ULCERATIVE COLITIS. Chronic inflammatory bleeding ulcers in the colonic mucosa of unknown etiology. Low rate of spontaneous remission.

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2 ULCERATIVE COLITIS Chronic inflammatory bleeding ulcers in the colonic mucosa of unknown etiology. No pathogen identified, contagious transmission never documented. Low rate of spontaneous remission. Lack of eradication therapy. Prevalence rates of 0.3% in Europe and North America.

3 GWAS STUDIES IN ULCERATIVE COLITIS Meta-analysis of GWA datasets (6,687 UC cases and 19,718 controls) have identified 47 susceptibility loci. 28 loci shared between Crohn s disease and UC. Candidate genes within associated loci include IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1 Anderson et al, Nat Genet 2011

4 PREVALENCE OF ULCERTIVE COLITIS Cases per India Hindu (UK) 100 Sikh (UK) European (UK) 0 Probert et al, Gut 1993 Probert el al, Digestion 1990

5 Genetic Susceptibility Dysregulated Immune Response Commensal Gut Microbes Xavier & Podolsky, Nature 2007 Strober et al, J Clin Invest 2007 Asquith & Powrie, J Exp Med 2010

6 INTERACTION OF NONPATHOGENIC BACTERIA WITH HUMAN INTESTINAL MUCOSA TNFα pg/ml Health Crohn s disease 428± * 0 Blank L. CASEI l. CRISP E. COLI Blank Viable Heat-killed L. crisp E. COLI ATCC L. CASEI DN * p < 0.05 vs. Blank Borruel et al, Gut 2002

7 Cytokine/Chemokine expression by human intestinal mucosa in organ culture IL-6 TGF-β IL-23p19 IL-12p35 IL-17F IL-8 CXCL1 CXCL2 Inflamed vs. Health 31,0±5,9 1,3±0,2 5,1±1,5 1,6±0,1 10,6±6,8 20,1±6,9 4,7±1,2 5,0±0,8 In vitro cytokine release by inflamed intestinal mucosa ER vs. Blanco IL-12p35 * Blanco E.coli L.casei IL TGF-β Blanco E.coli L.casei IL-6 * Blanco E.coli L.casei IL-23 p19 * BlancoE.coli L.casei IL-4 IL-10/ TGF-β IL IL-17F * Blanco E.coli L.casei Llopis et al, IBD 2009

8 INTERACTION OF NONPATHOGENIC BACTERIA WITH INFLAMED INTESTINAL MUCOSA IL-6,TNF, IL-12p35 IL-17F, IL-23p19 Villoosity Peyer s plaque M E. coli Crypt Dendritic cell Paneth cells T B CD3 TNF % TUNEL + Borruel et al, Gut 2002 Carol et al, J Leuk Biol 2005 Llopis et al, IBD 2008

9 IBD: CURRENT DRUG THERAPY Immunosuppression Strong Mild Drug Anti-TNF Anti-integrins Aminosalicylate Budesonide Mucosal healing High Mucosal lesions are caused by an aggressive immune Azathioprine response against gut Moderate Methotrexate Medium bacteria in genetically susceptible Corticosteroids individuals. Low

10 Severe tissue injury by inflammation: role of TNF-α Cell detachment MMP Macrophage TNF-α Inflammation Ulcers TNF-α IL-12 Activated T cell IFN-γ Th1 cell Inflammatory cells TNF-α Coagulation (increased production of thrombin) Adapted from Holtmann et al. Z Gastroenterol. 2002;40:

11 INTESTINAL HOMEOSTASIS

12 Decision making in the adaptive (acquired) immune system is instructed by the microbial impact on APCs and T cells Foreign antigen (Ag) + Naive T-cell activation + CD4 Th1, Th17 TNF-α, IFN-γ IL-17 Cytokines MHC II TCR Tr1 IL-10 APC CD4 T CD4 Treg PRR Ligation Th3 TGF-β + Signatures via pattern recognition receptors CD4 Th2 (PRRs): TLRs & NLRs IL-4, IL-5, IL-13 (NOD+LRR) By Brandtzaeg in Guarner et al, Nature Clin Practice 2006

13 A MODEL FOR THE RESOLUTION OF INFLAMMATION AFTER INFECTION 2 1 Infection by a pathogen expansion of Th1/Th2 cells Clearance of the pathogen and expansion of Treg by commensals Th1/Th2 TNFα / IL-4 IL-6 Treg 3 Resolution TNF / IL-4 IL-6 IL-10/TGF-β ISAPP 2007

14 The Human Gut Metagenome # of genes Non-redundant gene set 3,299,822 Average gene set per individual 590,384 Common (>50% of individuals) 294,110 Rare (<20% of individuals) 2,375,655 Qin et al, Nature 2010

