Diverses études, dont celles menées par l’équipe de MC CUTURI, ont montré l’efficacité de la thérapie cellulaire à l’aide de cellules dendritiques tolérogènes dans le contrôle du rejet de greffe chez le rongeur. L’originalité de nos travaux réside dans l’utilisation de cellules autologues, les ATDC (Autologous Tolerogenic Dendritic Cells). Dans l’optique de leur application clinique en transplantation rénale, les ATDC ont été générées chez des volontaires sains. Le premier objectif de ma thèse a été de valider le procédé de fabrication de grade clinique des ATDC humaines. De plus, ces travaux ont montré que les ATDC générées à partir de monocytes de patients atteints d’insuffisance rénale présentent les mêmes propriétés tolérogènes in vitro que celles issues de volontaires sains. Enfin, les ATDC humaines, à l’instar de leurs analogues murines, sont capables de réaliser une présentation croisée d’antigène. Le second objectif a été d’étudier le rôle du récepteur à la fractalkine, le CX3CR1, fortement exprimé par nos cellules. Mes résultats indiquent que l’absence de CX3CR1 ne modifie pas la génération des ATDC murines, ni leur survie, ou leur capacité de régulation des LT in vitro. Le troisième objectif de cette thèse a été d’élargir l’utilisation des ATDC murines à d’autres pathologies inflammatoires. Bien qu’elles ne semblent pas inhiber le développement de la colite, les ATDC protègent les souris de l’induction de l’asthme allergique. Ces résultats ont permis à l’équipe de réaliser le premier essai clinique de thérapie cellulaire à l’aide de TolDC en transplantation rénale. Cet essai de phase I/II permettra d’étudier l'innocuité et l'efficacité des ATDC puis potentiellement d’étendre l'utilisation clinique des ATDC, notamment chez des patients asthmatiques. / Various studies, including those done by our team, have demonstrated the efficacy of cell therapy using tolerogenic dendritic cells in the control of rodent transplant rejection. The originality of our work comes from the use of autologous cells, ATDCs (Autologous Tolerogenic Dendritic Cells). In a purpose of their clinical application in renal transplantation, the ATDCs were generated in healthy volunteers. The first objective of my thesis was to validate the clinical grade manufacturing process of human ATDCs. Furthermore, this work has shown that ATDCs generated from monocytes in patients with renal insufficiency have the same in vitro tolerogenic properties as those from healthy volunteers. Finally, as in mice, human ATDCs are able to cross-present antigen. The second objective was to study the role of the fractalkine receptor, CX3CR1, strongly expressed by our cells. My results indicate that the absence of CX3CR1 does not alter the generation of murine ATDCs, nor their survival, nor their ability to regulate LTs in vitro. The third objective of this thesis was to broaden the use of murine ATDCs to other inflammatory pathologies. Although they do not seem to inhibit the development of colitis, ATDCs protect mice from the induction of allergic asthma. These results allowed the team to perform the first clinical trial of cell therapy using TolDC in renal transplantation. This phase I/II trial will investigate the safety and efficacy of ATDCs and potentially extend the clinical use of ATDCs, particularly in asthmatic patients.

