京都大学 / 新制・課程博士 / 博士(医学) / 甲第24517号 / 医博第4959号 / 新制||医||1065(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 妹尾 浩, 教授 松田 道行, 教授 小濱 和貴 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the United States. Aldo-keto reductase 1 B10 (AKR1B10) is highly expressed in colon and small intestine of normal humans, but its expression is lost or markedly down-regulated in tissues of patients with ulcerative colitis (UC) and CRC. AKR1B10 is a monomeric cytosolic enzyme with strong enzymatic activity to α, β-unsaturated carbonyl compounds, protecting cells from carbonyl lesions; AKR1B10 also mediates de novo synthesis of long chain fatty acids and membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2). To study the etiopathogenic role of AKR1B10 in UC and CRC, our lab generated AKR1B8 deficient (AKR1B8 -/-) mice. AKR1 B8 is the orthologue in mice of human AKR1B10,

The mammalian intestine harbors an estimated 100 trillion microorganisms, which normally maintain a mutually beneficial relationship with the host. The intestinal epithelium consists of a single layer of intestinal epithelial cells (IECs) that provides a physical barrier as well as innate immune defense, preventing this vast community of microbes from entering host tissues. Secretory immunoglobulin A (SIgA) acts as the first line of antigen-specific immunity at the interface between the gut microbiota and the intestinal epithelium. Polymeric IgA secreted by plasma cells in the intestinal lamina propria is transported across IECs by the polymeric immunoglobulin receptor (pIgR). Defects in epithelial barrier and immune functions can lead to infections with opportunistic and pathogenic microbes and contribute to the etiology of inflammatory bowel disease (IBD). Here we investigate the ability of IEC biomarkers to define the mechanism and severity of intestinal inflammation, as well as provide insight into the function of IEC in regulating intestinal homeostasis and inflammation. Importantly, down-regulation of pIgR expression was a common feature in human IBD and mouse models of experimental colitis. One molecule of pIgR is consumed for every molecule of SIgA transported, thus high expression of pIgR is required to maintain sufficient supply of SIgA. Accordingly, we investigate the mechanisms by which IECs regulate pIgR expression in response to colonic bacteria. Cross-talk between the microbiota and IECs is mediated by pattern recognition receptors, including Toll-like receptors (TLR), leading to expression of gene products that enhance epithelial barrier function and innate immunity. The cytoplasmic adaptor protein MyD88 transduces signals from TLRs that recognize bacterial products. We show that pIgR induction by colonic bacteria is dependent on TLR4-MyD88 activation of NF-κB signaling. We examined the role of epithelial-specific MyD88 signaling in antibacterial immunity and epithelial expression of key gene products that participate in innate immunity in the gut by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88ΔIEC). MyD88ΔIEC mice display immunological and antimicrobial defects resulting in increased susceptibility to experimental colitis. We conclude that cross-talk between bacteria and IECs via MyD88-dependent signaling is crucial for maintenance of gut homeostasis.

Le projet de ma thèse a consisté à étudier chez les nouveau-nés la réponse des cellules épithéliales intestinales (IEC) en microARN (miR) et en peptides antimicrobiens (PAM) dans le contexte de l’infection par Cryptosporidium parvum. Ce protozoaire monoxène se développe uniquement dans les IEC et affecte plus particulièrement les nouveau-nés et les individus immunodéprimés. Nous avons étudié la réponse en miR dans les IEC après infection par C. parvum. La comparaison des réponses obtenues dans les IEC in vitro et in vivo nous a permis de montrer que l’expression du miR-181d-5p est diminuée durant l’infection et que cette diminution d’expression lèverait l’inhibition de l’expression des facteurs anti-apoptotiques OPG et BCL2, favorisant la survie du parasite dans les IEC. Nous avons également caractérisé l’impact de C. parvum sur l’expression des PAM chez le nouveau-né et leur rôle durant l’infection. Nous avons mis en évidence que l’infection entraine une forte modification de l’expression des PAM. Notre attention a été retenue par la diminution, surprenante, de l’expression de la chimiokine antimicrobienne CCL20 et de la cathélicidine CRAMP au cours de l’infection. Pour ces deux peptides, nous avons montré qu’une administration aux souriceaux réduisait significativement la charge parasitaire. Nous avons pu montrer que la protection induite par ces deux molécules antimicrobiennes résultait de leur activité parasiticide sur C. parvum. Ainsi, leur diminution d’expression semble être favorable au développement du parasite et nous avons suggéré qu’elle puisse être induite par le parasite pour échapper à cette activité parasiticide avec notamment la modulation de certains miR. / The aim of my thesis was to study in the mouse model, the intestinal epithelial cell (IEC) response during neonatal Cryptosporidium parvum infection with a focus on microRNAs (miR) and antimicrobial peptides (AMP) response. C. parvum is a protozoan parasite that affects preferentially newborn, young or immunocompromised adult and completes its life cycle only in IECs. In a first part, we studied the expression of miRs in IEC during C. parvum infection. We compared the responses between in vitro infected IEC and IECs purified from infected neonatal mice and observed a decrease of miR-181d-5p expression. This reduced expression of miR-181d-5p was associated with an upregulation of the mRNA coding for two putative targets OPG and BCL2 which are anti-apoptotic agents that may favor parasite survival in IEC. This functional relation between miR-181d-5p and OPG was next demonstrated by using reporter dual-luciferase assay. In a second part of my thesis, we characterized the AMP expression profile and studied their role during C. parvum infection in neonates. We showed that infection up-regulates a broad expression of AMP except for CCL20 and CRAMP cathelicidin for which mRNA expression was decreased. We next choose to focus our work on these two molecules and reported that administration of CCL20 and CRAMP to infected neonatal mice significantly reduced the number of parasites in the intestine through a direct killing activity on free stages of the parasite. As the decreased expression of these two AMPs during infection seems to favor the development of the parasite, this could be an escape mechanism developed by C. parvum that may occur through the modulation of miR.

No description available.

Ce travail de thèse est consacré à la préparation de glycopolymères porteurs de groupements pendants mannoside d’heptyle et à l’évaluation de la capacité de ces ligands multivalents à inhiber la fixation bactérienne sur les cellules humaines. Nous avons synthétisé, par polymérisation radicalaire contrôlée, une série de glycopolymères linéaires ou en étoile présentant des masses molaires, des densités en mannoside et des microstructures modulables dans le but d’évaluer l’influence de ces paramètres sur les processus d’interactions avec diverses souches de bactéries E coli (AIEC LF82 et UTI 89). Nous avons tout d’abord mis en évidence par diffusion dynamique et statique de la lumière, la formation d’agrégats entre ces glycopolymères et FimH, la lectine à l’origine de la fixation de souches de bactéries E coli, traduisant des interactions fortes entre les motifs mannosides et les sites de reconnaissance au mannose de la lectine. Nous avons ensuite évalué l’aptitude de ces ligands multivalents à bloquer l’adhésion bactérienne d’AIEC LF82 (impliquée dans la maladie de Crohn) sur des cellules épithéliales intestinales T84. Il a été démontré en conditions in vitro que l’ajout de 10 nM ou 100 nM d’unités mannoside (respectivement en pré- ou post-incubation) réduit de moitié l’adhésion des bactéries sur les cellules épithéliales. L’effet anti-adhésif de ces glycopolymères a été confirmé par des tests ex vivo réalisés sur des intestins isolés de souris transgéniques CEABAC10. Enfin, nous avons exploité la technique de nanoprécipitation pour l’élaboration de nanocapsules de glycopolymères à cœur huileux. Le procédé développé permet la synthèse de nanocapsules de dimensions contrôlées, porteuses de groupements fonctionnels (fluorophores, ligands) ou de particules métalliques et l’encapsulation de molécules actives à cœur en une seule étape. / This PhD work focuses on the preparation of glycopolymers bearing pendent heptyl mannose groups and the evaluation of the capability of such multivalent ligands to inhibit bacterial adhesion to human cells. Aiming at understanding the impact of various structural parameters on glycopolymer/ E coli interactions (AIEC LF82 et UTI 89 strains of E. coli), a series of linear and star-shaped glycopolymers with tunable molecular weight, mannoside density and microstructure (block copolymers, gradient copolymers, random copolymers) has been constructed. The association of the glycopolymers with FimH adhesin, a lectin which possesses a mannose-specific receptor site and is responsible for recognition and binding to host cells, was first confirmed by static and dynamic light scattering experiments. The propensity of the glycopolymers to prevent attachment of E. coli (AIEC LF82 involved in Crohn’s disease) to intestinal epithelial cells (T84 cells) was further investigated through adhesion assays. It was shown that under in vitro conditions, the addition of 10 nM or 100 nM of glycopolymer on a mannose unit basis (in pre-incubation and post-incubation respectively) decreases by half the bacterial adhesion to intestinal epithelial cells. The anti-adhesive effect of these multivalent ligands was further confirmed in ex vivo conditions for colonic loops of transgenic CEABAC10 mice (Crohn’s disease model mouse). Finally we took advantage of the nanoprecipitation process to generate glyconanocapsules with oily core. The employed strategy allowed for preparing well-defined nanocapsules bearing groups of interest (tags, ligands) or metal particles within the shell and loaded with active molecules in the core in one step.

No description available.