15 Reduced Microbial Gene Diversity in IBD Qin et al, Nature 2010

16 ULCERATIVE COLITIS Family controls UC Cohort study Sustained Remission Cross-sectional study Relapse 50% of patients expected to relapse during follow up analysis of faecal microbiota

17 Ulcerative colitis related microbiome 26 UC patients in remission 32 healthy first degree relatives Gene frequency measured by Illumina sequencing 1) ~30 million paired-end reads/individual determined 2) relative gene frequency calculated = (reads/kb)/σsample reads Genes different in frequency between patients & healthy searched by Ranksum analysis 7770 genes found different at p<10-3 BGI, Wang Jun et al; INRA, Ehrlich et al. Cross-sectional study

18 From UC-related genes to Meta-species by co-variance analysis Hypothesis: Genes from the same species should have similar abundance in an individual The genes that co-vary accross individuals should belong to the same species 5800 of 7770 UC-associated genes assigned to 24 groups >10 genes by covariance analysis Other catalog genes belonging to the same groups retrieved by covariance analysis INRA, Ehrlich et al.

19 Four meta-species only show a good discriminatory capacity between UC and health True positive rate Receiver operator characteristics (ROC) analysis SDC INRA, NN, Ehrlich Pedersen et al. et al. False positive rate Linear addtive model: (genes of meta-species correlated with healthy) - (genes of meta-species correlated with UC) 50 marker genes were used for each species

20 A small number of species diagnostic for the UC patients Bacterial species are associated to chronic disease!! Causal agents, contributors, consequence??

21 Faecal Mucin in UC 10 mg/g 5 * 0 * p<0.01 vs UC Healthy UC Remission UC AKK+ UC AKK-

22 The Mucous Gel Layer of the Human Colon 300 Mucous Layer Thickness 200 µm Caecum Ascending Transverse Sigmoid Rectum Matsuo et al, Gut 1997

23 Randomized, Double-blind, Placebo controlled Probiotic intervention BACKGROUND During remission, dominant gut microbial communities vary over time in UC patients. Temporal instability may be a consequence of low biodiversity, and enriching the ecosystem could favor stability. AIM To evaluate the effect of a fermented milk containing the probiotic B. animalis ssp. lactis DN in the stability of the gut ecosystem in UC patients in clinical remission as assessed by metagenomic analysis of faecal samples.

24 Co-variation of species Circles represent 155 most abundant species, lines the covariance, with R 0.4 Qin et al, Nature 2010

25 STUDY DESIGN Run-in Treatment: 3 months -1 wk 0 6 wk 12 wk UC Verum (n=24): stable drug therapy + probiotic yogurt UC Placebo (n=24): stable drug therapy + acidified milk Healthy controls (n=12): no intervention Clinical assessment, QoL, faecal sampling. Drop outs: 2 verum + 2 placebo for clinical relapse 3 placebo for patient decision

26 Demographic Data at Entry UC VERUM UC PLACEBO CONTROL Gender 14 F/ 10 M 15 F/ 9 M 5 F/ 7 M Age (y) 41 (25-68) 42 (24-64) 31 (20-44) BMI(Kg/m 2 ) 24( ) 24.7( ) 23.1 ( ) Smoking habit 12 no/5 yes/7 ex 12 no/4 yes/8 ex 7 no/4 yes/1 ex Montreal classification Disease duration (mo) 2 A1/ 18 A2/ 4 A3 1 A1/ 20 A2/ 3 A3-9 E1/8 E2/ 7 E3 13E1/4 E2/ 7 E (15-277) 103 (16-418) - Treatment 15 ASA/7 AZA/2 no 15 ASA/7 AZA/2 no - CAI Score 0-24 Score (remission <4) Score 1-0 Score 1-4 Score 2-0 Score 2-2

27 Bifidobacterium animalis DN * Basal (run-in) Final UC Verum 9/21 20/21 UC Placebo 9/19 1/19** Control 3/12 1/12** * Marker of compliance assessed by qpcr of faecal samples: positive out of total assayed. ** Two sided p value < vs. Final UC Verum (Fisher s exact test).

28 CALPROTECTIN* UC Verum UC Placebo µg/g Basal Final 0 Basal Final * Biochemical marker of intestinal inflammation. Values in healthy controls, median and range: 15 (5-70) µg/g.

29 Faecalibacterium prausnitzii copies/g Basal Final Basal Final Basal Final UC Verum UC Placebo Control

30 Acknowledgments MetaHIT Consortium INRA: E. Le Chatelier, M. Almeida, N. Pons, J.M. Batto, P. Renault. HUVH: Ch. Manichanh, E. Varela, M. Antolín, N. Borruel, A. Torrejón, Francesc Casellas. Danone Research: J. Van Hylckama Vlieg, M. Derrien.