Download Diet, microbiota and gut health – what five-a-day?

Diet, microbiota and gut health – what five-a-day?
Barry Campbell
Simms Lecture
RCP Regional meeting, York
5th Dec 2016
Gastroenterology Research Unit
Cellular & Molecular Physiology,
Institute of Translational Medicine
[email protected]
Diet, microbiota and gut health – what five-a-day?
Jon Rhodes
Barry Campbell
Simms Lecture
RCP Regional meeting, York
5th Dec 2016
Gastroenterology Research Unit
Cellular & Molecular Physiology,
Institute of Translational Medicine
[email protected]
Diet, microbiota and colonic health
– a long history
“ A full colon is the root
of all suffering”
An early advocate of dietary
prevention of cancer.
?
1856-1943
Sir William Arbuthnot Lane
Promoted whole foods, fruits
and vegetables, sunshine and
exercise: his plan to foster
health and longevity via 3
bowel movements daily.
By Photogravure after Elliott & Fry. - [1], CC BY 4.0,
https://commons.wikimedia.org/w/index.php?curid=33387516
Diet, microbiota and colonic health
– a long history
“90% of the diseases of
civilization are due to improper
functioning of the colon…”
“…poor diet favors harmful
bacteria that can then infect
other tissues in the body; that
the intestinal flora is changed
by the diet of the individual,
and is changed for the better…”
1852-1943
Dr John Harvey Kellogg
Diet, microbiota and colonic health
– a long history
1852-1943
Dr John Harvey Kellogg
Diet, microbiota and colonic health
– a long history
1852-1943
Dr John Harvey Kellogg
Vegetarianism, exercise, a
pint of yogurt (half eaten,
half as an enema to
replace toxic bacteria) for
a ‘squeaky clean’ intestine.
The importance of our microbiota
The Microbiome:
Joshua Lederberg argued that microorganisms
inhabiting the human body should be included as part
of the human genome, because of their influence on
human physiology. Lederberg & McCray 2001 Scientist 15: 8
We are only 10% human (more or less)
Revised estimates:
3 x 1013 Human cells
3.9 x 1013 Bacteria
Approx. 76% Human
10 trillion cells vs. ~100 trillion bugs
Sender et al. Plos Biol 2016;
doi:10.1371/journal.pbio 10002533
What do we know about our gut
microbial community (microbiota)?
• Established in the 1st year of life
Co-evolves with the immune system
• Highly variable between individuals
In 154 individuals no single shared abundant species
About 160 bacterial species in each faecal sample
gene set ~150 times human genome
• The range of bacteria appears to be fairly stable with time
Turnbaugh & Gordon 2009 J Physiol 587:4153-8
But influenced by diet
Turnbaugh et al. 2009. Nature 457:480-4
Qin et al. 2010. Nature 464, 59-65
Bacteria in the normal distal gut
% of bacteria
13,000 16S rRNA sequences
analysed from healthy young
adults and non-IBD controls.
Seven major phyla
Firmicutes
Bacteroidetes
>90%
Actinobacteria
Proteobacteria
Fusobacteria
Verrucomicrobia,
Cyanobacteria
Petersen et al. 2008 Cell Host & Microbe 3, 417-27
Inflammation dramatically re-shapes
our gut microbiota
Inflammation driven dysbiosis
% of bacteria
% of bacteria
5,405 16S rRNA
sequences from
patients with CD
and UC.
Petersen et al. 2008 Cell Host & Microbe 3, 417-27
Reduced microbiota diversity in Crohn’s disease
- metagenomic/macroarray approach
• fosmid vector approach to construct
2 genomic DNA libraries
• 25K clones each, screened for the
16S rRNA gene by DNA hybridisation
Manichanh et al. 2006 Gut 55, 205-11
Metagenomics of faecal microbiota also
differentiates UC from healthy individuals
Qin et al. 2010. Nature 464, 59-65
The importance of the mucus barrier
•
Colonic mucus is continuous with two
layers
– inner layer is normally free from bacteria
•
Small intestinal mucus is discontinuous
MUC2
FISH (bacteria)
Johansson et al. PNAS 2011; 108, 4659-65
Mucosa-associated bacteria differ from faecal bacteria!
