Transcript Document

The Gastrointestinal Microbiome in
Ulcerative Colitis.
Susan V. Lynch, PhD
Associate Professor of Medicine
Director Colitis and Crohn’s Disease Research Core
Division of Gastroenterology,
Department of Medicine,
University of California San Francisco.
BROAD FOUNDATION Investigator Meeting. LA, March 7-8, 2013
The Human Microbiome
The Human Microbiome
• Culture-independent microbial
profiling
• Composition
Function
• Contribute functions critical to host
health:
• Metabolism of indigestible carbohydrates
• Vitamin and hormone production
• Immune development
• Immune homeostasis
• Ancillary mucosal protection
• Microbiome dysbiosis associated with
chronic inflammatory/autoimmune diseases
Spor, A. et al. Nat Rev Microbiol. 2011 Apr;9(4):279-90.
Selective Pressures Shaping the Microbiome
Host Genetics
Environment
Gut Microbiome
Immune response
Relationship between IBD and GI Microbiome
• Antibiotic use can reduce or prevent inflammation both in patients and in
murine models of disease
Kang SS, et al. PLoS Med. 2008;5:e41.
Swidsinski A, et al, J Clin Microbiol. 2005;43:3380–3389.
• Depleted microbiome diversity – a hallmark of both Crohn’s disease and
Ulcerative colitis
Walker A. et al. BMC Microbiol. 2011 Jan 10;11:7.
• Fecal transfer - disease remission within a week; complete recovery noted
after 4 months. No clinical, colonoscopic, or histologic evidence of UC in any
patient (1-13 years later).
Borody, T.J. et al., J Clin Gastroenterol. 2003 Jul;37(1):42-7.
• Mutations in NOD2 and ATG16L1 associated with shifts in the relative
abundance of members of the Faecalibacterium and Escherichia genera.
Frank, D. et al. Inflamm Bowel Dis. 2011 Jan;17(1):179-84.
Role of Gut Microbes in Immunomodulation
• Mice from Jackson or Taconic labs had marked differences in Th17 cell numbers in
• Fecal transfer or co-housing induced Th17 phenotype.
Jackson
Th17 deficient
Taconic
Th17 sufficient
~100 taxa significantly altered in relative abundance
Ivanov et al. Cell. 139: 1-14. 2009
Role of GI microbes in immunomodulation
Ivanov et al. Cell. 139: 1-14. 2009
Segmented Filamentous Bacterium
• Segmented Filamentous Bacterium (SFB)
• Uncultured, commensal, gram-positive,anaerobic, spore-forming bacteria
• Adhere tightly to epithelium in the ileum
• Abundance correlates with reduced colonization and growth of pathogenic
bacteria
Garland et al., Microb Ecol, (198) 2181-190; Heczko et al., The Journal of infectious diseases (2000) 181,
1027-1033.
• Colonization
of Jackson
B6 mice
with SFB leads to IL-17 and IL-22 expression in
• Other bacteria
do not induce
IL-17
TCR+CD4+ small intestine lamina propria lymphocytes
Jax
Jax + SFB
IL-17
20
IL-22
18
10
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IL-17
IL-22
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0
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Jax
Jax
+ SFB
Jax
Jax
+ SFB
Sinotypes
• Sinus mucosal microbiota cluster by dominant pathogen
• Distinct co-colonization patterns
• Opportunity to clinically characterize patients by microbiotype
• Tailored treatment
p=0.001
Pseudomonaceae
Fusobacteriaceae
Relatively even community
Bacteriodales
Staphylococcaceae
Corynebacteriaceae
IBD Microbiome
• Prevalence of IBD is higher in migrant populations
• Microbiome perturbation
Host Genetics
Environment
Gut Microbiome
• Adoption of a Western Diet?
• Genetically predisposed to disease?
Immune response
• Relationships between the microbiome (bacterial and
fungal), host risk gene profile and diet
Study Cohort
• Examine a cohort of (first generation) migrant South Asian
and European UC patients
• 120 UC subjects:
• 60 European descent
• 60 South Asian
• 30 control subjects
• UC patients - Stable disease – gradient of disease severity
• Controls – family members not known to have UC
• Stool samples (Microbiome – bacterial and fungal species)
• Saliva collection (Risk allele SNP profile)
• Long-term dietary information (Block Food Frequency Questionnaire)
Aims
• Determine whether an ulcerative colitis microbial enterotype
exists across migrant populations of distinct ethnic
backgrounds compared to control populations
• Do distinct disease sub-enterotypes exist?
• Which microbial species (bacterial or fungal) dominate these
communities?
• What are the patterns of microbial co-colonization?
• Do relationships exist between sub-enterotypes and disease
severity/distinct phenotypes
• How is the intestinal microbiome related to host genetics,
long-term dietary habits?
To date
• Study commenced in May 2012
• Enrolled 47 subjects to date
Study Subjects Enrolled to Date
UC patients
Healthy subjects
South
Asian
European
South
Asian
European
16
15
9
7
• Paired stool and saliva samples and FFQ’s received from 30 participants
• Extracted nucleic acids from 25 paired samples – quantified
• Q-PCR for bacterial burden
• Bacterial microbiome profile – PhyloChip
• 60,000 bacterial taxa in a single assay
• Fungal profiling - MiSeq
• Risk allele sequencing commenced for 8 loci
Index SNP
Gene/Locus of
interest
Rs1317209
RNF186
Rs11209026
IL23R
Rs10800309
FCGR2A
Rs6706689
PUS10
Rs4957048
CEP72
Rs4077515
CARD9
Rs1558744
LOC341333
Rs971545
IL26
Bacterial Burden is lower in UC Patients
0.16
Anova P P
<<
0.09
16S rRNA PhyloChip Analysis
• All 25 samples amplified across a gradient of annealing temperatures
• Pooled, quantified, labeled and applied to PhyloChip
Extracted total DNA from a
bacterial community
Fluorescence data
analyzed and microbes
identified
15
16S rRNA gene is amplified
using universal primers in 12
replicate reactions across a
gradient of annealing
temperatures
Microarray stained,
washed and scanned
Amplicon pool fragmented,
biotin labeled and
hybridized to PhyloChip
Fluorescently labeled 16S fragments
hybridize to complementary probes on the
array surface
Next Steps
• Continue enrollment – currently on target
• Microbiome analysis
• PcoA - disease enterotypes
• Multi-variate regression (Adonis)
• Diet
• Host risk allele profile
• Disease severity
• Specific taxa related to variables
• Co-colonization patterns
• Dietary comparisons across ethnic groups and patients vs controls
• Risk allele comparisons across ethnic groups and patients vs controls
Acknowledgements
Uma Mahadevan
Michelle Nazareth
Morgan McCormick
Jordan Mar
Mark Seielstad
Mark Segal
Broad Foundation