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Hervé Blottiere-El impacto de las ciencias ómicas en la medicina, la nutrición y la biotecnología

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El 29 de marzo de 2016 celebramos un Simposio Internacional sobre el 'Impacto de las ciencias ómicas en la medicina, nutrición y biotecnología'. Organizado por la Fundación Ramón Areces en colaboración con la Real Academia Nacional de Medicina y BioEuroLatina, abordó cómo un mejor conocimiento del genoma humano está permitiendo notables avances hacia una medicina de precisión.

Published in: Health & Medicine
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Hervé Blottiere-El impacto de las ciencias ómicas en la medicina, la nutrición y la biotecnología

  1. 1. MICROBIOME FOR HUMAN HEALTH LESSONS OF A METAGENOMIC SCAN « IMPACT OF OMIC SCIENCES IN MEDICINE, NUTRITION AND BIOTECHNOLOGY » MADRID, MARCH 29TH 2016 Hervé M. Blottière FInE lab « Functionality of the Intestinal Ecosystem», Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay & MetaGenoPolis 78350 Jouy en Josas
  2. 2. « People are not just people. They are an awful lot of microbes, too » ME, MYSELF, US The Economist , August 18th 2012
  3. 3. THE HUMAN INTESTINAL MICROBIOTA Faecalibacterium prausnitzii Ruminococcus spp Clostridium difficile From mice cecumPhotos INRA Bacteroides dorei Escherichia coliBacteria anchored in a Peyer patch Mouse intestine  100 trillion microorganisms ;  10 times the number of cells in the human body;  Hundreds of different species predominantly not yet cultured (~70% of dominant species);  a few dozen conserved between individuals (core); a stable community.  A true organ ; geared to « protecting our health and well- being »« throughout all stages of our life »  A key organ, interacting with food (fermentation,…); interacting with our cells (Immune & nervous systems,…); protecting against pathogens (barrier function);… 3,9·1013 en 2016 x 1.3
  4. 4. Microbiota as an asset  Utilisation of indigestible carbohydrates  Epithelial nutrients (e.g. SCFAs)  Synthesis of B & K vitamins  Conversion of prodrugs  Metabolism of dietary carcinogens  Control motility/Peristalsis  Defenses - bacterial antagonism  Priming of mucosal immunity Microbiota as a liability  Procarcinogens  carcinogens  Overgrowth syndromes  Opportunism - Translocation  Essential ingredient for IBD  Implicated in obesity, metabolic syndrome and colorectal cancer Globally conserved : With a few exceptions : - Microbial metabolites - Methanogenesis - Microbial enzymes- cholesterol reduction - Barrier effect - lignan metabolism - polyphenol metabolism FUNCTIONS OF THE INTESTINAL MICROBIOTA barrier Physio defensesIntestinal Microbiota Nutrition
  5. 5. METAGENOME : genomes of all dominant microbes in an ecosystem Whole Genome Shotgun sequencing Assembly and annotation Reference gene catalog and gene counts A technical revolution since the turn of the century DNA extraction Lessons from early human intestinal tract metagenomics: • Qin Nature 2010 => 3.3 million gene catalog ; both core metagenome & rare genes • Arumugam Nature 2011 => 3 enterotypes, preferred ecological arrangements • Schloissnig Nature 2012 => stability at SNPs level (strains) • Qin Nature 2012 => metagenomic dysbiosis in T2D • Le Chatelier Nature 2013 => metagenomic dysbiosis in obesity ; diagnostic signatures • Cotillard Nature 2013 => metagenomic dysbiosis in obesity ; low gene count as stratifier • Qin Nature 2014 => metagenomic dysbiosis in liver cirrhosis • Li Nat Biotech 2014 => 10 million gene catalog ; core metagenome unchanged • Nielsen Nat Biotech 2014 => co-abundant gene clustering and metagenomic species • Xiao Nat Biotech 2015 => Mouse gene catalog ; environment dependent • Shoaie Cell Metabol 2015 => nutrition and intestinal metabolome • Forslund Nature 2015 => Metformin signature in T2D