includes bacteria that are asaccharolytic (eg some Firmicutes)
Or aerobic/ microaerophilic bacteria typically absent from the
faeces (eg E. coli)
Aldenburg et al. Gastroenterology 2014; 147, 1055-63
Increased ileal mucosa-associated
‘adherent, invasive E. coli’ in CD
Look like ‘harmless’ resident strains lacking major
pathogenic factors of toxigenic/diarrhoeagenic E. coli
Belong mainly to phylogenetic groups B2 and D
more commonly associated with ExPEC
Darfeuille-Michaud et al. Gastroenterology 1998; 115:1405-13
Darfeuille-Michaud. Int. J. Med. Microbiol. 2002; 292:185-93
Darfeuille-Michaud et al. Gastroenterology 2004;127:412-21
Increased colonic mucosa-associated bacteria were
cultured more commonly in CD and colon cancer
Pullan et al. Gut 1994; 35:353-9
>53% were E. coli
The (paediatric) Crohn’s disease pre-treatment
mucosa-associated microbiota
Terminal
ileum
447 CD patients aged 3 to 17
221 controls (abdo. pain and diarrhoea)
Increased include:
E. coli, Fusobacteria
Reduced include:
Faecalibacterium prausnitzii
(anti-inflammatory;
Protective against ileal Crohn’s relapse)
Gevers et al. Cell Host Microbe 2014;15:382-92
Early Crohn’s lesions arise at sites of
Peyer's patches or lymphoid follicles
TEM
Fujimura et al. 1996 Gut 38,724-32
Confocal laser endomicroscopy
Krauss et al. 2012.
Int. J. Clin. Exp. Path. 5, 411-421
Incidence of CD
Number of PP
Van Kruiningen et al. 1997 J Clin Gastro 25,470
Long polar fimbriae expressed by Crohn’s E. coli target
M cells in the FAE of Peyer’s patches
- linking colonization & the presence of early lesions overlying lymphoid
follicles
Peyer’s patch
FISH Cy3-EUB338
Chassaing et al. 2011. J. Clin Invest. 121, 966-75
Dogan et al. 2014. Inflamm Bowel Dis. 20, 1919-32
Crohn’s E. coli replicate within host
macrophages and are pro-inflammatory
• persisting within mucosal macrophages (human
mouse) they promote increased levels of TNF release
Double
around
vesicle
coli.
and
membrane
macrophage
containing
E.
Mpofu et al. Gastroenterology 2007; 133 1487-98
Subramanian et al. 2008 AAC 52, 427-34
The mucosal microbiota is also altered
in adenoma and colon cancer
Mucosa-associated bacteria and colorectal cancer
Key increased mucosal
bacteria include:
Strep. (bovis) gallolyticus
Bacteroides fragilis
E. coli
Fusobacteria nucleatum
Enterococcus faecalis
Akkermansia mucinophilia
Cipe et al. World J Gastro Oncol 2015; 7:233-40
Intracellular E. coli in human colon cancer tissue
• Intracellular E. coli (and Bacteriodes) within
the
colonic
mucosa
(tumour
and
histologically normal tissue) in colon cancer
• Healthy patient bowel tissue is relatively
free from bacteria
Alexander Swidsinski
Gastroenterology 1998;115: 281-6
Modelling interactions of AIEC in inflammationassociated colorectal carcinogenesis
• AOM treated germ-free Il10-/- mice, monoassociated with bacteria to induce colitis
Arthur et al. 2012. Science 338: 120-3
Science 2012: 338: 52
• pks associated with decreased tumour burden
Concept of colorectal cancer as a bacterial disease
1. Bacteria-epithelial interaction may be crucial by:
(i) stimulation of epithelial (Toll-Like) receptors and consequent
inhibition of apoptosis
(ii) DNA-damaging (genotoxic) effects.
2. This interaction is much more likely once
an adenomatous polyp is present
3. E. coli may be particularly important –
because they (a) adhere and invade to epithelial cells,
(b) tolerate relatively high oxygen environment (microaerophilic)
(c) produce genotoxins (pks PAI  colibactin)
(d) proangiogenic (afa)
4. Adhesin interactions occur with carbohydrate receptors on epithelial
cells which may be preventable by dietary polysaccharides
Maybe this is how fruit and vegetables protect against colorectal cancer?
Diet also shapes the gut microbiota
Impact of habitual diet in shaping gut microbiota
16S rRNA gene surveys reveal a clear separation of two child
populations from Burkina faso and EU
High fruit/
legume
fibre diet
More Gram +ve
saccharolytic spp.
