Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that a disintegrin and metalloproteinase 10 (ADAM10) is a critical regulator of Notch and that its deletion is embryonically lethal, we generated transgenic mice that overexpress ADAM10 at early stages of lymphoid and myeloid development (A10Tg). ADAM10 transgene expression alters hematopoiesis post-hematopoietic Lineage-Sca-1+c-kit+ (LSK) subset differentiation but prior to lineage commitment of progenitor populations. This results in delayed T cell development, abrogated B2 cell development, and dramatic expansion of functionally active myeloid derived suppressor cells (MDSCs) in A10Tg mice. Given ADAM10’s role in Notch signaling, we hypothesized that the observed hematopoietic alterations may be a consequence of perturbed Notch signaling. In fact, blockade of ADAM10 (S2) rescues B cell development and reduces myeloid cells in A10Tg LSKs. Inhibition of γ-secretase (S3) in wild type (WT) LSKs results in enhanced myelopoiesis, mimicking the phenotype of A10Tg mice. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical for hematopoietic cell-fate determination. Albeit arising in a tumor-free host, A10Tg MDSCs are functionally and phenotypically analogous to tumor-derived MDSCs. A10Tg MDSCs inhibit T cell activation in vitro, and inhibit adoptive immunotherapy (AIT) of metastatic melanoma in vivo, which can be reversed with MDSC depletion. Intriguingly, A10Tg mice are resistant to parasitic infection upon inoculation of Nippostrongylus brasiliensis. However, depletion of MDSCs abrogates this response, while adoptive transfer (AT) of MDSCs into WT mice increases their resistance. This polarized activity of MDSCs is heavily dependent upon interaction with mast cells (MCs). In fact, B16 melanoma cells metastasize more rapidly in WT mice infused with MDSCs when compared to MC-deficient mice (Kit Wsh/Wsh), with or without MDSC AT. Parallel to B16 progression, the ability of MDSCs to promote anti-Nb immunity is significantly diminished in MC-deficient (Kit Wsh/Wsh) mice even with MDSC AT. This augmentation of MDSC activity in the presence of MCs is further corroborated by in vitro co-culture assays that demonstrate a synergistic increase in cytokine production. Furthermore, MDSCs preferentially migrate to the liver in a MC-dependent manner. This interaction is mediated by MC-released histamine. In fact, MDSCs express histamine receptors (HR) and histamine induces MDSC survival, proliferation, and activation. We demonstrate that MDSC activity is abrogated with histamine blockade. Moreover, in humans, allergic patients present with an increase in MDSC population, and MDSCs purified from a stage I breast cancer patient exhibit increased survival in the presence of histamine. Taken together, our studies indicate that MCs and MC-released histamine are critical for the observed functional duality of MDSCs, ranging from immunosuppressive to immunosupportive, depending on the disease state.

1 Abstract Liver fibrosis is a condition described by extensive accumulation of scar tissue in the liver. With further progression, it leads to cirrhosis or even to hepatocellular carcinoma. Liver fibrosis accompanies every chronic liver disease and its prevalence in adult European population is estimated to be around 4%. During my dissertation work, I studied the function of three members of Metzincin family of metalloproteinases - ADAM17, ADAM10 and MMP-19, in liver fibrosis and liver regeneration using mouse genetic models. ADAM17 and ADAM10 are important regulators of signalling pathways which are involved in immune response as well as differentiation. Both proteases are able to cleave ectodomains of their substrates from cell membrane, affecting bioavailability of ligands and functionality of receptors. Several of their substrates are involved in liver pathologies. MMP-19 on the other hand, is a metalloprotease mainly involved in extracellular matrix cleavage, important process in fibrosis development, as well as resolution of fibrosis. Our results demonstrate that ablation of ADAM10 results in increased susceptibility to liver fibrosis in mice, both spontaneous and toxin induced. ADAM10 deficiency affected biliary epithelium, as we detected higher markers of biliary damage in serum of ADAM10 deficient...

No description available.

