Étude de l'effet de l'antibiothérapie et de l'anticoagulothérapie sur le développement de la sclérodermie expérimentale chez la souris

La sclérose systémique (SSc) est une maladie auto-immune chronique incurable caractérisée par une présentation clinique complexe et hétérogène. Notre laboratoire a développé un modèle murin de fibrose pulmonaire et cutanée qui est induit par l’immunisation répétitive avec des cellules dendritiques chargées avec des peptides de la topoisomérase I, et qui partage de nombreuses caractéristiques avec la SSc humaine. Premièrement, nous avons caractérisé la maladie expérimentale quant à sa persistance à long terme (objectif 1) et son caractère progressif (objectif 2). Une cascade de coagulation dérégulée est impliquée dans le développement de la fibrose dans la SSc. La thrombine, un médiateur clé de la coagulation, semble contribuer à ce processus. Deuxièmement, nous avons étudié l’efficacité d’un inhibiteur de la thrombine, i.e. dabigatran, dans ce modèle (objectif 3). Le microbiote intestinal semble jouer un rôle déterminant dans plusieurs pathologies, y compris les maladies auto-immunes. Troisièmement, nous avons évalué l’effet de la manipulation du microbiote des souris par l’administration de streptomycine (objectif 4). Les souris immunisées développent une maladie persistante et la fibrose observée est précédée d’une phase inflammatoire. Le dabigatran aggrave la fibrose pulmonaire et cutanée lorsqu’administré durant la période inflammatoire et n’a aucun effet protecteur durant la phase fibrotique. La manipulation du microbiote par la streptomycine aggrave l’atteinte pulmonaire lorsque l’antibiothérapie est donnée en début de vie et exacerbe l’atteinte cutanée lorsqu’administrée à l’âge adulte. Notre modèle expérimental représente donc un outil important pour évaluer différentes approches thérapeutiques pour la SSc de par sa persistance et son caractère progressif. En se basant sur nos résultats, le dabigatran ne semble pas constituer un choix thérapeutique adéquat pour traiter la fibrose chez les patients atteints de SSc. L’exposition à la streptomycine à certaines périodes de la vie affecte différentiellement le développement et les manifestations cliniques de la maladie expérimentale.Systemic sclerosis (SSc) is an incurable and chronic autoimmune disease characterized by a complex and heterogeneous clinical presentation. Our laboratory has developed a mouse model of lung and skin fibrosis that shares many features with human SSc, and is induced by repeated immunization with dendritic cells loaded with peptides of topoisomerase I. First, the long term persistence (objective 1) and progressive nature (objective 2) of this experimental disease model was characterized. A dysregulated coagulation cascade is implicated in the development of fibrosis in SSc. Thrombin, a key mediator of coagulation, appears to contribute to this process. Next, the efficacy of dabigatran, a thrombin inhibitor, to ameliorate lung and skin fibrosis was studied in this model (objective 3). Intestinal microbiota appears to play a key role in several diseases including autoimmune diseases. Finally, the effect of manipulating gut microbiota by administration of streptomycin on disease pathogenesis was evaluated in this model (objective 4). Immunized mice developed persistent fibrosis that was preceded by an inflammatory phase. Dabigatran aggravated pulmonary and skin fibrosis when administered during the inflammatory period and was not protective when given during the fibrotic phase. Manipulation of intestinal microbiota by streptomycin aggravated lung fibrosis when it was given early in life and exacerbated skin disease when administered in adulthood. Our model of experimental SSc with progressive and persistent disease represents an important tool to evaluate different therapeutic approaches for SSc. Furthermore, our results caution against the use of dabigatran as a therapeutic option to treat fibrosis in patients with SSc. Exposure to streptomycin for certain periods of life differentially affects the development and clinical manifestations of experimental SSc

Electric potential across epidermis and its role during wound healing can be studied by using an in vitro reconstructed human skin

Background : After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined. The Problem : Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of an in vitro model to study this phenomenon. Basic Science Advances : We carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. The in vitro TEP measurements over time in the wound were corroborated with histological changes and with in vivo TEP variations during porcine skin wound healing. The expression of a crucial element implicated in Na+ transport, Na+/K+ ATPase pumps, was also evaluated at the same time points during the re-epithelialization process. The ascending and decreasing TEP values were correlated with changes in the expression of these pumps. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Further, inhibition of the pump activity induced a significant decrease of the TEP and of the re-epithelization rate. Clinical Care Relevance : A better comprehension of the role of EEF could have important future medical applications regarding the treatment of chronic wound healing. Conclusion : This study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process

Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent

Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity-depleted environments by gene silencing or in\ua0situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co-expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield. A fusion protein-based system involving protease-sensitive peptide linkers to attach inactive variants of tomato cystatin SlCYS8 was then designed to relate the effects of M2 on protein levels with altered protease activities in\ua0situ. Secreted versions of the cystatin fusions transiently expressed in leaf tissue showed variable 'fusion to free cystatin' cleavage ratios, in line with the occurrence of protease forms differentially active against the peptide linkers in the secretory pathway. Variable ratios were also observed for the fusions co-expressed with M2, but the extent of fusion cleavage was changed for several fusions, positively or negatively, as a result of pH increase in the Golgi. These data indicating a remodelling of endogenous protease activities upon M2 expression confirm that the stability of recombinant proteins in the plant cell secretory pathway is pH-dependent. They suggest, in practice, the potential of M2 proton channel to modulate the stability of protease-susceptible secreted proteins in planta via a pH-related, indirect effect on host resident proteases

pH gradient mitigation in the leaf cell secretory pathway attenuates the defense response of Nicotiana benthamiana to agroinfiltration

