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Cited by 383Inflammatory Bowel DiseaseDrug Transport and Resistance MechanismsGastrointestinal motility and disorders

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The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of Intestinal Barrier and Immune Response to Experimental Colitis

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Cipriani, Sabrina, Mencarelli, Andrea, Chini, Maria Giovanna, Distrutti, Eleonora, Renga, Barbara, Bifulco, Giuseppe, Baldelli, Franco, Donini, Annibale, Fiorucci, Stefano·2011·PLoS ONE
DOI 10.1371/journal.pone.0025637PMC PMC3203117
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Abstract

BACKGROUND: GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation. AIMS: To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis. METHODS: Colitis was induced in wild type and GP-BAR1(-/-) mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies. RESULTS: GP-BAR1(-/-) mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn's disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1. CONCLUSIONS: GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn's disease. Ciprofloxacin is a GP-BAR1 ligand.

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Figure 1 GP-BAR1 gene deletion alters colon structure and function. Panel A. Upper panels: H&E staining of colonic sections obtained from 12-month old GP-BAR1+/+ and GP-BAR1−/− mice. A significant reduction in colon cellularity and crypt distortion is observed in the colon of GP-BAR1−/− mice. Lower panels: immunohistochemical analysis of GP-BAR1 in wild type mice showing expression in epithelial cells. No staining is observed when the primary antibody is omitted. Original magnification 20× and 40× (insect). Panel B. Alcian blue staining of colon sections from 12-month old mice showing a severe reduction of mucous cells and their impaired maturation in GP-BAR1−/− mice. Note the marked reduction of alcian blue positive cells in the crypts. Original magnification 20×. Panel C. RT-PCR analysis of mRNA expression of junctional proteins (zonulin-1, occludin and E-cadherin) in wild type and GP-BAR1−/− mice (n = 6; P<0.05). 12-months GP-BAR1−/− mice also show a significant increase in intestinal permeability to dextrane FITC (n = 6–8; P<0.05 versus naive). Panel D. Immuno-histochemical detection and immuno-fluorescence localization of zonulin-1. The staining of the protein is increased in GP-BAR1−/− animals but its cells localization appears to be discontinuous (arrows). Original magnification 20×.
Figure 1 GP-BAR1 gene deletion alters colon structure and function. Panel A. Upper panels: H&E staining of colonic sections obtained from 12-month old GP-BAR1+/+ and GP-BAR1−/− mice. A significant reduction in colon cellularity and crypt distortion is observed in the colon of GP-BAR1−/− mice. Lower panels: immunohistochemical analysis of GP-BAR1 in wild type mice showing expression in epithelial cells. No staining is observed when the primary antibody is omitted. Original magnification 20× and 40× (insect). Panel B. Alcian blue staining of colon sections from 12-month old mice showing a severe reduction of mucous cells and their impaired maturation in GP-BAR1−/− mice. Note the marked reduction of alcian blue positive cells in the crypts. Original magnification 20×. Panel C. RT-PCR analysis of mRNA expression of junctional proteins (zonulin-1, occludin and E-cadherin) in wild type and GP-BAR1−/− mice (n = 6; P<0.05). 12-months GP-BAR1−/− mice also show a significant increase in intestinal permeability to dextrane FITC (n = 6–8; P<0.05 versus naive). Panel D. Immuno-histochemical detection and immuno-fluorescence localization of zonulin-1. The staining of the protein is increased in GP-BAR1−/− animals but its cells localization appears to be discontinuous (arrows). Original magnification 20×.CC BY · PubMed Central
Figure 2 GP-BAR1 gene ablation predisposes to development of severe colitis in response to DSS in 3 months old mice C57Bl/6 mice. Panel A–C. GP-BAR1−/− mice develop a severe disease (n = 6; P<0.05), while changes of intestinal permeability were increased in GP-BAR1−/− mice in comparison to wild type mice (n = 10; *P<0.05 versus naive; **P<0.05 versus wild type DSS). Panel D. Immuno-histochemical detection of GP-BAR1 expression in the colon of wild type mice challenged with DSS. GP-BAR1 is abundantly expressed in mononuclear cells infiltrating the lamina propria. Original magnification 20× and 40× (inset). Panel E. Colon expression of GP-BAR1 mRNA in wild type mice challenged with DSS (n = 6; P<0.05). Panel F. Treatment of GP-BAR1−/− mice with DSS results in a dramatic and diffuse loss of alcian blue-positive mucous cells. Original magnification 10×.
Figure 2 GP-BAR1 gene ablation predisposes to development of severe colitis in response to DSS in 3 months old mice C57Bl/6 mice. Panel A–C. GP-BAR1−/− mice develop a severe disease (n = 6; P<0.05), while changes of intestinal permeability were increased in GP-BAR1−/− mice in comparison to wild type mice (n = 10; *P<0.05 versus naive; **P<0.05 versus wild type DSS). Panel D. Immuno-histochemical detection of GP-BAR1 expression in the colon of wild type mice challenged with DSS. GP-BAR1 is abundantly expressed in mononuclear cells infiltrating the lamina propria. Original magnification 20× and 40× (inset). Panel E. Colon expression of GP-BAR1 mRNA in wild type mice challenged with DSS (n = 6; P<0.05). Panel F. Treatment of GP-BAR1−/− mice with DSS results in a dramatic and diffuse loss of alcian blue-positive mucous cells. Original magnification 10×.CC BY · PubMed Central
Figure 3 Ciprofloxacin is a GP-BAR1 ligand. Panel A. Chemical structure of taurolithocholic acid (1) a natural GP-BAR1 ligand and ciprofloxacin (2). Panel B. The predicted GP-BAR1 three dimensional structure was used to analyze the interactions of taurolithocholic acid (lefts) and ciprofloxacin (right) with its binding domain. Both taurolithocholic acid (purple) and ciprofloxacin (yellow) accommodates on the GP-BAR1 binding site (lower panel). Panel C. Ciprofloxacin and TLCA increases [cAMP]i in GLUTag cells. N = 4; P<0.05. Panel D. Ciprofloxacin and taurolithocholic acid , 10 µM, caused a 2–3 fold increase in [cAMP]i in spleen-derived monocytes isolated from GP-BAR1 wild type mice (n = 4–5; P<0.05), but not in cells isolated from GP-BAR1−/− mice. Panel E. Ciprofloxacin, 10 µM, inhibits LPS-induced TNFα release in GP-BAR1+/+ monocytes but not in cells isolated from GP-BAR1−/− mice (n = 6; P<0.05 versus naive).
Figure 3 Ciprofloxacin is a GP-BAR1 ligand. Panel A. Chemical structure of taurolithocholic acid (1) a natural GP-BAR1 ligand and ciprofloxacin (2). Panel B. The predicted GP-BAR1 three dimensional structure was used to analyze the interactions of taurolithocholic acid (lefts) and ciprofloxacin (right) with its binding domain. Both taurolithocholic acid (purple) and ciprofloxacin (yellow) accommodates on the GP-BAR1 binding site (lower panel). Panel C. Ciprofloxacin and TLCA increases [cAMP]i in GLUTag cells. N = 4; P<0.05. Panel D. Ciprofloxacin and taurolithocholic acid , 10 µM, caused a 2–3 fold increase in [cAMP]i in spleen-derived monocytes isolated from GP-BAR1 wild type mice (n = 4–5; P<0.05), but not in cells isolated from GP-BAR1−/− mice. Panel E. Ciprofloxacin, 10 µM, inhibits LPS-induced TNFα release in GP-BAR1+/+ monocytes but not in cells isolated from GP-BAR1−/− mice (n = 6; P<0.05 versus naive).CC BY · PubMed Central
Figure 4 Effects of ciprofloxacin on colitis. Panel A. Systemic administration of ciprofloxacin (30 mg/kg/day) protects against development of signs and symptoms of colitis induced by TNBS in GP-BAR1+/+ mice (n = 6–8; p<0.05 versus naïve). Panel B and C. Ciprofloxacin attenuates histopathology changes induced by TNBS (n = 6–8; p<0.05 versus naïve). Original magnification 10× and 20×. Panel D and E. TNBS colitis associates with a robust influx of GP-BAR1 positive cells into the lamina propria of the colon and increased expression of GP-BAR1 mRNA. Mononuclear cells infiltrating the lamina propria show a robust staining for GP-BAR1 (arrow). This upregulation was partially attenuated by ciprofloxacin. Panel F. Immuno-hisyochemistry and RT-PCR analysis of GP-BAR1 expression in the colon of Crohn's disease patients. n = 6; P<0.05 versus control subjects. Panel G. RT-PCR analysis of expression of signature cytokines in wild type mice exposed to TNBS. Treatment with ciprofloxacin reduces significantly the expression of IL-6, TNF-α, IL-1β and INF-γ mRNAs (n = 6; P<0.05).
Figure 4 Effects of ciprofloxacin on colitis. Panel A. Systemic administration of ciprofloxacin (30 mg/kg/day) protects against development of signs and symptoms of colitis induced by TNBS in GP-BAR1+/+ mice (n = 6–8; p<0.05 versus naïve). Panel B and C. Ciprofloxacin attenuates histopathology changes induced by TNBS (n = 6–8; p<0.05 versus naïve). Original magnification 10× and 20×. Panel D and E. TNBS colitis associates with a robust influx of GP-BAR1 positive cells into the lamina propria of the colon and increased expression of GP-BAR1 mRNA. Mononuclear cells infiltrating the lamina propria show a robust staining for GP-BAR1 (arrow). This upregulation was partially attenuated by ciprofloxacin. Panel F. Immuno-hisyochemistry and RT-PCR analysis of GP-BAR1 expression in the colon of Crohn's disease patients. n = 6; P<0.05 versus control subjects. Panel G. RT-PCR analysis of expression of signature cytokines in wild type mice exposed to TNBS. Treatment with ciprofloxacin reduces significantly the expression of IL-6, TNF-α, IL-1β and INF-γ mRNAs (n = 6; P<0.05).CC BY · PubMed Central
Figure 5 Flow-cytometry analysis of GP-BAR1 expression in unfractioned lamina propria mononuclear cells (LPMC) and LPMC-derived CD3+(A) and CD14+ cells (B) obtained from colons of naïve mice and mice administered TNBS alone and in combination with ciprofloxacin (n = 6; *P<0.05 versus naïve mice, **P<0.05 versus TNBS mice). Panel C. Bar graphs of quantitative expression of GP-BAR1 in unfractioned LPMC. Panel D. Flow cytometry histogram showing GP-BAR1 protein expression in LPMC in different treatment settings. Panel E. Representative Dot plot (forward scatter/ side Scatter) of LMPC obtained from colons of naïve and colitic mice treated with ciprofloxacin. Representative dot plot graphs of relative expression of GP-BAR1 CD3+ and CD14+ cell obtained from LPMC. The numerical values indicate the means ± SE of % double positive cells.
Figure 5 Flow-cytometry analysis of GP-BAR1 expression in unfractioned lamina propria mononuclear cells (LPMC) and LPMC-derived CD3+(A) and CD14+ cells (B) obtained from colons of naïve mice and mice administered TNBS alone and in combination with ciprofloxacin (n = 6; *P<0.05 versus naïve mice, **P<0.05 versus TNBS mice). Panel C. Bar graphs of quantitative expression of GP-BAR1 in unfractioned LPMC. Panel D. Flow cytometry histogram showing GP-BAR1 protein expression in LPMC in different treatment settings. Panel E. Representative Dot plot (forward scatter/ side Scatter) of LMPC obtained from colons of naïve and colitic mice treated with ciprofloxacin. Representative dot plot graphs of relative expression of GP-BAR1 CD3+ and CD14+ cell obtained from LPMC. The numerical values indicate the means ± SE of % double positive cells.CC BY · PubMed Central
Figure 6 Anti-inflammatory effects of oleanolic acid. Oleanolic acid (panel A), a natural GP-BAR1 agonist, exerts anti-inflammatory activity and protects against colitis development in wild type mice. Panel B. Oleanolic acid increases [cAMP]i in RAW cells in a concentration-dependent manner. N = 4; *P<0.05 versus basal. Panel C. Oleanolic acid reduces TNFα release from murine macrophages exposed to LPS. N = 4. *P<0.05; **P<0.05 versus LPS alone. Panel D–F. Oleanolic acid dose-dependently attenuates sign and symptoms of colitis induced by TNBS in wild type mice. N = 5 per group. *P<0.05 versus naive: ** P<0.05 versus TNBS alone. Panel G–I. Oleanolic acid treatment leads to a significant reduction in the colonic expression of inflammatory mediators including TNF-α and IL-6. *P<0.05 versus naive: ** P<0.05 versus TNBS alone.
Figure 6 Anti-inflammatory effects of oleanolic acid. Oleanolic acid (panel A), a natural GP-BAR1 agonist, exerts anti-inflammatory activity and protects against colitis development in wild type mice. Panel B. Oleanolic acid increases [cAMP]i in RAW cells in a concentration-dependent manner. N = 4; *P<0.05 versus basal. Panel C. Oleanolic acid reduces TNFα release from murine macrophages exposed to LPS. N = 4. *P<0.05; **P<0.05 versus LPS alone. Panel D–F. Oleanolic acid dose-dependently attenuates sign and symptoms of colitis induced by TNBS in wild type mice. N = 5 per group. *P<0.05 versus naive: ** P<0.05 versus TNBS alone. Panel G–I. Oleanolic acid treatment leads to a significant reduction in the colonic expression of inflammatory mediators including TNF-α and IL-6. *P<0.05 versus naive: ** P<0.05 versus TNBS alone.CC BY · PubMed Central
Figure 7 Anti-inflammatory activities of ciprofloxacin are GP-BAR1 dependent. Panel A. Ciprofloxacin fails to attenuates colitis induced by TNBS in GP-BAR1−/− mice. Colitis was induced by administering TNBS to GP-BAR1+/+ and GP-BAR1−/− mice on a C57BL6J background. Treatment with TNBS results in epithelial degeneration and in intense inflammatory infiltrate. Co- treatment with ciprofloxacin, 30 mg/kg/day, reduces inflammatory infiltrate and epithelial degeneration in wild type mice but not in GP-BAR1−/− mice. Panel B. Ciprofloxacin, 30 mg/kg/day, failed to attenuates signs and symptoms of colitis and expression of signature cytokines in GP-BAR1−/− mice challenged with TNBS. (n = 6–8; p>0.05 versus naïve; **P<0.05 versus TNBS). Panel C. Systemic administration of ciprofloxacin exerts a comparable antibiotic effect in GP-BAR1+/+ and GP-BAR1−/− mice (n = 5 mice per group). Values are not statistically different.
Figure 7 Anti-inflammatory activities of ciprofloxacin are GP-BAR1 dependent. Panel A. Ciprofloxacin fails to attenuates colitis induced by TNBS in GP-BAR1−/− mice. Colitis was induced by administering TNBS to GP-BAR1+/+ and GP-BAR1−/− mice on a C57BL6J background. Treatment with TNBS results in epithelial degeneration and in intense inflammatory infiltrate. Co- treatment with ciprofloxacin, 30 mg/kg/day, reduces inflammatory infiltrate and epithelial degeneration in wild type mice but not in GP-BAR1−/− mice. Panel B. Ciprofloxacin, 30 mg/kg/day, failed to attenuates signs and symptoms of colitis and expression of signature cytokines in GP-BAR1−/− mice challenged with TNBS. (n = 6–8; p>0.05 versus naïve; **P<0.05 versus TNBS). Panel C. Systemic administration of ciprofloxacin exerts a comparable antibiotic effect in GP-BAR1+/+ and GP-BAR1−/− mice (n = 5 mice per group). Values are not statistically different.CC BY · PubMed Central

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Authors
Cipriani, S., Mencarelli, A., Chini, M. G., Distrutti, E., Renga, B., Bifulco, G., Baldelli, F., Donini, A., & Fiorucci, S.
Affiliations
  • University of Perugia (IT)
  • University of Salerno (IT)
  • Azienda Ospedaliera di Perugia (IT)
Corresponding author
Fiorucci, Stefano
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-10-27
Volume
6
Issue
10
Pages
e25637
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0025637
PMC
PMC3203117
OpenAlex
W1970465623
Cited by
382
References
30
Citations by year
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FWCI
10.58
Type
journal article
Language
en
Update notices
correction (2013-01-29)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • National University of Singapore (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
  • Archivio Istituzionale della Ricerca - Università degli Studi del Molise (submittedVersion)
Topics
  • Health Sciences › Medicine › Gastroenterology › Inflammatory Bowel Disease
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
Keywords
TGR5, bile acid receptors, colitis, intestinal barrier integrity, tight junctions, intestinal permeability, ciprofloxacin, oleanolic acid, cAMP signaling, TNF-α, TLR4, monocytes, Crohn's disease, DSS-induced colitis, TNBS-induced colitis, lamina propria
MeSH terms
Animals, Cholera Toxin, Ciprofloxacin, Colitis, Haptoglobins, Immunity, Intestinal Mucosa, Ligands, Permeability, Protein Precursors, Mice, Knockout, Tight Junctions, Receptors, G-Protein-Coupled, Mice

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