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Cited by 126Gastrointestinal motility and disordersImmune Response and InflammationGut microbiota and health

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Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

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Belmonte, Liliana, Beutheu Youmba, Stéphanie, Bertiaux-Vandaële, Nathalie, Antonietti, Michel, Lecleire, Stéphane, Zalar, Alberto, Gourcerol, Guillaume, Leroi, Anne-Marie, Déchelotte, Pierre, Coëffier, Moïse, Ducrotté, Philippe·2012·PLoS ONE
DOI 10.1371/journal.pone.0042777PMC PMC3461726
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Abstract

BACKGROUND: The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder whose pathogenesis is not completely understood. Its high prevalence and the considerable effects on quality of life make IBS a disease with high social cost. Recent studies suggest that low grade mucosal immune activation, increased intestinal permeability and the altered host-microbiota interactions that modulate innate immune response, contribute to the pathophysiology of IBS. However, the understanding of the precise molecular pathophysiology remains largely unknown. METHODOLOGY AND FINDINGS: In this study our objective was to evaluate the TLR expression as a key player in the innate immune response, in the colonic mucosa of IBS patients classified into the three main subtypes (with constipation, with diarrhea or mixed). TLR2 and TLR4 mRNA expression was assessed by real time RT-PCR while TLRs protein expression in intestinal epithelial cells was specifically assessed by flow cytometry and immunofluorescence. Mucosal inflammatory cytokine production was investigated by the multiplex technology. Here we report that the IBS-Mixed subgroup displayed a significant up-regulation of TLR2 and TLR4 in the colonic mucosa. Furthermore, these expressions were localized in the epithelial cells, opening new perspectives for a potential role of epithelial cells in host-immune interactions in IBS. In addition, the increased TLR expression in IBS-M patients elicited intracellular signaling pathways resulting in increased expression of the mucosal proinflammatory cytokines IL-8 and IL1β. CONCLUSIONS: Our results provide the first evidence of differential expression of TLR in IBS patients according to the disease subtype. These results offer further support that microflora plays a central role in the complex pathophysiology of IBS providing novel pharmacological targets for this chronic gastrointestinal disorder according to bowel habits.

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Crossref · 10.1371/journal.pone.0042777

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Figure 1 Expression levels of TLR2 and TLR4 in the colonic mucosa of controls and IBS patients. ( A ) Correlation between TLR2 and TLR4 mRNA expression in the whole group of IBS patients (rs = 0.78, p<0.0001). ( B ) Changes in mRNA expression levels of TLR2 and ( C ) TLR4 in controls and in patients with predominant diarrhea (IBS-D), or constipation (IBS-C) or mixed (IBS-M) assessed by real-time PCR. Values are expressed as mean ± SEM. * Represents p<0.05 using Mann-Whitney test.
Figure 1 Expression levels of TLR2 and TLR4 in the colonic mucosa of controls and IBS patients. ( A ) Correlation between TLR2 and TLR4 mRNA expression in the whole group of IBS patients (rs = 0.78, p<0.0001). ( B ) Changes in mRNA expression levels of TLR2 and ( C ) TLR4 in controls and in patients with predominant diarrhea (IBS-D), or constipation (IBS-C) or mixed (IBS-M) assessed by real-time PCR. Values are expressed as mean ± SEM. * Represents p<0.05 using Mann-Whitney test.CC BY · PubMed Central
Figure 2 Correlation analysis of TLR2 and TLR4 mRNA levels and symptoms. Correlation between TLR2 ( A ) and TLR4 ( B ) expressions in the colonic mucosa and the duration of symptoms of IBS patients (n = 46, rs = 0.34, p = 0.02 for TLR2 and rs = 0.37, p = 0.01 for TLR4). Data were correlated by using non-parametric Spearman test.
Figure 2 Correlation analysis of TLR2 and TLR4 mRNA levels and symptoms. Correlation between TLR2 ( A ) and TLR4 ( B ) expressions in the colonic mucosa and the duration of symptoms of IBS patients (n = 46, rs = 0.34, p = 0.02 for TLR2 and rs = 0.37, p = 0.01 for TLR4). Data were correlated by using non-parametric Spearman test.CC BY · PubMed Central
Figure 3 Immunofluorescence microscopy analysis of TLR2 and TLR4 in the colonic mucosa of IBS subgroups. Representative photomicrographs show the distribution of TLR2 and TLR4 proteins in the colonic mucosa of controls and IBS patients according to the disease subtype (IBS-Constipated, IBS-Diarrhea or IBS-Mixed alternating constipation with diarrhea) Red, TLR staining; blue, DAPI nuclear staining. Original magnification, ×20.
Figure 3 Immunofluorescence microscopy analysis of TLR2 and TLR4 in the colonic mucosa of IBS subgroups. Representative photomicrographs show the distribution of TLR2 and TLR4 proteins in the colonic mucosa of controls and IBS patients according to the disease subtype (IBS-Constipated, IBS-Diarrhea or IBS-Mixed alternating constipation with diarrhea) Red, TLR staining; blue, DAPI nuclear staining. Original magnification, ×20.CC BY · PubMed Central
Figure 4 TLR2, TLR4 and CD14 expression in IECs (Epcam+ cells). Epithelials cells obtained from the colonic mucosa were assessed by flow cytometry as described in Patients and Methods. Mucosal colonic cells were double-stained with isotype IgG control or mAbs to TLR2, TLR4 or CD14 together with APC-conjugated anti Epcam mAbs. ( A ) Representative histogram showing the fluorescence intensity of surface TLR4 expression in gated Epcam+ cells from all IBS subtypes and controls patients. Changes in TLR4 surface expression ( B ), intracellular TLR4 expression ( C ), CD14 expression ( D ) and TLR2 surface expression ( E ) in IECs in control and IBS subtypes. Values are expressed as mean intensities fluorescence ± SEM. (*) Represents p<0.05 and (**) represents p<0.005 using Mann-Whitney test. Kruskal-Wallis p values are *p = 0.01 (B); p = 0.5 (C); p = 0.95 (D) and *p = 0.03 (E) respectively.
Figure 4 TLR2, TLR4 and CD14 expression in IECs (Epcam+ cells). Epithelials cells obtained from the colonic mucosa were assessed by flow cytometry as described in Patients and Methods. Mucosal colonic cells were double-stained with isotype IgG control or mAbs to TLR2, TLR4 or CD14 together with APC-conjugated anti Epcam mAbs. ( A ) Representative histogram showing the fluorescence intensity of surface TLR4 expression in gated Epcam+ cells from all IBS subtypes and controls patients. Changes in TLR4 surface expression ( B ), intracellular TLR4 expression ( C ), CD14 expression ( D ) and TLR2 surface expression ( E ) in IECs in control and IBS subtypes. Values are expressed as mean intensities fluorescence ± SEM. (*) Represents p<0.05 and (**) represents p<0.005 using Mann-Whitney test. Kruskal-Wallis p values are *p = 0.01 (B); p = 0.5 (C); p = 0.95 (D) and *p = 0.03 (E) respectively.CC BY · PubMed Central
Figure 5 Relative PPARγ protein expression in colonic mucosa. ( A ) Representative western blot for PPARγ expression in colonic tissue from IBS subgroups and control. ( B ) Western blot analysis was performed for PPARγ expression on colonic biopsy lysates of control (n = 25) and IBS-C (n = 13), or IBS-D (n = 12), or IBS-M (n = 14). Data are expressed as means ± SEM; *p = 0.0033 vs. Controls, using Mann Whitney test.
Figure 5 Relative PPARγ protein expression in colonic mucosa. ( A ) Representative western blot for PPARγ expression in colonic tissue from IBS subgroups and control. ( B ) Western blot analysis was performed for PPARγ expression on colonic biopsy lysates of control (n = 25) and IBS-C (n = 13), or IBS-D (n = 12), or IBS-M (n = 14). Data are expressed as means ± SEM; *p = 0.0033 vs. Controls, using Mann Whitney test.CC BY · PubMed Central
Figure 6 Levels of pro-inflammatory cytokines and chemokines in colonic mucosa. Cytokines were measured by multiplex cytokine bead immunoassays as described previously in Patients and Methods. Protein extracts of colonic mucosa were evaluated in duplicate for the expression of IL-8 ( A ), IL1β ( B ), TNF-α ( C ), IL-6 ( D ) and IFN γ ( E ) in IBS patients and controls. Results were expressed as pg/mg protein. Differences in protein secretion were determined to be statistically significant by Mann-Whitney test with a value of p<0.05 (*), p<0.005 (**) and p<0.0005 (***).
Figure 6 Levels of pro-inflammatory cytokines and chemokines in colonic mucosa. Cytokines were measured by multiplex cytokine bead immunoassays as described previously in Patients and Methods. Protein extracts of colonic mucosa were evaluated in duplicate for the expression of IL-8 ( A ), IL1β ( B ), TNF-α ( C ), IL-6 ( D ) and IFN γ ( E ) in IBS patients and controls. Results were expressed as pg/mg protein. Differences in protein secretion were determined to be statistically significant by Mann-Whitney test with a value of p<0.05 (*), p<0.005 (**) and p<0.0005 (***).CC BY · PubMed Central

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Authors
Belmonte, L., Beutheu Youmba, S., Bertiaux-Vandaële, N., Antonietti, M., Lecleire, S., Zalar, A., Gourcerol, G., Leroi, A.-M., Déchelotte, P., Coëffier, M., & Ducrotté, P.
Affiliations
  • Academia Nacional de Medicina (AR)
  • Centre Hospitalier Universitaire de Rouen (FR)
  • Inserm (FR)
Corresponding author
Belmonte, Liliana
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-08-17
Volume
7
Issue
8
Pages
e42777
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0042777
PMC
PMC3461726
OpenAlex
W2080290298
Cited by
126
References
38
Citations by year
2026: 1 · 2025: 1 · 2024: 9 · 2023: 3 · 2022: 15 · 2021: 10 · 2020: 11 · 2019: 6 · 2018: 10 · 2017: 7 · 2016: 11 · 2015: 14 · 2014: 18 · 2013: 10
FWCI
13.04
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
Keywords
irritable bowel syndrome, Toll-like receptors, TLR2, TLR4, colonic mucosa, IBS subtypes, intestinal epithelial cells, mucosal inflammation, inflammatory cytokines, interleukin-8, IL-1β, host-microbiota interactions, innate immune response, intestinal permeability
MeSH terms
Adult, Aged, Colon, Female, Humans, Intestinal Mucosa, Male, Middle Aged, RNA, Messenger, Up-Regulation, Gene Expression, Case-Control Studies, Cytokines, Inflammation Mediators, Lipopolysaccharide Receptors, Irritable Bowel Syndrome, PPAR gamma, Toll-Like Receptor 2, Toll-Like Receptor 4

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