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Vibrio cholerae modulates the immune defense of human gut mucosa
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The key function of innate immunity is to sense danger signals and initiate effective responses as a defense mechanism against pathogens. Simultaneously, effector responses must be regulated to avoid excessive inflammation with resulting tissue damage. microRNAs (miRNAs), are small endogenous molecules, that has recently gained attention as important regulatory elements in the human inflammation cascade. The control over host miRNA expression may represent a previously uncharacterized molecular strategy exploited by pathogens to mitigate innate host cell responses.

Vibrio cholerae is a Gram-negative bacterium that colonizes the human small intestine and causes life-threatening secretory diarrhea, essentially mediated by cholera toxin (CT). It is considered a non-invasive pathogen and does not cause clinical inflammation. Still, cholera is associated with inflammatory changes of the small intestine. Furthermore, CT-negative strains of V. cholerae cause gastroenteritis and are associated with extra-intestinal manifestations, suggesting that other virulence factors than CT are also involved in the pathogenesis.

The innate immune response to V. cholerae is poorly investigated and the potential role of miRNA in cholera had not been studied before. Therefore, this thesis explores the role of intestinal epithelial cells in response to V. cholerae infection with a focus on regulatory miRNA as a potential contributor to the pathogenesis. The in vivo material was small intestinal biopsies from patients suffering from V. cholerae infection. As an in vitro model for V. cholerae attack on intestinal epithelium, we used tight monolayers of T84 cells infected with V. cholerae and their released factors. We analyzed changes in levels of cytokines, immunomodulatory microRNA and their target genes.

We showed that miRNA-146a and miRNA-155 reached significantly elevated levels in the intestinal mucosa at acute stages of disease in V. cholerae infected patients and declined to normal levels at the convalescent stage. Low-grade inflammation was identified at the acute stage of V. cholerae infection, which correlated with elevated levels of regulatory miRNA. Furthermore, outer membrane vesicles (OMVs) released by the bacteria were shown to induce miR-146a and live bacteria induced miR-155 in intestinal epithelial cells. In addition, OMVs decreased epithelial permeability and caused mRNA suppression of pro-inflammatory cytokines, including immune cell attractant IL-8 and CLL20, and the inflammasome markers IL-1b and IL-18. These results propose that V. cholerae regulates the host expression of miRNA during infection and may set the threshold for activation of the intestinal epithelium.

Moreover, we showed that V. cholerae also harbors inflammatory-inducing capabilities, by secreting a pore-forming toxin, Vibrio cholerae cytolysin (VCC). By using genetically modified strains as well as soluble protein challenge experiments, VCC was found solely responsible for the increased epithelial permeability and induction of several pro-inflammatory cytokines in intestinal epithelial cells. In contrast to OMVs, VCC displayed strong upregulation of the pro-inflammatory cytokines IL-8, TNF-a, CCL20 and IL-1b and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. This suggest that VCC is an important virulence factor in the V. cholerae pathogenesis, particularly in CT-negative strains. Furthermore, we showed that the bacterium could control the inflammatory actions of VCC by secreting the PrtV protease, which degraded VCC and consequently abolished inflammation.  

In summary, we showed that V. cholerae harbors immunomodulating capabilities, both at the gene level, through induction of host regulatory miRNA, and at the protein level, through secretion of VCC and PrtV. These strategies may be relevant for V. cholerae to promote survival in the gut and cause successful infections in the human host.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2018. , p. 82
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1962
Keywords [en]
Vibrio cholerae, intestinal epithelial cell, duodenum biopsy, acute cholera, immunomodulation, microRNA, OMV, cytokine, T84 monolayer, miR-146, miR-155, VCC, PrtV, hemolysis, IL-8, TNF-a, IL-1b, IL-18, CCL20, IRAK2, inflammation
National Category
Immunology in the medical area
Research subject
Microbiology; Immunology
Identifiers
URN: urn:nbn:se:umu:diva-147538ISBN: 978-91-7601-881-1 (print)OAI: oai:DiVA.org:umu-147538DiVA, id: diva2:1204191
Public defence
2018-06-05, Astrid Fagraeus hörsal A103, byggnad 6E, Umeå Universitet, Målpunkt R, byggnad 6B, våning 1, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-05-15 Created: 2018-05-07 Last updated: 2018-06-09Bibliographically approved
List of papers
1. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease
Open this publication in new window or tab >>Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease
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2009 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 4, no 11, article id e7806Article in journal (Refereed) Published
Abstract [en]

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2009
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-41945 (URN)10.1371/journal.pone.0007806 (DOI)000271721400007 ()19907657 (PubMedID)
Available from: 2011-04-04 Created: 2011-04-04 Last updated: 2018-06-08Bibliographically approved
2. Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera
Open this publication in new window or tab >>Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera
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2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 3, article id 0173817Article in journal (Refereed) Published
Abstract [en]

The potential immunomodulatory role of microRNAs in small intestine of patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) infection was investigated. Duodenal biopsies were obtained from study-participants at the acute (day 2) and convalescent (day 21) stages of disease, and from healthy individuals. Levels of miR-146a, miR-155 and miR-375 and target gene (IRAK1, TRAF6, CARD10) and 11 cytokine mRNAs were determined by qRT-PCR. The cellular source of microRNAs in biopsies was analyzed by in situ hybridization. The ability of V. cholerae bacteria and their secreted products to cause changes in microRNA- and mRNA levels in polarized tight monolayers of intestinal epithelial cells was investigated. miR-146a and miR-155 were expressed at significantly elevated levels at acute stage of V. cholerae infection and declined to normal at convalescent stage (P<0.009 versus controls; P = 0.03 versus convalescent stage, pairwise). Both microRNAs were mainly expressed in the epithelium. Only marginal down-regulation of target genes IRAK1 and CARD10 was seen and a weak cytokine-profile was identified in the acute infected mucosa. No elevation of microRNA levels was seen in ETEC infection. Challenge of tight monolayers with the wild type V. cholerae O1 strain C6706 and clinical isolates from two study-participants, caused significant increase in miR-155 and miR-146a by the strain C6706 (P<0.01). One clinical isolate caused reduction in IRAK1 levels (P<0.05) and none of the strains induced inflammatory cytokines. In contrast, secreted factors from these strains caused markedly increased levels of IL-8, IL-1β, and CARD10 (P<0.001), without inducing microRNA expression. Thus, miR-146a and miR-155 are expressed in the duodenal epithelium at the acute stage of cholera. The inducer is probably the V. cholerae bacterium. By inducing microRNAs the bacterium can limit the innate immune response of the host, including inflammation evoked by its own secreted factors, thereby decreasing the risk of being eliminated.

National Category
Microbiology in the medical area Immunology
Identifiers
urn:nbn:se:umu:diva-133013 (URN)10.1371/journal.pone.0173817 (DOI)000398945800031 ()28319200 (PubMedID)
Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2018-06-09Bibliographically approved
3. Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response ofhuman intestinal epithelial cells by inducing microRNA-146a
Open this publication in new window or tab >>Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response ofhuman intestinal epithelial cells by inducing microRNA-146a
(English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322Article in journal (Refereed) Submitted
Abstract [en]

The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulencefactors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wild-type and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α(TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not causeany changes. Notably, OMVs from the VCC deficient mutant caused significant decreases in CCL20 and IL-18 mRNA levels. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing thestrength of an epithelial innate immune defence reaction and also preventing inflammation inthe mucosa that factors like VCC can evoke.

Keywords
Vibrio cholerae, intestinal epithelial cells, immunomodulation, microRNA, OMV, T84 monolayer, miR-146, miR-155, VCC, IL-8, TNF-a, IL-1b, IL-18, CCL20, IRAK2, inflammation
National Category
Immunology in the medical area
Research subject
Microbiology; Medicine; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-147536 (URN)
Available from: 2018-05-07 Created: 2018-05-07 Last updated: 2018-06-09

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