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Malaria affects 200 million people annually, resulting in 584,000 - 1,238,000 deaths. The majority of these deaths occur in children, less than 5 years of age, in sub-Saharan Africa and are due to cerebral malaria (CM), a neuropathology induced primarily by the species Plasmodium (P.) falciparum. The pathogenesis of CM remains poorly understood and the mechanisms involved in acquired protection against the syndrome in malaria-endemic regions are undefined. Utilising the well characterised P. berghei ANKA experimental infection model of cerebral malaria (ECM), results presented in this thesis show that the development of ECM is associated with the accumulation and arrest of pathogenic CD8+ T cells within the perivascular spaces of the brain. Accumulation of activated CD8+ T cells, without arrest, was observed in the perivascular spaces of the brains of mice infected with the non-ECM causing P. berghei NK65 strain. These data show that the behaviour of intracerebral CD8+ T cells specifies their pathogenic function during malaria infection. The development of ECM was associated with extensive disruption to the BBB, which developed in the absence of extensive CD8+ T cell-dependent endothelial cell apoptosis. We modified the ECM model, establishing an infection-drug cure strategy, to investigate the immunological basis of parasite exposure-induced resistance to ECM development. Three rounds of infection-drug cure promoted resistance to ECM, which was associated with reduced intracerebral expression of genes involved in defence response, regulation of apoptosis, chemotaxis, CTL activity, antigen processing and presentation and cell adhesion, compared with ECM susceptible mice. Additionally, CD8+ T cell activation was suppressed in exposure-induced resistant mice and was associated with the antibody dependent expansion of a splenic plasmacytoid DC population, with a regulatory phenotype. The infection-induced protection against ECM was critically dependent upon secreted antibody production. A long standing problem in studying the immune response to malaria infection has been the inability to track parasite-specific CD4+ T cell responses. To address this, we generated and validated new transgenic P. berghei parasites expressing the model antigen, ovalbumin (OVA), either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). We found that cellular location and expression level of the antigen influence the induction and magnitude of parasite-specific T-cell responses. These parasites thus provide knowledge on the factors that influence the recognition of parasite antigens by the immune system and represent useful tools to study the development and function of antigen-specific T-cell responses during malaria infection. The results in this thesis improve our understanding of the events that lead to the development of CM, and the host immune responses that develop following parasite exposure to protect against it. The results should contribute towards the rational development of adjunctive therapies and effective vaccines for human CM.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20242号 / 医博第4201号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 岩井 一宏, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies. The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice. In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Résumé des travaux de thèse Contrairement au dogme original décrit par van Furth sur le système des phagocytes mononucléés (MP), il est maintenant suggéré que les monocytes ne représentent plus uniquement des précurseurs de macrophages mais qu'ils exercent des fonctions immunologiques en tant que tel. Non seulement les monocytes sont capables de circuler dans les tissus sans se différencier en macrophages mais la plupart des macrophages tissulaires ont en réalité une origine embryonnaire. Je me suis intéressée à l'activité migratoire et aux fonctions effectrices des monocytes et des macrophages grâce à l'utilisation de l'imagerie intravitale. Cette approche nous a permis d'appréhender la dynamique des MP entre le réseau vasculaire et le parenchyme tissulaire. De cette façon nous avons pu mettre en évidence le rôle de CX3CR1 dans le contrôle du déploiement monocytaire dans un modèle d'inflammation aiguë. En parallèle, dans un modèle de métastases pulmonaires, nous avons démontré que les monocytes ne contribuent qu'à une partie des macrophages associés aux tumeurs (TAM) et que l'autre partie dérive de la prolifération des macrophages interstitiels du poumon. Ces différentes sous-populations de TAM semblent avoir des contributions différentes dans le développement tumoral et la réponse à la chimiothérapie. Mon travail a permis de mieux appréhender la biologie des monocytes au-delà de leur fonction comme simples précurseurs de macrophages et apportent des concepts nouveaux sur l'origine des TAM. Ces travaux ouvrent de nouvelles perspectives dans la compréhension des mécanismes biologiques gouvernés par les MP et dans la régulation la réponse inflammatoire. / Contrariwise to the original dogma established by van Furth on the Mononuclear Phagocyte System (MPs), it is now suggested that monocyte fate and functions should not be restricted to macrophage precursors. Experimental evidences show that monocytes can circulate in tissues without differentiating and most of the tissue associated macrophages have embryonic origin. My thesis work focused on migratory activity and effective functions of monocytes and macrophages based on intravital imaging technology. This approach represent a unique tool to study the dynamic of MPs between the vasculature and tissue parenchyma. Our work provided new insights in the role of CX3CR1 in the monocyte deployment during acute inflammatory response.In a model of pulmonary metastases, we also demonstrated that monocytes represent only one part of tumor associated macrophages (TAMs) and the other part is constituted by lung interstitial macrophages. These different subsets of TAMs seem to contribute differently in tumor development and response to chemotherapy.Overall my work provides new insights about monocyte biology beyond their macrophage precursor fate and presents new concepts on the origin of TAMs. It also adds new perspectives in the understanding of biological mechanism regulated by MPs and in the control of the inflammatory response.