1. 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

2. The Human Microbiome

3. The Human Microbiome Culture-independent microbial profiling
Composition Function
Spor, A. et al. Nat Rev Microbiol Apr;9(4):
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

4. Selective Pressures Shaping the Microbiome
Environment
Host Genetics
Gut Microbiome
Immune response

5. 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 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 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 Jan;17(1):

6. Role of Gut Microbes in Immunomodulation
Th17 deficient Th17 sufficient
~100 taxa significantly altered in relative abundance
Jackson
Taconic
Ivanov et al. Cell. 139:
Mice from Jackson or Taconic labs had marked differences in Th17 cell numbers in
Fecal transfer or co-housing induced Th17 phenotype. 6

7. Role of GI microbes in immunomodulation
Ivanov et al. Cell. 139:

8. 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) ; Heczko et al., The Journal of infectious diseases (2000) 181,
Other bacteria do not induce IL-17 10 2 3 4 5
10.1
2.1
0.1
IL-17
IL-22
5.2
Jax
+ SFB
Jax + SFB
Colonization of Jackson B6 mice with SFB leads to IL-17 and IL-22 expression in TCR+CD4+ small intestine lamina propria lymphocytes

9. 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

10. IBD Microbiome Prevalence of IBD is higher in migrant populations
Microbiome perturbation
Adoption of a Western Diet?
Genetically predisposed to disease?
Relationships between the microbiome (bacterial and fungal), host risk gene profile and diet
Host Genetics
Gut Microbiome
Immune response
Environment

11. 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)

12. 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?

13. Study Subjects Enrolled to Date Gene/Locus of interest
Study commenced in May 2012
Enrolled 47 subjects to date
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
Study Subjects Enrolled to Date
UC patients
Healthy subjects
South Asian
European 16 15 9 7
Index SNP
Gene/Locus of interest Rs
RNF186 Rs
IL23R Rs
FCGR2A Rs
PUS10 Rs
CEP72 Rs
CARD9 Rs
LOC341333
Rs971545
IL26

14. Bacterial Burden is lower in UC Patients
Anova P < 0.09

15. 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
16S rRNA gene is amplified
using universal primers in 12 replicate reactions across a gradient of annealing temperatures
Amplicon pool fragmented, biotin labeled and hybridized to PhyloChip
Fluorescence data analyzed and microbes identified
Microarray stained, washed and scanned
Fluorescently labeled 16S fragments hybridize to complementary probes on the array surface 15

16. 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

17. Acknowledgements Uma Mahadevan Michelle Nazareth Morgan McCormick
Jordan Mar
Mark Seielstad
Mark Segal
Broad Foundation