Anne-Laure Mausset-Bonnefont

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3Y260X homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3Y260X mice by using rotating rod and vestibular behavioral tests. Tmprss3Y260X mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

IntroductionRegulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.MethodsCol-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.ResultsCol-Treg clones are characterized by their specific cytokine profile (IL-10highIL-4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.ConclusionsOur results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.

Cellular senescence impact on immune cell fate and function.

Cellular senescence occurs not only in cultured fibroblasts, but also in undifferentiated and specialized cells from various tissues of all ages, in vitro and in vivo. Here, we review recent findings on the role of cellular senescence in immune cell fate decisions in macrophage polarization, natural killer cell phenotype, and following T‐lymphocyte activation. We also introduce the involvement of the onset of cellular senescence in some immune responses including T‐helper lymphocyte‐dependent tissue homeostatic functions and T‐regulatory cell‐dependent suppressive mechanisms. Altogether, these data propose that cellular senescence plays a wide‐reaching role as a homeostatic orchestrator.

Immunohistochemistry as a tool for topographical semi-quantification of neurotransmitters in the brain

Immunohistochemistry is a powerful tool to detect neurotransmitter (NT) presence in different brain structures with a high spatial resolution. However, it is only scarcely used in quantitative approach due to lack of reproducibility and sensitivity. We developed a protocol of NT detection based on immunohistochemistry and image analysis to show that this approach could also be useful to evaluate NT content variations. We focused our study on the GABAergic system in the cerebellum and measured different accurate parameters, namely the optical density (O.D.), the stained area and the number of immunoreactive cells in each cerebellar cell layer. In order to modify the GABA content, we used gamma-vinyl-GABA (GVG), an inhibitor of GABA-transaminase, known to dramatically increase GABA concentration in the central nervous system (CNS) and especially in the cerebellum. We observed a significant increase in the three parameters measured in the molecular and the granular layers of the cerebellum after treatment with GVG, reflecting the well-established increase in GABA content after such a treatment. Therefore, our technical approach allows not only a precise determination of the effects in particular cell layers but also a semi-quantification of GABA content variations. This technique could be suitable for monitoring NT variations following any treatment.

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.

A8.26 Inducible IL-10 secreting CD49b +Treg cells as cell based-therapy for rheumatoid arthritis

Background Adoptive transfer of regulatory T cells (Tregs) is a promising approach to restore tolerance in autoimmune diseases. However based on the heterogeneity of the Tregs, we need to precisely establish which Tregs will be able to dampen efficiently the immune response in the various settings. We previously showed the potential of CD49b+ Treg cells to protect and prevent an experimental model of arthritis. Nevertheless the optimal injection dose, the phenotype and the in vivo-suppressive mechanism of these Treg cells remain unknown. In our study, we investigated and compared the therapeutic potential of CD25+FoxP3+ and induced IL10-secreting CD49b+ Treg cells in an experimental model of arthritis, the collagen-induced arthritis (CIA). Materials and Methods IL-10 secreting CD49b+ Treg cells were generated in naïve mice following repetitive injections of immature DCs (iDCs). Treg purification was based on the negative selection of CD4 T cells isolated from spleen and liver of the iDC-vaccinated mice. Cell sorting was performed to obtain 98% pure CD49b+ or CD25+ Treg cells. Several doses of CD49b+ were intravenously (i.v.) injected at day 28 in established CIA. Clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in sera and the cytokine profile of bCII specific T cells. Phenotypes of both Treg cells were compared as well as their suppressive activity in vitro and in vivo. Results Several doses of CD49+ Treg cells were tested in curative settings experiments. The dose of 105 CD49b+ or CD25+ cells reverse clinical symptoms of arthritis while interestingly, a lack of efficacy was observed after higher doses. In vitro suppressive experiments confirmed the similar efficiency of both populations and phenotype analyses of CD49b+ Treg cells showed expression profile of several Treg specific markers (LAP+, LAG+, CTLA-4high). Moreover, in an OVA-specific model of inflammation, we demonstrated the high impact of the CD49b Treg cells on the proliferation of effector cells in vivo. Conclusions Altogether, our results confirm the therapeutic potential of IL-10 secreting T cells in experimental model of arthritis in curative settings and unravel their mechanism of suppression.

