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Role of mast cells and probiotics in the regulation of intestinal barrier function
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The intestinal mucosa is the largest contact area and one of the most important barriers to the outside environment. It is highly specialized in aiding us digest and absorb nutrients. It is daily exposed to several potentially dangerous substances and microorganisms, which if they were allowed to pass into the body, could give rise to diseases. Throughout the small intestine certain sites specialized in antigen sampling are found. These sites are named Peyer’s patches and are lymphoid follicles. The epithelium covering the Peyer’s patches is called follicle-associated epithelium and is specialized in antigen sampling and uptake. The special epithelium enables presentation of luminal antigen to immune cells in the underlying follicle.

Persistent life stress and stressful life events affect the course of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) through largely unknown mechanisms. Regulation of epithelial permeability to antigens is crucial for the balance between inflammation and immune-surveillance, and increased intestinal permeability has been shown in patients with ulcerative colitis and Crohns disease. Vasoactive intestinal polypeptide (VIP) and corticotropin-releasing factor have been implicated as important mediators of stress-induced abnormalities in intestinal mucosal functions in animal models. Both of these mediators have been reported to regulate bowel ion secretion in humans during stress and uptake of horseradish peroxidase in rodents. Probiotics have been shown to ameliorate the deleterious effects of stress on intestinal function, but mechanisms remain to be elucidated.

The aim of this thesis was to elucidate whether mast cells play an important role in intestinal barrier function during stress and inflammation. Moreover, we wanted to determine whether probiotics can ameliorate the mucosal barrier integrity during stress and inflammation.

To study the function of mast cells we conducted in vitro experiments on cell lines and ex vivo experiments in Ussing chambers on mouse, rat and human intestinal tissue. The Ussing chamber technique measures electrophysiological properties of the tissue and also gives the possibility to study transcellular and paracellular passage of markers and bacteria. Immunohistology and confocal microscopy have been used to identify mast cells and receptors of interest.

Our results show that stress affects the follicle-associated epithelium barrier by mechanisms involving VIP and mast cells. These findings were corroborated by the localization of VIP receptors on mucosal mast cells. Furthermore, pretreatment with probiotics was effective in protecting the gut against stress-induced intestinal barrier dysfunction and mucosal inflammation. This protection appeared to involve a mast cell and peroxisome proliferatoractivated receptor-γ dependent mechanism.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. , 62 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1363
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-100770DOI: 10.3384/diss.diva-100770ISBN: 978-91-7519-630-5 (print)OAI: oai:DiVA.org:liu-100770DiVA: diva2:663534
Public defence
2013-12-12, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 13:15 (Swedish)
Opponent
Supervisors
Available from: 2013-11-12 Created: 2013-11-12 Last updated: 2013-11-12Bibliographically approved
List of papers
1. Vasoactive intestinal polypeptide regulates barrier function via mast cells in human intestinal follicle-associated epithelium and during stress in rats
Open this publication in new window or tab >>Vasoactive intestinal polypeptide regulates barrier function via mast cells in human intestinal follicle-associated epithelium and during stress in rats
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2013 (English)In: Neurogastroenterology and Motility, ISSN 1350-1925, E-ISSN 1365-2982, Vol. 25, no 6, e406-e417 p.Article in journal (Refereed) Published
Abstract [en]

Background Vasoactive intestinal polypeptide (VIP) has been implicated as a regulator of intestinal barrier function and inflammation. Our aim was to elucidate the role of VIP in follicle-associated epithelium (FAE) and villus epithelium (VE) permeability following stress in rats and on human intestinal barrier function. Methods Rats were injected intraperitoneally (i.p.) with VIP receptor-antagonists (anti-VPACs), a mast cell stabilizer, doxantrazole (DOX), or NaCl, and submitted to acute water avoidance stress. Ileal segments were mounted in Ussing chambers to assess 51chromium-edta (51Cr-edta) and Escherichia (E.) coli (strain K-12) permeability. Rat ileal and human ileal and colonic segments were exposed to VIP +/- anti-VPACs or DOX. An in vitro co-culture model of human FAE was used to study epithelial-VIP effects. VIP/VPACs distribution was assessed by microscopy. Key Results Stress increased 51Cr-edta and E.coli permeability in VE and FAE. The increases were abolished by i.p. injection of DOX or anti-VPACs. Ileal VIP-exposure ex vivo increased bacterial passage and this was reduced by DOX. In human FAE ex vivo, VIP treatment doubled bacterial uptake, which was normalized by DOX or anti-VPACs. No barrier effects were observed in human colonic tissue. VPACs were found in rat and human ileal follicles, with partial mast cell co-localization. The co-culture model confirmed VIPmast cellepithelial interactions in the regulation of barrier function. Conclusions andamp; Inferences Stress affects the FAE barrier by mechanisms involving VIP and VPACs on mucosal mast cells. We suggest a regulatory role for VIP in the control of ileal permeability that may be relevant to bacterialepithelial interactions in stress-related intestinal disorders.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keyword
Inflammatory bowel disease, permeability, Peyers patch, Ussing chamber
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-94315 (URN)10.1111/nmo.12127 (DOI)000318945400005 ()
Note

