Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis

BACKGROUND: Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis. RESULTS: Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFβ and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker. CONCLUSION: Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis

CD9+ Regulatory B Cells Induce T Cell Apoptosis via IL-10 and Are Reduced in Severe Asthmatic Patients

CD9 was recently identified as a marker of murine IL-10-competent regulatory B cells. Functional impairments or defects in CD9+ IL-10-secreting regulatory B cells are associated with enhanced asthma-like inflammation and airway hyperresponsiveness. In mouse models, all asthma-related features can be abrogated by CD9+ B cell adoptive transfer. We aimed herein to decipher the profiles, features, and molecular mechanisms of the regulatory properties of CD9+ B cells in human and mouse. The profile of CD9+ B cells was analyzed using blood from severe asthmatic patients and normal and asthmatic mice by flow cytometry. The regulatory effects of mouse CD9+ B cells on effector T cell death, cell cycle arrest, apoptosis, and mitochondrial depolarization were determined using yellow dye, propidium iodide, Annexin V, and JC-1 staining. MAPK phosphorylation was analyzed by western blotting. Patients with severe asthma and asthmatic mice both harbored less CD19+CD9+ B cells, although these cells displayed no defect in their capacity to induce T cell apoptosis. Molecular mechanisms of regulation of CD9+ B cells characterized in mouse showed that they induced effector T cell cycle arrest in sub G0/G1, leading to apoptosis in an IL-10-dependent manner. This process occurred through MAPK phosphorylation and activation of both the intrinsic and extrinsic pathways. This study characterizes the molecular mechanisms underlying the regulation of CD9+ B cells to induce effector T cell apoptosis in mice and humans via IL-10 secretion. Defects in CD9+ B cells in blood from patients with severe asthma reveal new insights into the lack of regulation of inflammation in these patients

From food allergy to asthma : role of CCR9

Aujourd’hui, l’allergie est classée 4ème maladie mondiale en termes de morbidité par l’OMS. Les allergies et leur évolution naturelle (marche atopique) sont devenues un problème majeur de santé publique particulièrement dans les pays industrialisés. La marche atopique se manifeste par l’évolution de la dermatite atopique et/ou des allergies alimentaires chez le jeune enfant vers des allergies respiratoires comme l’asthme ou la rhinite allergique plus tard dans l’adolescence. Ce passage pourrait impliquer un chimiotactisme contrôlé par le système chimiokine/récepteurs de chimiokine. A l’aide d’un modèle murin mimant la marche atopique composé d’un modèle d’allergie alimentaire au gluten et d’un modèle aigu d’asthme aux acariens, nous avons pu caractériser le rôle du récepteur de chimiokine CCR9 dans la maladie. Ainsi, des souris déficientes pour le gène de CCR9 montrent un phénotype atténué de la maladie démontrant une implication de ce récepteur dans la pathogénèse. De plus nous avons démontré que CCR9 agirait sur la balance TH17/Treg car sa délétion entraine une augmentation des T régulateurs. En parallèle, dans un modèle d’allergie alimentaire au gluten par sensibilisation cutanée, nous avons analysé l’inflammation intestinale en réponse à plusieurs allergènes. Ces derniers travaux ont été réalisés en collaboration avec un laboratoire du National Food Institute au Danemark. Ainsi, nos résultats démontrent donc l’importance de l’axe intestin-poumon et l’importance d’aborder l’allergie comme une maladie de l’ensemble de l’organisme et non pas comme une maladie d’organe.Allergic diseases are now considered as the fourth worldwide diseases in terms of morbidity, according to the World Health Organization. Allergic diseases and their natural evolution (atopic march) are a major health issue, particularly among developed countries. Indeed, the atopic march is characterized by an evolution from atopic dermatitis and/or food allergies in young children (6 months to 2 years) to respiratory allergies such as asthma and rhinitis later in life. This natural history could involve the chemotaxis, controlled by the chemokine/chemokine receptor system. Using a murine model of atopic march combining a food allergy model to gluten and a model of acute asthma to house dust mite, we analyzed the role of the chemokine receptor CCR9 in the evolution of the disease. Using knock-out mice for CCR9, we observed a decrease of the symptoms of the disease, suggesting a role for this receptor in the pathology. Moreover, we showed that CCR9 seems to act on the Treg/TH17 balance; indeed its deletion induces an increase of the T regulators cell level. Meanwhile, using a food allergy model to gluten based on cutaneous sensitizations, we analyzed the intestinal inflammation to different gluten products. This work was done in collaboration with a lab of the National Food Institute, in Denmark. Our results prove the great significance of the gutlung axis and more generally the importance of approaching the allergy as a whole disease and not as an organ-specific disease

