Fondazione Edmund Mach

Kieran Tuohy Fondazione Edmund Mach,

San Michele all’Adige, Trento, Italy

Impact of microbiome on liver disease

Ask not what your diet can do to your microbiome

but what your microbiome can do to your diet.

Liver fibrosis and repair: immune regulation of wound healing in a solid organ

Antonella Pellicoro, Prakash Ramachandran, John P. Iredale & Jonathan A. Fallowfield

Nature Reviews Immunology 14, 181–194 (2014)

Gut microbiome and liver disease

• Increased microbiota over-growth, dysbiosis and modified metabolic output

• Increased gut permeability – metabolic endotoxemia, hepatic inflammation

Cellular & Molecular Gastroenterology & Hepatology,

(2015) 1:275-284.

Cross talk between gut microbiota

and liver

• Metabolites – metabolic end products of ingested foods

(SCFA, small phenolic compounds, amino acids/amino acid

derivatives e.g. TRP metabolites, amines, phenols, ammonia)

• Enterohepatic circulation of bile acids – bile acid deconjugation,

dehydroxylation, 1° to 2° bile acids

• Bacterial translocation and inflammatory signals (e.g. LPS)

• Fats, lipids and choline metabolism

(e.g. TMA, Modified cholesterol species, microbial

biohydrogenation)

Impact of traditional diets rich in fiber on colonic fermentation

Proximal colon ~ saccharolytic

SCFA

Acetate Propionate

Butyrate

Energy source Apoptosis

Differentiation Epigenetics

Gene expression Gut hormones

Gut permeability

Distal colon ~ proteolytic

Amines Indoles

Ammonia Sulphides N-nitroso

DNA damage

Tumours Cytotoxicity

Leaky gut Liver disease

Modified from George Macfarlane

Impact of Western style diet on colonic fermentation

Proximal colon ~ saccharolytic

SCFA

Acetate Propionate

Butyrate

Energy source Apoptosis

Differentiation Epigenetics

Gene expression Gut hormones

Gut permeability

Distal colon ~ proteolytic

Amines Indoles

Ammonia Sulphides N-nitroso

DNA damage

Tumours Cytotoxicity

Leaky gut Liver disease

Modified from George Macfarlane

Table 1. Cellular actions described for TGR5 in different cell types. ∗Macrophages include alveolar macrophages,

Kupffer cells and THP-1 cells.

BA, fat & glucose

homeostasis

Inflammation (NF-κB)

Enterohepatic BA circulation

microbiota

(deconjugation, 1°→ 2°)

Journal of Hepatology (2011)

FXRα

VDR

PXR

• Lb. reuteri selected for Bile Salt Hydrolase activity (2 capsules/day at 2 x 109 CFU/capsule) for 9 weeks

• Randomized, double-blind, placebo-controlled, parallel-arm, multicenter study

• N=127 hypercholesterolemic patients

• Probiotic reduced plasma – TC by 9.14%

– LDL-C by 11.64%

– LDL-C/HDL-C ratio by 13.39%

– Non-cholesterol plant sterols

– Increased circulating deconjugated bile acids

• Proposed new cholesterol lowering activity of probiotics via modified absorption of lipids from the gut

Apple pectin and polyphenols increase faecal

excretion of bile acids and cholesterol derivatives

Apple pectin and polyphenols impact on lipid

metabolism and storage

TMA/TMAO confirmed strong link with CVD in patients •confirmed microbiota metabolism of L-carnitine/choline → TMA→TMAO •TMA not produced in vegans •confirmed inflammatory activity & linked to macrophage reverse cholesterol transport •TMAO reduced bile acid pool

Koeth et al., 2013 Nature Medicine

Gut flora metabolism of phosphatidylcholine

promotes cardiovascular disease

Wang et al., 2011 Nature

Established Therapies

• Unabsorbed antibiotics

(rifaxamin)

• Lactulose (prebiotic)

• Adjunt therapies: probiotics

Hepatic Encaphalopathy, a complication

of end stage liver failure

Cani et al., 2007 Diabetes

Plasma endotoxin (LPS) increased

with HF diet

• Upon high fat feeding or LPS injection inflammatory markers increased in liver and adipose tissue

– TNF-α, IL-1, IL-6

– insulin resistance and obesity

• In CD14 mutant mice this inflammatory response was blunted

Prebiotic relief of metabolic endotoxemia

through improved mucosal barrier function

LPS LPS LPS

High fat, low CHO diets induce microbiota dysbiosis

Low SCFA

•↑Inflammation •↑Insulin resistance •↑Hepatic fat deposition

Activation of WAT & liver inflammatory pathways

Prebiotic induced bifidogenesis & microbiota biosis

↑GLP-1, GLP-2, PYY ↑Tight junction proteins

High

SCFA

•↑ Satiety •↓Food intake •Maintenance of gut barrier function •Immune homeostasis

Mucosal

barrier

Metabolite Normal faecal concentration range*

Evidence for beneficial or harmful effects

Short chain fatty acid

•Acetate •Butyrate • Propionate

61-120 µmoles/g wet wt, 190-600 µmoles/g dry wt

Acetate: provides energy, substrate for lipogenesis and cholesterol Butyrate: provides energy for colonocytes, stimulates apoptosis and cell proliferation, inhibits histone deacetylase, regulates gut barrier and immune function Propionate: increases satiety, inhibits lipogenesis, increases absorption of water, regulates gut barrier and immune function

Branched chain fatty acids

2-3 µmoles/g wet wt Approx. 30 µmoles/g dry wt

Potentially harmful but also act against some less desirable bacteria

Hydrogen sulfide

Total sulfide 0.05-1.6 μmoles/g wet wt

Limited evidence in humans. Pro- and anti-inflammatory effects on colonic cells. Potentially toxic to colonic cells. Inhibits butyrate oxidation in vitro.

Ammonia 14-660 µmoles/g faeces No convincing evidence of harmful effects in healthy humans

Phenols

•p-Cresol

•Phenol

•Indole

Colonic generation rate is often

estimated from urinary

excretion/day

10-55 mg/d (urine), 5-8 mg/d

(faeces)

4-7.5 mg/d (urine), 0.25-0.66 mg/d

(faeces)

<50 mg/d (urine)

• Rapidly absorbed by colonic mucosa and excreted in urine

• Do not accumulate in healthy subjects

• Often considered as potentially toxic but no evidence for toxicity

in healthy humans

From polyphenols

•Simple phenols •Glycinated benzoic acids •Derivatives of

• Benzoic acid • Phenyl acetic acid • Phenylpropionic acid • Mandelic acids • Cinnamic acids

• Equol

varying concentration from

µmoles/l

(e.g. hydroxybenzoic acids)

to mmoles/l (e.g. phenyl propionic

acid)

Possible benefits in colon (anti-inflammatory, anti-oxidant)

but not well tested

*The normal concentration range is based on adults. This range may differ in stages of life as well as regions in the world. For detailed information on the references of the above-mentioned concentration values, please contact info@ilsieurope.be

Obese vs lean gut microbiota

Lean –open diet (LOD ■) Obese open diet (OOD ■) Obese on a saturated fat diet for 1 month (OHSFA ■) n=13

•Bacteroides uniformis and Prevotella copri more common in the microbiota of LOD than OOD – not present in the OHSFA

•Bacteroides vulgatus and Bacteroindes stercoris very frequently found in OHSFA, less frequent in OOD – not present in LOD

-5

0

5

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

PC

2

PC1

DGGE bands pattern (PCA)

[PC2]/[PC1]

%[1] = 0.092199 %[2] = 0.0642478

LOD

OHS

OOD

Fava et al in preparation

Fermentation end-products

• Faecal SCFA measured by GC

• Higher acetate in obese irrespective of diet

• Higher butyrate in obese (open diet)

0,0

5,0

10,0

15,0

20,0

25,0

30,0

35,0

40,0

ACETIC

PRO

PIO

NIC

I-BUTY

RIC

N-B

UTY

RIC

I-VALE

RIC

N-V

ALE

RIC

N-C

APRO

IC

mm

ol/g

faeces

LOD

OOD

OHSFA

SCFA as biomarker of healthy gut Increased faecal SCFA excretion due to decreased SCFA uptake?

MCT transporters

expression and

apical location is

promoted by

luminal SCFA

Increased faecal SCFA excretion could be due to decreased MCT active uptake in high fat/low CHO diets

Borthakur et al., 2012 Am J Physiol Gastrointest Liver Physiol

Goncalves et al., 2012 J Cell Biochemistry

Bile salt CDCA

and E. coli EPEC

inhibit butyrate

uptake

Biohydrogenation

• Recognised & studied in

rumenants

• Under studied & bearly

recognised in humans

Rosburia

Butryvibrio

Lactobacillus

Bifidobacterium

Lipids 2010

Chen et al. (2015)

Gastroenterology 148:203-214

Chen et al. (2015)

Gastroenterology 148:203-214

Chen et al. (2015)

Gastroenterology 148:203-214

•POLYPHENOLS….. 90% resistant to digestion and reach the colon, plant secondary metabolites, usually antioxidant, antimicrobial activities, enzyme/nutrient binding properties and possibly prebiotic type properties, e.g. red-wine polyphenols, apple tannins

Polyphenols

When to drink red wine, the French

paradox revisited?

Natella et al. Brit J Nutr (2011)

“the modality of drinking wine (during the

meal) could represent a decisive factor”

Natella et al. Brit J Nutr (2011)

American Journal of Clinical Nutrition, 2012

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Basline

De-alcoholized red wine

Red wine

Gin

* * * *

* *

*

* *

Targeting Microbiota Polyphenol

metabolism

• Targeted MS based quantitative metabolite analysis

•139 fruit polyphenols

•UPLC/QqQ-MS/MS

•Modified to accurately quantitify about 150 common fruit polyphenols

and their catabolites

Microbiota modulation - in vitro

faecal batch cultures

•4 commercial apples

•Simulated gastric and small intestinal digestion

•Fermentation pH and temperature controlled anaerobic

faecal batch cultures

•FISH microbial enumeration

•Profile of microbial polyphenol catabolites

GASTRO-DUODENAL DIGESTION IN VITRO vs IN VIVO

pH 2.5 Add gastric pepsin & lipases 37°C/ 2h

pH 6 (6N NaOH) Bile salts & enzymes (amylase, trypsin,

chymotrypsin,

colipase ) 37°C/ 1h

•Strawberry (commercial var.) •Wild Strawberry

In collaboration with

Dr Chris Gill,

Ulster University

200 g Wild Strawberry “smoothy”

•Ileal fluid (n=5) collected from ileostomy patients before (T0) and after (T8) consuming 200 g Wild Strawberry smoothy (WS). •5 healthy controls fed in parallel

Ileostomy bag

GASTRO-DUODENAL DIGESTION IN VITRO vs IN VIVO

In collaboration with

Dr Chris Gill,

Ulster University

Increasing fruit and vegetable intake

in vivo – FLAVURS project

Flavonoid-poor F&V

Flavonoid-rich F&V +2 + 4

+2

+ 6

+ 4 + 6

Habitual diet

Wk 0

Visit 1 Wk 6

Visit 2

Wk 12

Visit 3 Wk 18

Visit 4

?

Metabolomics workflow

Sample preparation:

extraction of all analytes

Statistical analysis Untargeted HR mass

spectrometry

Biomarker identification

Samples: urine,

plasma, fecal water

C+

NHO

F

CH3

NN

CH3

Separation on

LC column

Measuring the effect of apples (2 per day)

on the gut microbiome and heart health.

...from Trentino with love!

“Conslusions: Adherence to an MD pattern is associated with better HRQL. The

association is stronger with mental health than with physical health. Dietary total

antioxidant and fibre content independently explain this relationship”.

• Children with Prader-Willi syndrome (PWS), n=17 and children with diet associated “standard” obesity (n=21)

• Weight loss induced by reduced calorie intake and high fiber & polyphenol diet (whole grain/legume “grule”, plus 20g/prebiotic per day, fruit/veg, medicinal Chinese herbs)

Dietary intervention in obese children Before 30 day 60 day 90 day

Total Energy Intake 1559.56 (3577.4-916.2) 1232.7 (1992.4-858.3) 1167.9 (1648.8-74.2) 1169.2 (1919.3-940.7)

Protein (g) 46.9 (103.8-19.6) 46.2 (83.1-18.6) 38.2 (61.5-26.1) 36.9 (74.3-20.9)

Protein (%) 13. 6±1.1 13.7±0.5 13.0±0.5 12.7±0.5

Lipid (g) 64.6 (136.5-15.8) 26.6 (40.2-14.0) 25.2 (41.4-18.3) 24.8 (45.8-14.5)

Lipid (%) 34.0±4.0 19.7±0.4*** 20.3±0.7*** 19.7±0.6***

Carbohydrates (g) 218.3 (484.7-54.8) 191.9 (294.7-141.1) 189.7 (252.4-106.7) 187.1 (261.6-142.9)

Carbohydrates (%) 52.4±4.4 62.4±0.7* 62.6±0.7* 62.5±0.9*

Fiber (g) 6.2 (16.6-1.6)a 44.9 (58.7-30.7)b 48.5 (59.9-24.4)b 49.4 (66.9-37.3)b

Soluble fiber (g) 2.7 (7.2-0.7) 29.2 (40.9-10.7) 31.6 (39.3-11.1) 32.7 (45.6-23.7)

Zhang et al., 2015

Significantly reduced obesity and improved metabolic parameters in both genetically obese and diet associated obese children.

Zhang et al., 2015

Specific microbiota modulation

Zhang et al., 2015

•Dietary interventions modulate

gut microbiota (increased relative

abundance of Bifidobacterium

and other fiber degraders.

•Dietary interventions reduced

relative abundance of organisms

involved in TMAO, TRP and choline

metabolism

Summary • Metabolites derived from microbial activities in the gut play an

important role in liver disease risk (BA, lipid metabolism, inflammation)

• Probiotics (BSH), prebiotics/fiber and polyphenols all have potential to modulate both the flux of these metabolites and gut permeability/inflammatory output

• High resolution omics technologies have provided the necessary tools for tracking microbiome metabolic output and community structure/function

• Currently lack appropriately designed human studies to confirm theories generated from animal studies and human «case / control» observations

Fondazione Edmund Mach

• Thank you: ILSI Euroope, Prof Ian Rowland

• Fulvio Mattivi, Marynka Ulaszewska, Claudio Donati,

Fondazione Edmund Mach

• NN Group: Francesca Fava, Elena Franciosi, Athanasios Koutsos, Ilaria Caraffa,

Florencia Ceppa, Andrea Mancini

• University of Ulster: Dr Chris Gill

• University of Reading, Ian Rowland, Glenn Gibson, Julie Lovegrove, Parveen

Yaqoob, Christine Williams, John Swann