No description available.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer death in Western countries. The PDAC tumour microenvironment (TME) is characterized by a dense stromal reaction, consisting of many cell types including fibroblasts and immune cells. The chemokine receptor, CX3CR1 forms a high-affinity axis with its unique ligand CX3CL1 and is expressed on monocytes, macrophages and T cells. CX3CR1 is also present on pancreatic malignant cells, where it has been associated with metastasis formation. The aim of my project is to investigate the role of CX3CR1 in the progression and development of pancreatic cancer in a genetically engineered mouse model of PDAC, the CX3CR1GFP/GFPLSL-KRASG12D/+LSL-Trp53R172H/+Pdx1-Cre (CKPC) mouse. In these mice, the CX3CR1 protein is not functional but they express GFP. I have found that the absence of CX3CR1 in KPC mice has no effect in their lifespan and response to chemotherapy. Comparison of the immune infiltrate of the tumours revealed that the lack of CX3CR1 causes a significant decrease in T cells and a possible increase in myeloid cells in CKPC mice compared to KPC mice. Expression analysis of several inflammatory cytokines in the TME showed a significant difference in IL-10 between KPC and CKPC mice. There was also a significant increase in levels of, CX3CL1, both locally and in the plasma. Finally, we performed RNA-seq on KPC and CKPC tumours. My analysis revealed 607 differentially-expressed genes, some of which encoded other chemokines or protein regulating the immune system. In particular, I observed the upregulation of Cxcl10 and Cxcl12, and the downregulation of Gata3 and S100a4, which could explain the decrease in T cells in the TME of CKPC mice. In conclusion, although the lack of CX3CR1 modifies the TME in this genetic model of PDAC, these changes do not affect the lifespan or the response to chemotherapy.

Les chimiokines (CKs) jouent un rôle important dans l’orchestration de la réponse immunitaireen contrôlant notamment la mobilisation des cellules immunitaires. Les cellules myéloïdes et, enparticulier, les monocytes sont impliquées dans le processus inflammatoire et notamment dansle développement des cancers. En effet, les cellules dérivées des monocytes ou macrophagesassociés aux tumeurs sont fortement représentés dans le micro environnement tumoral et sontsouvent associés à un mauvais pronostic. La caractérisation des mécanismes aboutissant aurecrutement des monocytes dans la tumeur représentent donc un enjeu majeur pourl’optimisation des protocoles thérapeutiques anti-cancéreux. Chez la souris, deux populationsmonocytaires sont caractérisées sur la base de l’expression différentielle des récepteurs auxchimiokines CCR2 et CX3CR1 et interviennent dans leur recrutement et leur différenciation enmacrophages dans les tissus, les monocytes inflammatoires (CCR2+, CX3CR1low) et les monocytesdits résidents (CCR2-, CX3CR1high). L’objectif principal de mon projet de recherche a été de mieuxcomprendre les mécanismes contrôlant la mobilisation des monocytes suite à un traitementchimiothérapeutique. J’ai entrepris d’étudier le rôle des récepteurs aux chimiokines, enparticulier CX3CR1, dans la reconstitution monocytaire après traitement chimiothérapeutiquepar le cyclophosphamide (CP), un agent alkylant reconnu pour son activité myélosuppressive. LeCP provoque un renouvellement des monocytes et une forte infiltration de la tumeur par les LTspécifiques de la tumeur (issus d’un transfert adoptif) associés à la réactivation de la réponseimmune anti-tumorale. Cependant, les LTs spécifiques de l’antigène de la tumeur se localisentpréférentiellement dans des zones riches en cellules dendritiques associées à la tumeur (TuDCs)et sont piégés par ces dernières. Ces interactions diminuent potentiellement le nombre decontacts entre les LT et les cellules tumorales suggérant un rôle pro tumoral des TuDCs. Letraitement au CP