Role of chemokine receptors in allergic diseases

Immune cell trafficking is orchestrated by a family of small proteins named chemokines and leucocytes express chemokine receptors on their surface. Once chemokines are attached to these receptors, they trigger transendothelial migration. During basal conditions as well in various diseases, leukocyte migration is a crucial step for the immune system. Excessive or inappropriate expression of chemokine receptors in allergy is thus one of the key factors, which trigger the allergic response. Consequently, many studies have been made on the importance of chemokines and their receptors in allergic diseases such as asthma, food allergy and atopic dermatitis. Moreover, as these receptors can easily be blocked by an antagonist, this has led to the development of new therapeutic approaches aimed at suppressing the recruitment of immune cells during an allergic reaction. In this review, we will detail which chemokines and which chemokine receptors are important in the three main allergic diseases mentioned above and describe the chemokine receptor antagonists, which have been developed to date

Hypothèse hygiéniste : où en est-on ? Compte rendu de l’atelier « Allergies » du DHU 2020 « Médecine personnalisées des maladies chroniques »

National audienceAllergic diseases have been increasing constantly over the past few years. With the population aging, the rising cost of healthcare is inevitable.The increasing cost of curative medicine, which has developed considerably during recent decades, will no longer be accepted. Thus, the treatmentof many chronic diseases, including allergies, must be reconsidered. Current treatments such as allergen immunotherapy are not effective enoughfor all the different types of allergies and no valid preventive treatment exists. New strategies for the treatment of chronic diseases are now beingfocused on the development of personalized medicine and on 4P (preventive, personalized, predictive, and participative) medicine. For instance,vaccines or health food products are currently being developed for populations at risk.Les maladies allergiques sont en constante augmentation depuis quelques années. Avec le vieillissement de la population, les dépenses de santévont s’accroître également. Le coût de la médecine curative, qui a considérablement été développée ces dernières décennies ne pourra plus êtresupporté demain. Ainsi les traitements de nombreuses maladies chroniques telles que les allergies doivent être reconsidérés. Les thérapies existantestelles que l’immunothérapie ne sont pas efficaces pour tous les types d’allergies et il n’existe aucun moyen validé de prévenir ces maladies. Lesnouvelles stratégies thérapeutiques des maladies chroniques se focalisent aujourd’hui sur le développement de la médecine personnalisée oumédecine 4P (prédictive, préventive, personnalisée et participative). Ainsi, l’élaboration de vaccins ou encore d’aliment-santé à destination despopulations à risque est en cours

Perinatal exposure to galactooligosaccharides/inulin prebiotics prevent food allergy by protecting intestine and promoting tolerance

International audienceFood allergies are increasing, and no treatment exists, thus a strategy to prevent or reduce allergies would consist of modulating the host microbiota using either allochthonous bacteria (probiotics) or nondigestible food ingredients that can regulate the autochthonous microbiota (composition and metabolism), such as prebiotics. This study aimed to analyze the preventive effects of prebiotic feeding during perinatal and postweaning periods in a mouse model of food allergy

Food allergen-sensitized CCR9+ lymphocytes enhance airways allergic inflammation in mice

International audienceBACKGROUND: The mechanisms of the atopic march, characterized by a natural progression from food and cutaneous allergies to rhinitis and asthma, are still unknown. However, as several organs can be involved, chemokines and their receptors might be implicated in this process and may be instrumental factors. OBJECTIVES: We hypothesized that the T-cell gut-homing receptor CCR9 could be implicated in the evolution of allergic diseases. METHODS: We characterized the immune response and the role of CCR9 in a murine model combining food allergy to wheat gliadin and a model of acute airways inflammation in response to house dust mite. RESULTS: Compared with solely asthmatic-like mice, we demonstrated that the aggravation of pulmonary symptoms in consecutive food and respiratory allergies, characterized by an increase in pulmonary resistance and a higher Th17/ Treg ratio, was abrogated in CCR9 knock-out mice. Moreover, transfer of food-allergic CD4+ T cells from wild-type but not from CCR9-/- aggravated airways inflammation demonstrating that CCR9 is involved in food allergy-enhanced allergic airway inflammation to unrelated allergens. CONCLUSION: Taken together, our results demonstrated a crucial role of the T-cell homing receptor CCR9 in this model and validated its potential for use in the development of therapeutic strategies for allergic diseases. This article is protected by copyright. All rights reserved