presentación de powerpoint - uma 2016... · level: epithelial cells . tjs, ie lymphocytes, amp....

Agustín Albillos Hospital Universitario Ramón y Cajal Universidad de Alcalá, Ciberehd Madrid, Spain

“Barrera intestinal en la cirrosis”

XV Jornada de Avances en Hepatología Málaga

20 y 21 de Mayo de 2016

Modified from BA Duerkop et al. Immunity 2009

The intestinal barrier in health

1st level: External Host bacteria, mucus layer, Igs

3rd level: Subepithelial layer Immune system (DC, T cells, NK)

2nd level: Epithelial cells TJs, IE lymphocytes, AMP

Levels of defence

Com

parti

men

taliz

atio

n St

ratif

icat

ion

Urin

ary

excr

etio

n o

f DTP

A (%

)

↑Intestinal permeability in rats with cirrhosis and ascites and …

Control Cirrhosis

P<0.01

M Pérez-Páramo et al. Hepatology 2003

… patients with cirrhosis and ascites

A Parlesak et al. JHEP 2000

Abnormalities of intestinal mucosa in human and experimental cirrhosis

.

Structural • Congestion, edema, ↓ villous/crypt ratio • Inflammation, oxidative stress (↑ROS) • BBM lipid peroxidation • Alterations in TJ proteins Functional • ↑ small intestine (18 studies) and colonic permeability (Norman, 2012) • More severe in cirrhosis with ascites • Measured with sugar probes, and DTPA, EDTA, PEG

PEG

400

0

↑endotoxinemia in patients with cirrhosis

Increased intestinal permeability in cirrhosis

CCl4 cirrhosis Culture of MLN

48%

0%

Range 41-83% 10 studies

0%

Control Cirrhosis with

ascites

Cirrhosis without ascites

M Perez-Páramo et al. Hepatology 2000 G García-Tsao et al. Gastroenterology 2000

Increased bacterial translocation to the mesenteric lymph nodes (MLN)

in experimental and human cirrhosis with ascites

80

60

40

20

0 (%

)

Control Cirrhosis (Child)

A B C

3.6% 3% 8%

31%

Human cirrhosis Culture of MLN

I Cirera et al. J Hepatol 2001

MLN

Increased passage of enteric bacteria

Positive culture

(viable bacteria)

Impaired immune system

response

Increased intestinal permeability

Intestinal bacterial overgrowth

+

Blood Spontaneous bacterial infection

Gut bacterial translocation: Growth of viable bacteria in a mesenteric lymph node (MLN) culture

Healthy microbiota

Environmental and genetic factors ?

Intestinal hypomotility

Altered immunity

Altered bile flow

Increased gastric pH

Cirrhosis-associated dysbiosis

Gut bacterial translocation

Increased intestinal

permeability

Factors contributing to intestinal dysbiosis and gut bacterial translocation in cirrhosis

Phenobarbital & oral CCl4

weeks

Phenobarbital

CCl4 cirrhosis with ascites n=22

Experimental design

2-week oral course: • Obeticholic acid (5 mg/kg.d) or • Vehicle (carboxy-methyl-cellulose 0.5%)

Controls n = 14

Samples from: • blood • mesenteric lymph nodes • ileum • liver • ascitic fluid • spleen

Sprague Dawley rats

Sprague Dawley rats

2 weeks

2 weeks

Increased gut bacterial translocation and enteric bacterial load in cirrhotic rats with ascites

Gut bacterial translocation to

mesenteric lymph nodes

Total bacterial in ileum feces by

conventional culture

Total bacterial attached to the

ileum mucosa by qPCR

0%

25%

50%

75%

100%

Control Cirrhosis Cirrhosis OCA

Deferribacteres

Actinobacteria

Proteobacteria

Bacteroidetes

Firmicutes

0.6% 11%

2%

No. OTUs 4620 7509 5082

Intestinal dysbiosis in cirrhotic rats with ascites

Phyla distribution Principal

component analysis

“clusters”

↓↓ FXR reporter gen

↓ ↓ epithelium TJ proteins

↓ ↓ antimicrobial peptides

Lower expression of the FXR reporter gene, greater TJ damage and greater reduction of antimicrobial peptides in

cirrhotic rats with bacterial translocation

ZO-1 immunofluorescence

Control Cirrhosis

Reduced secretion of intestinal α-defensins by Paneth cells contributes to bacterial translocation in cirrhosis

Defensins in experimental cirrhosis • Rats with cirrhosis, ascites and GBT • ↓↓ Paneth cell α-defensins cryptidins 5&7 • More pronounced in the ileum and cecum • Not in pre-hepatic portal hypertension Z Telschik et al. Hepatology 2012

Functions of intestinal antimicrobial peptides

RL Gallo et al. Nat Rev Gastroenterol 2012

↑ ↑ intestinal inflammation

↑ ↑ intestinal permeability

More severe intestinal inflammation and greater increase in intestinal permeability in cirrhotic rats with bacterial translocation

J Turner et al. Nat Rev Gastroenterol 2009

Pro-inflammatory cytokines regulate tight junction permeability through MCLK

r=0.648 p=0.000

Correlation between fecal albumin loss and IFN-γ secreting T cytotoxic lymphocytes in cirrhotic rats with ascites

Control

%

Cirrhosis Cirrhosis +

Antibiotics

50

100

50

Control Cirrhosis Cirrhosis +

Antibiotics

2,5

5

Control Cirrhosis Cirrhosis +

Antibiotics

1

Tc-cells IFN-γ production

IFN-γ mRNA expression

TNF-α mRNA expression

P<0.05 P<0.01 P<0.01

Bowel decontamination reduces activated immune system cells and the production/expression of pro-inflammatory cytokines

in the ileal lamina propria of CCl4-cirrhotic rats

fold

exp

ress

ion

fold

exp

ress

ion

2,5

5

1

cell/

cm.1

0-3

100

150

50

10

20

15

30

5 5

Th-cells Activated Th-cells Tc-cells

P<0.01 P<0.01 P<0.01

Antibiotics Antibiotics Antibiotics

Antibiotics

Antibiotics

Antibiotics

Defects in the intestinal barrier in advanced cirrhosis: Contribution to GBT


Dysbiosis/IBO

Altered immunity ↓AMP

↓ bile flow/composi

tion

Intestinal inflammation

Altered immunity

“exhaustion”

Cirrhosis


↑ Intestinal permeability

?

Cirrhosis Culture - bDNA-

Cirrhosis Culture - bDNA+

Cirrhosis Culture + bDNA+

Control Cirrhosis Culture - bDNA-

Cirrhosis Culture - bDNA+

Cirrhosis Culture + bDNA+

Control

Antibiotics Antibiotics

L Muñoz et al. Hepatology 2012

Uptake of latex microbeads (phagocytic activity)

LPS-stimulated TNFα production

Antibiotics improve dendritic cell function of intestinal lamina propria of cirrhotic rats with bacterial translocation

Defects in the intestinal barrier in advanced cirrhosis: Contribution to GBT

Defects in the intestinal barrier in cirrhosis

• Dysbiosis and bacterial overgrowth - ↑ enteric aerobic bacterial load - dysbiosis: ↓ Firmicutes, ↑Proteobacteriaceae

• Damage of the epithelial barrier - structural damage -functional damage with↑permeability and ↑ endotoxinemia

• Intestinal inflammation - ↑ activated immune cells in the lamina propria with↑pro-inflammatory cytokines

• Exhaustion/tolerance of immune cells - Exhaustion of intestinal DCs → → immunodeficiency

• Impaired innate defence - ↓ antimicrobial peptides (α-defensin-5) by Paneth cells


Dysbiosis/IBO

Altered immunity ↓AMP

↓ bile flow/composi

tion

Intestinal inflammation

Altered immunity

“exhaustion”

Cirrhosis


↑ Intestinal permeability

?

CDCA - Enterobacteriaceae r= -0.57, p=0.008 CDCA - Bacterioidaceae r= 0.50, p=0.02

Secretion of bile in rats with cirrhosis (µL/kg.min)

G Kakiyama et al. JHEP 21013

V Lorenzo-Zúñiga et al. Hepatology 2003

Cirrhosis associates with changes in bile flow and composition

Correlation between microbiota composition and fecal bile acids in

human cirrhosis

• ↑ bile acid secretion • ↓ intestinal bacterial overgrowth • ↓ bacterial translocation (30 vs 66%) • ↓ endotoxinemia

Cecum bacterial overgrowth

Bacterial translocation to MLN

Oral bile acids or FXR agonist inhibit bacterial overgrowth, bacterial translocation, and ileal mucosal injury in cirrhotic rats

Bile duct ligated mice wild type and FXR-KO Treated with vehicle or GW404 FXR agonist

T Inagaki et al. PNAS 2006

Oral conjugated bile acids ( cholylsarcosine, cholylglycine)

in CCl4 cirrhotic rats

V Lorenzo-Zuñiga et al. Hepatology 2003

FXR agonist (GW404) in bile duct ligated cirrhotic mice

Enterohepatic effects of the farnesoid X receptor, FXR

Hepatocyte

Ileal cell

FXR functions: In the liver: • ↓BA synthesis • ↑BA export • Lipid and glucose metabolism In the intestine: • Antibacterial defence (angiogenin, iNOS) • Modulates innate immunity (↓NF-kB)

Moderador

Notas de la presentación

Figure 1 | Enterohepatic actions of FXR. Bile acids (exemplified as the primary bile acid CDCA) are produced in the liver by CYP7A1-initiated conversion of cholesterol in primary bile acids. Bile salts are secreted via BSEP into the canalicular lumen. In the ileum, bile salts are reabsorbed via ASBT in terminal ileum enterocytes. Here, they bind and activate FXR and this stimulates the transcription of FGF19, which encodes a protein that is secreted into the portal circulation. In the liver, FGF19 binds to its receptor FGFR4, which activates a signalling pathway involving MAP kinases and causes repression of CYP7A1, thus downregulating bile acid synthesis. After OSTα/β- mediated secretion into the portal circulation, bile acids are taken up by the liver via NTCP, thus, completing the enterohepatic cycle. In the liver, bile acids bind to FXR, which transcriptionally upregulates a protein called SHP (not shown) that interferes with expression of CYP7A1. Oral FXR agonists will affect FXR in both liver and intestine and this strongly downregulates CYP7A1 both by FGF19-dependent and FGF19- independent effects. FGF19 additionally affects lipogenesis, gluconeogenesis and liver regeneration. Abbreviations: ASBT, apical sodium-dependent bile salt transporter; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; CYP7A1, cholesterol 7‑α-monooxygenase; FGF19, fibroblast growth factor 19; FGFR4, fibroblast growth factor receptor 4; FXR, farnesoid X receptor; NTCP, Na+-taurocholate cotransporting polypeptide; OST, organic solute transporter; SHP, small heterodimer partner.

Phenobarbital & oral CCl4

weeks

Phenobarbital

CCl4 cirrhosis with ascites n=22

Experimental design

2-week oral course: • Obethicolic acid (5 mg/kg.d) or • Vehicle (carboxy-methyl-cellulose 0.5%)

Controls n = 14

Samples from: • blood • mesenteric lymph nodes • ileum • liver • ascitic fluid • spleen

Sprague Dawley rats

Sprague Dawley rats

2 weeks

2 weeks

Obeticholic acid (OCA) reduces gut bacterial translocation in cirrhotic rats with ascites

Gut bacterial translocation to

mesenteric lymph nodes

Total bacterial in ileum feces by

conventional culture

Total bacterial attached to the

ileum mucosa by qPCR

0%

25%

50%

75%

100%

Control Cirrhosis Cirrhosis OCA

Deferribacteres

Actinobacteria

Proteobacteria

Bacteroidetes

Firmicutes

0.6% 11%

2%

No. OTUs 4620 7509 5082

Phyla distribution Principal component

analysis “clusters”

Obeticholic acid (OCA) lowers the relative abundance of Proteobacteria in cirrhotic rats

SHP

Obeticholic acid (OCA) increases the synthesis of intestinal antimicrobial peptides in cirrhotic rats

Alpha-5-defensin Angiogenin-1

Obeticholic acid (OCA) restores intestinal epithelial integrity in cirrhotic rats

ZO-1 immunofluorescence

Control

Cirrhosis

Cirrhosis + OCA

ZO-1 Occludin

Obeticholic acid (OCA) ameliorates intestinal inflammation in cirrhotic rats with ascites

IFN-γ TNF-α IL-10

M Ubeda et al. JHEP 2016

Healthy microbiota

Environmental and genetic factors ?


Altered immunity

Altered bile flow

Increased gastric pH

Cirrhosis-associated dysbiosis


Increased intestinal

permeability

Factors contributing to intestinal dysbiosis and GBT in cirrhosis: Therapeutic targets

β-blockers Procinetics

Bile acids FXR agonists

Antibiotics Probiotics

?

• Intestinal inflammation in cirrhosis: - more severe in rats with GBT - consequence of dysbiosis - contributes to intestinal permeability • FXR agonists (i.e. obeticholic acid): - reduced GBT - ameliorate intestinal inflammation

Take home messages

presentación de powerpoint - uma 2016... · level: epithelial cells . tjs, ie lymphocytes, amp....

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