Injection of antigen-specific regulatory Tr1 lymphocytes protects mice from severe collagen-induced arthritis

Introduction Tr1 cells have been characterised as induced T regulatory lymphocytes (Treg) inhibiting inflammation in various chronic inflammatory models. Based on these data, a phase I/II clinical trial is currently under investigation in Crohns disease (TxCell). However, the therapeutic potential of these cells has not yet been evaluated in rheumatoid arthritis. In this study, the authors investigated the therapeutic potential of bovine type II collagen (bCII) specific Tr1 cells, isolated from TBC mice, in the experimental model of collagen-induced arthritis (CIA). Methods Collagen type II specific Tr1 clones were obtained from T cell receptor transgenic mice and expanded in vitro. Selected clones showed in vitro antigen specificity, Tr1 cytokine profile (IL-10high/IL-4neg) and IL-10- and transforming growth factor β-dependent suppressive activity. Male DBA/one mice were immunised with bCII and 10×106, 3×106, 1×106, 0.3×106 of Tr1 cells were injected (intravenously) 28 days postimmunisation. Hind paws swelling and clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in the sera of treated mice and the cytokine profile of bCII specific T cells. Results One single injection of 3×106 or 1×106 of Tr1 cells at day 28, in ongoing arthritis, significantly inhibits the development of arthritic disease, shown by reduction of disease severity and incidence. In contrast the injection of 0.3×106 and 10 M of Tr1 cells did not improve the clinical signs of arthritis. The analysis of the bCII specific T cell responses following euthanasia of the mice injected with 3×106 and 1×106 of Tr1 cells revealed a decrease of anti-bCII specific antibodies in the sera, a decrease of proliferation of bCII specific T cells and a slight increase of IL-10 secreted by activated splenocytes. Importantly, these preliminary data indicate that a single injection of Tr1 cells at disease onset could reduce disease severity and incidence in experimental arthritis. Conclusions Single dose 3×106, 1×106 of Tr1 cell administration showed a reduction of disease incidence and severity in CIA demonstrating the therapeutic potential of Tr1 cells in arthritis and confirming the clinical potential of these induced Treg.

Early Retinal Defects in Fmr1−/y Mice: Toward a Critical Role of Visual Dys-Sensitivity in the Fragile X Syndrome Phenotype?

Fragile X Syndrome (FXS) is caused by a deficiency in Fragile X Mental Retardation Protein (FMRP) leading to global sensorial abnormalities, among which visual defects represent a critical part. These visual defects are associated with cerebral neuron immaturity especially in the primary visual cortex. However, we recently demonstrated that retinas of adult Fmr1−/y mice, the FXS murine model, present molecular, cellular and functional alterations. However, no data are currently available on the evolution pattern of such defects. As retinal stimulation through Eye Opening (EO) is a crucial signal for the cerebral visual system maturation, we questioned the precocity of molecular and functional retinal phenotype. To answer this question, we studied the retinal molecular phenotype of Fmr1−/y mice before EO until adult age and the consequences of the retinal loss of Fmrp on retinal function in young and adult mice. We showed that retinal molecular defects are present before EO and remain stable at adult age, leading to electrophysiological impairments without any underlying structural changes. We underlined that loss of Fmrp leads to a wide range of defects in the retina, settled even before EO. Our work demonstrates a critical role of the sensorial dysfunction in the Fmr1−/y mice overall phenotype, and provides evidence that altered peripheral perception is a component of the sensory processing defect in FXS conditions.

Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis

Progressive loss of tissue homeostasis is a hallmark of numerous age-related pathologies, including osteoarthritis (OA). Accumulation of senescent chondrocytes in joints contributes to the age-dependent cartilage loss of functions through the production of hypertrophy-associated catabolic matrix-remodeling enzymes and pro-inflammatory cytokines. Here, we evaluated the effects of the secreted variant of the anti-aging hormone α-Klotho on cartilage homeostasis during both cartilage formation and OA development. First, we found that α-Klotho expression was detected during mouse limb development, and transiently expressed during in vitro chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Genome-wide gene array analysis of chondrocytes from OA patients revealed that incubation with recombinant secreted α-Klotho repressed expression of the NOS2 and ZIP8/MMP13 catabolic remodeling axis. Accordingly, α-Klotho expression was reduced in chronically IL1β-treated chondrocytes and in cartilage of an OA mouse model. Finally, in vivo intra-articular secreted α-Kotho gene transfer delays cartilage degradation in the OA mouse model. Altogether, our results reveal a new tissue homeostatic function for this anti-aging hormone in protecting against OA onset and progression.