A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8+ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24675号 / 農博第2558号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5456(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Periodontitis is a chronic inflammatory disease initiated by gram negative anaerobic bacteria. These bacteria possess pathogen-associated molecular patterns (PAMPs) that interact with various receptors including Toll like receptors (TLRs). Bacterial DNA (bDNA) is one of the PAMPs mainly recognized by TLR9. Interaction of bDNA and its receptors leads to activation of inflammatory signaling pathways potentially resulting in periodontal bone destruction. The aim of this study was to determine the production of IL- 6 and IL-8 in response to periodontal bDNA from human osteoblastic cells (MG-63). MG- 63 cells were stimulated in duplicate for 20 hours with 100ng/μl of bDNA from various pathogens including Porhyromonas gingivalis, Esherichia coli, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans as well as heat killed whole bacteria (1:100). E.coli LPS (10ng/μl) was used as a positive control in each experiment. To block TLR9 signaling, further experiments were carried out by treating MG-63 cells with chloroquine (10ng/μl) for 2 hours at 37ºC prior to stimulations. Cytokine levels were determined using enzyme linked-immunosorbent assay. Although IL-6 and IL-8 production was increased in response to periodontal bDNA in MG-63 cells, the results were not significant compared to unstimulated controls. As expected, E.coli DNA, E.coli LPS and heat killed whole bacteria stimulated significantly increased cytokine production (p<0.05). Blocking TLR9 with chloroquine did not affect the amount of cytokine production in bDNA stimulated cells suggesting that TLR9 may not be operant in triggering IL-6 and IL-8 production from MG- 63 cells. In conlusion, periodontal bDNA did not trigger significantly increased IL-6 and IL-8 production from MG-63 cells. Considering the involvement of several inflammatory mediators in periodontal bone destruction, further studies are warranted to assess the production of other cytokines in response to periodontal bDNA in human osteoblastic cells.

The aim of this thesis was to obtain some insights into mechanisms by which the immune system affects melanoma cells after anchoring agonists of phagocytic receptors (laminarin and f-MLF) to their surface. To verify the hypothesis that innate immune system plays a critical role, in vivo experiments were performed on SCID mice. To elucidate the importance of CR3, CD11b-deficient mice were used. In in vitro experiments production of inflammatory cytokines in tumor tissue was examined as well as the release of myeloperoxidase from neutrophil granules after incubation with malignant cells.

The main goal of this thesis was to study cancer immunotherapy based on combination of ligands of TLR and phagocytic receptor agonists. This study is focused on looking for proper and save TLR stimulation and specific anchoring of phagocytic ligands.

L'interaction entre le système immunitaire et le métabolisme du fer est bien illustrée par l'anémie des maladies chroniques (ACD), qui est fréquemment rencontrée dans les infections chroniques, l'inflammation et le cancer. La majorité des modifications dans les paramètres du fer observées dans l’ACD tient compte des modifications de l’homéostasie du fer, avec la délocalisation du métal de la circulation et les sites de l'érythropoïèse au compartiment de stockage dans les macrophages. Les mécanismes de la réponse hyposidérémique impliquent des cytokines, notamment TNF-alpha et IL-6, qui régulent les niveaux de plusieurs gènes du métabolisme du fer, y compris les transporteurs de fer et de l'hepcidine, un régulateur négatif de l’absorption du fer, ce qui entraîne l'inhibition de l'exportation du fer à travers la ferroportine 1 (FPN1) au niveau de l'intestin et les macrophages. Des études antérieures ont montré que l'IL-6 induit l’expression d’hepcidine dans les hépatocytes, mais il y a très peu de données concernant la façon par laquelle