José Manuel Fernández-Real, Hospital de Girona “Dr Josep Trueta”.

Institut d’Investigació Biomèdica de Girona

Universitat de Girona i CIBEROBN

Barcelona, 30 de novembre de 2018

Esteatosi hepàtica i el Microbioma

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Diabetes Care 2011

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Bouter et al. Gastroenterology 20017

J Henao-Mejia et al. Nature , 1-7 (2012) doi:10.1038/nature10809

Increased severity of NASH in Asc- and

Il18-deficient mice is transmissible to co-housed

wild-type animals.

Gut microbiota produce alcohol in patients with NASH

Loomba et al., Cell Metabolism 2017;25(5):1054-1062.

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Clinical need :Biomarkers of liver fibrosis

obese patients FLORINASH cohort

Liver Fibrosis

Biomarkers based on blood microbiota

Blood bacterial taxa assessed by 16S metagenomic

sequencing in buffy coats of control individuals or

patients with liver fibrosis

Lelouvier et al. Hepatology 2016

Blood microbiota sequences

➔ A diagnostic biomarker of the disease

Lelouvier et al. Hepatology 2016

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Nature Medicine 2018

• Morbidly obese non-diabetic patients (metformin-naïve)

• Recruited in Girona, Spain and Rome, Italy

• No antibiotics

• Stable weight for 3 months prior to sampling

• Hepatic biopsy

• Deep clinical phenotyping (OGTT, clamps)

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Age, country and BMI as confounders in FLORINASH

Nature Medicine 2018

Association of Low Microbial Gene Richness and Steatosis

Metagenomic richness decreased according to

degree of liver steatosis

Nature Medicine 2018

Correlations: Bacterial taxa/steatosis

Gram negative bacteria (LPS+) were associated with

liver steatosis

Nature Medicine 2018

Association between liver steatosis,

Microbial Gene Richness (MGR) and metagenomic

Data in obese women

Nature Medicine 2018

Metagenomic biochemical pathways

The metabolisms of LPS, peptidoglycans and branched amino acids

are correlated with steatosis

Nature Medicine 2018

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Liver Transcriptomics: Affected Pathways

Cluster of Insulin resistance,

inflammation, amino acid degradation

especially connected

Nature Medicine 2018

Transcriptomic networks of the liver:

Microbiota filter➔ 2200 genes

➔ Search for « core centers »➔ ~30

Cluster of Insulin resistance, inflammation, amino acid

degradation especially connected

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

1H NMR Urine Metabolomics:

Associations with Microbial Gene Richness and Steatosis

Nature Medicine 2018

1H NMR Plasma Metabolomics:

Associations with Microbial Gene Richness and Steatosis

Nature Medicine 2018

Phenylacetate accumulates in plasma with steatosis score

Branched amino acids and hepatic steatosis

Nature Medicine 2018

Clinical and Phenomic ROC Curves

Nature Medicine 2018

Phenome-Wide Crosstalk and Predictive Modelling

Nature Medicine 2018

1. Low Microbial Gene Richness

2. Increased genetic potential for processing of dietary

lipids and endotoxin biosynthesis (Proteobacteria)

3. Hepatic inflammation

4. Dysregulation of BCAA metabolism

5. Increased level of PAA

Les dones amb obesitat i esteatosis mostren :

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació en hepatòcits humans i en ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Quines són les possibles consequències de

la modificació del microbioma (i els seus

productes) sobre l’expressió de diferents

gens al fetge?

Microbial Phenylacetate (PAA) indueix esteatosi a

Hepatòcits humans

Nature Medicine 2018

Nature Medicine 2018, in press

Microbial Phenylacetate (PAA) indueix esteatosi a

Hepatòcits humans

Microbial Phenylacetate (PAA) indueix esteatosi a

Hepatòcits humans

Nature Medicine 2018

Microbial Phenylacetate (PAA) indueix esteatosi a

Hepatòcits humans

Nature Medicine 2018

PAA Indueix utilització de BCAA a

Hepatocits humans I a ratolins

CTRL PAA

p = 0.0435

Tri

gly

ceri

des (

mg

/g liv

er)

0

2

4

6

CTRL PAA8

10

12

14

16

Uri

nary

Iso

leu

cin

e (A

U)

p = 0.0003

PA

Nature Medicine 2018

• Introduccció– Com hem arribat aquí?

• Fetge i microbiota

• Microbiota circulant i fibrosi hepàtica

• The FLORINASH study• Troballes clíniques

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validació a hepatòcits humans i a ratolins

• Fecal microbiota transplantation

• Conclusions

Esteatosi hepàtica i el Microbioma

Faecal Microbiota Transplantations Impact the

Steatosis Phenome

Nature Medicine 2018

Faecal Microbiota Transplantations Impact the

Steatosis Phenome

Nature Medicine 2018

Faecal Microbiota Transplantations Impact the

Steatosis Phenome

Nature Medicine 2018

Faecal Microbiota Transplantations Impact the

Steatosis Phenome

Nature Medicine 2018

• Introduction– How we arrived here

• Antecedents

• Circulating microbiota and liver fibrosis

• The FLORINASH study• Clinical findings

• Liver transcriptomics-microbiome relationships

• Plasma and urine metabolomics

• Validation in human hepatocytes and mice

• Fecal microbiota transplantation

• Conclusions

Microbiome in non-alcoholic fatty liver disease

Conclusions

ASSOCIATIVE• Low microbial gene richness associates with steatosis• Increased genetic potential for processing of dietary lipids and

endotoxin biosynthesis (Proteobacteria)• Hepatic inflammation• Dysregulation of AAA and BCAA metabolism• Increased level of PAA

Conclusions

ASSOCIATIVE• Low microbial gene richness associates with steatosis• Increased genetic potential for processing of dietary lipids and

endotoxin biosynthesis (Proteobacteria)• Hepatic inflammation• Dysregulation of AAA and BCAA metabolism• Increased level of PAA

FUNCTIONAL• FMT triggers steatosis and impacts recipient mouse phenome• PAA induces steatosis in primary human hepatocytes and in mice• High amount of shared information content in phenomic and

metagenomic signatures• High predictivity of combined phenomic and metagenomic

signatures

Acknowledgments

University of Rome Tor Vergata

Marina Cardellini, Francesca Davato, Iris Cardolini, Ottavia Porzio, Paolo Gentilieschi, Massimo Federici

INSERM, ToulouseMatteo Serino, Julie Charpentier, Christophe Heymes, Vincent Azalbert, Vincent Blasco-Baque, Frédéric Lopez, Remy Burcelin

University of GironaJèssica Latorre Luque, José Maria Moreno-Navarrete, Josep Puig, Gemma Xifra, Wifredo Ricart, Jose-Manuel Fernandez-Real

Imperial College London

Lesley Hoyles, James Abbott,

Richard Barton, Julien Chilloux,

Antonis Myridakis, Laura Martinez Gili,

Mark Woodbridge, Chris Tomlinson,

Sarah Butcher, Elaine Holmes,

Jeremy Nicholson,

Marc-Emmanuel Dumas

INSERM, Paris

Fabienne Foufelle, Elodie Anthony,

Fadila Rayah,

Catherine Postic

Hospital de Girona (Radiologia)

Dr. Josep Puig

Dr. Gerard Blasco

Dr. Salvador Pedraza

Hospital de Girona (UDEN-TG)

Dr Gemma Xifra

Dr Jose Maria Moreno-Navarrete

Dr Mercé Fernández-Balsells

Dr Francisco Ortega

Oscar Rovira

Maria Arnorriaga

Dr Wifredo Ricart

Acknowledgments

Hospital Tor Vergata de Roma

Dr. Massimo Federici

INSERM, Toulouse

Dr. Rémy Burcelin

Dr. Manuel Portero-Otin

Universitat de Girona I Universitat de Lleida

Dr. Pepus Daunis

Hospital de Bellvitge

Dr. Fernando Fernández-Aranda

Dra. Susana Jiménez

Dr. Oren Contreras