XV Jornada de Avances en Hepatología Málaga 20 y 21 de Mayo de 2016 Barrera intestinal en la cirrosis Agustín Albillos Hospital Universitario Ramón y Cajal Universidad de Alcalá, Ciberehd Madrid, Spain
The intestinal barrier in health Levels of defence Compartimentalization Stratification 1 st level: External Host bacteria, mucus layer, Igs 2 nd level: Epithelial cells TJs, IE lymphocytes, AMP 3 rd level: Subepithelial layer Immune system (DC, T cells, NK) Modified from BA Duerkop et al. Immunity 2009
Increased intestinal permeability in cirrhosis Intestinal permeability in rats with cirrhosis and ascites and P<0.01 Urinary excretion of DTPA (%) Control Cirrhosis M Pérez-Páramo et al. Hepatology 2003 endotoxinemia in patients with cirrhosis 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 PEG 4000 patients with cirrhosis and ascites A Parlesak et al. JHEP 2000 Functional small intestine (18 studies) and colonic permeability (Norman, 2012) More severe in cirrhosis with ascites Measured with sugar probes, and DTPA, EDTA, PEG
Increased bacterial translocation to the mesenteric lymph nodes (MLN) in experimental and human cirrhosis with ascites CCl 4 cirrhosis Culture of MLN Range 41-83% 10 studies 48% 80 60 Human cirrhosis Culture of MLN (%) 40 31% 0% Control 0% Cirrhosis without ascites Cirrhosis with ascites 20 0 3.6% 3% Control 8% A B C Cirrhosis (Child) G García-Tsao et al. Gastroenterology 2000 M Perez-Páramo et al. Hepatology 2000 I Cirera et al. J Hepatol 2001
Gut bacterial translocation: Growth of viable bacteria in a mesenteric lymph node (MLN) culture MLN Blood Spontaneous bacterial infection Positive culture (viable bacteria) Increased passage of enteric bacteria + Impaired immune system response Increased intestinal permeability Intestinal bacterial overgrowth
Factors contributing to intestinal dysbiosis and gut bacterial translocation in cirrhosis Environmental and genetic factors? Intestinal hypomotility Altered bile flow Increased intestinal permeability Healthy microbiota Increased gastric ph Altered immunity Cirrhosisassociated dysbiosis Gut bacterial translocation
Experimental design CCl 4 cirrhosis with ascites n=22 Sprague Dawley rats Phenobarbital & oral CCl 4 weeks 2-week oral course: Obeticholic acid (5 mg/kg. d) or 2 weeks Vehicle (carboxy-methyl-cellulose 0.5%) Samples from: blood mesenteric lymph nodes ileum liver ascitic fluid spleen 2 weeks Sprague Dawley rats Phenobarbital Controls n = 14
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
Intestinal dysbiosis in cirrhotic rats with ascites 100% Phyla distribution 2% 11% 0.6% Principal component analysis clusters 75% Deferribacteres Actinobacteria 50% Proteobacteria Bacteroidetes Firmicutes 25% 0% Control Cirrhosis Cirrhosis OCA No. OTUs 4620 7509 5082
Lower expression of the FXR reporter gene, greater TJ damage and greater reduction of antimicrobial peptides in cirrhotic rats with bacterial translocation FXR reporter gen epithelium TJ proteins antimicrobial peptides ZO-1 immunofluorescence Control Cirrhosis
Reduced secretion of intestinal α-defensins by Paneth cells contributes to bacterial translocation in cirrhosis Functions of intestinal antimicrobial peptides 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 RL Gallo et al. Nat Rev Gastroenterol 2012
More severe intestinal inflammation and greater increase in intestinal permeability in cirrhotic rats with bacterial translocation intestinal inflammation intestinal permeability
Pro-inflammatory cytokines regulate tight junction permeability through MCLK J Turner et al. Nat Rev Gastroenterol 2009
Correlation between fecal albumin loss and IFN-γ secreting T cytotoxic lymphocytes in cirrhotic rats with ascites r=0.648 p=0.000
Bowel decontamination reduces activated immune system cells and the production/expression of pro-inflammatory cytokines in the ileal lamina propria of CCl 4 -cirrhotic rats 150 Th-cells 20 Activated Th-cells 30 Tc-cells P<0.01 P<0.01 P<0.01 cell/cm.10-3 100 Antibiotics 10 Antibiotics 15 Antibiotics 50 5 5 100 Tc-cells IFN-γ production 5 IFN-γ mrna expression TNF-α mrna expression P<0.05 P<0.01 P<0.01 5 % 50 fold expression Antibiotics Antibiotics Antibiotics 2,5 fold expression 2,5 50 1 1 Control Cirrhosis Cirrhosis + Antibiotics Control Cirrhosis Cirrhosis + Antibiotics Control Cirrhosis Cirrhosis + Antibiotics
Defects in the intestinal barrier in advanced cirrhosis: Contribution to GBT Cirrhosis Intestinal hypomotility? Altered immunity AMP bile flow/composi tion Dysbiosis/IBO Intestinal inflammation Intestinal permeability Altered immunity exhaustion Gut bacterial translocation
Antibiotics improve dendritic cell function of intestinal lamina propria of cirrhotic rats with bacterial translocation LPS-stimulated TNFα production Uptake of latex microbeads (phagocytic activity) Antibiotics Antibiotics Cirrhosis Culture - bdna- Cirrhosis Culture - bdna+ Cirrhosis Culture + bdna+ Control Cirrhosis Culture - bdna- Cirrhosis Culture - bdna+ Cirrhosis Culture + bdna+ Control L Muñoz et al. Hepatology 2012
Defects in the intestinal barrier in advanced cirrhosis: Contribution to GBT Intestinal hypomotility Cirrhosis? Altered immunity AMP Dysbiosis/IBO Intestinal inflammation Intestinal permeability bile flow/composi tion Altered immunity exhaustion 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 Gut bacterial translocation Impaired innate defence - antimicrobial peptides (α-defensin-5) by Paneth cells
Cirrhosis associates with changes in bile flow and composition Secretion of bile in rats with cirrhosis (µl/kg.min) Correlation between microbiota composition and fecal bile acids in human cirrhosis CDCA - Enterobacteriaceae CDCA - Bacterioidaceae r= -0.57, p=0.008 r= 0.50, p=0.02 V Lorenzo-Zúñiga et al. Hepatology 2003 G Kakiyama et al. JHEP 21013
Oral bile acids or FXR agonist inhibit bacterial overgrowth, bacterial translocation, and ileal mucosal injury in cirrhotic rats Oral conjugated bile acids ( cholylsarcosine, cholylglycine) in CCl 4 cirrhotic rats FXR agonist (GW404) in bile duct ligated cirrhotic mice Cecum bacterial overgrowth Bacterial translocation to MLN bile acid secretion intestinal bacterial overgrowth bacterial translocation (30 vs 66%) endotoxinemia Bile duct ligated mice wild type and FXR-KO Treated with vehicle or GW404 FXR agonist V Lorenzo-Zuñiga et al. Hepatology 2003 T Inagaki et al. PNAS 2006
Enterohepatic effects of the farnesoid X receptor, FXR Hepatocyte FXR functions: In the liver: BA synthesis BA export Lipid and glucose metabolism Ileal cell In the intestine: Antibacterial defence (angiogenin, inos) Modulates innate immunity ( NF-kB)
Experimental design CCl 4 cirrhosis with ascites n=22 Sprague Dawley rats Phenobarbital & oral CCl 4 weeks 2-week oral course: Obethicolic acid (5 mg/kg. d) or 2 weeks Vehicle (carboxy-methyl-cellulose 0.5%) Samples from: blood mesenteric lymph nodes ileum liver ascitic fluid spleen 2 weeks Sprague Dawley rats Phenobarbital Controls n = 14
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
Obeticholic acid (OCA) lowers the relative abundance of Proteobacteria in cirrhotic rats 100% Phyla distribution 2% 11% 0.6% Principal component analysis clusters 75% Deferribacteres Actinobacteria 50% Proteobacteria Bacteroidetes Firmicutes 25% 0% Control Cirrhosis Cirrhosis OCA No. OTUs 4620 7509 5082
Obeticholic acid (OCA) increases the synthesis of intestinal antimicrobial peptides in cirrhotic rats SHP Angiogenin-1 Alpha-5-defensin
Obeticholic acid (OCA) restores intestinal epithelial integrity in cirrhotic rats ZO-1 Occludin ZO-1 immunofluorescence Control Cirrhosis Cirrhosis + OCA
Obeticholic acid (OCA) ameliorates intestinal inflammation in cirrhotic rats with ascites IFN-γ TNF-α IL-10
M Ubeda et al. JHEP 2016
Factors contributing to intestinal dysbiosis and GBT in cirrhosis: Therapeutic targets β-blockers Procinetics Bile acids FXR agonists? Environmental and genetic factors? Intestinal hypomotility Altered bile flow Increased intestinal permeability Healthy microbiota Increased gastric ph Altered immunity Cirrhosisassociated dysbiosis Gut bacterial translocation Antibiotics Probiotics
Take home messages 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