Funding Agencies|Swedish Research Council|K2012-55X-12618-16-3|

Available from: 2013-06-24 Created: 2013-06-24 Last updated: 2017-12-06
2. Protective Effects of Probiotics on Chronic Stress-Induced Intestinal Permeability in Rats are mediated via Mast Cells and PPARγ
Open this publication in new window or tab >>Protective Effects of Probiotics on Chronic Stress-Induced Intestinal Permeability in Rats are mediated via Mast Cells and PPARγ
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2013 (English)Manuscript (preprint) (Other academic)
Abstract [en]

BACKGROUND: Chronic stress, which may affect in the clinical course of inflammatory and functional bowel diseases, disrupts intestinal barrier function by routes involving mast cells. Probiotics have been shown to ameliorate the deleterious effects of stress on intestinal function, but mechanisms remain to be elucidated. Peroxisome proliferator-activated receptor (PPAR)-γ signaling is activated as an endogenous defense mechanism during chronic stress and evidence suggests that probiotics reduce the degradation of PPAR-γ. As a source of the endogenous agonist for PPAR-γ, 15d-PGJ2, and as an important mediator of the stress response, mast cells may have both a beneficial and a deleterious role in the effects on intestinal function by probiotics.

AIM: Our aim was to study if mast cells contribute to the positive effects of probiotic therapy on intestinal function in a rat model of chronic stress.

METHODS: 32 Mast cell deficient (Ws/Ws) and 32 wild-type (+/+) rats were subjected to water avoidance stress (WAS) or sham stress (SS) 1hr/day for 10 days. Seven days prior to the onset of stress, probiotics (PB, multispecies combination of 10 different lactic acid bacteria) were added to the standard diet (St) in half of the animals. To determine dependence of PPAR-γ, 8 probiotic-fed wild-type rats subjected to WAS were injected daily with the specific PPAR-γ antagonist T0070907. The colonic mucosa was exposed to E. coli HB101 incorporated with green fluorescent protein and permeability was assessed in Ussing chambers. Mesenteric lymph nodes (MLN) were cultured to determine bacterial translocation.

RESULTS: Chronic stress induced a marked increase in ileal permeability to E.coli HB101 in +/+ rats (0.17±0.1 x106CFU/hr in SS/St/++ vs. 2.13±0.4 in WAS/St/++; P<0.001). This breach in barrier integrity was less pronounced in Ws/Ws rats (2.13±0.4 in WAS/St/++ vs. 1.19±0.3 in WAS/St/WsWs; P<0.01). Probiotics prevented stress-induced effects in E.coli HB101 passage only in wild-type rats (82% decrease in +/+ vs. 0% in Ws/Ws rats). Furthermore, only in the presence of mast cells did probiotics reduce the enhanced bacterial translocation to MLNs during chronic stress. In wild-type rats treated with a PPAR-γ antagonist, the barrier protective effects of probiotics were diminished.

CONCLUSIONS: Mast cells acting via a PPAR-γ dependent pathway contribute to the beneficial effects of probiotics on chronic stress-induced mucosal dysfunction in rats.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100767 (URN)
Available from: 2013-11-12 Created: 2013-11-12 Last updated: 2014-03-25
3. Probiotics modulate mast cell degranulation and reduce stress-induced barrier dysfunction in vitro
Open this publication in new window or tab >>Probiotics modulate mast cell degranulation and reduce stress-induced barrier dysfunction in vitro
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2013 (English)Manuscript (preprint) (Other academic)
Abstract [en]

BACKGROUND: Stress has well-established deleterious effects on intestinal barrier function and stressful life events are known to contribute to the development and perpetuation of inflammatory bowel diseases. Mast cells play a pivotal role in pathogenesis of stressinduced barrier dysfunction due to the release of barrier-disruptive content. Conversely, they also have recently been suggested to contribute to barrier protective properties of probiotics, through the release of 15d-PGJ2 and enhanced epithelial PPAR-γ activity. However, mechanisms remain to be elucidated.

