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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 Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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Page 1: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

The Gastrointestinal Microbiome in Ulcerative Colitis.

Susan V. Lynch, PhD

Associate Professor of MedicineDirector 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

Page 2: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

The Human Microbiome

Page 3: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

The Human Microbiome

• Culture-independent microbial profiling

• CompositionFunction

• Microbiome dysbiosis associated with chronic inflammatory/autoimmune diseases

• Contribute functions critical to host health:

• Metabolism of indigestible carbohydrates• Vitamin and hormone production• Immune development• Immune homeostasis• Ancillary mucosal protection

Spor, A. et al. Nat Rev Microbiol. 2011 Apr;9(4):279-90.

Page 4: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Selective Pressures Shaping the Microbiome

Host Genetics

Gut Microbiome

Immune response

Environment

Page 5: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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.

Page 6: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Role of Gut Microbes in Immunomodulation

Th17 deficient Th17 sufficient

~100 taxa significantly altered in relative abundance

Jackson Taconic

Ivanov et al. Cell. 139: 1-14. 2009

• Mice from Jackson or Taconic labs had marked differences in Th17 cell numbers in• Fecal transfer or co-housing induced Th17 phenotype.

Page 7: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Ivanov et al. Cell. 139: 1-14. 2009

Role of GI microbes in immunomodulation

Page 8: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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.

0 102 103 104 105

0

102

103

104

105

10.1

0 102 103 104 105

0

102

103

104

105

2.1

0.1

IL-17

IL-2

2

5.2

IL-17 IL-22

Jax Jax+ SFB

Jax Jax+ SFB

Jax 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

• Other bacteria do not induce IL-17

Page 9: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Corynebacteriaceae

Fusobacteriaceae

Relatively even communityBacteriodales

Pseudomonaceae

Staphylococcaceae

Sinotypes

p=0.001

• Sinus mucosal microbiota cluster by dominant pathogen• Distinct co-colonization patterns• Opportunity to clinically characterize patients by microbiotype

• Tailored treatment

Page 10: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

• 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

IBD Microbiome

Host Genetics

Gut Microbiome

Immune response

Environment

Page 11: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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)

Page 12: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

• 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?

• Determine whether an ulcerative colitis microbial enterotype exists across migrant populations of distinct ethnic backgrounds compared to control populations

Aims

Page 13: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

To date

• 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 South Asian

European

16 15 9 7

Index SNP Gene/Locus of interest

Rs1317209 RNF186

Rs11209026 IL23R

Rs10800309 FCGR2A

Rs6706689 PUS10

Rs4957048 CEP72

Rs4077515 CARD9

Rs1558744 LOC341333

Rs971545 IL26

Page 14: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Bacterial Burden is lower in UC Patients

Anova P < 0.09P < 0.16

Page 15: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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

Microarray stained, washed and scanned

Fluorescently labeled 16S fragments hybridize to complementary probes on the

array surface

Fluorescence data analyzed and microbes

identified

16S rRNA PhyloChip Analysis

15

• All 25 samples amplified across a gradient of annealing temperatures• Pooled, quantified, labeled and applied to PhyloChip

Page 16: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

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

Page 17: The Gastrointestinal Microbiome in Ulcerative Colitis. Susan V. Lynch, PhD Associate Professor of Medicine Director Colitis and Crohns Disease Research

Acknowledgements

Uma MahadevanMichelle NazarethMorgan McCormickJordan Mar

Mark Seielstad Mark Segal

Broad Foundation