Die Multiple Sklerose (MS) ist eine entzündliche Autoimmunerkrankung des Zentralnervensystems. Die klinisch und pathologisch heterogene Erkrankung wird im Tiermodell am besten durch eine experimentelle Autoimmunenzephalomyelitis (EAE) abgebildet. Entzündliche Läsionen einer EAE sind neben Lymphozyten durch Monozyten/ Makrophagen gekennzeichnet. Der in dieser Arbeit untersuchte Chemokinrezeptor CX3CR1 findet sich auf murinen Monozyten des Blutes und wird dort unterschiedlich hoch exprimiert. Im ZNS werden der Rezeptor auf Mikroglia und der Ligand Fraktalkin (CX3CL1) konstitutiv und somit entzündungsunabhängig auf Neuronen exprimiert. In verschiedenen Tiermodellen neurologischer Erkrankungen wurden unterschiedliche Auswirkungen dieser Interaktion beschrieben. Die vorliegende Studie untersucht die Fragestellung, ob sich in einem spontanen EAE-Modell CX3CR1-defiziente OSE-Mäuse von CX3CR1-kompetenten OSEMäusen hinsichtlich spontaner EAE-Inzidenz, Erkrankungsverlauf und Läsionspathologie unterscheiden. CX3CR1-defiziente OSE-Mäuse zeigen in diesem Modell eine erhöhte Inzidenz (54% vs. 32%), aber einen milderen Krankheitsverlauf gegenüber Rezeptor-Wildtypen. Dem milderen Krankheitsverlauf entsprechend weisen OSE CX3CR1-defiziente Mäuse histopathologisch in der akuten Phase kleinere demyelinisierte Läsionen der spinalen weißen Substanz und geringere meningeale spinale entzündliche Infiltrate auf mit einer signifikant geringeren Makrophageninfiltration in den Läsionen. In der spinalen grauen Substanz zeigen sie eine mildere neuronale Schädigung. In der chronischen Krankheitsphase findet sich eine reduzierte Infiltration von Entzündungszellen ohne signifikanten Unterschied zwischen OSE CX3CR1-defizienten Mäusen und Rezeptorwildtypen. Molekularbiologisch zeigen OSE CX3CR1-defiziente Mäuse eine verstärkte Expression von IL-17a in der akuten Krankheitsphase. Die erhöhte EAE-Inzidenz in CX3CR1-defizienten OSE-Mäusen ist am ehesten auf ein vermehrte Generierung von enzephalitogenen 2D2 T-Zellen im Darm in diesem spontanen EAE-Modell zurückzuführen. Der mildere Krankheitsverlauf bei CX3CR1- defizienten Mäusen weist auf eine Bedeutung von CX3CR1 in der Migration von Monozyten in das entzündete ZNS hin. Die vorliegende Arbeit ist die Grundlage für weiterführende Studien, in welchen die Bedeutung der Fraktalkin-CX3CR1-Interaktion für kortikale Pathologie bei der EAE untersucht werden wird.