High SCFA levels
High milk
fat/Animal
protein diet
Low SCFA levels
More Gram -ve
Proteobacteria
De Filippo et al. 2010 PNAS 107, 14691-6
Short-term dietary intervention alters the human gut
microbiota and microbial activity.
Bacterial 16S ribosomal RNA gene sequencing
• Microbial  diversity (the
difference between each
subject’s baseline and dietassociated gut microbiota)
changed within 1 day on
animal-based diet reaching
the colon.
David et al. 2014 Nature 505:559-63
Diet, interventions and the gut microbiota
Simpson & Campbell 2015. Aliment Pharmacol Ther. 42:158-79
Geographical variation in colorectal cancer incidence
WHO 2015
Colon cancer and diet - estimating the size of the risk
Colon cancer incidence in <65 year old in Connecticut
=10 times that in Nigeria, therefore about 90% of USA
colon cancer risk is environmentally determined,
largely by diet
Doll & Peto. J Natl Cancer Inst. 1981; 66:1191-308
Epidemiology of colon cancer
Factors correlated with increased risk:
red meat
calories
alcohol
body mass index
exercise
dietary fibre
Abstracted from World Cancer Research Fund/AICR 2016
http://www.wcrf.org/sites/default/files/WCRFI-Matrix-for-all-cancers.pdf
Meta-analysis of 21 prospective (cohort) studies
shows 14% increased risk for colorectal cancer
per 100g red/processed meat per day
(8oz = 227g)
Chen et al. Plos One 2011: e20456
Fibre and colorectal cancer – not a simple story
cereal fibre?
3 studies show protective effect
7 no association
3 increased risk
Fruit & vegetables ? 23/28
studies show protective effect
Legumes? …..
EPIC Oxford study also shows
increased risk for colorectal
cancer in vegetarians!
Soluble fibre intake and colorectal cancer
WCRF, 1997
EPIC 2003; Bingham et al. Lancet 2003; 361:1496-501
Key et al. Am J Clin Nutr 2009; 89; 1620S-6S
Diet swap experiment reveals junk food's harm to gut
Over 2 weeks - US African American volunteers (n=20) moved to
a low-fat, high-fibre diet while 20 volunteers from rural Africa
were asked to eat a more Western "junk" food.
In rural Africans on diet swap:
Inflammation increased.
Microbiome and metabolome altered.
Increased Fusobacterium spp. (genera associated with CRC)
In the African Americans (AA) on diet swap:
Increased saccharolytic fermentation and butyrogenesis,
Suppressed secondary bile acid synthesis
Decrease in Proteobacteria
(i.e. potential decrease in CRC risk)
O’Keefe SJ et al. Nat Commun. 2015; 6: 6342
Can
single
dietary
components
block
potentially harmful interactions between
bacteria and the gut epithelium
….and thus reduce risk for inflammatory bowel
disease and colon cancer?
Single dietary constituents may alter
host response to bacteria, if not the microbiome
% attachment
160
140
NO PLANTAIN
120
PLANTAIN
100
80
60
40
20
• Soluble plantain fibre (non
starch polysaccharides) blocks
E. coli attachment & invasion
to bowel cells
0
HM427
HM545
E. coli strain
14
% invasion
12
NO PLANTAIN
10
PLANTAIN
8
Musa spp.
6
Jon Rhodes
4
2
0
HM427
HM545
E. coli strain
Martin et al. 2004 Gastroenterology 127, 80-93
Soluble plant fibres block
E. coli translocation across M cells
Plantain NSP
Not all soluble fibres are equal – Broccoli NSP good
Leek and apple NSPs had little effect
Roberts et al. 2010 Gut 59:1331-39
Crohn’s E. coli translocation across human ileal Peyer’s
patches is blocked by soluble plant fibres
Ussing chamber culture to measure bacteria translocation across ex vivo
Peyer’s patches
Soluble plant fibres block bacteria translocation
Contrabiotics’ ‘Contrabiotics’ – a new therapeutic strategy?
Roberts et al. 2010 Gut 59:1331-39
Plantain NSP blocks interactions of Salmonella
with the intestinal epithelium
Roberts et al. J. Nutrit. Biochem. 2013; 24(1): 97-103
Parsons et al. PLoS ONE 2014, 9(2), e87658
control
banana
pectin
High intake of fruit fibre (but not cereal or legumes)
protects against Crohn’s (but not UC)
Prospective Nurses health study – 170,776 women followed up for 3,317,425 person years
1.4g/d vs.
6.4 g/d
fruit fibre
Ananthakrishnan et al. 2013 Gastroenterology 145:970-77
Whilst some dietary components block potentially
harmful bacteria-epithelial interactions other
dietary components may increase them.
Permitted food emulsifiers enhance E. coli
translocation across the epithelium
Ex vivo human ileal tissue mounted in Ussing chambers
• Resist digestion
• Alter lipid bilayer fluidity
at low levels
• Mucus in small intestine is
discontinuous so relatively
exposed to emulsifiers
• Acceptable daily intake of 25 mg/kg body wt (FAO/WHO data), 0.01% v/v would
represent a persistence of ~7% into terminal ileum in a 60kg human, assuming 1L
intestinal contents/day passing to the caecum
Roberts et al. Gut 2010; 59, 1331-9
Increasing incidence of Crohn's disease vs.
emulsifier consumption
Japan
Roberts et al. 2013; J. Crohns Colitis 7(4):338-41
Dietary emulsifiers impact mouse gut microbiota
promoting colitis and metabolic syndrome (and also CRC)
Dietary carboxymethyl cellulose and polysorbate 80 impact on mucus barrier
Causing colitis
…and metabolic syndrome
Effects dependent on altered microbiota; (incl. increased
mucolytic bacteria, increased LPS and flagellin)
Exacerbated tumour development in mice Chassaing et al. Nature 2015; 519, 92-69
Viennois et al. Cancer Res 2016; epub.;
And is lecithin (predominantly phosphatidyl choline) bad too?
TMAO=trimethylamine-N-oxide
A pro-atherogenic metabolite of choline
Lecithin also increases bacterial translocation in Balb/c mice
0.1% v/v in drinking
water for 4d
Ani et al.,
in progress, 2016
In patients with Crohn’s disease where bacterial
invasion into tissue macrophages/ lymph nodes
has already occurred, antibiotics are therapeutic…
Could dietary supplements be useful?
Vitamin D supports bacterial killing
Vitamin D deficiency results in impaired macrophage
function and inability to generate defensins
TLR2 & TLR4 stimulation lead to VDR
expression
1,25(OH)2Vit.D3 bound VDR induces:
• immune cell function
• NOD2
• Antimicrobial peptides
(2 defensins/Cathelicidin)
Adams & Hewison 2008. Nat. Clin. Pract. Endocrinol. Metab.; 4, 80-90
Hewison 2010. Endocrinol. Metab. Clin. North Am.; 39: 365
Wang et al. 2010 J Biol Chem; 285: 2227
Vitamin D enhances killing of Crohn’s
colonic mucosa-associated E. coli
Murine macrophages
Human MDM
Data for colonic CD isolate HM605
Similar results seen for ‘paradigm’ ileal CD isolate LF82
Flanagan et al. IBD 2015; 21(7):1499-510
Vitamin D deficiency in Crohn’s
•
•
•
•
Active Crohn's disease is associated with low vitamin D levels
70% of quiescent patients are deficient
Crohn’s less common with higher predicted levels
Vit D receptor (VDR) polymorphisms associated with Crohn’s
Clinical trial: vitamin D3 treatment in
Crohn's disease – a randomized
double-blind placebo-controlled study.
• Vitamin D (1200 IU/day) reduces
risk of clinical relapse
Jorgensen et al. APT 2010; 32: 377-83
IBD patients (CD & UC)
with low vitamin D levels
more likely to need surgery
If receiving vit. D supplements,
they are less likely to require
interventions (“health care
utilization”)
Kabbani et al. Am J Gastro 2016;111:712-9
What is the evidence for “Five A Day”
http://www.whatdotheyknow.com/request/five_a_day_evidence_base
Susan Davis made this Freedom of Information
request to Department of Health
The request was partially successful.
23 February 2010
“Dear Department of Health,
I am interested in learning more about the evidence base behind
the
"5 a day" campaign to promote a diet richer in fresh fruit
and
vegetables. In particular I am interested in finding out how
the figure of 5
was arrived at (rather than, say, 4, or 10).
Yours faithfully, Susan Davis”
Department of Health
24 February 2010
Thank you for your email.
Where a reply is appropriate we aim to send one within 20 working days.
If your enquiry is about a medical matter, please contact NHS Direct on
0845 4647 or visit [1] NHS Choices, or contact your GP surgery.
For the latest on swine flu, please visit [2] the National Pandemic Flu
Service (NPFS) or call 0800 1 513 100
.
For general health information you may also find it helpful to refer to
[3] Directgov, the UK Government's Official information website, or the
Department of Health website's [4] Frequently Asked Questions.
Basis of “5 a day”
Joyce Hughes (2000). The case for increasing the population consumption
of fruit and vegetables and the evidence for the effectiveness of
interventions.
“Internationally an intake of at least five portions (total 400g) of fruit and
vegetables per day has become an established “healthy eating” message.”
Refers to: Health Education Authority (1997): Eight Guidelines for a Healthy Diet.
London: Health Education Authority. (out of print)
Based upon levels of fruit and vegetable consumption in Southern
Mediterranean countries with low levels of Coronary heart disease.
(…..emphasized olive oil)
History of “5 a day”
“Invented” by Ken Kizer in California,
1980’s
"Beginning in the mid and late
1970s, the evidence became quite
clear about the role of diet in
preventing cancer and heart disease
and other conditions."
Adopted by USA National Cancer Institute,
1991
Adopted by UK Department of Health, 2003
“5 A day”: What counts?
“Potatoes are a vegetable, but they don't count towards your 5 A DAY.
That’s because the main nutrient in potatoes is starch.
Other vegetables that don’t count towards your 5 A DAY are
yams, cassava and plantain: they are also usually eaten as starchy foods.
However, other root vegetables such as sweet potatoes,
parsnips, swedes and turnips do count toward your 5 A DAY,
because they are usually eaten in addition
to the starchy food part of the meal.”
(“BOGSAT” guidance?)
The Mediterranean diet “pyramid”
Bach-Faig et al. Pub Health Nutr 2011;14:2274-84
The Mediterranean diet – quantified!
Tong et al. BMC Medicine 2016;14:135
PAF – population attributable fraction – preventable mortality by
increasing adherence to Mediterranean diet to top third
(i.e. 9.1 out of 15 points in “pyramid”)
Tong et al. BMC Medicine 2016;14:135
My “5 a day” for (gut) health
‘Mediterranean’ pyramid
Tree nuts
Red wine (2 glasses)
Olives
‘Cancer preventing /
contrabiotic’
Broccoli
‘Contrabiotic’
Plantains/green bananas
Plus …Aspirin/Vit D supplements
And exercise
And don’t forget to drink plenty of coffee!
Association of Coffee Drinking (4 or more cups/day) with Total and
Cause-Specific Mortality
Freedman et al. NEJM
2012;366:1891-1904
229,119 men
173,141 women
Age 50-71
Followed over 14 years
After correction for smoking:
≥ 6 cups coffee/day:
Men: 10% decreased deaths
Women: 15% decreased
deaths
Caffeinated = decaffeinated
And chocolate!
NEJM 2012; 367:1562-1564
Regression coefficient (r) = 0.0791
P<0.0001
r excluding Sweden = 0.0862
0.4kg chocolate/year/person/nobel prize/country
“minimally effective chocolate dose = 2kg/year
but dose response reveals no apparent ceiling up to highest dose of 11kg/year”
And cocoa flavonoids improve cognitive performance in elderly humans and rats
With thanks to:
Jon Rhodes
Looking for the environmental causes of IBD!
Crohns Colitis UK
CORE
Wellcome Trust
NIHR
MRC
BBSRC
NWCR
Provexis UK
Tony Hart
Keith Leiper
Craig Winstanley
Steve Edwards
Neil Hall
Helen Martin
Carol Roberts
Melissa Friswell
Maelle Prorok-Hamon
Yvette Merga
Hannah Simpson
Bryony Parsons
Chiedzo Mpofu
Sree Subramanian
Paul Flanagan
Paul Collins
Paul Knight
Fei Song
Richard Evans
Abdullah Alswied
 Julian Marchesi (Cardiff)
 Johan Soderholm/Asa Keita (Linkoping, Sweden)
 Arlette Darfeuille-Michaud (Clermont-Ferrand, France) & Benoit Chassiang
(Atlanta, USA)
 Kenny Simpson & Belgin Dogan (Cornell , USA)
 Christian Jobin & Janelle Arthur (Chapel Hill NC, USA)
 Alison Simmons (University of Oxford)
 Niahm O’Kennedy (Provexis, Aberdeen)
 Steve Fry (Univ of Edinburgh)