AIM: To study if probiotics can modulate mast cell mediator release, resulting in amelioration of stress-induced barrier dysfunction in vitro.

METHODS: Confluent monolayers of the human colon-derived T84 epithelial cell line were co-cultured with rat basophilic leukemia (RBL)-2H3 mast cells and pretreated with probiotics (125x104 CFU/ml, 1hr) before addition of 100nM CRF to activate mast cells. Release of beta hexosaminidase, TNF-α and 15d-PGJ2 from mast cells was determined. Transepithelial resistance (TER), and permeability to microspheres (0.2μm) were measured over a 24h period. To determine dependence of PPAR-γ, monolayers were incubated with the specific PPAR-γ antagonist T0070907 before treatment with probiotics.

RESULTS: CRF-induced activation of mast cells resulted in decreased TERs and increased permeability to microspheres. Both pretreatment with probiotics and filter-sterilized probiotic supernatant resulted in lower levels of mast cell-released beta hexosaminidase and TNF-α, and increased 15d-PGJ2. Furthermore, probiotics ameliorated epithelial barrier dysfunction in monolayers exposed to CRF-activated mast cells. However, when T84 monolayers were exposed to conditioned medium of CRF-activated mast cells or were incubated with T0070907, probiotics showed little or no effect.

CONCLUSIONS: Probiotics modulate mast cell mediator release to a more barrier protective profile, resulting in amelioration of stress-induced epithelial barrier dysfunction, which is putatively mediated by a PPAR-γ dependent pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100768 (URN)
Available from: 2013-11-12 Created: 2013-11-12 Last updated: 2014-03-25Bibliographically approved
4. Faecalibacterium prausnitzii supernatant improves intestinal barrier function in mice DSS colitis
Open this publication in new window or tab >>Faecalibacterium prausnitzii supernatant improves intestinal barrier function in mice DSS colitis
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2013 (English)In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 48, no 10, 1136-1144 p.Article in journal (Refereed) Published
Abstract [en]

Objective. The intestinal microbiota plays a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Faecalibacterium prausnitzii (FP) is underrepresented in IBD patients and have been suggested to have anti-inflammatory effects in mice. Increased intestinal permeability is common in IBD but the relationship between FP and intestinal barrier function has not been investigated. Our aim was to study treatment with FP supernatant on intestinal barrier function in a dextran sodium sulfate (DSS) colitis mice model. Material and methods. C57BL/6 mice received 3% DSS in tap water ad libitum during five days to induce colitis. From day 3 the mice received a daily gavage with FP supernatant or broth during seven days. Ileum and colon were mounted in Ussing chambers for permeability studies with Cr-51-EDTA and Escherichia coli K-12. Colon was saved for Western blot analyses of tight junction proteins. Results. DSS-treated mice showed significant weight loss and colon shortening. Gavage with FP supernatant resulted in a quicker recovery after DSS treatment and less extensive colonic shortening. Ileal mucosa of DSS mice showed a significant increase in Cr-51-EDTA-passage compared to controls. Cr-51-EDTA passage was significantly decreased in mice receiving FP supernatant. No significant differences were observed in passage of E. coli K12. Western blots showed a trend to increased claudin-1 and claudin-2 expressions in DSS mice. Conclusions. Supernatant of FP enhances the intestinal barrier function by affecting paracellular permeability, and may thereby attenuate the severity of DSS-induced colitis in mice. These findings suggest a potential role of FP in the treatment of IBD.

Place, publisher, year, edition, pages
Informa Healthcare, 2013
Keyword
dextran sodium sulfate, inflammatory bowel disease, permeability, probiotics, tight junctions
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-99406 (URN)10.3109/00365521.2013.828773 (DOI)000324761000005 ()
Note

Funding Agencies|Swedish Research Council|VR-M: K2012-55X-12618-16-3|

Available from: 2013-10-17 Created: 2013-10-17 Last updated: 2017-12-06

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