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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
The Human Microbiome
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.
Selective Pressures Shaping the Microbiome
Host Genetics
Gut Microbiome
Immune response
Environment
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
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.
Ivanov et al. Cell. 139: 1-14. 2009
Role of GI microbes in immunomodulation
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
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
• 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
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)
• 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
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
Bacterial Burden is lower in UC Patients
Anova P < 0.09P < 0.16
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
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 MahadevanMichelle NazarethMorgan McCormickJordan Mar
Mark Seielstad Mark Segal
Broad Foundation