l'hepcidine et la FPN1 sont régulées dans les macrophages. Récemment, nous avons constaté que l'induction de l'hepcidine dans le foie par le lipopolysaccharide (LPS) dépend de la voie de signalisation médiée par le récepteur Toll-like 4 (TLR4). Le but de ce travail est d’identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages et de déterminer l’exigence des TLRs dans l’induction de l’hepcidine et le développement d’hyposidérémie. En plus, nous voulons étudier l’effet de l’inflammation causée par les ligands des TLRs sur le taux de fer sérique, la production des cytokines et l'expression de l’hepcidine et de la ferroportine. D’autre part nous voulons étudier l’effet du taux du fer sur la production d’IL-6 macrophagique en réponse à la stimulation par le TLR4. D'abord, pour identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages, nous avons traité les macrophages RAW 264.7 et les macrophages péritonéaux de souris (MPMs) avec différents ligands TLRs et on a mesuré l’expression de l'hepcidine par qRT-PCR. Nous avons observé que Pam3CSK4 (Pam), un ligand de TLR2/1; LPS, un ligand de TLR-4 et FSL1 un ligand de TLR2/6 induisent l’expression de l'hepcidine dans les cellules RAW 264.7 et les MPMs, contrairement au polyinosinic: polycytidylic acid (Poly I: C), un ligand de TLR3. De plus, LPS était capable de réprimer l’expression de la ferroportine dans les cellules RAW 264.7. Afin de mieux définir la nécessité des TLRs pour assurer cette expression, nous avons utilisé les souris TLR-2 knock-out et on a établi que l'expression de l'hepcidine dans les macrophages par LPS, Pam ou FSL1 est dépendante du TLR2. En accord avec les expériences in vitro, les études effectuées in vivo ont montré que LPS réprime l’expression de la ferroportine, ainsi que PolyI:C n’est pas capable de stimuler l'expression d'hepcidine hépatique, par contre il était efficace pour déclencher une hyposidérémie. Ensuite, on voulait déterminer la voie de signalisation utilisée dans l’induction de l’hepcidine dans les macrophages. Comme il y deux voies majeures connues pour la signalisation des TLRs : une dépendante et l’autre indépendante de la protéine MyD88, on a étudié l’expression de l’hepcidine dans les MPMs isolés des souris MyD88-/- et nous avons constaté que l'absence de signalisation MyD88 abolit l'induction de l'hepcidine déclenchée par Pam, LPS et FSL1. D’autre part, la stimulation avec du LPS induisait in vivo la production d’IL-6 et de TNF-alpha, et la stimulation d’IL-6 était renforcée in vitro par la présence du fer. Ces observations indiquent que l’expression de HAMP (Hepcidin Antimicrobial Peptide) dans les macrophages peut être régulée par différents TLRs, ce qui suggère que la production d'hepcidine macrophagique fait partie d'une réponse immunitaire activées par les TLRs. / The interaction between the immune system and iron metabolism is well exemplified in the anemia of chronic disease (ACD), which is frequently encountered in chronic infections, inflammation and cancer. The major changes in iron parameters observed in ACD ultimately reflect modifications in iron trafficking, with relocation of the metal from both the circulation and sites of erythropoiesis to the storage compartment in macrophages. Mechanisms in the hypoferremic response involve cytokines, including TNF-alpha and IL-6. These pro-inflammatory cytokines regulate the levels of several iron metabolism genes, including iron transporters and hepcidin, a negative regulator of iron absorption, resulting in the inhibition of iron export by ferroportine 1 (FPN1) from the intestine and macrophages. Previous studies showed that IL-6 upregulates hepcidin in hepatocytes, but there are very few data regarding how hepcidin and FPN1 expression is regulated in macrophages. More recently, we found that hepcidin induction in the liver by lipopolysaccharide (LPS) is dependent on the signaling pathway mediated by toll-like receptor 4 (TLR4). The aim of this work is to identify TLR ligands able to induce hepcidin in macrophages and to determine the requirement for TLRs in hepcidin expression and the development of hypoferremia. In