  6. 6. A REFERENCE CATALOG OF 10 MILLION GUT GENES Rare genes are increasing Common genes are not Individuals from MetaHIT, Chinese and HMP studies, n=1267 Li et al. Nature Biotech, 2014 124 MetaHIT subjects 3.3 million gut genes Qin, Nature 2010 Total >5% >10% >20% >50% 3.3
  7. 7. HUMANS SHARE A CORE MICROBIOME, AND YET DIFFER AT THE METAGENOMIC LEVEL On average, each individual carries ~540 000 genes of the initial 3.3 million genes catalog Similarity: Core metagenome genes : ~50 % of an individual’s genes are shared by at least 50 % of individuals of the cohort We are all rather similar ! but not identical!! Yet, individuality: Rare genes : genes shared by less than 20 % of individuals = 2.4 million genes Qin, Raes et al, Nature 2010 Mouse catalogue : Xiao et al, Nature Biotech, 2015 Very different from mice : 4% shared genes in the 2 catalogues
  8. 8. HUMANS MICROBIOMES DIFFER AT THE LEVEL OF ECOLOGICAL ARRANGEMENT : THE ENTEROTYPES In a attempt to characterize the intestinal microbiome of ‘the average human’, we observed … an organization of intestinal metagenomes in 3 assemblages of genes and microbial taxa : order in chaos !?! Qin et al, Nature 2010 Arumugam, Raes et al, Nature 2011 May 2011 the enterotypes
  9. 9. Ecology underlying enterotypes should be better understood Bacteroides Prevotella Ruminococcus Impact of diet ? Wu Science 2011 Tap EM 2015
  10. 10. HUMAN GUT MICROBIOMES DIFFER AT THE LEVEL OF GENE RICHNESS (DIVERSITY) n=277 High gene count Low gene count Le Chatelier, et al, Nature 2013; Cottillard et al, Nature, 2013 LGC ≈15 % of lean individuals ≈ 25% Overweight- moderate obese ≈ 75% Morbid obese before bypass surgery 68 « species » significantly linked to gene count
  11. 11. Nielsen, Almeida, et al. Nature Biotech, 2014 GENE CATALOG CLUSTERED IN METAGENOMIC UNITS BY CO-ABUNDANCE BINNING MGS MGU Interaction network CAG: Co-abundant Gene Group • All genes of a species should have the same frequency in an individual • Although proportions of a species vary greatly between individuals • Genes of a species will precisely co-vary in abundance in each 741 large MGU (>700 Genes) correspond to bacterial species (MetaGenomic Species; 85% previously unknown) 238 high quality genomes reconstructed 6640 small MGU (phages, plasmids, CRISPR…)
  12. 12. QUANTITATIVE METAGENOMIC PIPELINE AT METAGENOPOLIS: SAMBO-METAQUANT-INFOBIOSTAT sample collection sequencing reference construction gene profiling sequence mapping onto gene catalogue stool sample total DNA library preparation SOLiD/Illumina/Proton sequencing short sequences 30-50 million reference gene catalogue gene counts genes bioinformatics /statistics analyses preprocessing / normalization & dimension reduction relation with clinical data Identify clinically relevant groups build and test prediction models catalogue structuration individuals 1 2 3 4 1 2 3 4 catalogue annotation MetaHIT & iMOMi Databases • 4 - 8 million genes • 6000 + genomes Standardization is critical http://www.microbiome-standards.org
