Insulin-Regulated Amino Peptidase (IRAP) contrôle la signalisation et les réponses inflammatoires médiées par les FcRs

Dans cette thèse nous avons étudié le rôle de l’Insulin-Regulated Amino Peptidase (IRAP) dans la signalisation médiée par les récepteurs Fc aux immunoglobulines (FcR). IRAP est une protéine membranaire multifonctionnelle exprimée sur les endosomes de recyclage et la membrane plasmique. Outre son activité protéolytique pouvant réguler l’activité physiologique des peptides, IRAP est impliqué dans la signalisation des cellules immunitaires en régulant le trafic intracellulaire des effecteurs de signalisation. En utilisant des souris déficientes en IRAP versus des souris sauvages dans des expériences in vivo nous montrons que, les souris déficientes en IRAP développent une anaphylaxie systémique passive dépendante des IgE, une anaphylaxie systémique active médiée par les IgG et une arthrite auto-immune moins sévère. En utilisant un modèle de mastocytes (MC) nous avons confirmé que les endosomes de recyclage IRAP+ sont rapidement recrutés à la membrane plasmique après stimulation via les récepteurs aux IgE (FcERI). En concordance avec les données in vivo, nous montrons que les MC déficientes en IRAP exhibent un signal calcique plus faible et une sécrétion diminuée de médiateurs inflammatoires. La phosphorylation des tyrosines de plusieurs effecteurs de la signalisation était également diminuée dans les MC déficientes en IRAP. En particulier, la forme active SykY524/525 est diminuée et moins présente à la membrane plasmique des MC IRAP déficientes après stimulation des FcERI, ce qui a été confirmé également dans les neutrophiles et monocytes activées via les FcYR. L’inhibition de Syk ne dépendait pas des évènements précoces de signalisation médiés par la kinase Lyn mais de l'activité phosphatase de SHP-1 comme dans les MC déficientes en IRAP stimulées via FcERI et les neutrophiles et monocytes stimulés via FcYR où l'inactivation sur S591 était diminuée, augmentant ainsi les réponses inflammatoires induites par les FcR. Ces découvertes soutiennent le rôle des compartiments de recyclage IRAP+ dans la signalisation en modifiant la balance kinase/phosphatase vers une activation de la signalisation induite par la kinase et une amélioration des réponses inflammatoires.In this thesis we investigated the role of Insulin-Regulated Amino Peptidase (IRAP) in the signaling of Fc receptors for Immunoglobulins (FcR). IRAP is a type II multifunctional membrane protein that distributes between recycling endosomes and the plasma membrane. Besides its proteolytic activity that can regulate the activity of physiological peptides, IRAP has been implicated in immune cell signaling regulating the trafficking of involved intracellular signaling effectors. Using IRAP-deficient versus wild-type mice we show in in vivo experiments that, in the context of IRAP-deficiency, mice exhibit a less severe IgE-dependent passive systemic anaphylaxis, IgG-triggered active systemic anaphylaxis and autoimmune arthritis. Using mast cells (MC) as a model we confirmed that IRAP+ recycling endosomes get rapidly recruited to the plasma membrane upon stimulation through IgE receptors (FcERI). In agreement with the in vivo data, we show that IRAP-deficient MC exhibited a reduced calcium signal and reduced inflammatory mediator secretion. Tyrosine-phosphorylation of several signaling effectors was also diminished in IRAP-deficient MC. In particular, active SykY524/525 was decreased and less prominent at the plasma membrane in IRAP-deficient FcERI-stimulated MC, a finding also confirmed in FcYR-stimulated neutrophils and monocytes. Syk inhibition did not depend on early signaling events mediated by Lyn kinase but relied on SHP-1 phosphatase activity as in IRAP deficient FcERI-stimulated MC and FcYR-stimulated neutrophils and monocytes the inactivation on S591 was decreased, hence enhancing FcR-induced inflammatory responses. Our findings support a role of IRAP+ recycling compartments in FcR signaling shifting the kinase/phosphatase balance to activating kinasedriven signaling and enhanced inflammatory responses

Allergy, Anaphylaxis and Non-Allergic Hypersensitivity: IgE, Mast Cells and Beyond

International audienceIgE-mediated type I hypersensitivity reactions have many reported beneficial functions in the immune defense against parasites, venoms, toxins etc. However, they are best-known for their role in allergy affecting nowadays almost one third of the population worldwide. IgE-mediated allergic diseases result from a maladaptive type 2 immune response that promotes the synthesis of IgE antibodies directed to a special class of antigens called allergens. IgE antibodies bind to type I high affinity IgE receptors (FcepsilonRI) on mast cells and basophils licensing them to get triggered in a subsequent encounter with the cognate allergen. This promotes the release of a whole set of inflammatory mediators including histamine responsible for the symptoms of immediate hypersensitivity. The development of type 2 driven allergies are dependent on a complex interplay of genetic and environmental factors at barrier surfaces including the host microbiome that builds up during early life. While without doubt IgE-mediated immediate hypersensitivity reactions are at the origin of the majority of allergies it has become clear that similar responses and symptoms can be triggered by other types of adaptive immune responses mediated via IgG or complement involving other immune cells and mediators. Likewise, various innate triggers via receptors expressed on mast cells have been found either to directly launch an allergic reaction and/or to amplify existing IgE-mediated responses. This review summarizes recent findings on both IgE-dependent and IgE-independent mechanisms in the development of allergies and provides an update on allergy diagnosis

Mast Cell Chymase and Kidney Disease

A sizable part (~2%) of the human genome encodes for proteases. They are involved in many physiological processes, such as development, reproduction and inflammation, but also play a role in pathology. Mast cells (MC) contain a variety of MC specific proteases, the expression of which may differ between various MC subtypes. Amongst these proteases, chymase represents up to 25% of the total proteins in the MC and is released from cytoplasmic granules upon activation. Once secreted, it cleaves the targets in the local tissue environment, but may also act in lymph nodes infiltrated by MC, or systemically, when reaching the circulation during an inflammatory response. MC have been recognized as important components in the development of kidney disease. Based on this observation, MC chymase has gained interest following the discovery that it contributes to the angiotensin-converting enzyme’s independent generation of angiotensin II, an important inflammatory mediator in the development of kidney disease. Hence, progress regarding its role has been made based on studies using inhibitors but also on mice deficient in MC protease 4 (mMCP-4), the functional murine counterpart of human chymase. In this review, we discuss the role and actions of chymase in kidney disease. While initially believed to contribute to pathogenesis, the accumulated data favor a more subtle view, indicating that chymase may also have beneficial actions

TLR4 Receptor Induces 2-AG–Dependent Tolerance to Lipopolysaccharide and Trafficking of CB2 Receptor in Mast Cells

International audienceMast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo