Toxoplasma gondii, protozoaire à l’origine de la toxoplasmose, cause de nombreux avortements ou malformations fœtales, chez l’homme et l’animal. Aucun vaccin n’étant disponible, le développement d’approches vaccinales efficaces est d’actualité. L’immunisation de souris CBA/J avec des cellules dendritiques pulsées avec de l’extrait de T. gondii induit une réponse immunitaire Th1 spécifique du parasite et une protection significative. La déplétion in vivo des LTCD4+ ou CD8+, suite à un challenge par des kystes de T. gondii, nous a permis de décrire les rôles décisif des LTCD8+ et accessoire des LTCD4+ dans la protection des souris chroniquement infectées. Nous avons également étudié deux voies de signalisation intracellulaires induites par la reconnaissance du toxoplasme. Nous avons décrit le rôle délétère de l’IL-17R dans la réponse inflammatoire chez la souris C57BL/6 ainsi que le caractère essentiel de MyD88 dans la survie et la protection de souris BALB/c infectées chroniquement. / Toxoplasma gondii, an obligate intracellular protozoan, is the etiologic agent of toxoplasmosis, responsible for numerous abortions or fetal malformations, both in humans and animals. No vaccine is available so the design of efficient vaccine strategies is a topical question. Immunization of CBA/J mice with T. gondii extract-pulsed dendritic cells induces a parasite-specific Th1 immune response and a significant protection. In vivo depletion of CD4+ or CD8+ T lymphocytes following a challenge with T. gondii cysts, allowed us to establish the main effector role of CD8+ T cells and the secondary role of CD4+ T cells in the protection of mice chronically infected. We next studied two intracellular signaling pathways induced following the parasite recognition. We described a deleterious role of IL-17R in the inflammatory response in C57BL/6 mice and the crucial role of MyD88 in the survival and the protection of BALB/c mice chronically infected with T. gondii.

Abnormal epigenetic control is a common early event in tumour progression, and aberrant acetylation in particular has been implicated in tumourigenesis. Histone deacetylases (HDACs) are enzymes that regulate acetylation of chromatin and a variety of other non-histone substrates. Significantly, HDAC inhibitors are potent anti-proliferative agents and exhibit clinical activity in lymphoproliferative and haematological maligancy. However, the mechanistic details by which HDAC inhibitors affect proliferation remain to be elucidated. I have explored the cellular processes affected by HDAC inhibitors, and begun to illuminate a new pathway, regulated by HDAC, which impinges on the cellular effect of HDAC inhibitors. My results suggest that the proteins HR23B and Myd88 are important sensitivity determinants for HDAC inhibitor treatment, and that their interplay with HDAC6 dictates cell fate choice between survival by autophagy or apoptosis.

Les Apicomplexes constituent une vaste famille de parasites protozoaires responsables de nombreuses maladies chez l’Homme et chez les animaux. C’est le cas de Toxoplasma gondii, agent de la toxoplasmose et de Cryptosporidium parvum, responsable de la cryptosporidiose. Ces pathogènes représentent un réel problème de santé publique et vétérinaire. A l’heure actuelle, les seuls moyens de lutte contre ces parasites demeurent la chimiothérapie car il n’existe aucune stratégie prophylactique efficace. L’identification de cibles vaccinales repose sur le décryptage des mécanismes de défense mis en jeux vis-à-vis de ces agents infectieux. Dans le cadre de cette thèse, nos recherches se sont tournées vers l’étude du bras inné de la réponse immunitaire et plus particulièrement vers les voies de signalisation intracellulaires conséquentes de la reconnaissance de ces parasites. / No summary available

The TAK1 and canonical IKK complexes are the two master protein kinases of the innate immune system that control the production of inflammatory mediators, but the mechanisms by which they are activated in this system are still unclear. In this thesis, I present the research I have carried out to solve these problems. The IKKb component of the canonical IKK complex is required to activate the transcription factors NF-kB and IRF5 and the protein kinase Tpl2, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the first part of this thesis, I describe my work that has resolved this controversy by demonstrating that the activation of IKKb induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 catalysed phosphorylation of Ser177 and, secondly, the IKKb-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKk to phosphorylate exogenous substrates. I also present genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of Met1-linked/linear ubiquitin (Met1-Ub) chains and their interaction with the NEMO (NF-kB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKb at Ser177 and IKKa at Ser176. These findings may be of general significance for the activation of other protein kinases. The activation of the TAK1 complex by inflammatory stimuli is thought to be triggered by the binding of Lys63-linked ubiquitin chains to the TAB2 or TAB3 components of the TAB1-TAK1-TAB2 and TAB1-TAK1-TAB3 complexes. In the second part of the thesis I tested whether this broadly accepted model was correct by knocking out the genes encoding TAK1 and its regulatory subunits TAB1, TAB2 and TAB3 by CRISPR/Cas9 gene-editing technology, alone and in combination, in an IL-1 receptor expressing human cell line. These genetic studies led me to discover that the IL-1-dependent activation of TAK1 occurs by two different mechanisms. The first, involves the previously described interaction of Lys63-linked ubiquitin chains with TAB2 and TAB3, while the second can take place in the complete absence of TAB2 and TAB3. The second mechanism, which involves activation of the TAB1-TAK1 heterodimer is more transient than the first, but is sufficient for the IL-1-dependent transcription of immediate early genes (A20, IkBa). I show that the activation of the TAB1-TAK1 complex requires the expression of the E3 ubiquitin ligase TRAF6 and the TRAF6-generated formation of Lys63-linked ubiquitin chains, which leads to the phosphorylation of TAK1 at Thr187 and activation. However, neither TAB1 nor TAK1 bind directly to Lys63-linked ubiquitin chains. I identify one novel IL-1-dependent phosphorylation site on TAB1 and two on TAK1 and propose that Lys63-linked ubiquitin chains activate an as yet unidentified protein kinase, which phosphorylates one or more of the novel phosphorylation sites on the TAB1-TAK1 heterodimer inducing a conformational change that permits TAK1 to autophosphorylate Thr187.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第19001号 / 工博第4043号 / 新制||工||1622 / 31952 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 今堀 博, 教授 森 泰生 / 学位規則第4条第1項該当

Waldenström's Macroglobulinemia (WM) is a rare, indolent, non-Hodgkin's lymphoma whose molecular pathology remains poorly understood. This disease is characterized by the accumulation of IgM-secreting lymphoplasmacytic cells in the bone marrow, and is often histopathologically indistinguishable from marginal zone lymphoma, IgM-secreting myeloma, and chronic lymphocytic leukemia with plasmacytic differentiation. To better understand the genomic landscape of this disease, whole genome sequencing was performed on bone marrow samples from thirty WM patients, ten of which were paired with germline tissue. This study identified two genes that are frequently mutated in WM: MYD88 and CXCR4. MYD88 was somatically mutated in 90% of WM samples, which displayed a single nucleotide variant resulting in a leucine to proline substitution at position 265. As prev iously demonstrated in activated B-cell subtype of diffuse large B-cell lymphoma, this mutation results in constitutive activation of the Toll-like receptor pathway and activation of nuclear factor kappa B (NF𝜅B). Highly sensitive allele specific polymerase chain reaction assays were developed to detect MYD88L265P in WM and related hematological malignancies. These studies demonstrated that MYD88L265P could be used to aid in the differential diagnosis, response assessment, and detection of minimal residual disease in WM. Moreover, MYD88L265P was observed in 50% of the precursor condition, IgM monoclonal gammopathy of undetermined significance, suggesting that it is an early event in the pathogenesis of WM. Blocking MYD88 dimerization or the use of downstream IRAK1/4 kinase inhibitors decreased the phosphorylation and nuclear localization of NF𝜅B. Somatic mutations in CXCR4 were only found in the regulatory C-terminal tail and were present in 29% of WM patients. These mutations were similar or identical to those found in the germline of patients with the autosomal dominant disease Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome. CXCR4 somatic WHIM-like mutations were found nearly exclusively in MYD88L265P mutated patients. These mutations impaired receptor internalization, increased signaling downstream of CXCR4, and instilled resistance to several WM directed therapeutics. WM patients who were wild type for both CXCR4 and MYD88 demonstrated inferior overall survival. These studies evidence highly recurring somatic events, and provide a genomic basis for the molecular pathogenesis of WM.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble serum stimulation-2 (sST2) are elevated during human and murine aGVHD and are correlated to a type 1 T cell response. Membrane-bound ST2 (ST2) on donor T cells has been shown to be protective against aGVHD. ST2 signals through the adapter protein myeloid differentiation primary response 88 (MyD88). The role of MyD88 signaling in donor T cells during aGVHD remains unknown. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of interleukin 1 receptor (IL-1R) and toll-like receptor 4 (TLR4) signaling, both of which also signal through MyD88, in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells, leading to a decreased type 1 response without affecting T cell proliferation, apoptosis, or migration. In our aGVHD models, loss of intrinsic MyD88 signaling is not responsible for the observed protection. However, transplanting donor MyD88-/- T conventional cells (Tcons) with wild type (WT) or MyD88-/- T regulatory cells (Tregs) ameliorated aGVHD severity and lowered aGVHD mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine ten days post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, and Jak2, among others. Decreased sST2 was confirmed at the protein level with less secretion of sST2 and more expression of ST2 compared to WT T cells. Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs mirrored observations when using donor MyD88-/- Tcons. This suggests that Treg suppression from lack of MyD88 signaling in Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways. The results of our study confirm that ST2 represents an aGVHD therapeutic target that spares Treg function.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19001号 / 工博第4043号 / 新制||工||1622(附属図書館) / 31952 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 今堀 博, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

MyD88 est une protéine adaptatrice du système immunitaire inné, impliquée dans la défense de l’organisme contre les agents microbiens. Elle est recrutée aux « Toll-like receptors » (TLRs) suite à la reconnaissance par ces derniers de motifs microbiens conservés, les PAMPs (Pathogens-associated molecular patterns). La voie de signalisation ainsi déclenchée va aboutir à la production de cytokines pro-inflammatoires, de chimiokines et d’espèces actives de l’oxygène. De cette façon, les TLRs, via MyD88, constituent la première ligne de défense contre les pathogènes.De nombreuses études ont permis de démontrer que MyD88 est nécessaire pour la réponse inflammatoire, qui promeut la carcinogenèse. Dans le cadre d’une étude sur les TLRs et le cancer, l’équipe a démontré, grâce à une étude in vivo, que MyD88 participe au processus de tumorigenèse médiée par l’oncogène ras et est nécessaire à l’activation de la voie canonique des MAPKs, ainsi qu’à la transformation cellulaire in vitro. Nous avons ensuite déterminé le mécanisme par lequel MyD88 intervient dans la voie de signalisation Ras/MAPKs, en permettant le maintien de l’activation de cette voie. En effet, MyD88 interagit avec une MAPK clé de cette voie : la kinase ERK, et protège cette dernière de sa déphosphorylation par sa phosphatase spécifique MKP-3, MyD88 et MKP-3 se liant à ERK par le même domaine. Nous avons démontré la pertinence de ce mécanisme, grâce à la mise en évidence d’une surexpression de la protéine MyD88 et de son interaction avec la forme phosphorylée d’ERK dans des coupes de tissus tumoraux humains (estomac, poumon, colon).L’ensemble des résultats obtenus au cours de ma thèse ont permis de montrer qu’en plus de son rôle bien défini en tant qu’adaptateur des récepteurs de l’immunité innée dans les processus inflammatoires, MyD88 joue un rôle direct, qui semble être crucial dans la signalisation Ras, le contrôle du cycle cellulaire et la transformation cellulaire / MyD88 is an adaptator protein of the innate immune system, implicated in the défense against microbes. MyD88 is recruited by the Toll-Like Receptors (TLRs) upon there interaction with conserved microbial patterns (PAMPs). Therefore, TLR signaling pathway induces the production of pro-inflammatory cytokines, chemokines and reactive oxygen species. TLRs, via MyD88, form the first line of defense against pathogens. Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for Ras-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, ERK, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical Ras pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated ERK in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in Ras signaling, cell-cycle control, and cell transformation

Les protéines de chocs thermiques (HSP) sont des protéines très conservées au cours de l’évolution des espèces. Ce sont des chaperons moléculaires impliqués dans le repliement des protéines nouvellement synthétisée ou dénaturées. Les HSP sont fortement exprimées dans les cellules cancéreuses, où elles contribuent à la résistance à l’apoptose et aux chimiothérapies. Parmi les HSP, HSP110 est jusqu’à présent peu étudiée. Cependant, HSP110 a été récemment associée au lymphome diffus à grandes cellules B (DLBCL). Le DLBCL est le syndrome lympho-prolifératif agressif le plus fréquent chez l’adulte (30% des lymphomes non-Hodgkinien). Il existe trois principaux sous-type de DLBCL : le type B activé (ABC-DLBCL), le type centre germinatif (GC-DLBCL) et le type lymphome primaire du médiastin (PMBL). La forme activée est celle associée au plus mauvais pronostic clinique. Bien que les thérapies classiques de chimiothérapies associées aux anti-CD20 aient permis d’augmenter le pronostic de survie des patients souffrant de l’ABC-DLBCL, nombreux sont ceux qui développent des résistances ou qui ne répondent pas aux traitements. L’identification de nouvelles cibles thérapeutiques est donc nécessaire.Mes travaux présentent un rôle de HSP110 dans l’activité d’une voie oncogénique du lymphome ABC-DLBCL. En effet, ces travaux démontrent une forte expression d’HSP110 dans les échantillons patients ABC-DLBCL. De plus, ces travaux in vitro sur des lignées ABC-DLBCL montrent une interconnexion entre HSP110 et Myd88 L265P, protéine mutée responsable de l’activation de la voie NF-kB. HSP110 stabilise l’oncogène Myd88 L265P, et participe à l’amplification de la voie NFkB dans le lymphome ABC, voie responsable de la survie et de la prolifération des cellules ABC-DLBCL.Par le biais d’une collaboration, nous avons pu obtenir récemment des inhibiteurs de HSP110. Ainsi, mon travail à également consisté aux criblages in vitro de ces inhibiteurs pour évaluer leur capacité d’inhibition de HSP110. Deux composés ont alors été identifiés comme candidats inhibiteurs d’HSP110. Je me suis ensuite intéressé à l’utilisation de ces inhibiteurs in vitro dans l’ABC-DLBCL. Mes résultats tendent à montrer que ces inhibiteurs ont une action similaire à ceux observés lors de l’inhibition de HSP110 par siRNA ou shRNA.Mes travaux de thèse présente donc HSP110 comme une cible moléculaire potentielle de l’ABC DLBCL. / Heat shock proteins (HSPs) are highly conserved protein across species, and are expressed in all cell type. HSPs are molecular chaperones involved in the folding of newly synthesized or denaturated proteins. HSPs are overexpressed in cancer cells, where they contribute to cancer resistance to chemotherapies. Among HSPs, roles and functions of HSP110 are less described. Interestingly, HSP110 was recently associated with lymphoma aggressiveness in Diffuse Large B Cell Lymphoma (DLBCL). DLBCL is the most lymphoproliferative disease diagnosed in adult (30% of Non-Hodgkin Lymphoma). Three main subtypes of DLBCL are described: Activated-B-Cell lymphoma (ABC-DLBCL), Germinal Center lymphoma (GC-DLBCL), and Primary Mediastinal B Lymphoma (PMBL). ABC-DLBCL is the most aggressive form associated with a poor prognosis. Even if R-CHOP therapies had improve patient’s survival over the last decades, most of patients experiences relapses or treatment resistances. New molecular target are now necessary to treat efficiently these subtypes.My PhD work has highlighted the role of HSP110 in the NFkB signaling pathway, which is an oncogenic pathway in ABC-DLBCL. First, we show that HSP110 is overexpressed in ABC-DLBCL patient sample. We also show an interaction between HSP110 and Myd88 L265P, that is an oncogenic protein responsible for NFkB pathway activation. Consequently, HSP110 stabilizes Myd88 L265P, leading to a sustain NFkB pathway activation in lymphoma cells, and promoting ABC-DLBCL cell survival and proliferation.Finally, our team recently characterized the first known HSP110 inhibitors. I took the opportunity to test these putative inhibitors in my study. My results suggest that these compounds have similar effects than siRNA or shRNA inhibition of HSP110 on ABC-DLBCL survival. This result provide a ground for future in vivo testing of chemical inhibitors of HSP110.In conclusion, my work highlight HSP110 as a potential therapeutic target in ABC-DLBCL.