Gut-associated lymphoid tissues such as Peyer’s patches (PP) are inductive sites for immune response in the intestine. Unlike other peripheral lymphoid tissues, gut-associated lymphoid tissues lack afferent lymphatics and can directly sample mucosal antigens by specialized epithelial cells in the follicular associated epithelia (FAE), known as M cells. M cells derive from Lgr5+ intestinal stem cells in intestinal crypts, where the daughter cells of Lgr5+ cells differentiate into M cells after stimulation from the cytokine receptor activator of nuclear factor-κB ligand (RANKL). RANKL is produced by stromal cells within the sub-epithelial dome (SED) residing below the FAE. The transcytosis of antigens across the FAE by M cells is an important initial step in the induction of efficient mucosal immune responses against certain pathogenic bacteria as well as the commensal bacterial flora. However some pathogens, for example orally-acquired prions, may also exploit M cells to infect the host. M cells have been implicated in the uptake of orally acquired prions from the gut lumen. After oral exposure, the accumulation of prions in PP is important for their efficient spread to the nervous system. Previous studies have also shown that pathogen-induced inflammation increases M cell density and this effect can be mimicked by exogenous administration of RANKL. This has led to the hypothesis tested in this thesis that inflammation-related enhancement of M cell differentiation aids the delivery of prions into the lamina propria of villi. The administration of RANKL resulted in increased M cell density in the gut epithelium of mice. Consequently, RANKL treatment enhanced the accumulation of orally-administered prions in PP, decreased disease incubation time and increased prion disease susceptibility. These data indicate the importance of M cells in prion disease pathogenesis and highlight the potential of M cells as vaccine targets against prion disease. The fate and terminal differentiation of distinct intestinal epithelial cell lineages from their uncommitted precursors is dependent on their intrinsic expression of one or more specific transcription factors during their development. Alongside inducing M cell differentiation, RANKL stimulation can also induce the nuclear translocation of the NF-κB transcription factor subunit c-Rel. A comparison of the genes encoding the individual NF-κB subunits c-Rel, Rel-A and Rel-B revealed that they were expressed at the mRNA level in the FAE and by M cells. A c-Rel-deficiency in mice did not influence the expression of RANKL or RANK in PP. The subsequent induction of M cell maturation in the FAE was also unaffected in, indicating that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells. The factors implicated in Lgr5+ intestinal stem cell proliferation and their differentiation into M cells are poorly understood. Some reports have indicated that crypt-associated macrophages may provide extrinsic factors that assist Lgr5+ intestinal stem cell proliferation. In this thesis, the ablation of macrophages in the gut resulted in dysregulation of crypt microarchitecture, depleting Paneth cells and the Lgr5+ stem cells. This adversely affected the subsequent differentiation of intestinal epithelial cell lineages and impeded the functional development of M cells. These data reveal a previously unknown role for macrophages in the maintenance of intestinal crypts and intestinal stem cell proliferation and differentiation.

Abstract Beta-lactoglobulin (β-lg) is the main whey protein in bovine milk. It belongs to the lipocalin protein family, and it is one of the main milk allergens. Resistance to hydrolysis is a particular feature of β-lg making it possible that β-lg reaches the small intestine in its native form. Heat treatments during milk processing may change the native structure of bovine β-lg and change its intestinal transport properties. Heat induced conformational alterations may also expose new antigenic sites. However, there have been no previous studies on the effects of heat treatment on the transport of β-lg or on its sensitizing properties. Cow's milk allergy is one of the most important food allergies affecting about 2.4% of infants. Milk proteins, including β-lg, in breast milk substitute formulas are often the earliest foreign antigens in the diet of newborns. According to the hygiene hypothesis, natural infections and vaccinations may modify the immunological balance and decrease the risk of allergy. Isoelectric precipitations followed by anion exchange and gel filtration were used to purify bovine milk β-lg in its native form. Transport of native and heat-denatured β-lg was compared in two in vitro cell models, Caco-2 and M-cells. Sensitization properties of native and heat-denatured β-lg were studied with an animal model using Hooded-Lister rats. Effects of BCG vaccination in combination with the native β-lg were also studied. Effects of different sensitizations were assessed by antibody levels in serum and inflammation locally in the gastrointestinal tract. Heat denaturation of β-lg made its transport slower in both enterocytes and M-cells. M-cells were more effective transporters of both native and heat-denatured β-lg than caco-2 cells. Animals generated higher levels of IgE when sensitized with native β-lg, but heat-denatured β-lg induced a more intense inflammatory cell reaction in the gastrointestinal tract. Vaccination with BCG decreased serum IgE concentration and modified the predominant site of the inflammatory cell response in intestine. The results indicate that, heat denaturation of β-lg and BCG vaccination, change both the systemic and the mucosal response to bovine milk β-lg. The reasons for this remain speculative. The effect of BCG vaccination is consistent with the hygiene hypothesis. The observed alteration of transport properties could be one mechanism by which heat denaturation modifies the allergenic properties of this protein, but additional studies are necessary to assess whether other mechanisms, such as exposure of new antigenic determinants are also relevant.

<p>The crossing of host barriers (intestinal, blood-brain, and placental) is a critical step for systemic infections caused by entero-invasive pathogens. In the intestine, the epithelial cells are the first line of defense against enteric pathogens. <i>Listeria monocytogenes</i> is a facultative-intracellular foodborne pathogen that first crosses the intestinal barrier to cause a systemic infection. However, the underlying mechanism is not well understood.</p><p><br></p> <p>We demonstrate that <i>Listeria</i> adhesion protein (LAP) promotes the translocation of <i>L. monocytogenes </i>across the intestinal barrier in mouse models (A/J and C57BL/6). Relative to the wild-type (WT; serotype 4b) or the isogenic bacterial invasion protein Internalin A mutant (Δ<i>inlA</i>) strain, the <i>lap^─</i> strain showed significant defect in translocation across the intestinal barrier and colonization of the mesenteric-lymph nodes, liver and spleen in the early phase of infection (24 h and 48 h). LAP induces intestinal epithelial barrier dysfunction for increased translocation as evidenced by increased permeability to 4-kDa FITC-dextran (FD4), a marker of paracellular permeability, in the serum and urine of WT and Δ<i>inlA</i>- infected mice and across Caco-2 cell barrier, but not the <i>lap^─</i> mutant strain. Microscopic examination confirmed localization of the WT and Δ<i>inlA</i> strains in the tight junction, a crucial barrier of intestinal paracellular permeability, in the mouse ileal tissue but the <i>lap^─</i> strain remained confined in the lumen. LAP also upregulates TNF-α and IL-6 in intestinal epithelia of mice and in Caco-2 cells for increased permeability. </p><p><br></p> <p>Investigation of the underlying molecular mechanisms of LAP-mediated increase in intestinal permeability by using <i>lap^─</i> mutant strain, purified LAP and shRNA-mediated Hsp60 suppression, we demonstrate that LAP interacts with its host receptor, Hsp60, and activates the canonical NF-κB signaling, which in turn facilitates myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of major epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of NF-κB or MLCK in cells or genetic ablation of MLCK in mice (C57BL/6) prevents mislocalization of epithelial junctional proteins, intestinal permeability and <i>L. monocytogenes</i> translocation across the intestinal barrier.</p> <p><br></p><p>Furthermore, LAP also promotes <i>L. monocytogenes </i>translocation across the intestinal barrier and systemic dissemination in a Mongolian gerbil that are permissive to the bacterial invasion proteins; InlA-and InlB-mediated pathways; similar to that in humans. We show a direct LAP-dependent and InlA-independent pathway<i> </i>for <i>L. monocytogenes</i> paracellular translocation across the intestinal epithelial cells that do not express luminally accessible E-cadherin. Additionally, we show a functional InlA/E-cadherin interaction pathway that aids <i>L. monocytogenes</i> translocation by targeting cells with luminally accessible E-cadherin such as cells at the site of epithelial cell extrusion, epithelial folds and mucus-expelling goblet cells. Thus, <i>L. monocytogenes</i> uses LAP to exploit epithelial innate defense in the early phase of infection to cross the intestinal epithelial barrier, independent of other invasion proteins.</p><p><br></p> <p>This work fills a critical gap in our understanding of <i>L. monocytogenes </i>pathogenesis and sheds light to the complex interplay between host-pathogen interactions for bacterial crossing of the crucial intestinal barrier.</p> <br>