THE ROLE OF THE WISKOTT-ALDRICH SYNDROME PROTEIN (WASP) IN IMMUNE REGULATION

Le syndrome de Wiskott-Aldrich (SWA) est un syndrome d’immunodéficience primaire rare lié au chromosome X. Il se caractérise par une triade de signes cliniques : thrombocytopénie, eczéma et troubles auto-immuns. Le SWA est causé par un défaut d'expression ou de fonction de la protéine SWA (WASP), une protéine régulatrice de la réorganisation du cytosquelette exprimée dans toutes les cellules hématopoïétiques. Les études sur des patients SWA et des modèles de souris KO SWA ont révélé plusieurs anomalies de la différenciation et de la fonction des lymphocytes T et B, conduisant à une rupture de la tolérance immunitaire et au développement de troubles auto-immuns. Ce travail de thèse décrit certains des mécanismes cellulaires immunologiques associés aux lymphocytes B et T régulateurs et qui sont responsables des troubles auto-immuns et du défaut de maintien de la tolérance immunitaire associées au SWA. D’une part, grâce à un modèle de souris KO conditionnel aboutissant à un défaut d’expression de WASP et N-WASP dans les lymphocytes B seulement (B/DcKO), nous avons pu démontrer que l'expression de N-WASP, une protéine homologue de WASP, dans les lymphocytes B est nécessaire pour le développement des troubles auto-associés au SWA. Les cellules B double KO pour Was et N-Was présentent une réponse altérée contre les antigènes dépendants des lymphocytes T comparé aux cellules B KO pour Was uniquement. Contrairement aux souris simple KO pour WASP dans les lymphocytes B qui récapitulent certaines des caractéristiques auto-immunes associées au SWA, les souris B/DcKO présentent une absence de lésions rénales auto-immunes et une diminution de production d'auto-anticorps. D’autre part, la caractérisation d'un modèle de souris KO conditionnel (R/WcKO) dépourvu de WASP spécifiquement dans les lymphocytes T régulateurs (Treg) nous a permis de démontrer que l’absence de WASP affecte la différenciation, la distribution et la fonction des Treg, et que ces altérations sont suffisantes pour promouvoir des atteintes aux organes, tels qu'observés chez les patients atteints de SWA. Ce modèle de souris R/WcKO a permis de mettre en évidence l'impact des Treg KO pour WASP et leur rôle dans le développement de troubles auto-immuns. Pour finir, nous avons tenté d'étudier le rôle de WASP dans la différenciation des sous-types de lymphocytes T CD4 humains, en particulier les cellules Th1, Th17 et iTreg. Nous avons réussi à établir un système de différenciation in vitro de cellules souches pluripotentes induites humaines (iPSC) en cellules progénitrices hématopoïétiques. Nous avons également mis au point des méthodes permettant de différencier des cellules T in vitro à partir de progéniteurs hématopoïétiques ainsi que de polariser des lymphocytes T naïfs en cellules Th1 et Th2. En conclusion, nos résultats apportent de nouveaux éléments de compréhension des mécanismes pathologiques responsables des troubles auto-immuns observés dans le SWA, démontrant que l'expression N-WASP dans les cellules B et que le défaut d’expression de WASP dans les Tregs joue des rôles importants dans le développement de l'auto-immunité associée au SWA. -- Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, and autoimmunity. WAS is caused by the defective expression or function of the WAS protein (WASP), a regulatory protein of cytoskeleton organization expressed in all hematopoietic cells. Studies of WAS patients and WAS knock-out mouse models revealed several defects of T and B cell differentiation and function that lead to tolerance breakdown and development of autoimmune disorders. This PhD thesis describes some of the immunological B and Treg cell mechanisms responsible for autoimmunity and defective maintenance of tolerance in WAS. In one project, using a conditional knock-out mouse model of combined WASP and N-WASP deficiency in B lymphocytes (B/DcKO), we showed that the expression of N-WASP, an homolog protein of WASP, is required in B cells for the development of WASP deficiency-mediated autoimmunity. Was and N-Wasdouble knock-out B cells showed impaired response to T-cell-dependent antigens compared to Was single knock-out B cells. However, on the contrary of Was single knock-out mice that recapitulate some of the features of WAS-associated autoimmunity, the B/DcKO mice showed absence of autoimmune kidney damage and decreased autoantibodies production.In a second project, the characterization of a conditional knock-out mouse model lacking WASP expression specifically in regulatory T cells (Treg), allowed us to demonstrate how WASP deficiency affects Treg differentiation, distribution and function, and that it is sufficient to drive autoantibody production and organ damage, as observed in WAS patients. Using this R/WcKO mouse model, we revealed the intrinsic impact of WASP-negative Tregs and their role in autoimmunity. Finally we attempted to investigate the role of WASP in the differentiation of human CD4+ T cell subsets, in particular iTregs, Th1 and Th17 cells. We successfully established an in vitro differentiation system of human induced pluripotent stem cells (iPSC) into hematopoietic progenitor cells, as well as methods to obtain in vitro T cell differentiation, and Th1 and Th2 cell polarization. Taken altogether, our results increase the understanding of the pathological mechanisms responsible for the autoimmune features of WAS, proving that N-WASP expression in B cells and WASP deficiency in Tregs have important roles in the development of autoimmunity in WAS

Comparison of clinical and imaging findings in cats with single and mixed lungworm infection

Objectives: This study has compared clinical and imaging features in 52 cats naturally infected by respiratory nematodes Aelurostrongylus abstrusus, Troglostrongylus brevior and Capillaria aerophila, and in both monospecific and mixed infections. Methods: Medical records of cats with a lungworm disease were retrospectively reviewed. Cats with clinical examination findings, haematobiochemical analysis and thoracic radiography were included in the study and clinical and radiographic scores were assigned. For eight cats CT of the thorax was also available and analysed. A statistical analysis was performed to investigate the potential correlation between clinical and radiographic score, and to evaluate the effect of age, sex and infection on clinical and radiographic severity. Results: Monospecific infections by A abstrusus (32/52), T brevior (6/52) and C aerophila (5/52) and coinfectionsby T brevior/A abstrusus (7/52), T brevior/C aerophila (1/52) and A abstrusus/C aerophila (1/52) were diagnosed. Cats with mixed infections showed higher clinical scores compared with cats with monospecific parasitoses (P <0.05), while no differences were observed for radiographic scores. No correlation between clinical and radiographic scores was found (rs= 0.50), and these scores were not affected by patient age or sex. CT, performed on cats infected with A abstrusus, T brevior or A abstrusus/T brevior, provided additional information in cats with mild radiographic signs. Conclusions and relevance: This study indicates that clinical parameters may be more severe in mixed infections than in monospecific parasitoses. A significant correlation between clinical and radiographic score was not detected, while several subclinically infected cats showed radiographic changes. In cats with mild-to-moderate lung patterns, the ventrodorsal/dorsoventral projection showed lesions that are not visible in the lateral projections, especially in the caudal lobes

N-WASP is required for B-cell-mediated autoimmunity in Wiskott-Aldrich syndrome

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott- Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling bymodulating B-cell receptor (BCR) clustering and internalization.Wehave generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-celldependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS

Efficacy and Adverse Events During Janus Kinase Inhibitor Treatment of SAVI Syndrome

Objectives: Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib. Methods: We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis. Results: We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease. Conclusions: We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment