additional file 9. Summary of significant differences between cytokine-producing lymphocyte subsets isolated from control and H. meleagridis infected chickens. In case of H. meleagridis/E. coli stimulated results only significance of E. coli corrected values are displayed. Asterisks indicate p-value levels (*p ≤ 0.05, and **p ≤ 0.01).

The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination., Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses.

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.