L. BuénoL. BuénoUnité de Neurogastroentérologie INRAUnité de Neurogastroentérologie INRA

Toulouse, FranceToulouse, France

ALTERATIONS DE LA BARRIERE MUQUEUSE ALTERATIONS DE LA BARRIERE MUQUEUSE ETET

SYNDROME DE L'INTESTIN IRRITABLESYNDROME DE L'INTESTIN IRRITABLE

Symposium Mayoly sur le Syndrome de l'Intestin Irritable.Symposium Mayoly sur le Syndrome de l'Intestin Irritable.OrganisateurOrganisateur: Pr. Boucekkine : Pr. Boucekkine

Alger, 25 Avril 2010 Alger, 25 Avril 2010

TRILOGY OF IBS PERIPHERAL PATHOPHYSIOLOGY

Altered gut sensitivity to DistensionAltered gut sensitivity to Distension

Colonic mucosal micro-inflammationColonic mucosal micro-inflammation

Increased paracellular permeabilityIncreased paracellular permeability

• Increased number of mast cells, immune cells (Weston et Increased number of mast cells, immune cells (Weston et al.1993 and 10-12 ref.)al.1993 and 10-12 ref.)• Presence of pro-inflammatory cytokines (Gwee et al. 2003)Presence of pro-inflammatory cytokines (Gwee et al. 2003)• Release of pro-inflammatory (eicosanoRelease of pro-inflammatory (eicosanoïds)(Jones et ïds)(Jones et al.1982)al.1982) and pronociceptive agents (Barbara et al. 2005) and pronociceptive agents (Barbara et al. 2005)

• colonic or intestinal level in PI-IBS (Dunlop,2000), colonic or intestinal level in PI-IBS (Dunlop,2000), • intestinal in all Rome I (Marschall et al.2004)intestinal in all Rome I (Marschall et al.2004)• colonic in IBS-D patients (Gesce et al. 2008)colonic in IBS-D patients (Gesce et al. 2008)

• Lower threshold of sensitivity (pain) to distension Lower threshold of sensitivity (pain) to distension evidenced in 60-70% of IBS patientsevidenced in 60-70% of IBS patients• Increased perception of pain for a given visceral stimulus Increased perception of pain for a given visceral stimulus (Whitehead et al.1998, 20-25 ref.) (Whitehead et al.1998, 20-25 ref.)

Trimble et al.1995Trimble et al.1995

1995-2009: Evidence (25 articles) of allodynia in 60-70% of IBS patients 1995-2009: Evidence (25 articles) of allodynia in 60-70% of IBS patients but not confirmed for all gut segmentsbut not confirmed for all gut segments

Trimble et al.1995Trimble et al.1995

Dorsal root ganglionDorsal root ganglion

Mechanosensitive afferentMechanosensitive afferent

Sensitized spinal circuits

Sensitized spinal circuitsRepeated

balloon distention

Repeated balloon

distention

Repetitive Stimulation Sensitizes the Spinal Cord

Repetitive Stimulation Sensitizes the Spinal Cord

Wind-upWind-up

Munakata J, Gastroenterology 1997; 112:55 Munakata J, Gastroenterology 1997; 112:55

Rectal Pain Threshold (mm Hg)

Rectal Pain Threshold (mm Hg)

Post sigmoidstimulationPost sigmoidstimulation

BaselineBaseline4545

4040

3535

3030

2525

2020

00

IBSIBS ControlsControls

HYPERALGESIA IN IRRITABLE BOWEL SYNDROMEHYPERALGESIA IN IRRITABLE BOWEL SYNDROME(Use of “barostatic” distensions)(Use of “barostatic” distensions)

HYPERALGESIA IN IRRITABLE BOWEL SYNDROMEHYPERALGESIA IN IRRITABLE BOWEL SYNDROME(Use of “barostatic” distensions)(Use of “barostatic” distensions)

