Vers la modélisation physiopathologique de la granulomatose septique chronique pour de nouvelles approches thérapeutiques (développement de la culture des cellules souches pluripotentes induites issues de fibroblastes de patients)

La Granulomatose septique chronique (CGD) est une maladie immunitaire et héréditaire rare diagnostiquée devant des infections sévères et récidivantes. La forme majoritaire de transmission liée à l X (CGDX) est causée par des mutations dans le gène CYBB codant la sous-unité NOX2 du complexe NADPH oxydase, enzyme clé de la production de réactifs toxiques de l oxygène (ROS) bactéricides par les cellules phagocytaires. Afin de développer de nouvelles approches thérapeutiques, il est nécessaire de posséder des modèles cellulaires mimant la maladie. Les cellules souches pluripotentes induites (iPS) représentent un outil inestimable pour la modélisation physiopathologique. L objectif de ce projet était de mettre en place au laboratoire la culture et la caractérisation des cellules iPS pour modéliser la CGD. Pour cela, des cellules iPS reprogrammées à partir de fibroblastes d un sujet sain et d un patient atteint de CGDX ont été cultivées avec succès à la fois sur cellules nourricières et sur matrice extracellulaire. Leur capacité de pluripotence a été validée d une part par l expression de marqueurs de pluripotence par immunofluorescence et cytométrie en flux, et d autre part grâce à la formation de corps embryoïdes constitués de cellules dérivées des trois lignées embryonnaires. L analyse du caryotype n a pas montré d anomalie chromosomique. Enfin, la mutation du patient à l origine de la CGD a été retrouvée dans les cellules iPS-CGDX. Les cellules iPS témoin et CGDX pourront donc être différenciées en progéniteurs hématopoïétiques pour la génération de souris humanisées, modèles de CGDX et pour l obtention de cellules phagocytaires pour le développement de nouvelles thérapies.Chronic granulomatous disorder (CGD) is a rare immune inherited disorder in which patients suffer from recurrent life-threatening bacterial and fungal infections early in childhood. The most frequent form is the X-linked CGD (X-CGD) due to mutations in the CYBB gene encoding NOX2 one of the sub-units of the phagocytic NADPH oxidase complex, responsible for the production of microbicidal reactive oxygen species (ROS). To develop new therapeutic approaches, cellular models mimicking the pathology are needed. Induced Pluripotent Stem Cells (iPSCs) are an invaluable tool for physiopathological modelization. The purpose of this project was to develop in our laboratory the culture and the methods of characterization of iPSCs in order to model CGD. For this, iPSCs reprogrammed from healthy and X-CGD patient s fibroblasts have successfully been cultured on feeder cells or on extracellular matrix. On one hand, their pluripotency ability was assessed with the expression of pluripotency markers by immunofluorescence and flow cytometry and on the other hand, with the generation of embryoid bodies composed of cells derived from the three embryonic germ layers. The karyotype analysis showed no chromosomal abnormalities. Finally, the patient s mutation responsible for the X-CGD was found in the XCGD-iPS cell line. Control and X-CGD iPS cells will be differentiated into hematopoietic progenitors and use to generate X-CGD humanized mouse model and to obtain mature phagocytic cells for the development of new therapies.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Altered Humoral Immune Responses and IgG Subtypes in NOX2-Deficient Mice and Patients: A Key Role for NOX2 in Antigen-Presenting Cells

Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting from loss of function mutations in the reactive oxygen species generating phagocyte NADPH oxidase (NOX2). CGD patients are prone to infection, but also have an increased susceptibility to autoimmune diseases. The aim of this study was to investigate the role of NOX2 in the regulation of specific immunity. In both CGD patients and NOX2-deficient mice, we observed an alteration in the basal proportions of IgG subtypes. Upon immunization with curdlan—a dectin 1 agonist—NOX2-deficient mice showed increased production of IgG2c compared to controls, and restimulation of lymph node-derived cells led to increased production of IFNγ, but not IL-5, indicative hallmark of an enhanced Th1 response. T cell activation was increased in NOX2-deficient mice and a similar trend was observed in vitro when T cells were co-cultured with NOX2-deficient bone marrow-derived cells. In contrast, no difference in T cell activation was observed when NOX2-deficient T cells were co-cultured with wild-type BMDC. Following stimulation of NOX2-deficient dendritic cells (DCs), no difference in costimulatory molecules was observed, while there was an increase in the release of Th1-driving cytokines. In summary, both CGD patients and CGD mice have an altered IgG subtype distribution, which is associated with an increased IFNγ production. Thus, NOX2 within DCs appears to be an important regulator at the interface of innate and specific immunity, especially after activation of the dectin 1 pathway, limiting immune activation and the development of autoimmunity