  13. 13. Can the neglected organ inform on a risk of chronic diseases? Can it be a target for intervention? CHRONIC DISEASES INCREASE STEADILY IN INDUSTRIALIZED COUNTRIES Bach JF, N Eng J Med 2002
  14. 14. Crohn Seksik, 2003; Sokol, 2006, 2008, 2009; Mondot 2010 Ulcerative colitis Sokol, 2008; Martinez, 2008; Lepage 2011 IBS Rajilic-Stojanovic 2011; Saulnier 2011; Chassard 2012; Pouchitis Lim, 2009; Kühbacher, 2006 Obesity Ley, 2007; Furet, 2011, Le Chatelier, 2013; Cotillard, 2013 Type-2 diabetes Cani and Delzenne, 2009; Burcelin 2011, Qin, 2012 Type-1 diabetes Dessein, 2009; Wen, 2008 Coeliac disease Nadal, 2007; Collado, 2009 Allergy Björkstén 2009; Abrahamsson 2012, Russel 2012 Autism Finegold, 2002; Paracho, 2005 Colorectal cancer Mai, 2007; Scanlan, 2008; Sobhani 2011 Frailty in seniors Van Tongeren, 2005, Claessen et al., 2012 Breast cancer Velicer et al., 2004 HIV infection Gori, 2008 Liver Henao-Mejia 2012; Qin 2014 Cardiovascular Wang 2011; Karlsson 2012, Metacardis EU project Other…. PERTURBATION OF INTESTINAL MICROBIOTA AS A POSSIBLE CHRONIC DISEASE FACTOR Indications from metagenomic clinical studies Dysbiosis :  Loss of keystone species  Loss of richness  Increased pathobionts  Metabolic shift
  15. 15. METAGENOMIC SIGNATURES OF DYSBIOSIS IN IMMUNE DISEASES Bacterial genes and genomes specific of the microbiome of patients BMI Inflammatory bowel diseases and obesity 0.10.1 generans nalis.M50.1 f ormis occus.comes.SL7.1 dium.sp.SS2.1 ubacterium.rectale.M104.1 anisolv ens.XB1A 5 gum.subsp..inf antis.CCUG.52486 es.merdae olescentis coccus.torques.L2.14 p..D1 eroides.distasonis.ATCC.8503 ccus.obeum.A2.162 us.bromii.L2.63 cae .thermophilus.LMD.9 cola ium.v entriosum ccus.lactaris pillosusL2.50 ulum.v ariabile v ibacter.smithii.DSM2375 us.colihominis 1 .pamelaeae.gen.nov .sp.Nov C.BAA.835 ctus rof orme prophilus H1 842 rev e mutans.UA159 5586 mentum.IFO.3956es.odontoly ticus uinis.SK36 AA.381 antella.f ormatexigens y riv ibrio.f ibrisolv ens.16.4 a.stadtmanae.DSM.3091 aeruginosa.LESB58 .subsp..multocida.str..Pm70 senteroides.subsp..mesenteroides.ATCC.8293 oncisus.13826 ndidatus.Sulcia.muelleri.GWSS ardii.ATCC.8290 eri.SD2112 P76 is.stercorihominis idium.phy tof ermentans.ISDg nensis.DSM.16047eticus.DPC.4571 TCC.25302us.ATCC.15305 generans nalis.M50.1 f ormis occus.comes.SL7.1 dium.sp.SS2.1 ubacterium.rectale.M104.1 tena anisolv ens.XB1A 5 gum.subsp..inf antis.CCUG.52486 m.hallii es.merdae olescentis coccus.torques.L2.14 p..D1 eroides.distasonis.ATCC.8503 .siraeum.70.3 ccus.obeum.A2.162 us.bromii.L2.63 cae .thermophilus.LMD.9 cola ium.v entriosum usccus.lactaris pillosusL2.50 ulum.v ariabile v ibacter.smithii.DSM2375 us.colihominis 1 .pamelaeae.gen.nov .sp.Nov ansia.muciniphila.ATCC.BAA.835 ctus rof orme prophilus H1 842 densssotus rev e mutans.UA159 5586 mentum.IFO.3956es.odontoly ticus uinis.SK36 cter.hominis.ATCC.BAA.381 antella.f ormatexigens y riv ibrio.f ibrisolv ens.16.4 a.stadtmanae.DSM.3091 aeruginosa.LESB58 is.SH0165 .subsp..multocida.str..Pm70 senteroides.subsp..mesenteroides.ATCC.8293 oncisus.13826 plei.str..Twist ndidatus.Sulcia.muelleri.GWSS ardii.ATCC.8290 eri.SD2112 P76 is.stercorihominis idium.phy tof ermentans.ISDg nensis.DSM.16047eticus.DPC.4571 nsonii.NCC.533aracasei.subsp..paracasei.ATCC.25302saprophy ticus.subsp..saprophy ticus.ATCC.15305 s d = 5 Scores and classes N:N Y:CD Y:UC Axis3 d = 2 Y:UC p-value: 0.031 Crohn Patients UC Patients Healthy Controls Guarner (HUVH, Barcelona) Wang Jun (BGI, Shenzen) Ehrlich, Lepage, Tap (INRA) Pedersen (SDC, Copenhagen) Wang Jun (BGI, Shenzen) Ehrlich (INRA, Paris)
