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Human intestinal epithelial cells in innate immunity: interactions with normal microbiota and pathogenic bacteria
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rod-shaped bacteria were previously shown to be associated with the small intestinal epithelium of children with celiac disease (CD). Using culture-dependent and independent methods, we characterized the microbiota of small intestine in children with CD and controls. The normal microbiota constitutes an unique organ-specific biofilm. Dominant bacteria are Streptococcus, Neisseria, Veillonella, Gemella, Actinomyces, Rothia and Haemophilus. Altogether 162 Genus Level Operational Taxonomic Units (GELOTU) of six different phyla were identified in a total of 63 children. In biopsies collected during 2004- 2007 we did not find major differences in the microbiota between CD patients and controls. However, in biopsies collected earlier from children born during the “Swedish CD epidemic” and demonstrated to have rod-shaped bacteria by electron microscopy, we found that unclassified-Clostridales and Prevotella species were associated with CD. These anaerobic, rod-shaped bacteria showed marked affinity for the intestinal epithelium. Changes in breast-feeding practice and/or regiments for introduction of gluten containing food probably affect the composition of the bacterial flora in small intestine. We hypotesize that these bacteria contribute to contraction of CD.

An in vitro model for studies of immune mechanisms of the intestinal epithelium was established. Polarized tight monolayers of the human colon carcinoma cell lines, T84 and Caco2, were developed by culture in a two-chamber system. The two cell lines showed the features of mature- and immature columnar epithelial cells respectively. Polarized monolayers were challenged with bacteria and proinflammatory cytokines. Immune responses were estimated as quantitative changes in mRNA expression levels of a secreted mucin (MUC2), glycocalyx components (CEACAMs, MUC3), antimicrobial factors and cytokines (IFN-g, TNF-a, IL-6 and IL-8). Tight monolayer cells were more resistant to bacterial attack than ordinary tissue culture cells and only B. megaterium induced the defensin, hBD2. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-a induced markedly increased levels of IL-8 and TNF- a itself in both cell lines suggesting recruitment and activation of immune cells. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-g was selective for CEACAM1- L while TNF-a upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

As a pathogenic enteric bacterium, Vibrio cholerae secretes cholera toxin that is the major factor of cholera diarrhea. However, some strains of O1 serogroup lacking the cholera toxin still cause enterocolitis and most V. cholerae vaccines candidates exhibit reactogenicity in clinical trails. An extracellular metalloprotease PrtV was characterized. It was associated with killing of bacteria predators such as the nematode Caenorhabditis elegans. Its role in human intestine was addressed by using the T84 tight monolayer in vitro model. We found that Vibrio Cholera Cytolysin (VCC), a pore-forming toxin, induces an inflammatory response in intestinal epithelial cells that includes increased epithelial permeability and induction of IL-8 and TNF-a and hence could be responsible for enterocolitis. The inflammatory response was abolished by PrtV thus VCC is indeed an autologous substrate for PrtV. In protein rich environment PrtV degradation of VCC was inhibited, suggesting that the magnitude of the inflammatory response is modulated by the milieu in the small intestine. Thus, VCC is likely to be part of the pathogenesis of cholera diarrhea and the causative agent of enteropathy in V. cholerae strains lacking the cholera toxin.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2009. , 71 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1242
Keyword [en]
Immunology
National Category
Microbiology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-18388ISBN: 978-91-7264-721-3 (print)OAI: oai:DiVA.org:umu-18388DiVA: diva2:158827
Distributor:
Institutionen för klinisk mikrobiologi, 90185, Umeå
Public defence
2009-02-27, A103-by 6A (Norrlands universitetssjukhus), Umeå universitet, 901 85 Umeå, Astrid Fagraeussalen (A 103), 10:00 (English)
Opponent
Supervisors
Available from: 2009-02-10 Created: 2009-02-04 Last updated: 2013-03-25Bibliographically approved
List of papers
1. Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease
Open this publication in new window or tab >>Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease
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2009 (English)In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 104, no 12, 3058-3067 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Alterations in the composition of the microbiota in the intestine may promote development of celiac disease (CD). Using scanning electron microscopy (SEM) we previously demonstrated that rod-shaped bacteria were present on the epithelium of proximal small intestine in children with CD but not in controls. In this study we characterize the microbiota of proximal small intestine in children with CD and controls and identify CD-associated rod-shaped bacteria. METHODS: Proximal small intestine biopsies from 45 children with CD and 18 clinical controls were studied. Bacteria were identified by 16S rDNA sequencing in DNA extracted from biopsies washed with buffer containing dithiothreitol to enrich bacteria adhering to the epithelial lining, by culture-based methods and by SEM and transmission electron microscopy. RESULTS: The normal, mucosa-associated microbiota of proximal small intestine was limited. It was dominated by the genera Streptococcus and Neisseria, and also contained Veillonella, Gemella, Actinomyces, Rothia, and Haemophilus. The proximal small intestine microbiota in biopsies from CD patients collected during 2004-2007 differed only marginally from that of controls, and only one biopsy (4%) had rod-shaped bacteria by SEM (SEM+). In nine frozen SEM+ CD biopsies from the previous study, microbiotas were significantly enriched in Clostridium, Prevotella, and Actinomyces compared with SEM- biopsies. Bacteria of all three genera were isolated from children born during the Swedish CD epidemic. New Clostridium and Prevotella species and Actinomyces graevenitzii were tentatively identified. CONCLUSIONS: Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.

