Étude d'une nouvelle molécule neuroprotectrice et optimisation d'un modèle murin d'ischémie cérébrale focale

Les accidents vasculaires cérébraux (AVC) sont considérés comme la pathologie neurologique la plus sévère en terme de mortalité et d'infirmité. Elle constitue ainsi la troisième cause de mortalité dans les pays industrialisés (120000 cas par an en France), après les accidents cardiaques et les cancers, mais surtout la première cause de ahndicap acquis. Elle constitue donc un problème de santé public majeur. Un tiers des AVC est dû à l'occlusion de l'artère sylvienne modélisée chez la souris par occlusion de l'artère cérébrale moyenne (OACM) conduisant à une dégénérescence des cellules du cortex temporo-pariétal. Nous avons, dans ce modèle, étudié le comportement d'une protéase spécifique, la cathepsine B et montré qu'elle était précocement activée au cours de la première heure qui suit l'occlusion. Cette activation aboutissait à l'activation des caspases-1, -11 et -3. L'utilisation d'un inhibiteur spécifique de la cathepsine B, le CA-074, nous a permis de réduire le volume lésé à 24 heures parallèlement à une réduction de l'activité de la caspase-3. Dasn un but thérapeutique, nous avons construit des protéines chimériques contenant XIAP (puissant inhibiteur endogène des caspases), son domaine BIR2 ou ses domaines BIR3/RING précédées du domaine PTD (protein transduction domain) du VIH-1. Ce domaine permet la traversée de la barrière hémato-encéphalique ainsi que des membranes lipidiques.Cerebral ischemia is considered as the most severe neurological pathology in term of mortality and infirmity. It constitues the third cause of mortality in industrial nations (120000 cases a year in France), after the heart attacks and the cancers, but especially the first cause of acquired handicap. It thus constitutes a major public problem of health. A third party of stoke is due to the occlusion of the sylvius artery modelled to the mouse by occlusion os the middle cerebral artery (MCAO) inducing degeneracy of the temporo-parietal cortex neurons. We studied, in this model, a specific protease, the cathepsin B, and showed that it was activated prematurely during the first hour which follows the occlusion. This activation ended in the activation caspases-1, -11 and -3. The use of a specific inhibitor of the cathepsin B, the CA 074, allowed us to reduce the 24 h infarct volume at the same time as a reduction of caspase-3 activity. In a therapeutic aim, we built chimeric proteins containing XIAP (powerful endogenous inhibitor of caspases), its domain BIR2 or its domain BIR3 / RING preceded by the PTD domain (peotein transduction domain) of the HIV 1. This domain allows the crossing of the blood brain barrier as well as the lipidic membranes.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Receptor activating NF-κB ligand (RANKL) is a constitutive intracellular protein in resting human basophils and is strongly induced on their surface by interleukin 3

International audienceReceptor activating NF-B ligand (RANKL) is a member of the TNF superfamily that plays a pivotal role in bone homeostasis as being the major osteoclastogenesis factor. RANKL also has pleiotropic effects in the immune system in which it is expressed by activated T and B cells and some innate lymphoid cells. RANKL-RANK interactions mediate lymph node organogenesis and immunoregulatory functions in autoimmune disease and carcinogenesis as well as cross talk between the immune system and bone. In this study, we show that basophils were the strongest RANKL mRNA-expressing cells amongst major leukocyte subsets in human blood. RANKL was preformed as an intracellular protein in resting basophils and was rapidly and strongly expressed on their surface upon stimulation with IL-3, but not other stimuli. This expression was stable for at least 6 days. Activated basophils could also release soluble RANKL in small quantities upon interaction with DCs or monocytes. In the blood, basophils were the sole cells to express membrane RANKL in response to IL-3. This study indicates that basophils should be considered as new players in the pleiotropic and complex RANKL-RANK interaction system and suggests a role for RANKL in the interaction between basophils and immune cells in inflammatory allergic tissues and secondary lymphoid organs

Persistent deficiency of circulating mucosal-associated invariant T (MAIT) cells in ANCA-associated vasculitis

International audienceObjective: Mucosal associated invariant T cells (MAIT) and innate lymphoid cells (ILCs) have immuno-regulatory functions at mucosal sites and have been involved in various inflammatory and autoimmune diseases. The aim of this study was to assess their frequencies in blood in ANCA-associated vasculitis (AAV).Methods: The frequencies and function of MAIT cells, ILCs, gdT, iNKT, NK cells were analyzed by flow cytometry on PBMC of patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) without any treatment, in acute (AP) and remission phase (RP) and compared with healthy controls (HC).Results: The frequencies of MAIT cells were strongly decreased in GPA and MPA in AP compared to HC, both in never treated and in relapsing patients and independently of patient age. This was associated with an activated phenotype of patient MAIT cells, as shown by increased expression of CD69 and IFNg. MAIT cells remained decreased during RP in AAV patients. The frequencies of iNKT and gdT cells were unaffected compared to HC, whereas those of NK cells were slightly reduced during AP in MPA. We also observed a significant decrease in frequencies of total ILCs with decreased ILC2 and ILC3 and increased ILC1 during AP in both GPA and MPA compared to HC. These frequencies normalized during RP. Interestingly , we observed a significant correlation between the frequency of total ILCs and BVAS.Conclusion: We show for the first time that AAV are associated with a major decrease and an activated phenotype of blood MAIT cell. These features persisted during remission suggesting a role for MAIT cells in the pathogenesis of AAV

Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer’s disease mice

International audienceInterleukin-2 (IL-2)-deficient mice have cytoarchitectural hippocampal modifications and impaired learning and memory ability reminiscent of Alzheimer’s disease. IL-2 stimulates regulatory T cells whose role is to control inflammation. As neuroinflammation contributes to neurodegeneration, we investigated IL-2 in Alzheimer’s disease. Therefore, we investigated IL-2 levels in hippocampal biopsies of patients with Alzheimer’s disease relative to age-matched control individuals. We then treated APP/PS1ΔE9 mice having established Alzheimer’s disease with IL-2 for 5 months using single administration of an AAV-IL-2 vector. We first found decreased IL-2 levels in hippocampal biopsies of patients with Alzheimer’s disease. In mice, IL-2-induced systemic and brain regulatory T cells expansion and activation. In the hippocampus, IL-2 induced astrocytic activation and recruitment of astrocytes around amyloid plaques, decreased amyloid-β42/40 ratio and amyloid plaque load, improved synaptic plasticity and significantly rescued spine density. Of note, this tissue remodelling was associated with recovery of memory deficits, as assessed in the Morris water maze task. Altogether, our data strongly suggest that IL-2 can alleviate Alzheimer’s disease hallmarks in APP/PS1ΔE9 mice with established pathology. Therefore, this should prompt the investigation of low-dose IL-2 in Alzheimer’s disease and other neuroinflammatory/neurodegenerative disorders

Real-Time Monitoring of Exosome Enveloped-AAV Spreading by Endomicroscopy Approach: A New Tool for Gene Delivery in the Brain

International audienceExosomes represent a strategy for optimizing the adeno-associated virus (AAV) toward the development of novel therapeutic options for neurodegenerative disorders. However, in vivo spreading of exosomes and AAVs after intracerebral administration is poorly understood. This study provides an assessment and comparison of the spreading into the brain of exosome-enveloped AAVs (exo-AAVs) or unassociated AAVs (std-AAVs) through in vivo optical imaging techniques like probe-based confocal laser endomicroscopy (pCLE) and ex vivo fluorescence microscopy. The std-AAV serotypes (AAV6 and AAV9) encoding the GFP were enveloped in exosomes and injected into the ipsilateral hippocampus. At 3 months post-injection, pCLE detected enhanced GFP expression of both exo-AAV serotypes in contralateral hemispheres compared to std-AAVs. Although sparse GFP-positive astro-cytes were observed using exo-AAVs, our results show that the enhancement of the transgene expression resulting from exo-AAVs was largely restricted to neurons and oligodendro-cytes. Our results suggest (1) the possibility of combining gene therapy with an endoscopic approach to enable tracking of exo-AAV spread, and (2) exo-AAVs allow for widespread, long-term gene expression in the CNS, supporting the use of exo-AAVs as an efficient gene delivery tool

IL-22BP production is heterogeneously distributed in Crohn’s disease

International audienceCrohn’s disease (CD), a form of inflammatory bowel disease (IBD), is characterized by impaired epithelial barrier functions and dysregulated mucosal immune responses. IL-22 binding protein (IL-22BP) is a soluble inhibitor regulating IL-22 bioactivity, a cytokine proposed to play protective roles during CD. We and others have shown that IL-22BP is produced in IBD inflamed tissues, hence suggesting a role in CD. In this work, we extended the characterization of IL-22BP production and distribution in CD tissues by applying enzyme-linked immunosorbent assays to supernatants obtained from the culture of endoscopic biopsies of patients, and reverse transcription-quantitative polymerase chain reaction on sorted immune cell subsets. We reveal that IL-22BP levels are higher in inflamed ileums than colons. We observe that in a cell-intrinsic fashion, populations of mononuclear phagocytes and eosinophils express IL-22BP at the highest levels in comparison to other sources of T cells. We suggest the enrichment of intestinal eosinophils could explain higher IL-22BP levels in the ileum. In inflamed colon, we reveal the presence of increased IL-22/IL22BP ratios compared to controls, and a strong correlation between IL-22BP and CCL24. We identify monocyte-derived dendritic cells (moDC) as a cellular subtype co-expressing both cytokines and validate our finding using in vitro culture systems. We also show that retinoic acid induces the secretion of both IL-22BP and CCL24 by moDC. Finally, we report on higher IL-22BP levels in active smokers. In conclusion, our work provides new information relevant to therapeutic strategies modulating IL-22 bioactivity in CD, especially in the context of disease location

Inhibition of DYRK1A proteolysis modifies its kinase specificity and rescues Alzheimer phenotype in APP/PS1 mice

Abstract Recent evidences suggest the involvement of DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1 A) in Alzheimer’s disease (AD). Here we showed that DYRK1A undergoes a proteolytic processing in AD patients hippocampus without consequences on its kinase activity. Resulting truncated forms accumulate in astrocytes and exhibit increased affinity towards STAT3ɑ, a regulator of inflammatory process. These findings were confirmed in APP/PS1 mice, an amyloid model of AD, suggesting that this DYRK1A cleavage is a consequence of the amyloid pathology. We identified in vitro the Leucettine L41 as a compound able to prevent DYRK1A proteolysis in both human and mouse protein extracts. We then showed that intraperitoneal injections of L41 in aged APP/PS1 mice inhibit STAT3ɑ phosphorylation and reduce pro-inflammatory cytokines levels (IL1- β, TNF-ɑ and IL-12) associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-β plaque burden. Importantly, L41 treatment improved synaptic plasticity and rescued memory functions in APP/PS1 mice. Collectively, our results suggest that DYRK1A may contribute to AD pathology through its proteolytic process, reducing its kinase specificity. Further evaluation of inhibitors of DYRK1A truncation promises a new therapeutic approach for AD