addition, we want to study the effect of inflammation induced by TLR ligands on serum iron levels, cytokine production, hepcidin and ferroportin expression. On the other hand we want to study the effect of iron levels on IL-6 production by macrophages in response to TLR4 stimulation. First, to identify TLR ligands capable of inducing hepcidin in macrophages, we treated Raw 264.7 macrophages and thioglycollate-stimulated mouse peritoneal macrophages (MPMs) with various TLR ligands and measured hepcidin and ferroportin expression by real-time RT-PCR. We observed that Pam3CSK4 (Pam), a TLR1/2 ligand; LPS, a TLR-4 ligand; and FSL1 a TLR6/2 ligand, but not polyinosinic: polycytidylic acid (poly I:C), a TLR3 ligand, upregulate hepcidin expression in both Raw 264.7 cells and MPMs. Furthermore, LPS was able to repress ferroportine expression in RAW 264.7 macrophages. To further define the requirement for the identified TLRs, we used TLR-2 knockout mice and established that upregulation of macrophage hepcidin expression by Pam or FSL1 is TLR2 dependent, respectively. In agreement with the in vitro experiments, when tested in vivo LPS repressed ferroportine expression and polyI:C failed to induce hepatic hepcidin expression but was effective in triggering hypoferremia. We next investigated whether MyD88, the predominant but not exclusive intracellular signal transduction pathway for TLR-4, is necessary for hepcidin induction in macrophages. Using MyD88 knockout mice, we found that the absence of MyD88 signaling abolishes hepcidin induction triggered by Pam, LPS and FSL1. On the other hand, stimulation with LPS induced in vivo the production of IL-6 and TNF-alpha, and IL-6 stimulation was enhanced in vitro by high amount of iron in macrophages.These observations indicate that HAMP (Hepcidin Antimicrobial Peptide) expression in macrophages can be regulated through multiple TLRs, suggesting that macrophage hepcidin production is part of an immune response activated by the TLRs.

A interação entre a imunidade inata e adaptativa acontece durante diversas fases da resposta imune. Os Toll-like receptors (TLRs) tem importante papel na ativação de macrófagos e portanto, no conjunto de moléculas secretadas por estas células. Pouco se sabe sobre o papel destas substâncias no processo de contração da população de células T ativadas (Activation-induced cell death - AICD). Portanto, o objetivo do presente trabalho foi avaliar se macrófagos estimulados com diferentes agonistas de TLRs poderiam produzir fatores solúveis com capacidade modulatória da morte por AICD. Primeiramente, demonstramos que tanto a linhagem macrofágica J774, quanto os macrófagos derivados de medula óssea (BMDMs) expressam todos os TLRs, com excessão do TLR11. Comprovamos que estas proteínas são funcionais, uma vez que o estímulo com agonistas de TLRs leva à ativação de NF-<font face=\"Symbol\">&#954B nestes macrófagos. Finalmente, mostramos que os sobrenadantes gerados pelos macrófagos são capazes de proteger as células DO11.10 da AICD, via a regulação negativa de FasL, parcialmente mediada por PGE2. / The interaction between innate and adaptative immunity occurs in several phases of the immune response. The Toll-like receptors (TLRs) have an important role in the activation of macrophages directly acting on the molecules secreted by these cells. Little is known about the role of these secreted molecules on the survival control of activated T lymphocytes (Activation-induced cell death - AICD). Therefore, the objective of the present work was to evaluate the effects of soluble factors produced by macrophages activated with several TLRs agonists, on the survival of T lymphocytes. First we sought the expression of TRLs on both bone marrow-derived and J774 macrophage cell line and we could see that both cells express all TLRs, except for TLR 11. The stimulation of both cells with TLRs agonists leads to the expression of NF-<font face=\"Symbol\">&#954B and the production of soluble factors that are able to protect DO11.10 T lymphocyte cell line from AICD, via down regulation of FasL partially mediated by PGE2.

L'interaction entre le système immunitaire et le métabolisme du fer est bien illustrée par l'anémie des maladies chroniques (ACD), qui est fréquemment rencontrée dans les infections chroniques, l'inflammation et le cancer. La majorité des modifications dans les paramètres du fer observées dans l’ACD tient compte des modifications de l’homéostasie du fer, avec la délocalisation du métal de la circulation et les sites de l'érythropoïèse au compartiment de stockage dans les macrophages. Les mécanismes de la réponse hyposidérémique impliquent des cytokines, notamment TNF-alpha et IL-6, qui régulent les niveaux de plusieurs gènes du métabolisme du fer, y compris les transporteurs de fer et de l'hepcidine, un régulateur négatif de l’absorption du fer, ce qui entraîne l'inhibition de l'exportation du fer à travers la ferroportine 1 (FPN1) au niveau de l'intestin et les macrophages. Des études antérieures ont montré que l'IL-6 induit l’expression d’hepcidine dans les hépatocytes, mais il y a très peu de données concernant la façon par laquelle l'hepcidine et la FPN1 sont régulées dans les macrophages. Récemment, nous avons constaté que l'induction de l'hepcidine dans le foie par le lipopolysaccharide (LPS) dépend de la voie de signalisation médiée par le récepteur Toll-like 4 (TLR4). Le but de ce travail est d’identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages et de déterminer l’exigence des TLRs dans l’induction de l’hepcidine et le développement d’hyposidérémie. En plus, nous voulons étudier l’effet de l’inflammation causée par les ligands des TLRs sur le taux de fer sérique, la production des cytokines et l'expression de l’hepcidine et de la ferroportine. D’autre part nous voulons étudier l’effet du taux du fer sur la production d’IL-6 macrophagique en réponse à la stimulation par le TLR4. D'abord, pour identifier les ligands des TLRs capables d'induire l'hepcidine dans les macrophages, nous avons traité les macrophages RAW 264.7 et les macrophages péritonéaux de souris (MPMs) avec différents ligands TLRs et on a mesuré l’expression de l'hepcidine par qRT-PCR. Nous avons observé que Pam3CSK4 (Pam), un ligand de TLR2/1; LPS, un ligand de TLR-4 et FSL1 un ligand de TLR2/6 induisent l’expression de l'hepcidine dans les cellules RAW 264.7 et les MPMs, contrairement au polyinosinic: polycytidylic acid (Poly I: C), un ligand de TLR3. De plus, LPS était capable de réprimer l’expression de la ferroportine dans les cellules RAW 264.7. Afin de mieux définir la nécessité des TLRs pour assurer cette expression, nous avons utilisé les souris TLR-2 knock-out et on a établi que l'expression de l'hepcidine dans les macrophages par LPS, Pam ou FSL1 est dépendante du TLR2. En accord avec les expériences in vitro, les études effectuées in vivo ont montré que LPS réprime l’expression de la ferroportine, ainsi que PolyI:C n’est pas capable de stimuler l'expression d'hepcidine hépatique, par contre il était efficace pour déclencher une hyposidérémie. Ensuite, on voulait déterminer la voie de signalisation utilisée dans l’induction de l’hepcidine dans les macrophages. Comme il y deux voies majeures connues pour la signalisation des TLRs : une dépendante et l’autre indépendante de la protéine MyD88, on a étudié l’expression de l’hepcidine dans les MPMs isolés des souris MyD88-/- et nous avons constaté que l'absence de signalisation MyD88 abolit l'induction de l'hepcidine déclenchée par Pam, LPS et FSL1. D’autre part, la stimulation avec du LPS induisait in vivo la production d’IL-6 et de TNF-alpha, et la stimulation d’IL-6 était renforcée in vitro par la