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Thesis (Ph. D.)--University of Wisconsin. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 319-344).

Much of the research that has been done on the fish immune system has focussed on innate immunity. Very little is known about the adaptive immune system of fish and how it is regulated. This study has identified and characterised key cytokines that had not been found in any teleost species to date involved in T helper 2 (Th2) and T helper 17 (Th17) responses. By exploiting the synteny that exists between the human and zebrafish genomes two IL-4 homologues were identified, and their full length cDNA cloned and sequenced, which were termed IL-4 like (IL-4L) and IL-4 related (IL-4rel). This work describes the finding of what we consider to be the actual IL-4 homologue in fish and establishes reasons why the already published ‘IL-4’ gene in Tetraodon (Li, Shao et al. 2007b) may not in fact be the actual IL-4 homologue of mammals but an IL-4 related (IL-4rel) gene which we have also been able to locate within the zebrafish genome. To help with determining the identity of these genes within fish, recombinant proteins of the IL-4L and IL-4rel genes were produced in zebrafish and the IL-4rel molecule in trout. Preliminary expression studies have displayed some evidence of bioactivity in the zebrafish IL-4L and trout IL-4rel proteins, through their impact on the regulation of various immune related genes. These include IL-1β, IFN-γ, the IFN-γR1 and the IL-4 receptor-α chain. This work has also identified and characterised for the first time in any lower vertebrate, the zebrafish IL-23 p19 subunit, which is very important in Th17 responses. In addition the p19 sequence has been predicted from other available fish genomes (stickleback, Fugu, Tetraodon) for comparison. Expression analysis of this subunit along with the p40 subunit within a zebrafish disease model for tuberculosis shows up regulation of this gene after 1 day.

Les cellules dendritiques (DCs) sont essentielles pourconnecter l'immunité innée et adaptative et orienter lesréponses des lymphocytes T. Les récepteurs Lectin de type-Cprésents dans les DCs sont activés par des ligands exogènes etendogènes, ce qui dicte la réponse aux agents pathogènes parla modulation de la réponse T immunitaire. Nous avons déjàdécrit chez le rat, l'expression de CLEC-1 dans les DCs etnous avons démontré in vitro son rôle inhibiteur dansl'activation de la réponse T helper (Th17) ). Dans cette étude,nous avons examiné l'expression et la fonction de CLEC-1dans les DCs humaines et nous avons montré son expression àla surface cellulaire de la sous-population de DCs CD16- dansle sang et sur les DCs dérivées des monocytes (moDCs).L'expression de CLEC-1 sur les moDCs est diminuée par desstimuli inflammatoires et renforcée par le TGF-β. De plus,nous avons démontré que CLEC-1 est un récepteurfonctionnel sur les moDCs humains et que, bien qu'il nemodule pas la voie classique d’activation du facteur detranscription NFкB lié à la protéine kinase Syk, il réprime laréponse ultérieure Th17. De façon très importante, en utilisantdes rats déficients pour CLEC-1, nous avons montré que laperturbation de la signalisation de CLEC-1 conduit à unesurexpression de la sous-unité Il-12p40 dans les DCs, et à uneexacerbation des réponses CD4+ Th1 et Th17 in vitro et invivo. Collectivement, nos résultats établissent le rôleinhibiteur de CLEC-1 dans les DCs, capable d'amortir leuractivation et la réponse ultérieure Th17. CLEC-1 peutreprésenter une cible thérapeutique utile pour moduler lesréponses immunitaires T dans un contexte clinique. / Dendritic cells (DCs) represent essential antigen-presentingcells that are critical for linking innate and adaptive immunity,and influencing T-cell responses. Among pattern recognitionreceptors, DCs express C-type lectin receptors triggered byboth exogenous and endogenous ligands, therefore dictatingpathogen response, and also shaping T-cell immunity. Wepreviously described in rat, the expression of the orphan Ctypelectin-like receptor-1 (CLEC-1) by DCs anddemonstrated in vitro its inhibitory role in downstream Thelper 17 (Th17) activation. In this study,we examined theexpression and functionality of CLEC-1 in human DCs, andshow a cell-surface expression on the CD16+ subpopulation ofblood DCs and on monocytederived DCs (moDCs). CLEC-1expression on moDCs is downregulated by inflammatorystimuli and enhanced by TGF- β. Moreover, we demonstratethat CLEC-1 is a functional receptor on human moDCs andthat although not modulating the spleen tyrosine kinase (Syk)dependent canonical nuclear factor-kB (NFкB) pathway,represses subsequent Th17 responses. Importantly, usingCLEC-1–deficient rats, we showed that disruption of CLEC-1signaling led to an enhanced Il-12p40 subunit expression inDCs, and to an exacerbation of downstream in vitro and invivo CD4+ Th1 and Th17 responses. Collectively, our resultsestablish a role for CLEC-1 as an inhibitory receptor in DCsable to dampen activation and downstream effector Thresponses. As a cell-surface receptor, CLEC-1 may representa useful therapeutic target for modulating T-cell immuneresponses in a clinical setting.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19222号 / 医博第4021号 / 新制||医||1010(附属図書館) / 32221 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 長澤 丘司, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses. Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization. Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes. Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. / Ph. D.