  16. 16. 12 MGS AUC = 0.84 Linear additive model Metagenomic species show a good discrimination power between obese and lean, in contrast to human genome ROCs for individual MGS False positive True positive INTESTINAL MICROBIOTA AND OBESITY IN HUMAN n= 154 Danes 32 obesity risk loci AUC = 0.58 False positive rate n = 8,120 European Speliotes et al. Nature Genetics 2010 Le chatelier et al, Nature, 2013
  17. 17. Le Chatelier et al, Nature 2013 Low to High gene count (French or Danes) Known species n=10 Unknown species n=48 Signature species (n= 58) ROC analysis 4 species LGC HGC In overweight & obesity, Low Gene Count (low bacterial richness) individuals have less healthy metabolic & inflammatory traits: MICROBIOTA GENE COUNT / DIVERSITY IS A HEALTH-ASSOCIATED STRATIFIER Increased adiposity, insulin resistance, dyslipidaemia, inflammation, that predispose for type 2 diabetes, cardio- vascular disease, cancer
  18. 18. FUNCTIONAL SHIFTS IN THE LGC MICROBIOME Le chatelier et al, Nature, 2013 SCFA Butyrate • Energy source for IEC • Trophic & barrier functions • HDAC inhibitor (gene regulation) • GPR agonist – PYY & GLP1 • Immunomodulatory effects Blottière et al, PNS 2003; Segain et al, Gut, 2000
  19. 19. intervention : high protein, low fat and low glycemic index carb with a highly diverse fibre content (1200 to 1500 Kcal) 6 w-intervention 6w-stabilisation Low gene count High gene count DIETARY INTERVENTION AND MICROBIOTA RICHNESS Low gene count individuals lose less weight in response to diet + 25% Cotillard et al, Nature 2013 In low-gene count microbiota, a high-diverse fiber supply may promote an increase in dominant microbiota gene richness
  20. 20. DIETARY HABITS IMPACT GENE RICHNESS 3 dietary patterns are determined based on food frequency records, using 7days-means for 26 food categories Cluster 2 Cluster 3 Cluster 1 ‘richness in fibres’Canonical discriminant analysis Healthier’ dietary habits are associated with higher gene richness Kong et al, PLoS one 2014 yoghurt water vegetables fruits soups Potatoes including chips sweets Sweetened soft drinks - + Karine Clément, ICAN, & Danone Research .
  21. 21. DIETARY MODULATION OF GUT MICROBIOTA CONTRIBUTES TO ALLEVIATION OF BOTH GENETIC AND SIMPLE OBESITY IN CHILDREN diet based on whole-grains, traditional Chinese medicinal foods and prebiotics Phenotype improved Microbiota modulated PWS Prader Willi Syndrome SO Simple Obesity Zhang C-H et al, eBioMedicine, 2015 Functional level Composition
  22. 22. Tremaroli et Bäckhed, Nature 2012; McFall- Ngai et al, 2013 Brain Liver Adipose tissue Muscle Intestine Microbiote MECHANISMS LINKING DYSBIOSIS AND DISEASES
  23. 23. Functional Metagenomics  The intestinal microbiota plays a key role on Human Health, but how ?  Few bacterial compounds/metabolites modulating host cell response identified  An objective : identify bacterial compounds/genes with functional interest and potential applications in the gut microbiota  Mining the microbiome for different functions  How to study these interactions when 80 % of the commensals are not yet cultured ?