présence du fer. Ces observations indiquent que l’expression de HAMP (Hepcidin Antimicrobial Peptide) dans les macrophages peut être régulée par différents TLRs, ce qui suggère que la production d'hepcidine macrophagique fait partie d'une réponse immunitaire activées par les TLRs. / The interaction between the immune system and iron metabolism is well exemplified in the anemia of chronic disease (ACD), which is frequently encountered in chronic infections, inflammation and cancer. The major changes in iron parameters observed in ACD ultimately reflect modifications in iron trafficking, with relocation of the metal from both the circulation and sites of erythropoiesis to the storage compartment in macrophages. Mechanisms in the hypoferremic response involve cytokines, including TNF-alpha and IL-6. These pro-inflammatory cytokines regulate the levels of several iron metabolism genes, including iron transporters and hepcidin, a negative regulator of iron absorption, resulting in the inhibition of iron export by ferroportine 1 (FPN1) from the intestine and macrophages. Previous studies showed that IL-6 upregulates hepcidin in hepatocytes, but there are very few data regarding how hepcidin and FPN1 expression is regulated in macrophages. More recently, we found that hepcidin induction in the liver by lipopolysaccharide (LPS) is dependent on the signaling pathway mediated by toll-like receptor 4 (TLR4). The aim of this work is to identify TLR ligands able to induce hepcidin in macrophages and to determine the requirement for TLRs in hepcidin expression and the development of hypoferremia. In addition, we want to study the effect of inflammation induced by TLR ligands on serum iron levels, cytokine production, hepcidin and ferroportin expression. On the other hand we want to study the effect of iron levels on IL-6 production by macrophages in response to TLR4 stimulation. First, to identify TLR ligands capable of inducing hepcidin in macrophages, we treated Raw 264.7 macrophages and thioglycollate-stimulated mouse peritoneal macrophages (MPMs) with various TLR ligands and measured hepcidin and ferroportin expression by real-time RT-PCR. We observed that Pam3CSK4 (Pam), a TLR1/2 ligand; LPS, a TLR-4 ligand; and FSL1 a TLR6/2 ligand, but not polyinosinic: polycytidylic acid (poly I:C), a TLR3 ligand, upregulate hepcidin expression in both Raw 264.7 cells and MPMs. Furthermore, LPS was able to repress ferroportine expression in RAW 264.7 macrophages. To further define the requirement for the identified TLRs, we used TLR-2 knockout mice and established that upregulation of macrophage hepcidin expression by Pam or FSL1 is TLR2 dependent, respectively. In agreement with the in vitro experiments, when tested in vivo LPS repressed ferroportine expression and polyI:C failed to induce hepatic hepcidin expression but was effective in triggering hypoferremia. We next investigated whether MyD88, the predominant but not exclusive intracellular signal transduction pathway for TLR-4, is necessary for hepcidin induction in macrophages. Using MyD88 knockout mice, we found that the absence of MyD88 signaling abolishes hepcidin induction triggered by Pam, LPS and FSL1. On the other hand, stimulation with LPS induced in vivo the production of IL-6 and TNF-alpha, and IL-6 stimulation was enhanced in vitro by high amount of iron in macrophages.These observations indicate that HAMP (Hepcidin Antimicrobial Peptide) expression in macrophages can be regulated through multiple TLRs, suggesting that macrophage hepcidin production is part of an immune response activated by the TLRs.

Made available in DSpace on 2014-07-29T15:07:28Z (GMT). No. of bitstreams: 1 Adriano Queiroz de Mesquita -30-07-2010.pdf: 3684854 bytes, checksum: bc87bf213d6700cac487c2b4580b87a1 (MD5) Previous issue date: 2010-07-30 / A mastite tem sido considerada, mundialmente, a doença de maior impacto nos rebanhos leiteiros, devido à elevada prevalência e aos prejuízos econômicos que determina. As desordens decorrentes da mastite por agente etiológico de origem bacteriana são complexas, dependentes do microrganismo envolvido, e desencadeiam inúmeros processos de reconhecimento. As estruturas moleculares dos microrganismos são conhecidas como padrões moleculares associados aos patógenos (PAMPS) e os receptores nas células do hospedeiro como receptores de reconhecimento de padrões (PRR). O presente trabalho foi desenvolvido com o objetivo de identificar a presença de polimorfismos de nucleotídeo único no gene codificador do TLR4 em vacas leiteiras da raça holandesa em uma propriedade leiteira em Goiás, avaliando a relação dos alelos identificados, com a ocorrência de mastite subclínica e contagem celular somática. Foram coletadas 150 amostras de leite individual de vacas para identificação de microrganismos, contagem celular somática e composição centesimal, e 150 amostras de sangue para genotipagem em uma propriedade rural do Estado de Goiás. A discriminação alélica foi realizada por meio da técnica de PCR em tempo real, baseada em 4 SNPs de referência no gene codificador do TLR4 depositados no NCBI (rs8193046, rs8193047, rs8193060 e rs29017188). Os resultados obtidos revelam maior frequência de microrganismos Gram negativos na propriedade de estudo (52,47%) e que, animais identificados com os genótipos AACCCC, GGTCGG e GACCGC são os mais indicados para seleção assistida por marcadores moleculares. / Mastitis has been considered, worldwide, the disease of greatest impact in dairy herds because of the high prevalence and the economic losses that determines. The disorders caused by mastitis causative agent of bacterial origin are complex, depending on the microrganism involved, and trigger numerous processes of recognition. The molecular structures of microrganisms are known as Pathogen- Associated Molecular Patterns (PAMPs) and the receptors on host cells as pattern recognition receptors (PRR). This study was developed with the aim of identifying the presence of single nucleotide polymorphisms in TLR4 in Holstein dairy cows on a dairy farm in Goiás, evaluating the relationship between identified alleles, occurrence of subclinical mastitis and somatic cell count. 150 milk samples from individual cows were collected for identification of microrganisms, somatic cell count and composition, and 150 blood samples for genotyping on a farm in the State of Goiás. The allelic discrimination was performed by Real-time PCR, based on four reference SNPs in TLR4 gene from NCBI (rs8193046, rs8193047, rs8193060 and rs29017188). The results showed higher frequency of Gram negative microrganisms (52.47%) and that animals with the genotypes AACCCC, GGTCGG GACCGC are best suited for marker-assisted selection.

A interação entre a imunidade inata e adaptativa acontece durante diversas fases da resposta imune. Os Toll-like receptors (TLRs) tem importante papel na ativação de macrófagos e portanto, no conjunto de moléculas secretadas por estas células. Pouco se sabe sobre o papel destas substâncias no processo de contração da população de células T ativadas (Activation-induced cell death - AICD). Portanto, o objetivo do presente trabalho foi avaliar se macrófagos estimulados com diferentes agonistas de TLRs poderiam produzir fatores solúveis com capacidade modulatória da morte por AICD. Primeiramente, demonstramos que tanto a linhagem macrofágica J774, quanto os macrófagos derivados de medula óssea (BMDMs) expressam todos os TLRs, com excessão do TLR11. Comprovamos que estas proteínas são funcionais, uma vez que o estímulo com agonistas de TLRs leva à ativação de NF-<font face=\"Symbol\">&#954B nestes macrófagos. Finalmente, mostramos que os sobrenadantes gerados pelos macrófagos são capazes de proteger as células DO11.10 da AICD, via a regulação negativa de FasL, parcialmente mediada por PGE2. / The interaction between innate and adaptative immunity occurs in several phases of the immune response. The Toll-like receptors (TLRs) have an important role in the activation of macrophages directly acting on the molecules secreted by these cells. Little is known about the role of these secreted molecules on the survival control of activated T lymphocytes (Activation-induced cell death - AICD). Therefore, the objective of the present work was to evaluate the effects of soluble factors produced by macrophages activated with several TLRs agonists, on the survival of T lymphocytes. First we sought the expression of TRLs on both bone marrow-derived and J774 macrophage cell line and we could see that both cells express all TLRs, except for TLR 11. The stimulation of both cells with TLRs agonists leads to the expression of NF-<font face=\"Symbol\">&#954B and the production of soluble factors that are able to protect DO11.10 T lymphocyte cell line from AICD, via down regulation of FasL partially mediated by PGE2.