La broncho-pneumopathie chronique obstructive (BPCO) est une maladie respiratoire inflammatoire chronique caractérisée par une limitation progressive et irréversible des débits aériens causée par l’inhalation à long terme de particules nocives telles que le tabac. La BPCO présente un remodelage majeur et hétérogène des voies aériennes comportant une grande variabilité inter-individuelle. La réponse inflammatoire et immunitaire au cours de la BPCO est caractérisée par une infiltration du tissu respiratoire par les polynucléaires neutrophiles (PN), les macrophages et par les cellules T. La dégradation des fibres élastiques du poumon en peptides solubles d’élastine (PE) par le biais de la sécrétion de protéases par les cellules de l’immunité innée est une caractéristique constante de la BPCO. Ces PE participent à la physiopathologie de la BPCO comme cela a été démontré dans différents modèles murins de la maladie emphysémateuse. L’orientation de la réponse des cellules T helper (Th) et des cellules T cytotoxiques (Tc) au cours de la BPCO n’est pas élucidée et reste controversée. L’objectif principal de ce travail de thèse a été de définir l’orientation de la réponse Th et Tc ainsi que le rôle des PE dans le remodelage associé à la BPCO. Pour cela, nous avons étudié la signature cytokinique Th-1/Tc-1 (IFN-), Th-2/Tc-2 (IL-4), Th-17/Tc-17 (IL-17) spécifique du remodelage bronchique associé à la BPCO au niveau cellulaire, mais également transcriptionnel et fonctionnel. L’association entre les résultats expérimentaux obtenus et le phénotype des patients inclus a été analysé de manière à déterminer le rôle de ces mécanismes dans l’expression clinique de cette pathologie respiratoire chronique. Les résultats obtenus ont mis en évidence une diminution de l’expression de l’IL-4 (Th2) chez les patients BPCO par rapport aux sujets contrôles ainsi qu’une potentialisation de l’expression de cette cytokine en présence des PE. / Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory pathology characterized by a progressive and irreversible limitation of airflow caused by the long-term inhalation of harmful particles such as tobacco. COPD presents a major and heterogeneous remodeling of the airways with important inter-individual variability. The inflammatory and immune response during COPD is characterized by infiltration of pulmonary tissue by neutrophils (PN), macrophages, and T cells. The degradation of lung elastic fibers into soluble elastin peptides (EP) is caused by the secretion of proteases by innate immunity cells and it is a consistent feature of COPD. These EP participate in the pathophysiology of COPD as demonstrated in different murine models of the emphysematous disease. The T helper (Th) and the T cytotoxic (Tc) orientation during COPD is unclear andremains controversial. The main objective of this work was to define the Th and Tc responses as well as the role ofEP in airways remodeling associated to COPD. For this purpose, we studied the cytokine signature Th-1/Tc-1 (IFN- ), Th-2/Tc-2 (IL-4), Th-17/Tc-17 (IL-17) specific of the airway remodeling associated to COPD both at the cellular, transcriptional and functional level. Association between the experimental results obtained and the phenotype of the patients included in the study was analyzed in order to determine the role of these mechanisms in the clinical expression of this chronic respiratory pathology. The results obtained showed a decrease in the expression of IL-4 (Th2) in COPD patients compared to control subjects as well as a potentiation of this cytokine expression in the presence of EP.

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.

Clinical characteristics of idiosyncratic drug reactions (IDRs) suggest that they are immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats was used as a tool for mechanistic studies of this type of IDR. It has been shown that T helper 17 (Th17) cells play a central role in many types of autoimmune diseases. This study was designed to test whether Th17 cells are involved in the pathogenesis of penicillamine-induced autoimmunity. In sick animals, interleukin (IL) 6 and transforming growth factor-β1, known to be driving forces of Th17 differentiation, were consistently increased following penicillamine treatment. IL-17 and IL-22, characteristic cytokines produced by Th17 cells, were increased in sick animals. Furthermore, the percentage of IL-17-producing CD4 T cells was significantly increased, but only in sick animals. Retinoic acid, which has been reported to inhibit Th17 cell development, made the autoimmunity worse, increased IL-6 production, and did not decrease the number of Th17 cells. An infiltration of CD8 cytotoxic T cells in the liver suggests that they may be the key player in causing liver toxicity induced by D-penicillamine. Drug-induced liver injury (DILI) is one of the major causes of morbidity, mortality, and drug candidate failure. Recently, it has been suggested that Th17 cells may play an active role in inflammatory human liver diseases. In a study of patients being treated with isoniazid, some patients developed mild liver injury. The percentage of Th17 cells in the blood of these patients significantly increased when the ALT increased, and this suggests that they play a role in the mechanism of this liver injury. Furthermore, IL-10-producing T cells also increased and this may have prevented the development of severe liver injury. In another study, two hours after treatment of mice with acetaminophen there was a significant increase in Th17 cells in the liver. This rapid response suggests that Th17 cells can be part of the innate immune response to liver injury. Our data provided evidence that Th17 cells are involved in both “toxic” and idiosyncratic liver toxicity. This pathway could be a new target for the therapeutic interventions to treat DILI.