京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24046号 / 生博第472号 / 新制||生||63(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 上村 匡, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>

Allergic diseases are influenced by genetics and the environment. Maternal allergy appears to confer a higher risk for allergic sensitization than paternal allergy, suggesting an in utero influence. A decrease in particular infections or a lower exposure to microbial components during infancy is suggested to contribute to the high allergy prevalence in affluent societies. Toll-like receptors (TLR) 2 and 4 recognize peptidoglycan (PGN) and LPS respectively, are expressed on e.g. monocytes, and have been implicated in modulating the risk of IgE-sensitization. This thesis aimed to study the influence of maternal allergy and early microbial exposure on monocyte function and allergic sensitization during childhood. Blood samples from children participating in a prospective allergy cohort were used. Two-year old infants with allergic mothers had lower IL-6 production and reduced activation of the TLR-signalling intermediate p38-MAPK in response to PGN than children with non-allergic mothers. In 5-year old children, allergic disease and not maternal allergy influenced monocytic TLR2-regulation. Five-year olds who were seropositive for Epstein-Barr virus (EBV) at 2-years of age had a lower risk of persistent IgE-sensitization while EBV contraction after 2-years of age related to a higher risk of IgE-sensitization. Upon in vitro stimulation, NK cells from EBV+ 2-year olds produced lower IFN-g levels. EBV+ 2-year olds had also lower systemic IFN-g. In comparison to CD14++CD16- monocytes, CD14+CD16+ cells induced NK-cell IFN-g more potently in vitro, and EBV+ infants tended to have lower proportions of these CD14+CD16+ monocytes. This thesis highlights the importance of early-life microbial (EBV) exposure for a proper allergy-protective immunity. Also, maternal allergic heredity appears to influence monocytic microbial responses in early infancy. All these aspects relate to altered monocyte functionality, which suggest that they could have a role in allergic sensitization.

The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function. In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin. Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.

Introduction: Bone is a highly active tissue throughout life and is a subject to constant remodelling. Main cells responsible for continuous resorption and de novo synthesis of bone matrix are osteoclast, osteoblasts and osteocytes. Osteoclasts are the only known type of cells able to resorb bone. These cells are formed by fusion of precursor cells in bone marrow or peripheral blood in a process called osteoclastogenesis. Formation of osteoclasts may be of importance concerning chronic inflammatory diseases that are linked with higher risk of developing osteoporosis during lifespan. Celiac disease is one of those diseases, which is characterized by destruction of intestinal mucosa after ingestion of gluten by susceptible individuals followed by induction of chronic inflammation. In this work, we focused on the potential role of osteoclastogenesis in the development of osteoporosis in patients with celiac disease and we studied roles of selected inflammatory agents (TNF-α, IL-6, IFN-γ a cfDNA) with supposed or hypothesised effects on osteoclastogenesis. Material & Methods: We obtained plasma and serum samples from newly diagnosed patients with celiac disease, patients on gluten free diet and healthy controls and analysed concentrations of cfDNA and inflammatory cytokines TNF-α, IL-6 and IFN-γ in...

Magister Scientiae - MSc / "The aim of the study was to use in vitro human whole blood cultures to screen the water samples collected from the Eerste/Plankenbrug river system for cytotoxicity and inflammatory activity and for the first time investigate the impact on the cell- mediated and humoral immune pathways. Water samples were collected fronm the sites during the dry summer season and rainy winter season. Blood was collected from the healthy male volunteers and diluted with RPMI 1640. For cytotoxicity and inflammatory activity 2.5ul of blood for 18-20 hrs at 37 C... This study shows that waster from the Plankenbrug River is heavily polluted by contaminants from both the agricultural area and informal settlement of Kayamandi. These contaminants can be potentially immunotoxic during the summer season and they can result in inflammatory diarrheal disease and immunosuppression in exposed individuals..."

