Listeria monocytogenes is a facultative intracellular bacterium that causes foodborne disease in humans. L. monocytogenes invade the gut mucosa and then disseminate, causing systemic infections associated with high mortality rates in immunocompromised individuals. It is unknown how L. monocytogenes traffic to the mesenteric lymph nodes, which represent an important bottleneck for systemic spread. In addition, little is known about the gastrointestinal stage of infection due to the general resistance of mice to oral infection with L. monocytogenes. Our laboratory developed a novel foodborne mouse model of listeriosis utilizing a murinized strain of L. monocytogenes to investigate the gastrointestinal stage of infection. First, we found that the majority of L. monocytogenes isolated from the intestinal tissue and MLN were extracellular; however, the minimal fraction of intracellular L. monocytogenes was vital for persistence in the gut and spread to the MLN. The vast majority of cell-associated L. monocytogenes in the MLN were adhered to inflammatory monocytes, but these cells did not support the intracellular growth of L. monocytogenes. A minor proportion of L. monocytogenes were associated with migratory dendritic cells in the intestinal lamina propria and MLN, but like monocytes, these cells did not appear to serve as an intracellular growth niche for L. monocytogenes. Lastly, extracellular L. monocytogenes were observed migrating in mesenteric lymphatic vessels that drain from the intestine to the MLN, suggesting that L. monocytogenes can spread beyond the intestinal mucosa independent of migratory immune cells. Overall, these studies are the first to characterize the interaction of L. monocytogenes with immune cells in the intestine and MLN following foodborne infection and suggest that extracellular, and not cytosolic L. monocytogenes, primarily drive innate immune responses in the gut.

<p>One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. </p><p>Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. </p><p>The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation.</p><p>Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. </p><p>In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.</p>

One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation. Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.

The innate and adaptive immune processes are modulated by hormones including glucocorticoids and by microbiota. The exact mechanisms underlying the microbial and hormonal contributions to this control are not completely clear. Present study is therefore focused to crosstalk between microbiota and de novo biogenesis or local regeneration of glucocorticoids. In particular, the study analysed the effect of commensal microbiota on expression of genes encoding steroidogenic enzymes (Star, Cyp11a1, Hsd3b1, Cyp21a1, Cyp11b1) and regeneration of glucocorticoids (Hsd11b1) in adrenal glands, colon, spleen and mesenteric lymph nodes using conventional and germ-free mice. The expression of all 5 components of steroidogenesis was identified only in the adrenal gland and colon, whereas the lymphoid organs expressed predominantly Star, Cyp11a1 and Hsd3b1 indicating the ability to produce only progesterone but not corticosterone. Microbiota decreased the expression of Star in all studied tissues but the expression of other genes was insensitive to microbiota or did not respond homogenously depending on the tissue and gene. Hsd11b1 expression was upregulated by microbiota in the spleen but not in other tissues. Similarly, the in vitro treatment of immune cells isolated from mesenteric lymph nodes by microbial...

La tolérance orale permet la modulation de la réponse immunitaire à l’égard des antigènes exogènes présents dans la lumière intestinale. Essentiels à l’établissement d’une relation symbiotique entre le système immunitaire et la flore intestinale, l’induction et le maintien de la tolérance orale reposent sur différents mécanismes immunologiques. Parmi eux, l’induction de cellules T régulatrices par les cellules dendritiques et de mécanismes apoptotiques. Or, la glycoprotéine membranaire CD47 est impliquée, en périphérie, dans ces mécanismes. Cependant, le rôle de CD47 dans la tolérance orale n’est pas connu. À l’aide d’un modèle murin déficient en CD47, nous avons démontré principalement, que l’absence de CD47 est associée à une diminution de 50 % de la proportion de cellules dendrites myéloïdes CD11b+CD103- retrouvées dans les ganglions mésentériques. Suite au transfert adoptif de cellules T antigènes spécifiques dans nos différents modèles expérimentaux, on a, aussi, observé une diminution de 45 % de leur niveau d’activation dans les ganglions mésentériques. Malgré les effets observés, le CD47 n’est pas impliqué dans l’induction d’une réaction de tolérance orale secondaire à l’administration intragastrique de fortes doses d’ovalbumine. Cependant, nous avons démontré que CD47 est impliquée au niveau de la migration des cellules dendritiques de la peau et de certaines sous-populations retrouvées dans les ganglions mésentériques. / Oral tolerance allows the modulation of the immune response against exogenous antigens present in the intestinal lumen. Essential to establish a symbiotic relationship between the immune system and intestinal flora, the induction and maintenance of oral tolerance rests on different immunological mechanisms. Among them, induction of regulatory T cells by dendritic cells and apoptotic mechanisms. However, the membrane glycoprotein CD47 is involved in the periphery of these mechanisms. However, the role of CD47 in oral tolerance is unknown. With a mouse model deficient in CD47, we showed mainly that the absence of CD47 is associated with a decrease of 50% in the proportion of myeloid dendritic cells CD11b+ CD103- found in the mesenteric lymph nodes. Following the adoptive transfer of antigen specific T cells in our experimental models, we also observed a decrease of 45% of their level of activation in mesenteric lymph nodes. Despite the observed effects, CD47 is not involved in the induction of oral tolerance response secondary to intragastric administration of high doses of ovalbumin. However, we have shown that CD47 is involved in the migration of dendritic cells of the skin and some sub-populations found in mesenteric lymph nodes.

