Par2 Inactivation Inhibits Early Production of TSLP, but Not Cutaneous Inflammation, in Netherton Syndrome Adult Mouse Model

Netherton syndrome (NS) is a severe genodermatosis characterized by abnormal scaling and constant atopic manifestations. NS is caused by mutations in SPINK5 (Serine Protease INhibitor Kazal-type 5), which encodes LEKTI (LymphoEpithelial Kazal Type-related Inhibitor). Lack of LEKTI causes stratum corneum detachment secondary to epidermal proteases hyperactivity. Whereas a skin barrier defect is generally regarded as a major cause for atopy, we previously identified a cell-autonomous signaling cascade that triggers pro-Th2 cytokine thymic stromal lymphopoietin (TSLP) production in LEKTI-deficient epidermis. This signaling is initiated by unrestricted kallikrein 5 (KLK5) activity, which directly activates proteinase-activated receptor 2 (PAR2)-mediated expression of TSLP and favors a cutaneous proallergic microenvironment independently of the environment and of the adaptive immune system. To further confirm these results in vivo, we generated Spink5/Par2 double knockout (DKO) mice. At embryonic day 19.5, these mice display a dramatic decrease in TSLP expression, although stratum corneum detachment persists, confirming the role of the KLK5–PAR2 cascade in TSLP-mediated early proallergic signaling. However, deletion of Par2 in adult DKO-grafted skin does not rescue the inflammatory phenotype probably resulting from stratum corneum detachment. We conclude that several mechanisms trigger and maintain the inflammatory phenotype in NS. These include skin barrier impairment, mechanical stress secondary to stratum corneum detachment, as well as protease-induced proinflammatory and proallergic pathways, including PAR2-mediated overexpression of TSLP

Corneodesmosomal Cadherins Are Preferential Targets of Stratum Corneum Trypsin- and Chymotrypsin-like Hyperactivity in Netherton Syndrome

SPINK5 (serine protease inhibitor Kazal-type 5), encoding the protease inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor), is the defective gene in Netherton syndrome (NS), a severe inherited keratinizing disorder. We have recently demonstrated epidermal protease hyperactivity in Spink5−/− mice resulting in desmosomal protein degradation. Herein, we investigated the molecular mechanism underlying the epidermal defect in 15 patients with NS. We demonstrated that, in a majority of patients, desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) were dramatically reduced in the upper most living layers of the epidermis. These defects were associated with premature degradation of corneodesmosomes. Stratum corneum tryptic enzyme (SCTE)-like and stratum corneum chymotryptic enzyme (SCCE)-like activities were increased, suggesting that these proteases participate in the premature degradation of corneodesmosomal cadherins. SCTE and SCCE expression was extended to the cell layers where Dsg1 and Dsc1 immunostaining was reduced. In contrast, a subset of six patients with normal epidermal protease activity or residual LEKTI expression displayed apparently normal cadherin expression and less severe disease manifestations. This suggests a degree of correlation between cadherin degradation and clinical severity. This work further supports the implication of premature corneodesmosomal cadherin degradation in the pathogenesis of NS and provides evidence for additional factors playing a role in disease expression

Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

Netherton syndrome (NS) is a severe genetic skin disease with constant atopic manifestations that is caused by mutations in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lymphoepithelial Kazal-type–related inhibitor (LEKTI). Lack of LEKTI causes stratum corneum detachment secondary to epidermal proteases hyperactivity. This skin barrier defect favors allergen absorption and is generally regarded as the underlying cause for atopy in NS. We show for the first time that the pro-Th2 cytokine thymic stromal lymphopoietin (TSLP), the thymus and activation-regulated chemokine, and the macrophage-derived chemokine are overexpressed in LEKTI-deficient epidermis. This is part of an original biological cascade in which unregulated kallikrein (KLK) 5 directly activates proteinase-activated receptor 2 and induces nuclear factor κB–mediated overexpression of TSLP, intercellular adhesion molecule 1, tumor necrosis factor α, and IL8. This proinflammatory and proallergic pathway is independent of the primary epithelial failure and is activated under basal conditions in NS keratinocytes. This cell-autonomous process is already established in the epidermis of Spink5−/− embryos, and the resulting proinflammatory microenvironment leads to eosinophilic and mast cell infiltration in a skin graft model in nude mice. Collectively, these data establish that uncontrolled KLK5 activity in NS epidermis can trigger atopic dermatitis (AD)–like lesions, independently of the environment and the adaptive immune system. They illustrate the crucial role of protease signaling in skin inflammation and point to new therapeutic targets for NS as well as candidate genes for AD and atopy