provoque une déplétion des cellules myéloïdes suivie d’une reconstitutionmassive des réservoirs de monocytes (moelle osseuse et rate). Au cours de la reconstitutionmonocytaire, l’expression de CX3CR1 diminue et est corrélée à une diminution de l’adhérence exvivo des cellules médullaires. Nous avons mis en évidence une mobilisation accrue desmonocytes inflammatoires au sein des souris CX3CR1- /- comparée aux souris WT et CCR2-/-.L’imagerie in vivo de la moelle osseuse au sein de souris CX3CR1-/- ou à l’aide d’un antagonistede CX3CR1 nous a permis de montrer un rôle spécifique de CX3CR1 dans le « crawling » sur lescellules endothéliales et le confinement des cellules monocytaires au niveau des sinus et duparenchyme médullaire. Nous suggérons qu’au cours de la mobilisation cellulaire induite par leCP le récepteur CX3CR1 contrôle la rétention médullaire des monocytes. Nous pensons que lamodulation du taux de mobilisation cellulaire au cours de la reconstitution induite par CP et/oule ciblage de CX3CR1 pourrait, par augmentation du pool de cellules myéloïdes leucocytairesd’un hôte, contribuer à l’amélioration des réponses cellulaires à la suite d’une lésion tissulaireou d’un dysfonctionnement des défenses immunitaires. De plus, le ciblage de CX3CR1 pourraittrouver des applications dans le domaine de la greffe de HSCs. / Chemokines orchestrate immune response especially by leucocytes mobilization. Myeloidcells, notably monocytes, are involved in inflammation and cancer development. Indeedmonocyte-derived cells and macrophages are strongly represented in tumourmicroenvironment and are associated with a bad prognosis. Characterization of mechanismsleading to monocytes recruitment within the tumor is thus a major issue in anti-cancertherapeutic protocols optimization. Based on the differential expression of chemokinereceptors CCR2 and CX3CR1, two populations of monocytes, inflammatory monocytes(CCR2+, CX3CR1low) and resident monocytes (CCR2-, CX3CR1high) have been characterized inmice which are involved in monocytes recruitment and differentiation into macrophages.The main objective of my work was to better understand the mechanisms of monocytesmobilization following chemotherapeutic treatment. I started to study the role of chemokinereceptors with a focus on CX3CR1 in monocytes reconstitution following cyclophosphamide(CP) treatment. CP is an alkylant agent known for its myelosuppressive properties. Thischemotherapeutic agent induces a transitory anti tumour immune response associated witha monocyte renewal and a strong infiltration of the tumour by adoptively transferred Tlymphocytes. However, antigen-specific T cells are trapped by tumour-associated dendriticcells (TuDCs). This potentially decreases interactions between T lymphocytes and tumourcells suggesting an immunosuppressive role of TuDCs. CP induces a strong depletion ofmyeloid cells followed by a massive reconstitution of bone marrow and spleen monocytesreservoirs. CX3CR1 expression on bone marrow monocytes is decreased duringreconstitution and correlated with a decreasing adhesion of these cells to CX3CL1 ex vivo.We highlighted an increased mobilization of inflammatory monocytes in CX3CR1-/- micecompared to WT and CCR2-/-. Intra vital imaging of bone marrow within CX3CR1-/- mice orwith the help of a CX3CR1 antagonist allowed us to show a specific role of CX3CR1 in thelumen crawling and confinement of monocyte-derived cells in both sinusoid andparenchyma of the bone marrow. We suggest that CX3CR1 controls the release of bonemarrow monocytes during CP-induced mobilization. We think that modulating the rate ofcellular mobilization, by increasing the host’s leukocyte pool during CP inducedreconstitution and/or targeting CX3CR1, could contribute to improve cellular responsefollowing tissue damage or immune cell dysfunction. Furthermore, targeting CX3CR1couldprovide applications in the stem cell transplantation domain.

Chemokines and their receptors are involved in many aspects of immunity. Chemokine CX3CL1, acting via its receptor CX3CR1, regulates monocyte migration and macrophage differentiation as well as T cell-dependent inflammation. Two common, nonsynonymous polymorphisms in CX3CR1 have previously been shown to alter the function of the CX3CL1/CX3CR1 pathway and were suggested to modify the risk for asthma. Using matrix-assisted laser desorption/ionization time-of-flight technology, we genotyped polymorphisms Val249Ile and Thr280Met in a cross-sectional population of German children from Munich (n = 1,159) and Dresden (n = 1,940). For 249Ile an odds ratio of 0.77 (95% confidence interval 0.63–0.96; p = 0.017) and for 280Met an odds ratio of 0.71 (95% confidence interval 0.56–0.89; p = 0.004) were found with atopy in Dresden but not in Munich. Neither polymorphism was associated with asthma. Thus, amino acid changes in CX3CR1 may influence the development of atopy but not asthma in German children. Potentially, other factors such as environmental effects may modify the role of CX3CR1 polymorphisms. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La dégénérescence des photorécepteurs dans la pathologie de la dégénérescence maculaire liée à l'âge (DMLA) est associée à une infiltration et accumulation des phagocytes mononuclées (PM). Nous avons montré précédemment que les souris déficientes pour Cx3cr1 développent une accumulation des PM sous-rétiniens avec l'âge et avec le stress, qui est associée une dégénérescence des photorécepteurs. Dans le cerveau, la déficience de Cx3cr1 dans les PM induit une augmentation de la mort des neurones via IL-1β. La raison de l'augmentation de la sécrétion d'IL-1β par les PM déficients en Cx3cr1 reste inconnue. Nous montrons que les PM déficients en Cx3cr1 surexpriment le récepteur P2RX7 qui stimule la maturation et la sécrétion d'IL-1β. L'inhibition de P2RX7 et d'Il-1β diminuent la mort des photorécepteurs dans un modèle de cocultures de monocytes/rétine et avec le modèle d'illumination in vivo. Nos résultats suggèrent que l'inhibition de P2RX7 ou d'Il-1β peut diminuer l'inflammation sous-rétinienne qui est associée à la mort des photorécepteurs dans la pathologie de la DMLA, où il n'existe aucun traitement à l'heure actuelle pour la forme atrophique. / Photoreceptor degeneration in age-related macular degeneration (AMD) is associated with an infiltration and chronic accumulation of mononuclear phagocytes (MPs). We have previously shown that Cx3cr1 -deficient mice develop age- and stress- related subretinal accumulation of MPs, which is associated with photoreceptor degeneration. Cx3cr1 -deficient MPs have been shown to increase neuronal apoptosis through IL-1β in neuroinflammation of the brain. The reason for increased IL-1 β secretion from Cx3cr1 -deficient MPs, and whether IL-1β is responsible for increased photoreceptor apoptosis in Cx3cr1 -deficient mice, has not been elucidated. Here we show that Cx3cr1 -deficient MPs express increased surface P2X7 receptor (P2RX7), which stimulates IL-1β maturation and secretion. P2RX7 and IL-1_β inhibition efficiently blunted Cx3cr1 -MP-dependent photoreceptor apoptosis in a monocyte/retina coculture system and in light induced subretinal inflammation of Cx3cr1 -deficient mice in vivo. Our results provide an explanation for increased CX3CR1-dependent IL-1β secretion and suggest that IL-1β or P2RX7 inhibition can help inhibit the inflammation-associated photoreceptor cell loss in late AMD, including geographic atrophy, for which no efficient treatment currently exists.

<p>The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.</p><p>There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.</p><p>The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.</p> / Dissertation

In the central nervous system, the axons of neurons are protected from damage and aided in electrical conductivity by the myelin sheath, a complex of proteins and lipids formed by oligodendrocytes. Loss or damage to the myelin sheath may result in impairment of electrical axonal conduction and eventually to neuronal death. Such demyelination is responsible, at least in part, for the disabling neurodegeneration observed in pathologies such as Multiple Sclerosis (MS) and Spinal Cord Injury. In the regenerative process of remyelination, oligodendrocyte precursor cells (OPCs), the resident glial stem cell population of the adult CNS, migrate toward the injury site, proliferate and differentiate into adult oligodendrocytes which subsequently reform the myelin sheath. Existing research indicates that OPC migration is directed by chemomigratory signals released from the site of injury and that the absence of OPCs is a feature of some MS lesions, suggesting that increased recruitment of OPCs to injury sites might improve remyelination, eventually leading to treatments of patient pathologies. I hypothesized that as yet undiscovered migration cues for OPCs might be released at sites of demyelination, diffuse through the CNS tissue, activate distal OPCs and guide them back to sites of demyelination. In this thesis, I performed bioinformatics analysis of gene expression arrays and identified upregulated cell surface receptors on OPCs activated in a cuprizone model, and upregulated secreted factors in whole lesion sites from an LPC induced MS type injury model and a Spinal Cord Injury model. I then optimised the X-celligence system for the quantification of OPC migration in response to secreted factors identified in my bioinformatics screen. By combination of these techniques with immunofluorescent staining I discovered novel expression of the cell surface receptor CX3CR1 on OPCs, increased expression of the corresponding ligand CX3CL1 in both MS type injury and Spinal Cord Injury, increased directional migration of OPCs in response to low concentrations of CX3CL1, and increased maturation of OPCs into adult oligodendrocytes at high concentrations of CX3CL1. Taken together these results propose a system in which an increasing gradient of CX3CL1 released from the site of injury directs the recruitment, then maturation of OPCs, making CX3CL1 a master regulator of OPC led CNS regeneration.

Le sepsis est la réaction inflammatoire généralisée secondaire à une infection. C’est une pathologie fréquente et grave qui fait intervenir le système immunitaire. L’action de l’immunité innée se fait par l’activation et le recrutement des monocytes, des cellules mononuclées circulantes qui modulent le phénomène inflammatoire. La mobilisation des monocytes fait intervenir les cytokines chimiotactiques (chimiokines) et leurs récepteurs. Nous nous sommes spécifiquement intéressé dans ce travail au rôle de l’expression monocytaire des récepteurs aux chimiokines CCR2 et CX3CR1 au cours du sepsis. Pour ce faire, nous avons utilisé des modèles murins de sepsis et analysé le rôle d’un polymorphisme génétique de CX3CR1 dans une cohorte de malades atteints de sepsis. Nous avons montré qu’au cours du sepsis, les monocytes présentaient une augmentation de l’adhérence aux parois vasculaire contrôlée par le récepteur CX3CR1. Nous avons également montré que les monocytes inflammatoires jouaient un rôle essentiel dans la régulation du phénomène inflammatoire au cours du sepsis en protégeant le rein des lésions septiques. Cette protection est médiée par l’expression de CX3CR1. L’allèle I249 du gène Cx3cr1, à l’origine d’une augmentation des propriétés adhésives du couple CX3CR1/CX3CL1, est un facteur protecteur dans la survenue d’insuffisance rénale aiguë chez le malade atteint de sepsis. Collectivement, ces travaux confirment un rôle régulateur des monocytes inflammatoires au cours du sepsis et identifient de potentielles nouvelles cibles thérapeutiques. / Sepsis is the generalized inflammatory response secondary to an infection. This is a common and serious condition that involves the immune system. The action of innate immunity in sepsis is mediated by the activation and recruitment of monocytes, which are mononuclear circulating cells, which modulate the inflammatory process. The mobilization of monocytes involves chemotactic cytokines (chemokines) and their receptors. This work was specifically focused on the role of monocyte expression of chemokine receptors CCR2 and CX3CR1 in sepsis. To this end, we used mouse models of sepsis and analyzed the role of a common genetic polymorphism of CX3CR1 in a cohort of patients with sepsis.We have shown that in sepsis, monocytes’ motility was modified with an increase of their adhesion to vascular walls that was controlled in part by CX3CR1. We have also shown that inflammatory monocytes play a key role in the regulation of the inflammatory phenomenon in sepsis and that they protected the kidney from septic lesions via a CX3CR1 mediated adhesion mechanism. The I249 allele of CX3CR1, confering increased adhesive properties to monocytes, is a protective factor regarding the occurrence of acute kidney injury in septic patients. Collectively, these data confirm a a regulatory role for inflammatory monocytes during sepsis and identify potential new therapeutic targets.

No description available.