Les maladies inflammatoires intestinales (MII), maladie de Crohn (MC) et colite ulcéreuse (CU), représentent un problème de santé publique majeur en raison de leur prévalence, de leur chronicité et de l’absence de traitement curatif disponible. La physiopathologie de ces maladies implique des facteurs de prédisposition génétique, des facteurs environnementaux et une réponse anormale du système immunitaire. De par leur position à l’interface entre les facteurs environnementaux, les cellules épithéliales et les cellules de l’immunité adaptative, les cellules de l’immunité innée (phagocytes monocucléés (MNPs) et granulocytes) sont des acteurs importants dans l’initiation et le maintien de l’inflammation intestinale. Largement étudiés chez la souris, leur investigation chez l’humain restait parcellaire, souvent contradictoire dans le colon et rarement étudiée dans le ganglion mésentérique (MLN). Nous avons caractérisé par des méthodes de cytométrie de flux multi-couleurs, de cytométrie de masse (CyTOF) et de séquencage de l’ARN (total et à l’échelon de la cellule unique), les MNPs de la muqueuse colique et des ganglions mésentériques chez les patients atteints de MC et de CU. Nous avons également évalué la fonction des MNPs et des basophiles sur les réponses mémoires T CD4+ autologues tissulaires. Notre travail a mis en évidence des similitudes et des différences entre la MC et la CU, dans la distribution des MNPs et le profil de la réponse mémoire T CD4+ dans le colon et le ganglion. La sous-population de MNPs HLADR+SIRPα+CD14+CD64+CD163- qualifiée de monocytes inflammatoires, et non les macrophages HLADR+SIRPα+CD14+CD64+CD163+, s’accumule dans la muqueuse inflammatoire des patients atteints de MC et de CU, et promeut les réponses mémoires de type Th17 et Th17/Th1 d’une manière dépendante de l’IL-1β. La fréquence de cette population corrèle avec le score de sévérité endoscopique en MC. Cependant, la distribution des MNPs ganglionnaires diffère entre la MC et la CU. Nous montrons que, dans les ganglions des patients atteints de CU, les MNPs HLADR+SIRPα+CD14+CD64+ sont enrichis en cellules CD163+, qui incluent principalement des cellules ‘monocyte-like’ HLA-DRdim en plus de macrophages HLA-DRhi. Parmi les cellules dendritiques (DCs) HLADR+SIRPα+CD14-CD64-, les DCs plasmocytoides prédominent dans les deux MII, avec une fréquence supérieure en MC qu’en CU. Par ailleurs, l’IL-1β dans la MC et l’IL-12 dans la CU favorisent un profil pathogénique dans les lymphocytes T CD4+ (IFN-γ, TNF-α, GM-CSF, IL-6) de la muqueuse colique. Par sérendipité, nous avons aussi mis en évidence que l’IL-12 et les monocytes inflammatoires tissulaires induisent la production d’IL-8 par les lymphocytes T CD4+ mémoires de la muqueuse intestinale et des MLNs dans la CU mais pas dans la MC. Au cours de cette étude, nous avons également observé l’accumulation de basophiles, mais pas de mastocytes, dans la muqueuse colique et le MLN en MC et en CU, et montré qu’ils favorisaient également les réponses Th17 et Th17/Th1 et non Th1 dans les lymphocytes T CD4+ mémoires exprimant CCR7. En conclusion, la caractérisation des MNPs de la muqueuse intestinale et des MLNs dans les maladies inflammatoires intestinales (MII) permet de mieux appréhender la physiopathologie de la maladie, dans l’espoir d’optimiser la stratification des MII et de permettre ainsi une prise en charge thérapeutique personnalisée. / Crohn's disease (CD) and ulcerative colitis (UC), two common forms of inflammatory bowel disease (IBD), represent a major public health problem because of their prevalence, chronicity and lack of available curative treatment. The pathophysiology of these diseases involves predisposing genetic factors, environmental triggers, and a dysfunctional immune response. Innate immune cells, including mononuclear phagocytes (MNPs) and granulocytes, are important players in the initiation and maintenance of intestinal inflammation due to their position at the interface between the external environment, epithelium and adaptive immune cells. Although widely studied in mice, their investigation in humans remains fragmentary, often with contradictory findings reported in the colon, and they are rarely studied in the mesenteric lymph nodes (MLNs). MNPs from the colon and MLNs of patients with CD and UC were characterized by multi-color flow cytometry, mass cytometry (CyTOF) and RNA sequencing (bulk and single cell). The function of MNPs and basophils on autologous memory CD4+ T cell responses was also assessed. The results presented here highlight similarities and differences in the distribution of MNPs between CD and UC, and the profile of memory CD4+ T cell response in colon and MLNs. HLADR+SIRPα+CD14+CD64+CD163- MNPs, defined as inflammatory monocytes, but not HLADR+SIRPα+CD14+CD64+CD163+ macrophages, accumulated in the inflammatory mucosa of CD and UC patients, and promoted Th17 and Th17/Th1 memory responses in an IL-1β dependent manner. The frequency of this subpopulation correlated with endoscopic severity in CD. In contrast, the distribution of these two MNP populations in the MLNs differs between CD and UC. HLADR+SIRPα+CD14+CD64+ MNPs were enriched in CD163+ cells that predominantly included HLA-DRdim monocytes-like cells over HLA-DRhi macrophages in UC patients only. Among HLADR+SIRPα+CD14-CD64- dendritic cells (DCs), plasmocytoid DCs predominated in both UC and CD, with higher frequency in CD versus UC. IL-1β in CD and IL-12 in UC favor a pathogenic CD4+ T cell profile (IFN-γ, TNF-α, GM-CSF, IL-6 expression/production) in the colonic mucosa. It was also demonstrated that IL-12 and inflammatory tissue monocytes induced IL-8 production by memory CD4+ T cells in intestinal mucosa and MLNs of UC but not CD. In this study, it was also observed that basophils and not mast cells accumulated, in the colonic mucosa and MLNs of CD and UC patients, and favored Th17 and Th17/Th1, but not Th1, responses in CCR7+ memory CD4+ T cells. In conclusion, characterization of MNPs in the intestinal mucosa and MLNs of IBD patients contributes to a better understanding of IBD pathophysiology and opens avenues to optimize patient stratification, and thus, personalized treatment of IBD patients.

La tolérance orale permet la modulation de la réponse immunitaire à l’égard des antigènes exogènes présents dans la lumière intestinale. Essentiels à l’établissement d’une relation symbiotique entre le système immunitaire et la flore intestinale, l’induction et le maintien de la tolérance orale reposent sur différents mécanismes immunologiques. Parmi eux, l’induction de cellules T régulatrices par les cellules dendritiques et de mécanismes apoptotiques. Or, la glycoprotéine membranaire CD47 est impliquée, en périphérie, dans ces mécanismes. Cependant, le rôle de CD47 dans la tolérance orale n’est pas connu. À l’aide d’un modèle murin déficient en CD47, nous avons démontré principalement, que l’absence de CD47 est associée à une diminution de 50 % de la proportion de cellules dendrites myéloïdes CD11b+CD103- retrouvées dans les ganglions mésentériques. Suite au transfert adoptif de cellules T antigènes spécifiques dans nos différents modèles expérimentaux, on a, aussi, observé une diminution de 45 % de leur niveau d’activation dans les ganglions mésentériques. Malgré les effets observés, le CD47 n’est pas impliqué dans l’induction d’une réaction de tolérance orale secondaire à l’administration intragastrique de fortes doses d’ovalbumine. Cependant, nous avons démontré que CD47 est impliquée au niveau de la migration des cellules dendritiques de la peau et de certaines sous-populations retrouvées dans les ganglions mésentériques. / Oral tolerance allows the modulation of the immune response against exogenous antigens present in the intestinal lumen. Essential to establish a symbiotic relationship between the immune system and intestinal flora, the induction and maintenance of oral tolerance rests on different immunological mechanisms. Among them, induction of regulatory T cells by dendritic cells and apoptotic mechanisms. However, the membrane glycoprotein CD47 is involved in the periphery of these mechanisms. However, the role of CD47 in oral tolerance is unknown. With a mouse model deficient in CD47, we showed mainly that the absence of CD47 is associated with a decrease of 50% in the proportion of myeloid dendritic cells CD11b+ CD103- found in the mesenteric lymph nodes. Following the adoptive transfer of antigen specific T cells in our experimental models, we also observed a decrease of 45% of their level of activation in mesenteric lymph nodes. Despite the observed effects, CD47 is not involved in the induction of oral tolerance response secondary to intragastric administration of high doses of ovalbumin. However, we have shown that CD47 is involved in the migration of dendritic cells of the skin and some sub-populations found in mesenteric lymph nodes.