Molecular and functional characterization of a new X-linked chronic granulomatous disease variant (X91+) case with a double missense mutation in the cytosolic gp91phox C-terminal tail

AbstractWe report here two atypical cases of X-linked CGD patients (first cousins) in which cytochrome b558 is present at a normal level but is not functional (X91+). The mutations were localized by single-strand conformational polymorphism of reverse transcriptase–polymerase chain reaction amplified fragments and then identified by sequence analysis. They consisted in two base substitutions (C919 to A and C923 to G), changing His303 to Asn and Pro304 to Arg in the cytosolic gp91phox C-terminal tail. Mismatched polymerase chain reaction and genomic DNA sequencing showed that mothers had both wild-type and mutated alleles, confirming that this case was transmitted in an X-linked fashion. A normal amount of FAD was found in neutrophil membranes, both in the X91+ patients and their parents. Epstein–Barr virus-transformed B lymphocytes from the X91+ patients acidified normally upon stimulation with arachidonic acid, indicating that the mutated gp91phox still functioned as a proton channel. A cell-free translocation assay demonstrated that the association of the cytosolic factors p47phox and p67phox with the membrane fraction was strongly disrupted. We concluded that residues 303 and 304 are crucial for the stable assembly of the NADPH oxidase complex and for electron transfer, but not for its proton channel activity

A novel point mutation in the CYBB gene promoter leading to a rare X minus chronic granulomatous disease variant — Impact on the microbicidal activity of neutrophils

AbstractThis article reports an atypical and extremely rare case of X-linked CGD in an Italian family characterized by a low expression of gp91phox (X91− CGD). A novel point mutation in the CYBB gene's promoter (insertion of a T at position −54T to −56T) appeared to prevent the full expression of this gene in the patient's neutrophils and correlated with a residual oxidase activity in the whole cells population. The expression and functional activity of the oxidase in eosinophils appeared to be almost normal. Gel shift assays indicated that the mutation led to decreased interactions with DNA-binding proteins. The total O2− production in the patient's granulocytes (5–7% of normal) supported no microbicidal power after 45 min and 60 min of contact with S. aureus and C. albicans, respectively. Despite this residual oxidase activity, the patients suffered from severe and life-threatening infections. It was concluded that in these X91− CGD neutrophils, the O2− production per se was not sufficient to protect the patient against severe infections

Chronic granulomatous disease: the European experience.

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst", essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (approximately 1:250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91(phox) deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with gamma-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

Chapitre 6 - Tests cellulaires

International audienceCet ouvrage rédigé sous l'égide de l'Assim, offre un tour d'horizon complet et synthétique de toutes les méthodes actuelles en immunologie. Il en recense les différentes pratiques, partant des principes des méthodes à leurs applications. Il s'articule en deux grandes parties :- la première portant sur les techniques elles-mêmes ;- la seconde dédiée à des domaines d'applications spécifiques et mobilisant parfois une grande diversité de techniques.Chacun des 15 chapitres est étayé d'illustrations didactiques et de tableaux synthétiques, construit de telle sorte que le lecteur y retrouve :- une présentation générale de chaque technique ;- une description synoptique de la méthodologie ;- des recommandations spécifiques de mises en oeuvre ;- des indications sur le mode de présentation des résultats ;- des informations sur la validation des résultats ;- un point sur les avantages et les inconvénients de chaque technique ; - des exemples d'applications pratiques

[The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation]

International audienceChronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation