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Mechanisms of bacterial-epithelial interaction in Crohn’s disease
Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Crohn’s disease (CD) is believed to be initiated when an individual, who has agenetic predisposition either leading to a disturbance in the barrier functionand/or the innate immune system is exposed to triggering environmentalfactors, the most important being intraluminal bacteria. Genetic and functionalstudies have confirmed the Pattern-recognition receptors (PRRs), Nod2, TLR4and NALP3, as important mediators of the inflammatory process associatedwith disease progression. However, the mechanisms that link enteric bacteriaand barrier function in a background of genetic predisposition to CD are justbeginning to emerge. The general aim of this thesis was therefore to morethoroughly investigate the mechanisms of bacterial-epithelial interaction in CD.

Here we present evidence suggesting that the small bowel is able to inducetranscytosis of antigens after short term exposure to Yersinia pseudotuberculosis. This suggests that small bowel enterocytes are able toattain follicle associated epithelial (FAE) abilities and contribute to the barrierdysfunction observed in CD. Furthermore we report a positive effect of anti-TNFα treatment (infliximab) on the translocation of adherent invasive E.coli (AIEC) across the colonic mucosa of patients suffering from severe CD.

We also confirm the importance of the Nod-like receptors (NLRs) in thepathogenesis of CD by showing that combined polymorphisms in the genesencoding NALP3 and CARD8 confer susceptibility to CD among Swedish menand in addition to previous published results add a gender aspect on thegenotype-phenotype relationship in CD. Finally, we show that Nod2 is rapidlysubjected to ubiquitination followed by proteasomal degradation, henceproviding important clues about how NLR regulation might occur in the cell,suggesting that the ubiquitin-proteasome pathway is an important factor toconsider in the development of the disease.

In conclusion we report novel insights into the bacterial-epithelial interactionsoccurring in CD and contribute important clues about the origin of this disease.ISBN: 978-

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 71 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1113
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-21104ISBN: 978-91-7393-667-5 (print)OAI: oai:DiVA.org:liu-21104DiVA: diva2:240589
Public defence
2009-04-17, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2009-09-29 Created: 2009-09-29 Last updated: 2009-09-29Bibliographically approved
List of papers
1. Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis
Open this publication in new window or tab >>Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis
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2008 (English)In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 88, no 11, 1215-1226 p.Article in journal (Refereed) Published
Abstract [en]

Crohns disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta 1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv + Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P < 0.01) and ileal mucosa (268 +/- 47% of control; P < 0.01), whereas inv-bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size-and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta 1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta 1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.

Keyword
barrier function, beta 1-integrin, Caco-2 cells, confocal microscopy, Crohns disease, Ussing chambers
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16079 (URN)10.1038/labinvest.2008.86 (DOI)
Available from: 2009-01-07 Created: 2009-01-07 Last updated: 2017-12-14Bibliographically approved
2. Infliximab reduces bacterial uptake in mucosal biopsies of Crohn’s colitis viamicrotubule-dependent pathway
Open this publication in new window or tab >>Infliximab reduces bacterial uptake in mucosal biopsies of Crohn’s colitis viamicrotubule-dependent pathway
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: A defective intestinal barrier, shown by increased paracellular permeability is an importantpathogenic factor in Crohn’s disease (CD). TNFα is a key mediator in the regulation of the intestinal barrierfunction. Treatment with antibodies directed against TNFα, such as infliximab, has been established as animportant part of the therapeutic arsenal in severe Crohn’s disease, but the mechanisms of action have yet tobe elucidated. Part of infliximab’s effect has been suggested to be reduced apoptosis of epithelial cells andthereby reduced paracellular permeability. Our aim was to study how infliximab affects uptake of adherent E.coli across the colonic mucosa in CD.

Method: Seven patients with active CD colitis were examined before and after a four week treatment withinfliximab. Control biopsies were taken from healthy volunteers (4) and patients undergoing controlexamination for colonic polyps (4), aged 36 (range 25-81), coloscopy. Biopsies were taken from macroscopicallynon-inflamed descending colon and were mounted in Ussing chambers to study barrier function. Transmucosalpassage of green fluorescent protein (GFP) incorporated E. coli HM427, an adherent bacteria isolated from thecolon of a CD patient, was studied by quantifying the translocated fluorescent bacteria using flow cytometry.

Results: Bacterial passage across the colonic mucosa was increased in CD (2500 ± 300 arb. units) comparedwith controls (960 ± 280; p<0.05), and was reduced to 500 ± 200 units after the infliximab treatment (p<0.05).In biopsies treated with colchicine (a microtubuline inhibitor) uptake of E. coli HM427 was reduced by 2/3compared to non-treated biopsies.