L’importance des activités protéolytiques associées à la membrane plasmique dans divers processus biologiques fondamentaux est de mieux en mieux définie. Les protéases de la famille ADAM (A Disintegrin and Metalloprotease), et ADAM10 en particulier, ont suscité un intérêt tout particulier du fait de l’importance de leurs substrats (récepteur de l’EGF, TNFα, Notch, APP…). Néanmoins, peu d’études se sont intéressées aux mécanismes régulant le trafic d’ADAM10.Les tétraspanines sont une super-famille de protéines de surface impliquées dans de nombreux processus biologiques fondamentaux parmi lesquels la migration, les interactions intercellulaires, la réponse immunitaire, la fusion des gamètes… L’une des caractéristiques majeure des tétraspanines est leur capacité à organiser un réseau d’interactions moléculaires appelé le « tetraspanin web ». De précédentes études menées dans le laboratoire ont montré qu’ADAM10 est associé au « tetraspanin web ». Néanmoins, la tétraspanine en interaction directe avec ADAM10 permettant son association au réseau n’est pas encore connue. Dans cette étude nous nous sommes intéressés à la régulation d’ADAM10 par les tétraspanines. Nous avons ainsi pu identifier une sous-famille de tétraspanines à 8 cystéines, les TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 et Tspan33), comme étant capables d’interagir directement avec ADAM10 et de réguler sa sortie du réticulum endoplasmique. Nous avons montré que Tspan5, Tspan14, Tspan15 et Tspan33 sont capables de réguler l’expression de surface d’ADAM10 et que Tspan10 et Tspan17 entrainent l’accumulation d’ADAM10 dans un compartiment endosomal tardif. Les TspanC8 pourraient également contribuer à la régulation de la spécificité de substrat d’ADAM10 puisque nous avons montré que l’expression des TspanC8 humaines Tspan5 et Tspan14 augmente l’activation de la voie Notch alors que Tspan15 n’a pas d’effet. Par ailleurs, les TspanC8 de Drosophile sont capables d’interagir directement avec Kuzbanian (l’orthologue d’ADAM10), permettent son accumulation à la surface cellulaire et régulent l’activation de la voie Notch dans différents contextes développementaux. Nous proposons que les TspanC8 soient une nouvelle famille de protéines ayant une fonction très conservée dans la régulation de l’activité et du trafic d’ADAM10, capables de réguler l’activation de la voie Notch. / Increasing evidence suggests a critical implication of membrane-associated protease activities in numerous biological processes. ADAM (A Disintegrin and Metalloprotease) proteases, and especially ADAM10, are of particular interest because of the importance of their substrates (EGF receptor, TNF α, Notch, APP…). However, few studies focus on the mechanisms of ADAM10 trafficking. Tetraspanins are a super-family of proteins implicated in numerous biological processes including migration, intercellular interactions, immune response, gamete fusion… One of the most striking features of tetraspanins is their ability to organise multi-molecular complexes called « Tetraspanin Web ». Previous studies in the laboratory have shown that ADAM10 is associated to the « Tetraspanin Web ». Nevertheless, the tetraspanin in direct interaction with ADAM10 that drives its association to the web is not known. In this study, we focused on ADAM10 regulation by tetraspanins. We identified a subfamily of tetraspanins with 8 cysteines in their large extracellular domain that we called TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) that can directly interact with ADAM10 and regulate its egress from the endoplasmic reticulum. We have shown that Tspan5, Tspan14, Tspan15 and Tspan33 regulate the surface expression of ADAM10 and that Tspan10 and Tspan17 accumulate ADAM10 in a late endosomal compartment. TspanC8 could also contribute to substrate specificity since Tspan5 and Tspan14 can increase Notch activation when Tspan15 cannot. Drosophila TspanC8 directly interact with the Drosophila ADAM10 ortholog Kuzbanian, increase its accumulation at the cell surface and modulate Notch activation in several developmental contexts. We propose that TspanC8 constitute a new family of Notch regulators with conserved functions in the regulation of ADAM10 trafficking and activity.

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4+ T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry.

No description available.

La démyélinisation et la mort oligodendrocytaire sont bien connues dans la sclérose en plaques (SEP). Au cours de ces dernières années, plusieurs études ont également décrit ce type de lésion après un traumatisme crânien (TC), participant à l’aggravation des lésions de la substance blanche, responsables des dysfonctionnements cognitifs et moteurs. Malgré de nombreux efforts, aucune thérapie efficace n’est disponible à ce jour pour traiter les lésions de la substance blanche. Dans ce contexte, une stratégie thérapeutique prometteuse serait de freiner la neuro-inflammation et la démyélinisation, en plus de promouvoir la maturation des oligodendrocytes afin de favoriser la remyélinisation des axones et de limiter ainsi leur dégénérescence. Notre choix de stratégie porte sur la stimulation des processus de réparation endogène via la protéine neuroprotectrice et neurotrophique sAPPα, forme soluble de la protéine βAPP libérée par l’action des α-sécrétases (ADAM10). Dans ce contexte, l’objectif de mes travaux de thèse était d’étudier l’intérêt thérapeutique de l’étazolate, un activateur desα-sécrétases, sur les conséquences biochimiques, histologiques et fonctionnelles, dans différents modèles de TC et de SEP in vivo chez la souris, et ex vivo sur des tranches organotypiques de cervelet. Les résultats obtenus sur le modèle de TC par percussion mécanique chez la souris ont montré pour la première fois le potentiel anti-inflammatoire de l’étazolate, associé à la restauration du taux de la sAPPα. De plus, l’étazolate s’est également opposé aux troubles fonctionnels post-TC tels que l’hyperactivité locomotrice et le déficit cognitif à court et à long terme. Par la suite, j’ai développé un nouveau modèle ex vivo de TC par percussion mécanique sur des tranches organotypiques de cervelet. Nous avons montré pour la première fois que l’étazolate était neuroprotecteur dans le tissu cérébelleux, et qu’il s’opposait à la démyélinisation post-traumatique. Par ailleurs, les effets bénéfiques de l’étazolate sur les gaines de myéline ont été reproduits dans un modèle ex vivo de démyélinisation induite par la lysolécithine, modèle ex vivo de SEP. De façon intéressante nous avons montré que l’étazolate exerçait un effet remyélinisant en stimulant la différenciation des oligodendrocytes. Cet effet a été reproduit in vitro dans des cultures primaires mixtes de cellules gliales issues de souris PLP-eGFP, où la maturation morphologique des oligodendrocytes a été favorisée en présence d’étazolate. L’ensemble des effets bénéfiques exercés par l’étazolate a été inhibé en présence d’un inhibiteur pharmacologique spécifique d’ADAM10, le GI254023X, suggérant que l’effet remyélinisant de l’étazolate dépend, au moins en partie, d’ADAM10. Par la suite, l’effet remyélinisant de l’étazolate a été étudié dans un modèle in vivo de démyélinisation chronique induite par la cuprizone. Dans ce modèle, l’étazolate a été capable de promouvoir la remyélinisation en stimulant la différenciation des oligodendrocytes, confirmant nos résultats in vitro et ex vivo. L’ensemble de mon travail permet de considérer le potentiel thérapeutique de l’étazolate, en visant l’ADAM10 comme nouvelle cible thérapeutique neuroprotectrice et remyélinisante. Cela aura pour intérêt de limiter la neuro-inflammation, la démyélinisation, ainsi que de promouvoir la différenciation des oligodendrocytes et la remyélinisation, afin de favoriser la récupération fonctionnelle suite aux lésions de la substance blanche survenant après un TC ou la SEP chez l’homme. / Demyelination and oligodendrocyte cell death are well established in multiple sclerosis (MS) and are increasingly described after traumatic brain injury (TBI), participating in the aggravation of white matter injury responsible of motor and cognitive deficits. Despite many efforts in clinical research, no efficient therapy against white matter injury progression is available nowadays. Thus, promoting remyelination and counteracting neuroinflammation and demyelination are major therapeutic strategies in order to restore white matter integrity. Here, we studied the stimulation of endogenous repair mechanisms through the neuroprotective and neurotrophic protein sAPPα, the soluble form of βAPP protein released by the α-secretases (ADAM10). In this context, the aim of this work was to evaluate the therapeutic potential of etazolate, an α-secretase activator on short- and long-term biochemical, histological and functional outcome in different mouse models of TBI and MS in vivo, and ex vivo on organotypic cerebellar slices. The results obtained from the TBI mouse model by mechanical percussion showed for the first time the anti-inflammatory effect of etazolate associated to a restoration of sAPPα levels. The same treatment was able to attenuate functional deficits (hyperactivity, cognitive deficit). We also developed a new ex vivo model of TBI by mechanical percussion on organotypic cerebellar slices. We confirmed the neuroprotective effect of etazolate on cerebellar tissue reducing the lesion size. Interestingly, etazolate treatment attenuated post-traumatic ex vivo demyelination. Moreover, the beneficial effect of etazolate on myelin sheaths have been well reproduced after lysolecithin-induced demyelination, an ex vivo model of MS. Interestingly, etazolate was able to enhance remyelination promoting oligodendrocyte differentiation. This effect has been reproduced in the primary mixed glial culture from PLP-eGFP mice, enhancing oligodendrocyte morphological maturation. However, etazolate failed to promote its beneficial effects in the presence of GI254023X, a specific ADAM10 (α-secretase) inhibitor, suggesting that the mechanism of action of etazolate is partly through the activation of ADAM10. Furthermore, etazolate reproduced in vivo the oligodendrocyte differentiation, accompanied by an increase of the myelinated axons, observed by electron microscopy in a mouse model of cuprizone-induced chronic demyelination. Taken together, the findings of this work provide a first evidence for the therapeutic potential of etazolate, with ADAM10 as new therapeutic target in white matter repair. The interest of this approach is to attenuate neuroinflammation and demyelination and to enhance oligodendrocyte differentiation and thus remyelination, in order to promote functional recovery following white matter lesions arising after TBI or MS.