IBSIBS

pACCpACC

MCCMCC

ThalamusPf, Re, Cl, LiThalamus

Pf, Re, Cl, Li

Prefrontal CortexCing Cx included

Prefrontal CortexCing Cx included

Brain Activation with Noxious Visceral StimulationBrain Activation with Noxious Visceral Stimulation

Locus coeruleusLocus coeruleus

Subnucleus reticularis dorsalis

Subnucleus reticularis dorsalis

Spinal cordLamina 1Spinal cordLamina 1

Evidence for colonic mucosal immune alterations and increasedEvidence for colonic mucosal immune alterations and increaseddensity of mast cell and immunocytes in IBS density of mast cell and immunocytes in IBS

Mast cellsMast cells

CD3CD3++

““spastic colon”spastic colon” ++++ Hiatt 1962Hiatt 1962

Lymphocytes TLymphocytes T Neutrophils Neutrophils ECCECCICCICC

CD4CD4++

CD8CD8++

CD25CD25PatientsPatients Ref.Ref.

IBS- D/CIBS- D/C ++++ Weston et al. 1993Weston et al. 1993

Jejun/Jejun/ileumileum

Cecum/Cecum/coloncolon

IBS- D/C/AIBS- D/C/A ++

IBS- D/C/A +PIIBS- D/C/A +PI ++++ ++++ ++++ Thorbloom et al. 2002Thorbloom et al. 2002

O’sullivan et al.2000O’sullivan et al.2000

Dunlop et al. 2003Dunlop et al. 2003IBS-PIIBS-PI ++++++++

Spiller 2004Spiller 2004IBS-PIIBS-PI ++++++++

Park et al. 2003Park et al. 2003++++IBS- DIBS- D

Chadwick et al. 2002Chadwick et al. 2002IBS-D/CIBS-D/C ++++ ++++ ++++++

Barbara et al. 2004Barbara et al. 2004IBS- D+SII-CIBS- D+SII-C ++++

ECC: enteric chromaffin cells; ICC : interstitial cell of Cajal; IBS-C: constipated patients;ECC: enteric chromaffin cells; ICC : interstitial cell of Cajal; IBS-C: constipated patients; IBS-D: diarrheoic patients; IBC-A: alternated diarrhea-constipation; PI: post-infectious IBS.IBS-D: diarrheoic patients; IBC-A: alternated diarrhea-constipation; PI: post-infectious IBS.

Chang et al. 2004Chang et al. 2004IBS- D/ PIIBS- D/ PI ++++

Park et al. 2006Park et al. 2006IBS- DIBS- D ++++++++

Lymphocytose ganglionnaire dans le SIILymphocytose ganglionnaire dans le SII

Infiltration de lymphocytes dans les ganglions myentériques. La flèche noire indique un Infiltration de lymphocytes dans les ganglions myentériques. La flèche noire indique un neurone et quelques lymphocytes à la base du neurone.Il ya plus de lymphocytes dans neurone et quelques lymphocytes à la base du neurone.Il ya plus de lymphocytes dans la zône délimitée par les les flêches bleues. Hématoxiline-éosine, X 380la zône délimitée par les les flêches bleues. Hématoxiline-éosine, X 380

(d’après Thorbloom et al. 2002)(d’après Thorbloom et al. 2002)

Sang

Colon

(Ohman et al.2005)(Ohman et al.2005)

Activation des lymphocytes T circulants CD4Activation des lymphocytes T circulants CD4++ et CD8 et CD8++ et expression de MAdCAM par l’endothélium colique dans l’IBSet expression de MAdCAM par l’endothélium colique dans l’IBS

IBS: D+C+AIBS: D+C+AUCr: RCH en rémission; UCa: RCH active; CTRL: TémoinsUCr: RCH en rémission; UCa: RCH active; CTRL: Témoins

Increased mast cell tryptase labeling in the sub-mucosa of IBS Increased mast cell tryptase labeling in the sub-mucosa of IBS patientspatients

Proximal colon biopsies in IBS = Proximal colon biopsies in IBS = density of mast cellsdensity of mast cells amount of tryptaseamount of tryptase colocalisation nerve-mast cellscolocalisation nerve-mast cells

COLONIC MAST CELLS IN CLONIC BIOPSIES OF COLONIC MAST CELLS IN CLONIC BIOPSIES OF IBS PATIENTS IBS PATIENTS

IBSCTRL

( Barbara et al., 2004)( Barbara et al., 2004)

MastocyteMastocyteStressStress

AllergieAllergie

InflammationInflammation

InfectionInfection

activationactivationSystème Système

CRFCRF

IgE, SP,5-HTIgE, SP,5-HT

NGF,

NPY

NG

F, N

PY

DegranulationDegranulation

SecretionSecretion

minutesminutes

heuresheures

GM-CSF, IL-1

GM-CSF, IL-1

PAF, ATPPAF, ATP

HistamineHistamineLeukotrienesLeukotrienesCytokinesCytokines**proteasesproteasesNGFNGF

CytokinesCytokines**ChemokinesChemokines

** Cytokines: TNFCytokines: TNF, IL-3, IL-5, IL-4, IL-13, IL-10, GM-CSF, IL-3, IL-5, IL-4, IL-13, IL-10, GM-CSF

(adapted from Shakoory et al,2004, Penicci et al.2003)(adapted from Shakoory et al,2004, Penicci et al.2003)

Facteurs principaux produisant la dégranulation ou l’activation des mastocytes muqueux du tube digestif

Distribution of nerve terminals close to mast cellDistribution of nerve terminals close to mast cellin IBS patients.in IBS patients.

MC

MC

Red: nerve terminals (enolase labeling)Red: nerve terminals (enolase labeling)Blue: mast cells (alcian blue)Blue: mast cells (alcian blue)

( Wang et al. Gut 2004 )( Wang et al. Gut 2004 )

Mast cell number (mm2)Mast cell number (mm2)(ileal mucosa)(ileal mucosa)

(X1000)

PI-IBS……… 11.2 ± 2.8PI-IBS……… 11.2 ± 2.8** (n=27)(n=27)

Non PI-IBS…..10.8 ±1.2Non PI-IBS…..10.8 ±1.2** (n=29)(n=29)

Control………..6.1±0.5Control………..6.1±0.5 (n=12)(n=12)

**: : from control at p<0.01from control at p<0.01

PI-IBS CTRL

00 55 1010 1515 202000

11

22

33

44

00 55 1010 1515 202000

11

22

33

44

(Barbara et al. Gastro. 2004)(Barbara et al. Gastro. 2004)

RELATIONSHIP BETWEEN MAST CELL-NERVESRELATIONSHIP BETWEEN MAST CELL-NERVES CONNECTION AND PAIN IN IBS PATIENTSCONNECTION AND PAIN IN IBS PATIENTS

Number of mast cells at a distance < 5µm from nervesNumber of mast cells at a distance < 5µm from nerves

Pai

n in

ten

sisi

ty s

cori

ng

Pai

n in

ten

sisi

ty s

cori

ng

Pai

n f

req

uen

cy s

cori

ng

Pai

n f

req

uen

cy s

cori

ng

Epithelial cellsEpithelial cells

Is increased gut permeability able to initiate mucosalIs increased gut permeability able to initiate mucosal immune response and visceral hypersensitivity?immune response and visceral hypersensitivity?

Increased paracellular permeabilityIncreased paracellular permeability==

Entry of pathogens, toxins, antigens, bacteria Entry of pathogens, toxins, antigens, bacteria

- activation of immunocytes- activation of immunocytes- cytokines releasecytokines release- inflammatory mediatorsinflammatory mediators

NociceptiveNociceptivehypersensitivity hypersensitivity PAINPAIN

MotilityMotilitydisordersdisorders

ENSENSdisorders disorders

T-cellT-cell

B-cellB-cell

MastMastcellcell

GranulocyteGranulocyteSensory nervesSensory nerves(nociceptive fibers)(nociceptive fibers)In

test

inal

or

colo

nic

mu

cosa

Inte

stin

al o

r co

lon

ic m

uco

sa

EpithelialEpithelialcellscells

Tight junction Tight junction PathogensPathogens AllergensAllergens

( from Perdue et al. 2000 )( from Perdue et al. 2000 )

(LPS, DNA,peptidoglycans,…etc.)(LPS, DNA,peptidoglycans,…etc.)

WT PAR-2-/-

*

0

100

200

300

INFLUENCE OF FECAL SUPERNATANT FROM IBS PATIENTS INFUSEDINFLUENCE OF FECAL SUPERNATANT FROM IBS PATIENTS INFUSEDON PARACELLULAR PERMEABILITY OF MICE COLONIC STRIPS ON PARACELLULAR PERMEABILITY OF MICE COLONIC STRIPS

Fecal supernatants (n=6)

IBS-D fecal supernatants (n=4)

IBS-D fecal supernatants (n=3)

Increase in permeability triggered by Increase in permeability triggered by apical application of IBS-D fecal apical application of IBS-D fecal supernatant is reduced by ser-protease supernatant is reduced by ser-protease inhibitor and is absent in PAR-2 inhibitor and is absent in PAR-2 -/- -/- KO KO micemice

(Gecse et al. 2008)(Gecse et al. 2008)

(Gecse et al.(Gecse et al.2008)2008)

INFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS INFUSEDINFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS INFUSEDINTRACOLONICALLY IN MICE ON COLONIC SENSITIVITY TO DISTENSIONINTRACOLONICALLY IN MICE ON COLONIC SENSITIVITY TO DISTENSION

P-MLC (green)

ZO-1 (green)

INFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS ON INFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS ON COLONIC EPITHELIAL TJ INTEGRITY IN MICECOLONIC EPITHELIAL TJ INTEGRITY IN MICE

P-MLC in colonic mucosa

(Gecse et al. 2008)(Gecse et al. 2008)

P-MLC is over expressed in colonic mucosa P-MLC is over expressed in colonic mucosa infused with IBS-D supernatant reflecting a infused with IBS-D supernatant reflecting a EC cytoskeleton contractionCEC cytoskeleton contractionC

Resulting opening of TJs is associated with a Resulting opening of TJs is associated with a reduced apical expression of ZO-1reduced apical expression of ZO-1

Luminal ser- proteasesLuminal ser- proteasesPAR-2 activationPAR-2 activation

Long-termhypersensitivity

T-cellT-cell

Afferent neuronsAfferent neurons

SPSP

IFNIFN

Mast cellMast cell

tryptasetryptase

tryptasetryptase

Increasedpermeability

(mins to hours)

Mucosalmicro-inflammation(hours to weeks)

Nerve terminalsensitization

(weeks to months)

InflammatoryInflammatorymediatorsmediators

( Bueno et al. 2008)( Bueno et al. 2008)

Mechanisms involved in long-term sensitization of mucosal sensory nerves in IBS-D patients

enzymesenzymes

Biliary saltsBiliary salts bacteria (proteases?)bacteria (proteases?)Allergens, parasitesAllergens, parasites

stressstress sepsissepsis

Bacterial secetion or lysis (acetaldehyde, LPS…etc)Bacterial secetion or lysis (acetaldehyde, LPS…etc)

InflammationInflammation(gastroenteritis)(gastroenteritis)

Factors able to alter gut permeability/sensitivity in FGIDFactors able to alter gut permeability/sensitivity in FGID

Piche et al. Gut 2009Piche et al. Gut 2009

IN VITRO MEASUREMENT OF PARACELLULAR PERMEABILITY OF IN VITRO MEASUREMENT OF PARACELLULAR PERMEABILITY OF COLONIC BIOPSIES (Ussing chambers)COLONIC BIOPSIES (Ussing chambers)

The degree of porosity of biopsies The degree of porosity of biopsies from IBS patients is higher than from IBS patients is higher than that of healthy subjects that of healthy subjects independently of bowel habit independently of bowel habit alterations. alterations.

This altered permeability is associated This altered permeability is associated with a decrease in the expression of with a decrease in the expression of ZO-1, a protein linking the ZO-1, a protein linking the actinomyosin apical ring to the proteins actinomyosin apical ring to the proteins of the TJs of the TJs

EFFECTS OF COLONIC BIOPSY SUPERNATANT ON CaCo2 CELL PERMEABILITY AND CORRELATIONS WITH SYMPTOM SCORES

Increase of permeability of CaCo2 Increase of permeability of CaCo2 cells, 48h after mucosal exposure cells, 48h after mucosal exposure with supernatant of biopsies from with supernatant of biopsies from IBS patientsIBS patients

Changes in permeability of CaCo2 Changes in permeability of CaCo2 cells is correlated with the pain cells is correlated with the pain score of explored IBS patientsscore of explored IBS patients

Piche et al. Gut 2009Piche et al. Gut 2009

NormalNormal IBSIBS

10.010.0

7.57.5

5.05.0

2.52.5

0.00.0V

AS

sco

re (

visc

eral

)V

AS

sco

re (

visc

eral

)

VISCERAL PAIN* VISCERAL PAIN*

Zhou et al. Pain 2009Zhou et al. Pain 2009

0.300.30

0.250.25

0.200.20

0.150.15

0.100.10

0.050.05

0.000.00

Lact

ulos

e/ M

anni

tol

Lact

ulos

e/ M

anni

tol

NormalNormal IBSIBS

INTESTINAL PERMEABILITYINTESTINAL PERMEABILITY

RELATIONSHIPS BETWEEN GUT PERMEABILITY AND PAINFUL SENSATIONSRELATIONSHIPS BETWEEN GUT PERMEABILITY AND PAINFUL SENSATIONSTO DISTENSION MEASURED IN VIVOTO DISTENSION MEASURED IN VIVO

Pain measurement after repeated (2) 35mm Hg rectal distension performed during 30 sec. Pain measurement after repeated (2) 35mm Hg rectal distension performed during 30 sec. at 2 min. interval.at 2 min. interval.

**

Zhou et al. Pain 2009Zhou et al. Pain 2009

120120

100100

8080

6060

4040

2020

00

FB

DS

I sc

ore

FB

DS

I sc

ore

CONTROLCONTROL IBSIBS

RELATIONSHIPS BETWEEN GUT PERMEABILITY AND SOMATIC* SENSITIVITYRELATIONSHIPS BETWEEN GUT PERMEABILITY AND SOMATIC* SENSITIVITYIN IBS PATIENTSIN IBS PATIENTS

Patients with Patients with altered altered

permeabilitypermeability

skin thermal stimulus (Pelletier probe 3x3 cms) at 47°C applied to the left hand during 10 sec.skin thermal stimulus (Pelletier probe 3x3 cms) at 47°C applied to the left hand during 10 sec.**

CONCLUSIONS CONCLUSIONS

La douleur abdominale associée au SII à le plus souvent comme La douleur abdominale associée au SII à le plus souvent comme origine une hypersensibilité intestinale ou colique à la distension.origine une hypersensibilité intestinale ou colique à la distension.

Cette hypersensibilité est associée à une micro-inflammation de la Cette hypersensibilité est associée à une micro-inflammation de la paroi pouvant paroi pouvant être considérée comme résultant d'une augmentation de être considérée comme résultant d'une augmentation de la “porosité“ de la muqueuse colique associée au passage de bactéries la “porosité“ de la muqueuse colique associée au passage de bactéries et toxineset toxines..

Certains facteurs luminaux dont les protéases agissent sur les Certains facteurs luminaux dont les protéases agissent sur les récepteurs des cellules épithéliales pour augmenter cette perméabilité récepteurs des cellules épithéliales pour augmenter cette perméabilité

Cette augmentation de perméabilité à été montrée, in vivo et in vitro, Cette augmentation de perméabilité à été montrée, in vivo et in vitro, être être corrélée aux symptômes corrélée aux symptômes