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of Nicotiana benthamiana infiltrated with the bacterial gene vector Agrobacterium tumefaciens. An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector. Leaves infiltrated with the empty vector had a low soluble protein content compared to noninfiltrated control leaves, associated with increased levels of stress-related proteins but decreased levels of photosynthesis-associated proteins. M2 expression partly compromised these effects of agroinfiltration to restore soluble protein content in the leaf tissue, associated with restored levels of photosynthesis-associated proteins and reduced levels of stress-related proteins in the apoplast. These data illustrate the cell-wide influence of the Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana responding to microbial challenge. They also underline the relevance of assessing the eventual unintended effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites1[OA]

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment

Topoisomerase I peptide-loaded dendritic cells induce autoantibody response as well as skin and lung fibrosis

<p>DNA Topoisomerase I (TopoI) is a candidate autoantigen for diffuse cutaneous systemic sclerosis (dcSSc) associated with fatal lung disease. Dendritic cells (DCs) contribute to bleomycin-induced lung fibrosis. However, the possibility that TopoI-loaded DCs are involved in the initiation and/or perpetuation of dcSSc has not been explored. Here, we show that immunization with TopoI peptide-loaded DCs induces anti-TopoI autoantibody response and long-term fibrosis. Mice were repeatedly immunized with unpulsed DCs or DCs loaded with either TOPOIA or TOPOIB peptides, selected from different regions of TopoI. At week 12 after initial DC immunization, TOPOIA DCs but not TOPOIB DCs immunization induced mixed inflammation and fibrosis in lungs and skin. At a late time point (week 18), both TOPOIA DCs and TOPOIB DCs groups displayed increased alpha-smooth muscle actin expression in lungs and dermis along with skin fibrosis distal from the site of injection when compared with unpulsed DCs. Both TopoI peptide-DC-immunized groups developed IgG2a anti-TopoI autoantibody response. At week 10, signs of perivascular, peribronchial, and parenchymal pulmonary inflammation were already observed in the TOPOIA DCs group, together with transient elevation in bronchoalveolar lavage cell counts, IL-17A expression, and CXCL4 production, a biomarker of early human dcSSc. Collectively, TopoI peptide DCs induce progressive autoantibody response as well as development of protracted skin and lung dcSSc-like disease. Pronounced lung inflammation, transient IL-17A, and CXCL4 expression precede fibrosis development. Our immunization strategy, that uses self immune system and autoantigen, will help to further investigate the pathogenesis of this complex autoimmune disorder with unmet medical needs.</p

Restoration of the transepithelial potential within tissue-engineered human skin in vitro and during the wound healing process in vivo

Normal human epidermis possesses a transepithelial potential (TEP) that varies in different parts of the body (10–60 mV). The role of TEP in normal epidermis is not yet identified; but after skin injury, TEP disruption induces an endogenous direct current electric field (100–200 mV/mm) directed toward the middle of the wound. This endogenous electric field could be implicated in the wound healing process by attracting cells, thus facilitating reepithelialization. However, little is known on the restoration of the TEP during human skin formation and wound healing. In this study, the variations in TEP and Na+/K+ ATPase pump expression during the formation of the epithelium were investigated in vitro using human tissue-engineered human skin (TES) reconstituted by tissue engineering and in vivo with a porcine wound healing model. Results showed that TEP undergoes ascending and decreasing phases during epithelium formation in TES as well as during wound repair within TES. Similar results were observed during in vivo reepithelialization of wounds. The ascending and decreasing TEP values were correlated with changes in the expression of Na+/K+ ATPase pump. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Taken together, these results suggest that the variations in the expression of Na+/K+ ATPase pump over time and across epidermis would be a determinant parameter of the TEP, dictating a cationic transport during the formation and restoration of the epidermis. Therefore, this study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process. This might have important future medical applications regarding the treatment of chronic wound healing

Human keratinocytes respond to direct current stimulation by increasing intracellular calcium : preferential response of poorly differentiated cells

A direct current (DC) endogenous electric field (EF) is induced in the wound following skin injury. It is potentially implicated in the wound healing process by attracting cells and altering their phenotypes as indicated by the response to an EF of keratinocytes cultured as individual cells. To better define the signalization induced by a direct current electric field (DCEF) in human keratinocytes, we took advantage of an in vitro model more representative of the in vivo situation since it promotes cell–cell interactions and stratification. Human keratinocytes were grown into colonies. Their exposure to a DCEF of physiological intensity induced an increase of intracellular calcium. This variation of intracellular calcium resulted from an extracellular calcium influx and was mediated, at least in part, by the L-type voltage-gated calcium channel. The increase in intracellular calcium in response to a DCEF was however not observed in all the cells composing the colonies. The intracellular calcium increase was only detected in keratinocytes that didn't express involucrin, a marker of differentiated cells. These results indicate that DCEF is able to induce a specific calcium response in poorly differentiated keratinocytes. This study brings a new perspective for the understanding of the signaling mechanism of endogenous EF in reepithelialization, a critical process during skin wound healing

Wine phenolic compounds differently affect the host-killing activity of two lytic bacteriophages infecting the lactic acid bacterium oenococcus oeni

International audienceTo provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition