Background and Aim: Bacterial infections are a well described complication of cirrhosis and occur in 37% of hospitalized patients. Culture positive infections in addition to the presence of bacterial products and DNA lead to loss of liver function and decompensation in cirrhosis. The mechanisms and molecular pathways associated with Bacterial Translocation (BT) are unknown. The aims of this study were to determine: i. macrophage phenotype and molecular pathways associated with bacterial translocation ii. if intestinal macrophages in liver cirrhosis are capable of modulating intestinal permeability.iii. structural integrity of the epithelial barrier. Methods: Duodenal biopsies and serum samples were collected from 29 patients with decompensated cirrhosis, 15 patients with compensated and 19 controls. Duodenal macrophages were characterized by means of flow cytometry and IHC. Gene expression analysis was performed to determine molecular pathways involved in BT. Inflammatory cytokine determination was done in serum and culture supernatant by means of customized cytometric bead arrays. Results: Patients with decompensated cirrhosis demonstrated: increased frequency of CD33+/CD14+/TREM-1+ and iNOS+ macrophages in their duodenum, elevated mRNA levels of nitric oxide synthase 2 (NOS2), chemokine ligand 2 (CCL2), chemokine ligand 13 (CCL13) and interleukin 8 (IL8) and increased serum levels of interleukin 6 (IL6), IL8 and lipopolysaccharides (LPS). Additionally, patients with decompensated cirrhosis showed an increase in NO, IL6, IL8 and CCL2 levels in culture supernatant after short term duodenal biopsy culture. Although the epithelial barrier on EM seemed intact, significantly increased expression of the “pore” forming tight junction claudin 2 was observed. Conclusion: This study showed the presence of activated CD14+Trem- 1+iNOS+ intestinal macrophages and increased levels of NO, IL-6 and claudin-2 levels in the duodenum of patients with decompensated liver cirrhosis, suggesting that these factors enhance intestinal permeability to bacterial products. / Afrikaans: Inleiding: Bakteriele infeksie is ‘n beskryfde komplikasie van lewersirrose wat in 37% van gehospitaliseerde pasiente voorkom. Kultuur positiewe infeksies asook die teenwoordigheid van bakteriele produkte en DNA lei tot verlies van lewerfunksie en dekompensasie. Die molekulere meganismes wat verband hou met bakteriele translokasie is nog onbekend. Die doel van hierdie studie was om: i. Makrofaag fenotipe en molekulere meganismes geassosieerd met bakteriele translokasie te beskryf, ii. te bepaal of intestinale makrofage dermdeurlaatbaarheid beinvloed, asook iii. om die struktruele integriteit van die dermwand te bepaal. Methods: Serum en dunderm biopsies was verkry van 29 pasiente met gedekompenseerde lewer sirrose, 15 pasiente met gekompenseerde sirrose en 19 kontroles. Dunderm makrofage was gekarakteriseer met behulp van vloeisitometrie en immunohistochemie. Molekulere meganisms belangrik tydens bakteriele translokasie was bepaal met behulp van geneekspressie. Serum en selkultuur supernatant sitokien bepalings was met Bioplex assays gedoen. Resultate: Pasiente met gedekompenseerde sirrose demonstreer: ‘n verhoogde frekwensie van CD33+/CD14+/TREM-1+ en iNOS+ makrofage in hul dunderm, verhoogde mRNA vlakke van NOS2, CCL2, CCL13 en IL8 asook verhoogde serum vlakke van IL6, IL8, LPS. Addisioneel het pasiente met gedekompenseerde sirrose vehoogde supernatant vlakke van NO, IL6, IL8 and CCL2 na kort termyn dunderm biopsie kulture. Alhoewel elekronmikroskopie gewys het dat die dundermwand intak is, was daar statisties-beduidend verhoogde ekspressie van die “porie” vormende vasteaansluitings- proteien, claudin 2 sigbaar. Gevolgtrekking: Gesamentlik het die studie gewys dat geaktiveerde CD14+/Trem-1+/iNOS+ intestinale makrofage asook verhoogde vlakke van NO, IL-6 en claudin-2 teenwoordig is in die dunderm van pasiente met gedekompenseerde sirrose. Dit dui daarop dat diè faktore derm deurlaatbaarheid vir bakteriele produkte kan verhoog. / Dissertation (MSc)--University of Pretoria, 2011. / Immunology / MSc / Unrestricted

Les maladies inflammatoires chroniques de l’intestin (MICI), comprenant la maladie de Crohn (MC) et la colite ulcéreuse (CU), sont caractérisées par une inflammation persistante du tractus gastro-intestinal attribuable à un dysfonctionnement de la réponse immunitaire aux microbiotes de la lumière intestinale. L'épithélium intestinal est essentiel dans le maintien de l'homéostasie, servant de barrière entre les microbes intestinaux et le système immunitaire. La perte de l’intégrité physique de cette barrière est une caractéristique des MICI. Les études génomiques chez les individus atteints de MICI ont identifié plus de 200 régions génomiques (R2 ≥ 0.8) associées aux MICI, ce qui suggère que les voies biologiques influencées par les gènes situés dans ces régions pourraient jouer un rôle dans leur pathogenèse. Notre hypothèse suggère que l'identification de gènes cibles régulés en commun par ceux situés dans ces régions génomiques peut révéler des fonctions biologiques importantes impliquées dans la pathogenèse des MICI. Parmi les gènes associés aux MICI situés dans les régions génomiques pertinentes, on y retrouve DUSP1 et DUSP16, deux gènes appartenant à la famille des phosphatases à double spécificité (« Dual-specificity phosphatase », DUSP). Ces gènes ciblent spécifiquement les « Mitogen-activated protein kinase » (MAPK) en catalysant leur déphosphorylation. Mon projet visait à déterminer le rôle de DUSP1 et DUSP16 dans l'épithélium intestinal. Nous avons exploré leurs fonctions en modifiant l'expression de ces gènes dans des systèmes cellulaires en laboratoire mimant la fonction intestinale, à savoir les cellules HT-29 et les cellules Caco-2. Ces études nous ont permis de déterminer diverses fonctions biologiques importantes régulées par DUSP1 et DUSP16, qui pourraient être impliquées dans le développement des MICI, notamment la transcytose, la préservation de l'intégrité de la barrière épithéliale, l'activation de l'inflammasome et les processus d'autophagie. En identifiant les liens fonctionnels entre deux gènes situés dans des régions génomiques indépendantes associées aux MICI, à savoir DUSP1 et DUSP16, notre étude a mis en évidence leurs rôles significatifs dans le maintien de l'homéostasie intestinale et leur contribution au développement des MICI. / Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are characterized by persistent inflammation of the gastrointestinal tract attributable to a dysfunction of the immune response to microbiota of the intestinal lumen. The intestinal epithelium is essential in maintaining homeostasis, serving as a critical barrier between intestinal microbes and the immune system. The loss of the physical integrity of this barrier is a hallmark of IBD. Genomic studies in individuals with IBD have identified more than 200 genomic regions (R2 ≥ 0.8) associated with IBD, suggesting that biological pathways influenced by genes located in these regions could play a role in their pathogenesis. Our hypothesis suggests that the identification of genes regulated in common by those located in these genomic regions may reveal important biological functions involved in the pathogenesis of IBD. Among the genes associated with IBD located in the relevant genomic regions, we find DUSP1 and DUSP16, two genes belonging to the dual-specificity phosphatase (DUSP) family. These genes specifically target Mitogen-activated protein kinase (MAPK) by catalyzing their dephosphorylation. My project aimed to determine the role of DUSP1 and DUSP16 in the intestinal epithelium. We explored their functions by modifying the expression of these genes in laboratory cell systems mimicking intestinal function, specifically HT-29 and Caco-2 cells. These studies allowed us to determine various important biological functions regulated by DUSP1 and DUSP16, which could be involved in the development of IBD, including transcytosis, preservation of epithelial barrier integrity, inflammasome activation and autophagy processes. By identifying functional links between two genes located in independent genomic regions associated with IBD, namely DUSP1 and DUSP16, our study highlighted their significant roles in maintaining intestinal homeostasis and their contribution to the development of IBD.

<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>