Celiaki (glutenintolerans) är en kronisk, autoimmun sjukdom med diffusa symtom. Vid förtäring av glutenhaltig mat uppstår en allergisk reaktion hos glutenintoleranta individer. Gluten kan inte fullständigt brytas ned av kroppens enzymer, vilket betyder att icke nedbrutna peptidfragment (såsom glutamin) absorberas i tarmslemhinnan. Enzymet transglutaminas katalyserar omvandlingen av glutamin till glutamat. Glutenkänsliga T-celler aktiveras av glutamat att utsöndra proinflammatoriska cytokiner såsom interferon-gamma (IFN-γ) och tumörnekrosfaktor-alfa (TNF-⍺). Syftet med studien var att undersöka om gluten- och gliadinstimulering av celler i helsaliv in vitro kan inducera produktion av IFN-γ och TNF-⍺. Pilotstudier med 2 försökspersoner utfördes, där celler i salivprover stimulerades med enzymatiskt nedbrutet gliadin (&lt;40 mg gliadin), samt PHA (5 µg/mL), PMA (50 ng/mL) och LPS (1 µg/mL) 20 timmar vid 37 ˚C. Cytokinproduktionen i salivproverna kvantifierades med ELISA och uppreglering av IFN-γ och TNF-⍺ undersöktes med RT-qPCR. Efter metodutveckling upprepades stimulering och ELISA med salivprov från 12 försökspersoner (6 individer med och utan celiaki). Immunreaktionen som uppstår hos glutenintoleranta individer in vivo kunde inte återskapas i saliv in vitro med den framtagna metoden. Hos övervägande delen av salivproverna var cytokinproduktionen under detektionsgränsen, 4 pg/mL för IFN-γ och 15,6 pg/mL för TNF-⍺. Det finns risk för att outforskade detaljer eller agens saknades från reaktionskedjan och därmed kunde den förväntade immunreaktionen inte återskapas. En annan felkälla kan vara för låg koncentration av immunceller i saliven. / Celiac disease is a chronic, autoimmune disease that has diffuse symptoms. Upon consuming gluten containing food, an allergic reaction occurs in gluten-sensitive individuals. Gluten cannot be fully digested by human enzymes, which leads to non-digested peptide fragments (such as glutamine) to be absorbed in the gastrointestinal wall. The transglutaminase enzyme catalyzes the conversion of glutamine to glutamate. Glutamate activates gluten-specific T-lymphocytes to produce proinflammatory cytokines e.g., interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-⍺). The aim of this study was to investigate whether stimulation of cells in whole saliva in vitro with gluten and gliadin can induce production of IFN-γ and TNF-⍺. Pilot studies were conducted, where cells in saliva from 2 subjects was stimulated with enzymatically digested gliadin (&lt;40 mg gliadin) together with PHA (5 µg/mL), PMA (50 ng/mL) and LPS (1 µg/mL) for 20 hours at 37 ˚C. The production of cytokines was quantified by ELISA, and the upregulation of IFN-γ and TNF-⍺ was analyzed by RT-qPCR. After method development, the stimulations and ELISA quantifications of the proinflammatory cytokines were repeated in saliva samples from 12 subjects (6 individuals with and without celiac disease). The immune reaction that occurs in people with celiac disease could not be recreated in saliva in vitro with the developed method. In most of the samples the production of cytokines was under the detection range, 4 pg/mL for IFN-γ and 15,6 pg/mL for TNF-⍺. There is risk of unstudied details or agents missing from the reaction chain, and therefore the expected immune reaction could not be recreated. Another source of error could be low concentration of immune cells in saliva.

Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, I hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, I developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. I characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.

In patients after cardiac surgery, there is an increase in the level of molecules with both pro-inflammatory and anti-inflammatory effects. This increase is influenced by the patient's clinical condition, but also by the nature of the operation itself, which uses conventional extracorporeal circulation. This technique leads to damage to blood elements by direct contact with air and parts of the extracorporeal circulation, as well as to ischemia-reperfusion injury. The specifics of cardiac surgery then affect possible postoperative complications such as multiorgan failure or septic shock. The diploma thesis is divided into a theoretical and a practical part. The theoretical part describes the principle and influence of cardiopulmonary bypass on the human body and the complications that are associated with its use. Furthermore, IFN-γ is described herein as a potential marker of septic conditions that could reflect the clinical postoperative condition of patients. The practical part of the diploma thesis monitored the percentage change of selected cell populations and the production of IFN-γ in the peripheral blood of patients before and after cardiac surgery. Furthermore, the response of individual isolated populations of healthy volunteers to selected stimulators was investigated. The percentage of...

La barrière hémo-encéphalique (BHE) est formée des cellules endothéliales microvasculaires cérébrales reliées entre elles par des jonctions serrées. Grâce à sa perméabilité restreinte et sélective, la BHE entrave le passage des molécules et cellules du sang vers le système nerveux central (SNC). Chez les patients atteints de sclérose en plaques (SEP), une maladie inflammatoire du SNC, la rupture de la BHE permet aux cellules immunes actives d'infiltrer le tissu cérébral. Il s'ensuit une réaction inflammatoire excessive au cours de laquelle d'autres leucocytes sont recrutés dans le cerveau et qui culmine par la formation des plaques de démyélinisation caractéristiques de la SEP. On dénote au niveau de ces lésions une présence importante de lymphocytes T CD4⁺ activés et de cytokines pro-inflammatoires propres à une réponse de type TH1, tels l’IFN-γ et l’IL-1. Curieusement cependant, l’inhibition de la voie TH1 n’empêche pas l’apparition de la maladie dans le modèle murin de la SEP et en aggrave même les symptômes. On attribue maintenant aux lymphocytes TH17, nommées en raison de leur capacité à produire de l’IL-17, un rôle clé dans le développement de la maladie. L’objectif de ce travail de thèse visait à caractériser les lymphocytes TH17 chez l’humain et définir leur contribution exacte dans la fragilisation de la BHE, une étape décisive dans la formation des lésions de SEP. Pour ce faire, nous avons mis au point une méthode expérimentale permettant l’expansion in vitro de populations de lymphocytes TH17 à partir de cellules mononuclées du sang de donneurs sains. Nos travaux démontrent que l’IL-23 induit la production d’IL-17, d’IL-22 et de granzyme B par les lymphocytes T CD4⁺CD45RO⁺ mémoires humains et qu’une proportion des cellules exprime de manière concomitante de l’IL-17 et de l’IFN-γ. La fréquence des lymphocytes T CD4⁺ IL17⁺, IL-22⁺ et des doubles positifs IL-17⁺IFN-γ⁺ est significativement plus élevée dans les lignées de lymphocytes TH17 provenant de patientes en poussée que dans celles de contrôles. Nos analyses démontrent que les cellules endothéliales de la BHE expriment de faibles niveaux des récepteurs de l’IL-17 et de l’IL-22 à l’état basal mais que leur présence est accrue dans le cerveau de patients atteints de SEP. L’activation du récepteur de l’IL-17 entraîne une augmentation de la perméabilité de la BHE et une perturbation de l’organisation des protéines de jonction occludine et ZO-1. Finalement, nous démontrons que la migration des lymphocytes TH17 à travers la BHE est régie en grande partie par la molécule d’adhérence ICAM-1 et que les lymphocytes qui co-expriment l’IL-17 et l’IFN-γ sont plus aptes à franchir la BHE que ceux qui produisent uniquement l’une ou l’autre de ces cytokines. Nous retrouvons d’ailleurs des cellules qui expriment simultanément les facteurs de transcription T-bet et RORC, associés respectivement aux lymphocytes TH1 et aux TH17, au sein des infiltrats péri-vasculaires des lésions actives de SEP. Les travaux présentés dans cette thèse auront permis d’affiner nos connaissances sur les mécanismes d’entrée des lymphocytes TH17 dans le SNC et les propriétés délétères des cytokines qu’ils sécrètent, notamment dans l’activation et la déstabilisation de l’endothélium cérébral. / The blood-brain barrier (BBB) plays a crucial role in protecting the central nervous system (CNS) by restricting entry of cells and molecules into the brain. In the CNS disorder multiple sclerosis (MS), breakdown of the BBB allows activated leukocytes to infiltrate the brain parenchyma, leading to the formation of the characteristic demyelinated lesions. For decades, MS was viewed as a TH1-mediated disease, a notion that was largely supported by studies in its animal model and by the abundance of prototypical TH1-associated cytokines within active MS lesions. However, over the years, accumulating evidence has highlighted the involvement of another subset of CD4⁺ T cells that express IL-17, therefore named TH17 lymphocytes, in the pathology of the disease. The goal of the work presented herein was to characterize the human TH17 lymphocyte population and define their contribution to the disruption of the BBB and leukocyte infiltration into the CNS, both important early events in the formation of MS lesions. To do so, we developed and optimized a method to successfully generate human TH17 lines in vitro from peripheral blood mononuclear cells of healthy donors. We demonstrate that in response to IL-23, human memory CD4⁺CD45RO⁺ but not naïve CD4⁺CD45RA⁺ T lymphocytes produce IL-17, IL-22, and granzyme B, with a subset of cells simultaneously expressing IL-17 and IFN-γ. Interestingly, we measure a significant increase in the percentage of T CD4⁺ IL17⁺, of IL-22⁺, and of IL-17⁺IFN-γ⁺ dual producers in TH17 cell lines expanded from the peripheral blood of acutely relapsing MS women as compared to those generated from healthy controls and remitting MS patients. We show that both IL-17 and IL-22 receptors are upregulated on BBB endothelial cells in situ during inflammation and that IL-17 enhances BBB permeability by disrupting the integrity of tight junction proteins occludin and ZO-1. Finally, we provide evidence that TH17 lymphocytes transmigrate efficiently across human brain endothelial cells via the adhesion molecule ICAM-1 and show that IL-17⁺IFN-γ⁺ double producers have an increased propensity to do so. Accordingly, we detect lymphocytes that display immunoreactivity against both the TH1- and TH17-associated transcription factors T-bet and RORC within perivascular infiltrates of active MS lesions. The work presented in this thesis has refined our understanding of the mechanisms that drive TH17 lymphocyte recruitment into the CNS and shed light on the deleterious effect of TH17-secreted cytokines, specifically in the activation and breakdown of the BBB.