  24. 24. Bacterial fraction Large fragment (40 kb) Metagenomic DNA DNA extraction Fosmid vector cloning Transformation in E.coli Metagenomic Library in E.coli Identification of • clones • genes • enzymes • molecules HTS High throughput screening Reporter Human Intestinal Epithelial Cells Substrate of interest (fibers/carbohydrates) Enzymatic activities (hydrolyse) Reporter gene activities Ecosystem (feces) FUNCTIONAL METAGENOMICS Gloux et al., AEM, 2007 Lakhdari et al., Pone, 2010; de Wouters et al., Pone, 2014
  25. 25. ROBOTIC EQUIPMENTS FOR HTS Castor 1 & 2 – Human cells Pollux 1 & 2 – Bacteria culture Development of MetaFun
  26. 26. Metagenomiclibraries Identification of • Clones • Genes • Molecules Sampling & DNA extraction Cloning, recombinants selection Homogenous DNA library Screening 10 libraries = 340 000 clones → 13 million genes HTS Reporter human intestinal epithelial cells FUNCTIONAL METAGENOMICS TO STUDY MICROBIOTA – HOST CROSSTALK
  27. 27. Endocrine Metabolism Proliferation Immunity TSLP, TGF-β, IDO, PlgR, RALDH1,… Muc2, Cyclin D1 Fiaf/Angptl4PYY, GLP-1 Focusing on functional bacterial genes… … and metabolic pathways NF-κB, AP1, AhRAP1, p53PPARγCa2+ Epithelial cell Enteroendocrine cell TARGETED INTESTINAL FUNCTIONS
  28. 28. HTS OF METAGENOMIC CLONES Lakhdari et al, PLoS One, 2010 stimulators inhibitors Growth of metagenomic clones (DO 600nm)
  29. 29. AN EXEMPLE : CLONE 52B7 (FROM CROHN ‘S DISEASE LIBRARY) Random transposon mutagenesis : *52B7/A and C : transposon targeted the same gene encoding an ABC transporter permease *52B7/B transposon targeted a putative lipoprotein with unknown function. the 2 targeted genes code for : a lipoprotein and a lipoprotein transporter Lakhdari et al, PLoS one, 2010 Stimulates NF-kB in several cell lineages and IL-8 secretion in IEC Secreted in the supernatant, size 50 KD, Trypsin sensitive From Bacteroides vulgatus
  30. 30. 5 clones were identified and selected after a screening of 15,000 metagenomic clones from a healthy donor library Cellular reporter systems used: TGFb and IDO promoter assay in HT-29 cells. Work in progress , joint scientific program with Enterome. Aiming to: • Identify, purify the bacterial components and elucidate their structures • decipher the activation pathway, receptors involved • Validate bacterial components on human cellular models (in vitro) or mice models (in vivo) Activation via NF-kB. MyD88 and TLR independent pathway. Sequences from the selected clones belong to Firmicutes / Clostridia XIVa / Ruminococcus.sp Control ControlClone Clone Nicolas Lapaque et al, on going work OTHER EXEMPLE: MODULATION OF IDO/TGF EXPRESSION
  31. 31. TAKE HOME MESSAGES  The Human Gut metagenome is much more complex than expected. 10 million genes facing 23 000 genes;  Although most gut bacteria are still not cultured, MGS can be reconstituted from metagenomic sequencing;  Humans share a core microbiome and yet they differ by genes, species, enterotypes (ecology) and gene count (microbiota diversity).  Metagenomic revealed pathology- associated species;  Functional metagenomic reveals new bacterial genes involved in the cross-talk with human cells;  Human Gut Metagenome is a powerful reservoir of bioactive compounds.
  32. 32. Nicolas Lapaque, Catherine Juste, Christel Maillet-Béra Jean-Marc Lelièvre et al…. Joël Doré « Functionality of the Intestinal Ecosystem » FInE/Blottière lab, INRA UMR 1319 Micalis, Jouy en Josas Karine Clément (ICAN, CHU Pitié Salpétrière) & MetaCardis Consortium Harry Sokol (Hôpital Saint Antoine) Sven Pettersson et col. (Karolinska Institute) Maria Rescigno (IEO, Milan) Oluf Pederson (Novo Nordisk Fundation, Copenhague) Francisco Guarner (Val Hebron Hosp., Barcelone) S. Dusko Ehrlich & MetaHIT Consortium Florence Haimet Nicolas Pons Emmanuelle Le Chatelier Véronique Lejard Florence Levenez Jean-Michel Batto et al….

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