1. Evaluating human gut microbiota and microbe-host phenotype relationships Jeremiah Faith Icahn School of Medicine at Mount Sinai Immunology Institute Institute for Genomics and Multiscale Biology 2014 Advances in Inflammatory Bowel Diseases Orlando, FL Dec 6, 2014

2. Associations between our microbes and intestinal disease Crohn’s disease Ulcerative colitis Pouchitis Necrotizing enterocolitis Coeliac disease Diarrhea (C. diff) Colorectal cancer What must be done 1.Verify causation 2.Identify microbial effector strains 3.Modify effector strains to improve health What must be done 1.Verify causation 2.Identify microbial effector strains 3.Modify effector strains to improve health

3. Identifying human microbial effector strains in mice

4. Germ-free and gnotobiotic mice Germ-free: devoid of microbial life Gnotobiotic: “known life”, colonized with defined collections of microbes Humanized microbiota: gnotobiotic animals harboring a human community

5. Effector strain identification successes B. vulgatus accelerates colitis in gnotobiotic guinea pigs [A. Onderdonk et.al., 1980s] IL10-/- mice; HLA-B27 rats [B. Sartor 1990s-now] B. fragilis matures immune system [Mazmanian, et.al., Cell 2005] SFB increase Th17 in small intestine [Ivanov, et.al., Cell 2009] K. pneumoniae and P. mirabilis elicit colitis in T-bet-/- x Rag2-/- [Garrett et. al., Cell Host Microbe 2010] Clostridia increase Treg in colonic lamina propria [Atarashi, et.al., Science 2011] E. coli NC101 modulates colorectal cancer (AOM/IL10) [Arthur, et.al., Science 2012] We are getting better at this. But can we be more exhaustive

6. A systematic pipeline for identifying human effector strains in mice

7. Screening “humanized” mice Which bacteria are causing this?!? Faith, et.al., Sci Transl Med 2014 Diverse donors Gnotobiotic mice Humanized with the microbiota of a unique donor %FoxP3+ among CD4+ Tcells Donor1 Donor2 Donor3 Donor4 Donor5 Germ-free

8. High-throughput anaerobic bacterial isolation Anaerobic isolationProtein extractionIdentification (MALDI-TOF) Each well contains a unique bacterial strain Donor Goodman, et.al., PNAS 2011 Faith, et.al., Science 2013

9. Large scale bacterial isolation Each well contains a unique bacterial strain (17 total microbes) Donor Fractionate community Gnotobiotic mice Phenotype screen Phenotype response Community size % TregsSerineAdiposity

10. Identifying effector strains %FoxP3+ among CD4+ T cells Which bacteria are causing this?!? %FoxP3+ among CD4+ Tcells Donor1 Donor2 Donor3 Donor4 Donor5 Germ-free Faith, et.al, Sci Transl Med 2014

11. Next steps for understanding host/microbe interactions in IBD Diverse donors Gnotobiotic mice Humanized with the microbiota of a unique donor UC, Crohn’s, Pouchitis, PSC DSS, IL10, T-cell transfer Next steps Contributions of host/genotype? Diversity of effector strains Effector strains consistent across mouse colitis models?

12. Identifying human microbial effector strains in humans!

13. Identifying effector strains Hypothesis: members of human gut microbiota modulate complex disease risk

14. Microbial inheritance in complex disease 1.The “strain” is the key unit of measure a.acquired during the first three years of life. b.stable inhabitants for decades. c.decades to manifest clinical symptoms

15. Microbial inheritance in complex disease Microbiome’s Future: optimal colonization at birth H. pylori cagA-cagA+ 1.The “strain” is the key unit of measure a.acquired during the first three years of life. b.stable inhabitants for decades. c.decades to manifest clinical symptoms

16. Microbial inheritance in complex disease DiseaseAgentTime to manifest clinical symptoms LeprosyMycobacterium leprae10-30 years; (13% of household contacts are carriers; Araujo et.al., mem Inst Oswaldo Cruz 2012 ) Whipple’s diseaseTropheryma whipplei~10 years (many asymptomatic carriers) Peptic ulcerHelicobacter pylori~10 years (few infected progress) Gastric cancerHelicobacter pylori>50 years (few infected progress) 1.The “strain” is the key unit of measure a.acquired during the first three years of life. b.stable inhabitants for decades. c.decades to manifest clinical symptoms Evidence of microbial role: MZ < 40% concordant GWAS loci involved in immune regulation, microbial recognition/defense Antibiotics somewhat effective Germ-free models have no or significantly less pathology than colonized Hypothesized: Crohn’s disease Rheumatoid arthritis Multiple sclerosis Treatment: slow the progressive pathology (like Pre-1982 peptic ulcer)

17. Microbial inheritance in complex disease: a simple model P(transmission i ) is the probability of transmission of strain i P(access i ) is the probability of access to strain i P(resistance i ) is the resistance of the host to the colonization by strain i

18. Microbial inheritance in complex disease: consequences Family members share microbes! High Microbial diseases can “look” genetic Majority of family members share strains Faith, et.al., Science 2013

19. Microbial inheritance in complex disease: consequences Family members share microbes! Sibling microbiotas should be more similar than parents Turnbaugh et al, Nature 2009 *p<10 -5 ; **p<10 -14 ; ***p<10 -41 Schloss et al, Microbiome 2014 Low High

20. Microbial inheritance in complex disease: consequences Family members share microbes! Sibling microbiotas should be more similar than parents Increase in disease risk in siblings, especially sequential 1.Gastric Cancer risk 2x higher in sibships of 7 individuals compared to 1-3 [Blaser et.al., PLoS Med 2007] 2.Crohn’s disease risk higher in consecutive births relative to non-consecutive births [Hugot et.al., Eur J Hum Genet 2003] Low High

21. Microbial inheritance in complex disease: consequences Family members share microbes! Sibling microbiotas should be more similar than parents Increase in disease risk in siblings, especially sequential Very hard for cohabiting adults to pass microbes Disease takes years to manifest P(resistance) is HIGH Unrelated individuals do not! Low

22. Microbial inheritance in complex disease 1.The “strain” is the key unit of measure a.acquired during the first three years of life. b.stable inhabitants for decades. c.decades to manifest clinical symptoms We can use methods similar to human genetics! gene

23. Microbial inheritance in complex disease: using the rules to identify effector strains We can use methods similar to human genetics! Case-control association: difficulty unrelated individuals do not share microbial strains Crohn’sNo Crohn’s

24. Microbial inheritance in complex disease: using the rules to identify effector strains We can use methods similar to human genetics! Familial association: difficulty powering study

25. Microbial inheritance in complex disease: moving forwards Need methods to microbe-type at the strain-level

26. Thanks to Faith laboratory Eduardo Contijoch Sean Llewellyn Ilaria Mogno Cinkia Fermin Zhihua Li Ruby Ng Eddie Vazquez Immunology Institute Institute for Genomics and Multiscale Biology Icahn School of Medicine at Mount Sinai Jeffrey Gordon, Philip Ahern, Andrew Goodman, Jean-Frederic Colombel IBD Collaborators Jose Clemente Sergio Lira Miriam Merad Judy Cho Inga Peter Ari Grinspan Funding NIH GM108505 SUCCESS