Isolement, caractérisation et cibles de nouveaux Inhibiteurs de protéases pour la création de plantes transgéniques résistantes aux pucerons

Jury : Michel BREHELIN Philippe BULET Philippe GIORDANENGO Lise JOUANIN Thierry LANGIN Yvan RAHBEAmong the phytophagous insects, aphids show the particularity of feeding on sap. Their stylets allow them to reach the phloem tissues, a compartment which protein/amino acid ration is low. In this context, aphids are considered to be devoid of the enzymatic stock capable of hydrolysing protein. Strategies using proteinase inhibitor (PI) as entomotoxic polypeptide are considered at first inadequate to fight this kind of insects. Nevertheless, several PI have paradox toxicity against aphids. This study is aimed at increasing the number of potential novel PI, at understanding their function in order to introduce them in biologic fight program.During this work, new informations on digestive proteolysis in Homoptera were gained: cystein proteinase cathepsins (lysosomal enzyme) are specifically expressed in gut and in a specialized organ, the bacteriocyte. They are potential targets of cystein-PI. Seedrapes expressing Oryzacystatine (isolated from rice seed) induce a 25% reduction of Myzus persicea fecundity. This result shows the possibility to express PI in phloem to fight aphids.Improvement of a serine-PI (isolated from pea) via directed mutagenesis was performed but no active form was obtained neither in Pichia pastoris nor in Arabidosis thaliana expression system. We tried to enlarge the gene pool to fight aphids. Isolation of a new cystein-PI from aphid haemolymph was initiated at the molecular and biochemical level. These results show that PI constitute a new strategy to fight aphids.Parmi les insectes phytophages, les pucerons sont particuliers car ils se nourrissent de sève élaborée. Leurs pièces buccales leur permettant d'effectuer des piqûres dans la plante et d'atteindre les faisceaux du phloème, un compartiment dont le ratio protéines/acides aminés est très déséquilibré. C'est pourquoi, dans ce contexte, les pucerons sont réputés comme ne possédant pas l'arsenal enzymatique permettant une bonne utilisation des protéines. Aussi les stratégies utilisant les inhibiteurs de protéases (IP) comme polypeptide entomotoxique ne semblent pas adaptées a priori à ce groupe d'insectes. Or plusieurs IP ont montré des toxicités paradoxales contre les pucerons.Cette étude a pour objectif d'accroître la disponibilité des gènes d'IP originaux et d'en comprendre le mode d'action afin d'en maîtriser l'introduction raisonnée dans les programmes de lutte variétale. Au cours de ce travail de thèse, des informations sur la protéolyse digestive des Homoptères ont été apportées : des protéases à cystéine, les cathepsines (enzymes lysosomiales) sont spécifiquement exprimées dans le tube digestif et dans un organe spécialisé, le bactériocyte. Elles sont la cible potentielle d'inhibiteurs de protéases à cystéine. Lorsque l'Oryzacystatine (inhibiteur de protéase à cystéine, isolé du grain de riz) est exprimé dans le colza, la fécondité du puceron Mysus persicae diminue de 25%. Ce résultat démontre qu'il est donc possible d'exprimer des IP dans le phloème pour lutter contre les pucerons. L'amélioration, par mutagenèse dirigée, d'un inhibiteur de protéases à sérine, isolé du pois, a aussi été effectuée mais son expression hétérologue dans Pichia pastoris ou Arabidospsis thaliana n'a pas permis d'obtenir une forme active. Nous avons cherché à élargir le pool de gènes disponible pour lutter contre les pucerons. L'isolement d'un inhibiteur de protéases à cystéine de l'hémolymphe du puceron a été entrepris au niveau moléculaire et biochimique. Ces résultats démontrent que les IP constituent une nouvelle voie pour lutter contre les pucerons