Contrairement aux autres chimiokine, CX3CL1 a la particularité d'exister sous deux formes protéiques fonctionnelles : une forme soluble chimio-attractante impliquée dans le recrutement leucocytaire comme toutes les chimiokines et une forme membranaire qui confère au couple CX3CL1/CX3CR1, une propriété surprenante de molécule d'adhésion participant à l'arrêt et à la transmigration des leucocytes circulants. Le CX3CR1 appartient à la famille des Récepteurs Couplés aux Protéines Gi, c'est-à-dire qu'il inhibe l'enzyme de synthèse de l'AMP cyclique (AMPc), l'adénylate cyclase. L'étude de la cinétique et du rôle du signal AMPc déclenché en aval du CX3CR1, activé par la forme soluble ou membranaire du CX3CL1 font l'objet de ce travail de thèse. La réponse à la forme membranaire est de même amplitude que celle induite par la forme soluble, mais présente une cinétique considérablement ralentie. Ce ralentissement corrèle avec une moindre internalisation du CX3CR1, qui semble être retenu en surface par le ligand membranaire. Un recrutement plus lent des ? arrestines sur le CX3CR1 activé par le CX3CL1 membranaire renforce cette hypothèse d'une internalisation plus tardive du CX3CR1 comparé à une activation par la forme soluble. Le rôle physiologique de l'AMPc a été également exploré sur les deux fonctions principales du couple CX3CL1/CX3CR1 : le chimiotactisme et l'adhésion. Le rôle inhibiteur de l'AMPc sur ces deux fonctions, confirme une action immunosuppressive de ce messager secondaire. Ces résultats indiqueraient que, grâce à leurs réponses AMPc inhibitrices, les cellules activées par les chimiokines seraient sélectionnées pour une réponse efficace en milieu inflammatoire. / CX3CL1 is a particular chemokine which, in contrast to other chemokines exists in two physiological forms: a soluble form is implicated in chemotaxis and cellular migration like all chemokines; whereas a membranous form confers to the CX3CL1/CX3CR1 couple an adhesive role, contributing in particular to the arrest of circulating leukocytes and their migration to the site of inflammation. The CX3CR1 belongs to the family of Receptor Coupled with Proteins Gi (RCPG), which inhibits the enzyme that syntheses cyclic AMP (cAMP), the adenylyl cyclase. The study of the kinetics and the role of the cAMP signal triggered downstream of CX3CR1, activated by soluble or membranous form of the CX3CL1 are the aims of this work of thesis. The response induced by the membranous form has the same amplitude as the one induced by the soluble form, but has a significantly slowed kinetic. This slowdown correlates with less internalization of CX3CR1, which seems to be retained on the surface by the membranous ligand. A slower recruitment of β-arrestin on the CX3CR1-activated by membranous CX3CL1 strengthens the hypothesis of a later internalization of CX3CR1 compared to activation by the soluble form. The physiological role of cAMP was also explored in the two principal functions of the couple CX3CL1 / CX3CR1: chemotaxis and adhesion. The inhibiting effect of cAMP in these two functions confirms an immunosuppressive action of this second messenger. These results indicate that through their inhibitory cAMP responses, cells activated by chemokines are selected for an effective response in the inflammatory conditions.

Adult neurogenesis, is a lifelong process by which relatively few cells are added into two restricted regions of the brain. Integration of the cells into the existing neuronal circulatory, with the unique properties involved in the maturation of these cells, is possibly critical to the acquisition and retrieval of new memories. With the chronological aging of the organism a process of cellular senescence occurs throughout the body; a portion of which is independent of primary alterations to the stem cells; instead, it appears to be dependent on the environment where the cells reside, and is in part regulated by inflammation. Microglia, the resident immune cells in the brain, are neuroprotective but chronic activation of the microglia, such as the chronic activation that occurs with advanced age, can promote neurotoxic inflammation. However, it not clear if the aged-related increase in neuroinflammation is at least partly responsible for the aged related decrease in neurogenesis. To address the involvement in neuroinflammation in regulating neurogenesis we used 3 different potential therapeutically relevant manipulations. The first was a targeted approach directed at disrupting the synthesis of Interleukin-1beta (IL-1B), which is a proinflammatory cytokine that is consistently found elevated in the aged brain. The second was a cell therapy approach in which human umbilical cord blood cells were injected into the systemic circulation. The final approach was directed at a chemokine system, fractalkine/CX3CR1, which has been shown as an important paracrine signal, from neurons that regulates the activation state of microglia. While the three approaches used to manipulate, aging-rodent model system were different, a consistent finding was reached in all three studies. In the aged brain, microglia which are the predominate produces of IL-1B, negatively regulate neurogenesis. When IL-1B is decreased or microglia activation is decreased, neurogenesis can be partially restored in the aged brain. The results of these studies, demonstrate a key role for microglia in regulating the neurogenic neiche, which are amendable to therapeutic manipulations.