Conclusion: Patients with active Crohn’s disease showed a defect in the barrier to adherent E. coli, which waspartly mediated through a microtubule dependent uptake. The four week treatment with infliximab improvedthe intestinal barrier to these bacteria. This may constitute an important part of infliximab’s mechanisms ofaction in active colitis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21097 (URN)
Available from: 2009-09-29 Created: 2009-09-29 Last updated: 2010-01-14Bibliographically approved
3. Combined Polymorphisms in Genes Encoding the Inflammasome Components NALP3 and CARD8 Confer Susceptibility to Crohns Disease in Swedish Men
Open this publication in new window or tab >>Combined Polymorphisms in Genes Encoding the Inflammasome Components NALP3 and CARD8 Confer Susceptibility to Crohns Disease in Swedish Men
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2009 (English)In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 104, no 5, 1180-1188 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES : Crohns disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1 beta. Production of mature IL-1 beta is dependent on a caspase-1-activating protein complex called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required for bacteria-induced IL-1 beta secretion. The combination of the polymorphisms CARD8 (C10X) and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis. Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.

METHODS: The study included 498 CD patients in two cohorts from different regions and 742 control individuals from a Swedish population. DNA was isolated from whole blood. Polymorphisms of (Q705K) NALP3 and (C10X) CARD8, as well as the Nod2 variants, R702W and G908R, were genotyped using the Taqman single nucleotide polymorphism assay. The Nod2 frameshift mutation, L1007fs, was detected by Megabace SNuPe genotyping.

RESULTS: Our results show that men who have both the C10X and Q705K alleles in CARD8 and NALP3, and who express wild-type alleles of Nod2 are at an increased risk of developing CD (odds ratio, OR: 3.40 range: 1.32-8.76); P = 0.011). No association with these polymorphisms was found in women (OR: 0.89 (range: 0.44-1.77); P = 0.74).

CONCLUSIONS: We suggest a role for combined polymorphisms in CARD8 and NALP3 in the development of CD in men, with obvious sex differences in the genetic susceptibility pattern. These findings give further support to the importance of innate immune responses in CD.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18957 (URN)10.1038/ajg.2009.29 (DOI)
Available from: 2009-06-06 Created: 2009-06-05 Last updated: 2017-12-13Bibliographically approved
4. Ubiquitination and degradation of the Crohn’s Disease associated protein Nod2involves the E2 enzyme UBE2G2
Open this publication in new window or tab >>Ubiquitination and degradation of the Crohn’s Disease associated protein Nod2involves the E2 enzyme UBE2G2
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Mutations predisposing to Crohn’s disease (CD) have been mapped to the CARD15/Nod2 locus,which encodes a cytoplasmic receptor hereafter referred to as Nod2, a member of the NACHT-LRR (NLR) familyof pattern recognition receptors. The binding of bacterial muramyl dipeptide (MDP) to Nod2 triggers theactivation of the nuclear factor-κB (NFκB) pathway, thereby inducing a number of pro-inflammatory genes. Themost common variant of Nod2 associated with CD is the frame shift mutation L1007fs, which results in atruncated form of the protein unable to respond to MDP. Despite active research, little is known about howNod2 is regulated. The aim of this study was to investigate if the cellular Nod2 protein level is regulated by theubiquitin-proteasome pathway.

Material and Methods/Results: Nod2 was shown to be subjected to rapid turnover in the colorectal cancer cellline SW480 as measured by immunoprecipitation following inhibition of protein synthesis with cyklohexamide.Immunoprecipitation of Nod2 also revealed co-precipitation of ubiquitin, suggesting that Nod2 wasubiquitinated. In line with this observation, inhibition of the proteasome using MG-132 or lactacystin, resultedin increased levels of Nod2 in the cells. UBE2G2, an E2 enzyme, thus conferring specificity of ubiquitin binding,was identified to have affinity for the CARD domain of Nod2. Activation of Nod2 with MDP enhanced itsubiquitination, and increasing amounts of UBE2G2 in the cells abrogated NFB-activation, suggesting thatubiquitination of Nod2 may be important for the resolution of the inflammatory signal.

Conclusion: Taken together, our results show that the cellular level of Nod2 protein is regulated via theubiquitin-proteasome pathway, suggesting that Nod2-driven inflammation may be resolved through rapiddegradation of Nod2. Consequently, not only polymorphisms in Nod2 directly, but also in genes regulatingNod2 protein levels may contribute to the susceptibility to Crohn’s disease.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21099 (URN)
Available from: 2009-09-29 Created: 2009-09-29 Last updated: 2010-01-14Bibliographically approved

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