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22376号 / 医博第4617号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 林 康紀, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUCTION: Injury to the facial nerve can lead to functional and aesthetic sequelae in patients. Though surgical interventions are available to restore lost motor and sensory function, outcomes are often suboptimal due to inadequate or disorganized axonal regeneration. While engineering improvements to the standard of care are underway, gaps remain in our molecular understanding of peripheral nerve injury to translate these efforts clinically. Over the last few decades, advancements in intravital imaging such as the development of fluorescent reporter mice and use of multiphoton excitation techniques have allowed for markedly enhanced characterization of biological phenomena at higher resolutions, at greater depths, and for longer timescales. Challenges in reliably and serially imaging in vivo within murine models have been overcome through the development of chronic imaging windows in various settings of the body. However, there are very few techniques available presently for imaging the peripheral nerve microenvironment and no prior work detailing use in the facial nerve setting. OBJECTIVE: Longitudinal studies employing intravital imaging techniques carry potential to improve understanding of peripheral nerve regeneration and function. Using multiphoton microscopy and fluorescent reporter mice, we propose a prototype, surgical protocol of implantation, and initial safety and efficacy testing of a facial nerve window to enable chronic imaging for enhanced characterization of the peripheral nerve microenvironment. METHODS: A stainless-steel implant with an affixed glass coverslip and aluminum external fixation component was developed for implantation in a transgenic reporter mouse model to enable chronic intravital imaging of the facial nerve buccal and marginal mandibular branches. A qualitative observational study and clinical assessment scoring study was performed post-surgical implantation to monitor behavior, physical appearance, weight loss, and reactivity to animal handling over the typical time-course of nerve regeneration. Segments of facial nerve branches were harvested from control and window-implanted mice and imaged using widefield epifluorescence microscopy for axon quantification to determine any adverse effects from window compression onto axonal fibers. Two-photon microscopy (2PM) and Simulated Raman Scattering (SRS) were also performed through the window to visualize axon tracts, myelin sheaths, and surrounding collagen matrix in wild-type and transgenic mice models. RESULTS: Qualitative serial observational studies and assessment scoring indicated no obvious functional deficits over the time-course of typical nerve regeneration and normal scores for weight, behavior, physical appearance, and reactivity. Neural histomorphometric analysis indicated no significant difference in mean myelinated axon count of buccal (mean ± SD; control buccal, 947.6 ± 129.9; window-implanted buccal, 799.3 ± 128.6; p = .136) and marginal mandibular branches (control marginal mandibular, 801.3 ± 145.1; window-implanted marginal mandibular, 738.0 ± 197.2; p = .599) between control and window-implanted mice, suggesting that neuropathy was not induced from the window itself. High-resolution images of nerve morphology in healthy and injured transgenic and wild-type mice were obtained using 2PM and SRS. CONCLUSION: Herein, we describe a novel and replicable platform for longitudinal intravital imaging of murine facial nerve. Future studies will evaluate viability of this model for imaging the facial nerve microenvironment, particularly Schwann cell-axon interactions, in the setting of severe nerve injury over a period of several weeks to months. Improved understanding gained through such studies of the structural peripheral nerve microenvironment may allow for advancements in viral vector therapeutics, nerve graft scaffold design, as well as advanced injury diagnostics and tracking. / 2022-06-02T00:00:00Z

The juxtaglomerular niche occupied by renin cells (RCN) plays an important role in glomerular repair but the precise temporal and spatial interrelations remain unclear. This study proposes the hypothesis of a local intra-extraglomerular regenerative feedback system and establishes a new quantifiable system for RCN responses in individual glomeruli in vivo. A strictly intraglomerular twophoton laser-induced injury model was established. Labeled renin cells (RC) in transgenic renin reporter mice were fate-traced in healthy and injured glomeruli over several days by intravital microscopy and quantified via new three-dimensional image processing algorithms based on ray tracing. RC in healthy glomeruli demonstrated dynamic extraglomerular protrusions. Upon intraglomerular injury the corresponding RCN first increased in volume and then increased in area of dynamic migration up to threefold compared to their RCN. RC started migration reaching the site of injury within 3 hours and acquired a mesangial cell phenotype without losing physical RCN-contact. During intraglomerular repair only the corresponding RCN responded via stimulated neogenesis, a process of de novo differentiation of RC to replenish the RCN. Repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular RCN repair feedback system in individual glomeruli in vivo in a quantifiable manner.