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now the role of microglia in pruning remain under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the Purkinje cell layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in–depth histological analysis of the cerebellar cortex.:1 Introduction 6 1.1 Origin of microglia 6 1.2 Synaptic refinement 7 1.3 Fractalkine Receptor CX3CR1 9 1.4 Climbing fiber maturation and PCL development 10 1.5 Aim of the study 12 2 Materials and Methods 13 2.1 Materials 13 2.1.1 General material 13 2.1.1.1.1 Hardware 13 2.1.1.1.2 Consumable supplies 13 2.1.2 Chemicals 14 2.1.3 Solutions 14 2.1.4 Animals 14 2.1.5 Primary Antibodies 15 2.1.6 Secondary Antibodies 15 2.1.7 Software 15 2.2 Methods 15 2.2.1 Genotyping 15 2.2.2 Fixation and cryopreservation 16 2.2.3 Fluorescence Immunohistochemistry 16 2.2.4 Quantification of Microglia in the Cerebellum 16 2.2.5 Assessment of VGluT2 in the cerebellum 18 2.2.6 Statistical Analysis 19 3 Results 20 3.1 Postnatal enrichment of microglia cells in the cerebellar Purkinje cell layer 20 3.2 Microglial proximity to Climbing fibers 22 3.3 Population of the granular und molecular layer of CX3CR1 knock-out mice during early postnatal development 23 3.4 Influence of CX3CR1 knock-out on microglial morphology 25 3.5 Influence of CX3CR1 deletion on the VGluT2 expression during postnatal development 29 4 Discussion 31 4.1 Role of microglia in the developing cerebellum 31 4.2 CX3Cr1 Signaling and influence on microglial motility and morphology 32 4.3 CX3CR1 signaling and synaptic pruning 33 4.4 CX3CR1 signaling and formation of functional synapses 34 4.5 Correlation of immunohistological data with electrophysiological findings 35 5 Summary and conclusion 36 6 Zusammenfassung der Arbeit 38 7 References 41 8 Erklärung über die eigenständige Abfassung der Arbeit 49 9 Publications 50

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the PC layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in-depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX3CR1 signaling is not mandatory for correct cerebellar circuit formation. Main Points - Ablation of CX3CR1 results in a transient migration defect in cerebellar microglia. - CX3CR1 is not required for functional pruning of cerebellar climbing fibers. - Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX3CR1-deficient mice.

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Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. The crosstalk between the gut microbiota and the immune system plays an important role in the tolerance induction to self-antigens both in the intestinal mucosa and at the systemic level. The MRL/lpr mouse model exhibits lupus-like symptoms early in life due to multiple SLE susceptible loci of the MRL background, plus the Faslpr mutation that offers an accelerated model. Recently, we experienced a loss of disease phenotype in our in-house colony compared to the previous published phenotype of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX) with our in-house MRL/lpr mice and found that the phenotypic drift, most significantly the attenuation of glomerulonephritis, was present in both colonies. In addition, while JAX mice and mice in our colony are genetically identical, there were minor differences in disease that might be due to differences in splenic microRNAs and the gut microbiota. Once confirming that our MRL/lpr mouse model was as good as that from JAX, we continued our investigation of the role of Lactobacilli in the pathogenesis of lupus-like disease in MRL/lpr mice. We previously published that the mixture of Lactobacillus reuteri (L. reuteri), L. oris, L. johnsonii, L. gasseri, and L. rhamnosus significantly attenuated disease in MRL/lpr mice by restoring the imbalance between regulatory T cells and T helper-17 cells. To further understand the role of Lactobacillus spp., we treated MRL/lpr mice with the combined culture supernatant of the 5 strains containing secreted metabolites, given that the metabolites may induce an immunosuppressive response. The results showed significant attenuation of the inflammation of the spleen and renal lymph nodes similar to the effect of the bacteria themselves. There was also a trending decrease of double-stranded DNA autoantibodies with the combined supernatant. We thus tested the strains individually but none was able to recapitulate the effect of the bacterial mixture. This suggests cell-to-cell contact among different strains of lactobacilli may be required in ameliorating the disease. With these results, we now have a better understanding of the role of probiotic Lactobacillus spp. against SLE. Future investigations will focus on the potential therapeutic effect of Lactobacillus spp. as a combination. Additionally, our group generated a Cx3cr1-deficient MRL/lpr mouse which exhibits a distinct phenotype of exacerbated glomerulonephritis with concurrent change of the gut microbiota composition compared to Cx3cr1+/+ MRL/lpr littermates. Interestingly, upon correction of the gut microbiota with Lactobacillus administration, the phenotype of exacerbated glomerulonephritis was reversed, suggesting that CX3CR1 controls glomerulonephritis in MRL/lpr mice through a gut microbiota-dependent mechanism. In addition, a collaborative project revealed that Cx3cr1 deficiency-mediated pathogenic mechanisms also contributed to SLE-associated cardiovascular disease in MRL/lpr mice. The results of these studies will lead to the identification of new therapeutic targets for the treatment of two severe manifestations, glomerulonephritis and cardiovascular disease, that together account for most of the morbidity and mortality in SLE. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is an autoimmune disease with no known cure. Commensal microbiota, mostly bacteria living in our gut, and the immune system have a strong relationship in maintaining a healthy state of the gut as well as the whole body. Alterations in the gut microbiota, known as dysbiosis, can facilitate SLE in human and animal models. Current treatments for SLE are primarily focused on using immunosuppressants, but the side effects are still a concern. The use of long-term nonselective immunosuppressant conducts a higher incidence of severe infections in SLE patients. It is thus necessary to develop new approaches and treatments against SLE. My dissertation research is focused on understanding how commensal bacteria influence in the pathogenesis of SLE. My studies have shown that environmental factors can manipulate the gut microbiota leading to different disease outcomes. In addition, following upon previously published studies from our laboratory, I have delineated the mechanism how a mixture of probiotic Lactobacilli can exert a beneficial effect against lupus. Finally, I have revealed a new, CX3CR1-mediated mechanism through which the gut microbiota controls kidney disease in the MRL/lpr lupus-prone mouse model.

Neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, are a heterogeneous set of developmental disorders affecting the central nervous system. Studies into their etiology remain challenging, as neurodevelopmental disorders frequently present with a wide range of biological, behavioral, and comorbid symptomologies. Increasing epidemiological reports of antibiotic use during pregnancy as a significant correlate of subsequent mental disorder diagnosis in children suggest a mechanism of influence via the maternal gut-fetal brain axis. Importantly, antibiotics cause dysbiosis of the gut microbiome and disrupt the delicate composition of the microbial inoculum transferred from mother to child, which is critical for development of the immune system and holds implications for long-term health outcomes. The research objective of this dissertation is to reveal a causal mechanism of maternal microbial influence on neurodevelopment by examining the brain's resident immune cells, microglia, and corresponding behavioral outcomes in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We identify early gross motor deficits and social behavior impairments in offspring born to MMD dams, which paralleled hyperactivated microglia in brain regions specific to cognition and social reward. The MMD microglia also exhibited altered transcriptomic signatures reflective of premature cellular senescence that support evidence of impaired synaptic modeling found in MMD brains. We report that these deficits are rescued in the absence of Cx3cr1, a chemokine receptor expressed ubiquitously on microglia, to highlight a pathway in which maternal microbiota may signal to neonatal microglia to undergo appropriate neurodevelopmental actions. Finally, we characterize Lactobacillus murinus HU-1, a novel strain of an important gut bacterium found in native rodent microbiota, and demonstrate its use as a probiotic to restore microglial and behavioral dysfunction in MMD offspring. / Ph. D. / Population studies on neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, highlight antibiotic use during pregnancy as a major correlate of subsequent diagnoses in children. These findings support a growing body of evidence from animal and human studies that the microbial ecosystems (“microbiome”) found in and on our bodies play significant roles in mental health, including mood, cognition, and brain function. Importantly, antibiotics during pregnancy create an imbalance of the gut microbiome (“dysbiosis”) and disrupt the microbial inoculum transferred from mother to child, which is critical for maturation of the infant immune system and holds implications for long-term health outcomes. Thus, the research objective of this dissertation is to identify a mechanism of influence from the mother’s gut to the neonate’s brain by examining the brain’s resident immune cells (“microglia”) in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We uncover autism-like behavioral deficits and dysfunctional microglia in MMD offspring, and characterize signaling cues specific to microglia by which improper neurodevelopment may be taking place. We also reveal that the detrimental effects of MMD are reversed in mice born to mothers pretreated with a probiotic candidate, Lactobacillus murinus HU-1, to suggest maternally-derived Lactobacillus may help to mediate proper neurodevelopment.

Orientador : Alexandre Leite Rodrigues de Oliveira / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:27:25Z (GMT). No. of bitstreams: 1 Freria_CamilaMarques_D.pdf: 9799936 bytes, checksum: 9a9057c6ab5ca87ea1bc6767c4f71490 (MD5) Previous issue date: 2013 / Resumo: Lesões nervosas periféricas e centrais levam à inflamação local e retrógrada, resultando em alterações axonais, perdas neuronais e sinápticas significativas. Juntamente a tais alterações, as células gliais tornam-se reativas, influenciando na remodelação do SNC após lesão. Os mecanismos que desencadeiam tais mudanças não são completamente compreendidos, mas é evidente que as moléculas classicamente relacionadas com o sistema imune estão envolvidas em tais eventos diretamente ou através da modulação da reatividade glial. Assim, nossa hipótese é que o controle da sinalização inflamatória após a lesão central ou periférica possa afetar indiretamente nos mecanismos endógenos de reparação no SNC, resultando em maior preservação das conexões neurais e melhor recuperação funcional. Para isso, realizamos lesões periféricas e centrais expondo os animais a diferentes microambientes de lesão a fim de investigar o papel das células gliais na sobrevivência, capacidade regenerativa axonal e estabilidade sináptica de motoneurônios medulares. Os resultados mostraram que, após lesão, a modulação da sinalização inflamatória através da administração de citocinas ou deleção de moléculas expressas na superfície das células gliais podem influenciar direta ou indiretamente na estabilidade dos circuitos neuronais, na regeneração axonal e sobrevivência neuronal. Desse modo, conclui-se que o controle da inflamação e da reatividade glial são, provavelmente, críticos para a plasticidade no Sistema Nervoso viabilizando, assim, novas estratégicas de tratamentos / Abstract: Central or peripheral lesions result in local and retrograde inflammation, leading to axonal degeneration, synaptic and/or neuronal loss. Additionally, after injury, reactive glial cells are recruited to the lesion site, influencing the plasticity of the nervous system. The mechanisms which trigger such changes are not completely understood, but evidences have shown that molecules classically related to the immune system are involved in such events directly or indirectly by glial modulation. Based on this, our hypotheses is that the control of inflammatory signaling after central or peripheral injury may indirectly affect the endogenous repair mechanisms, resulting in a greater synaptic preservation and better functional recovery. In this sense, animals were submitted to both central and peripheral lesions in order to investigate the effects of glial cells on neuron survival, axonal regeneration and synaptic plasticity. The results showed that, after lesion, the modulation of inflammatory signaling by cytokines or knocking down molecules on glial surface, directly or indirectly influence the stability of neural circuits, neuronal survival and axonal regeneration. Thus, we believe that this is important findings that may be critical to the development of new therapeutic strategies following nervous system injury / Doutorado / Clinica Medica / Doutora em Ciências