Keyword
small intestinal microbiota, identification of rod-shaped bacteria, childhood celiac disease
National Category
Gastroenterology and Hepatology
Identifiers
urn:nbn:se:umu:diva-35059 (URN)10.1038/ajg.2009.524 (DOI)19755974 (PubMedID)
Available from: 2010-07-05 Created: 2010-07-05 Last updated: 2017-12-12Bibliographically approved
2. Contribution of intestinal epithelial cells to innate immunity of the human gut: studies on polarized monolayers of colon carcinoma cells
Open this publication in new window or tab >>Contribution of intestinal epithelial cells to innate immunity of the human gut: studies on polarized monolayers of colon carcinoma cells
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2009 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 69, no 2, 150-161 p.Article in journal (Refereed) Published
Abstract [en]

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram- bacteria from the apical side and the proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human beta-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-alpha. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-alpha levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-alpha induced significantly increased levels of IL-8 and TNF-alpha itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-gamma was selective for CEACAM1-L while TNF-alpha upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-21077 (URN)10.1111/j.1365-3083.2008.02208.x (DOI)19170965 (PubMedID)
Available from: 2009-04-02 Created: 2009-04-02 Last updated: 2017-12-13Bibliographically approved
3. A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing
Open this publication in new window or tab >>A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 24, 9280-9285 p.Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.

Keyword
Animals, Bacterial Proteins/genetics/metabolism, Biofilms, Caenorhabditis elegans/cytology/microbiology/physiology, Cell Communication, Cell Line; Tumor, Cholera Toxin/metabolism, Feeding Behavior, Fimbriae; Bacterial/metabolism, Humans, Interleukin-8/secretion, Intestines/cytology/microbiology, Peptide Hydrolases/genetics/metabolism, Predatory Behavior, Repressor Proteins/genetics/metabolism, Survival Rate, Trans-Activators/genetics/metabolism, Transcription Factors/genetics/metabolism, Vibrio cholerae/enzymology/genetics/pathogenicity
National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-6243 (URN)10.1073/pnas.0601754103 (DOI)16754867 (PubMedID)
Available from: 2008-12-14 Created: 2008-12-14 Last updated: 2017-10-24Bibliographically approved
4. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal cells that is modulated by the protease PrtV secreted by the same bacterium
Open this publication in new window or tab >>Vibrio cholerae cytolysin causes an inflammatory response in human intestinal cells that is modulated by the protease PrtV secreted by the same bacterium
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2009 (English)Manuscript (preprint) (Other academic)
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-18353 (URN)
Available from: 2009-02-04 Created: 2009-02-04 Last updated: 2015-11-11Bibliographically approved

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