Postoperative ileus: A pharmacological perspective

International audiencePostoperative ileus (POI) is a frequent complication after abdominal surgery. The consequences of postoperative ileus can be potentially serious such as bronchial inhalation or acute functional renal failure. Numerous advances in peri-operative management, particularly early rehabilitation, have made it possible to decrease postoperative ileus. Despite this, the rate of prolonged postoperative ileus remains high and can be as high as 25% of patients in colorectal surgery. From a pathophysiological point of view, postoperative ileus has two phases, an early neurological phase and a later inflammatory phase, to which we could add a 'pharmacological' phase during which analgesic drugs, particularly opiates, play a central role. The aim of this review article is to describe the phases of the pathophysiology of postoperative ileus, to analyse the pharmacological treatments currently available through published clinical trials and finally to discuss the different research areas for potential pharmacological targets

Isolement, caractérisation et cibles de nouveaux inhibiteurs de protéases pour la création de plantes transgéniques résistantes aux pucerons

Parmi les insectes phytophages, les pucerons sont particuliers car ils se nourrissent de sève élaborée. Leurs pièces buccales leur permettant d'effectuer des piqûres dans la plante et d' atteindre les faisceaux du phloème, un compartiment dont le ratio protéines/acides aminés est très déséquilibré. C'est pourquoi, dans ce contexte, les pucerons sont réputés comme ne possédant pas l'arsenal enzymatique permettant une bonne utilisation des protéines. Aussi les stratégies utilisant les inhibiteurs de protéases (IP) comme polypeptide entomotoxique ne semblent pas adaptées a priori à ce groupe d'insectes. Or plusieurs IP ont montré des toxicités paradoxales contre les pucerons. Cette étude a pour objectif d'accroître la disponibilité des gènes d'IP originaux et d'en comprendre le mode d'action afin d'en maîtriser l'introduction raisonnée dans les programmes de lutte variétale. Au cours de ce travail de thèse, des informations sur la protéolyse digestive des Homoptères ont été apportées: des protéases à cystéine, les cathepsines (enzymes lysosomiales) sont spécifiquement exprimées dans le tube digestif et dans un organe spécialisé, le bactériocyte. Elles sont la cible potentielle d'inhibiteurs de protéases à cystéine. Lorsque l'Oryzacystatine (inhibiteur de protéase à cystéine, isolé du grain de riz) est exprimé dans le colza, la fécondité du puceron Mysus persicae diminue de 25%. Ce résultat démontre qu'il est donc possible d'exprimer des IP dans le phloème pour lutter contre les pucerons. L'amélioration, par mutagenèse dirigée, d'un inhibiteur de protéases à sérine, isolé du pois, a aussi été effectuée mais son expression hétérologue dans Pichia pastoris ou Arabidospsis thaliana n'a pas permis d'obtenir une forme active. Nous avons cherché à élargir le pool de gènes disponible pour lutter contre les pucerons. L'isolement d'un inhibiteur de protéases à cystéine de l'hémolymphe du puceron a été entreprise au niveau moléculaire et biochimique. Ces résultats démontrent que les IP constituent une nouvelle voie pour lutter contre les pucerons.Among the phytophagous insects, aphids show the particularity of feeding on sap. Their stylets allow them to reach the phloem tissues, a compartment which protein/amino acid ration is low. In this context, aphids are considered to be devoid of the enzymatic stock capable of hydrolysing protein. Strategies using proteinase inhibitor (PI) as entomotoxic polypeptide are considered at first inadequate to fight this kind of insects. Nevertheless, several PI have paradox toxicity against aphids. This study is aimed at increasing the number of potential novel PI, at understanding their function in order to introduce them in biologic fight program. During this work, new informations on digestive proteolysis in Homoptera were gained: cystein proteinase cathepsins (lysosomal enzyme) are specifically expressed in gut and in a specialized organ, the bacteriocyte. They are potential targets of cystein-PI. Seedrapes expressing Oryzacystatine (isolated from rice seed) induce a 25% reduction of Myzus persicea fecundity. This result shows the possibility to express PI in phloem to fight aphids. Improvement of a serine-PI (isolated from pea) via directed mutagenesis was performed but no active form was obtained neither in Pichia pastoris nor in Arabidosis thaliana expression system. We tried to enlarge the gene pool to fight aphids. Isolation of a new cystein-PI from aphid haemolymph was initiated at the molecular and biochemical level. These results show that PI constitute a new strategy to fight aphids.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF