Dogma: no bacteria, no IBD!

Transcription

1 MICROBIOMA E MICI Mariangela Allocca, MD, PhD IBD Center, Dept. of Gastroenterology Humanitas Research Hospital Rozzano, Milan, Humanitas Gradenigo, Turin, Italy

2 Dogma: no bacteria, no IBD! Experimental models of colitis No inflammation in IL-10 deficient mice when kept germ-free Clinical models Faecal stream diversion prevents recurrence Madsen K et al Gastroenterology 1999 Rutgeerts P et al Lancet 1991

3 Proximal diversion N = 21 Ileocolonic anastomosis N = 75 Recurrence during diversion Recurrence 3-6 months after reanastomosis 0/21 (0%) - 13/14 (93%) 53/75 (71%)

4 After 6 weeks After 16 weeks Before infusion After 13 days D Haens GR et al. Gastroenterology 1998;114:262-7

5 Avoidance of antibiotics in childhood protects from IBD Author Journal N IBD N controls RR (95% CI) Card et al Gut ( ) Hildebrand et al Scand J Gastro ( ) Shaw SY et al Am J Gastro ( ) Hviid A et al GUT ( ) Virta L et al Am J Epidem ( )

6 Gastroenteritis predisposes to IBD Author Journal N cases N controls Med FU (yrs) HR (95% CI) Garcia Rodriguez LA Gradel KO Gastroenterology 2006 Gastroenterology ( ) ( ) Porter CK Gastroenterology IBD ( )

7 Basic components of IBD pathogenesis Fiocchi C. 1998

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9 Experimental evidence for the importance of microbial exposure Dirty pigs (Outdoor environment) Clean pigs (Indoor environment + antibiotics) Mulder Iet al. BMC Biol 2009;7:79 Gut flora 90% Firmicutes, mostly Lactobacillaceae Gene expression Balanced immunity Gut flora <70% Firmicutes, fewer Lactobacillaceae Gene expression inflammation, cholesterol synthesis

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11 We are only 10% human...and 90% microbial Lee YK & Mazmanian S. Science 2010;330:1768

12 IBD patients are different from healthy individuals in their core microbiome Qin J et al; Nature 2010;464:59-65

13 Dysbiosis (changes in the types and numbers of bacteria in the gut) in IBD 1. Lower diversity 2. Increased density of adherent mucosal bacteria 3. Altered composition

14 Reduced diversity in IBD Loss of normal anaerobic bacteria Ott SJ et al Gut 2004;53:685-93

15 High concentrations of mucosal bacteria in patients with IBD 40% of CD with concentrations > cfu/ml N = Swidsinski et al. Gastroenterology 2002

16 High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in CD Darfeuille-Michaud et al, Gastroenterology 2004

17 Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach Healthy Crohn s disease Bacteroidetes Firmicutes Proteobacteria Actinobacteria Bacteroides vulgatus Eubacteria Peptostreptococci Coprococci E. coli Bifidobacteria Lactobacilli Faecalibacterium prausnitzii Manichanh et al. Gut 2006

18 Faecalibacterium prausnitzii and IBD Author Journal Year Source Effect? Sokol PNAS 2008 Mucosa reduced Willing IBD 2009 Mucosa reduced Schwiertz J Pediatr 2010 Faeces Reduced Kang IBD 2011 Faeces Reduced Mondot IBD 2011 Faeces Reduced Joossens Gut 2011 Faeces Reduced Sokol et al PNAS 2008; 105: Ng IBD 2011 Faeces reduced

19 Dysbiosis signature in CD patients Bacterial species Unknown species of Clostridium cluster XIVa Ruminococcus gnavus Faecalibacterium prausnitzii Presence on DGGE (%) Crohn s disease patients (N = 68) Unaffected controls (N = 139) Corrected P-value* 60% 88% % 9% 2.1 x % 71% 1.4 x 10-5 Dialister invisus 16% 42% 0.04 Bifidobacterium adolescentis 37% 71% 5.4 x 10-6 *Mann-Whitney U test on the intensity per band-class per groups; two-tailed P-values after Bonferroni correction (number of band-classes = 75) Joossens M et al Gut 2011; 60: 631-7

20 Dysbiosis signature in CD patients and their unaffected relatives CD patients F. Prausnitzii B. Adolescentis UK species C. cluster XIVa D. Invisus R. gnavus Unaffected relatives C. Aerofaciens Member E coli-shigella group R. torques butyrate producing-bacteria mucin degradation capacity bacteria Joossens M et al Gut 2011; 60: 631-7

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24 The dominant impact of the gut microbiota: should microbial health be the ultimate goal in medicine? Bischoff S. BMC Biomedicine 2011; 9:24

25 Maintaining and restoring a natural lawn Maintaining and restoring a healthy gut microbiota A flourishing lawn Drought, pesticides, parasites Left alones, weeds run wild Healthy microbiota Antibiotics, poor diet, infections Abnormal microbiota Fertilizers Proper food New seeding Probiotics Prebiotics Balanced diet Restored lawn Restored microbiota Lozupone C et al. Nature 2012;489:220

26 Microbial manipulation strategies to maintain intestinal health Diet Probiotics: introduction of missing desirable microbes Antibiotics: removal or suppression of undesirable microbes Prebiotics: proliferation of beneficial microbes and probiotics Fecal transplant: replacement of pathologic microbiota with normal microbiota Defensins: replenishment of antimicrobial peptides controlling the gut microbiota Predeis GA & Versalovic J. Gastroenterology 2009;136:2015

27 How to change gut microbial composition? Diet and specific foods Antibiotics Pre-and probiotics Faecal bacteriotherapy: place in IBD?

28 Do foods condition the gut microbiota composition?

29 Influence of diet on the composition of gut flora Diet Burkina Faso EU (Italy) Fat content Low High Animal proteins Low High Fibers, plant polysaccharides High (2-4%) Low ( %) De Filippo et al, PNAS 2010

30 The impact of diet in shaping the gut microbiota: drastically different microbial patterns (enterotypes) in children from rural Africa and Europe Rural Africa Europe De Filippo C et al. PNAS 2010;107:14691

31 Influence of diet on composition of gut flora Phyla Burkina Faso EU Actinobacteria Bacteroidetes Firmicutes Proteobacteria BF EU De Filippo et al, PNAS total SCFA Acetic Propionic Butyric

32 Induction of increased endotoxin (LPS) plasma levels in healthy subjects on a Western-style (high fat) diet Pendyala S et al. Gastroenterology 2012;142:1100

33 Food intake and risk for IBD Increase in incidence of IBD associated with Western diet : high fat, polyunsaturated fatty acids, meat intake, low intake of fibers, fruits and vegetables Hou JK et al Am J Gastroenterol 2011; 106:

34 Case study Moroccan family - moving to Belgium 1965 Father 46 moves to Belgium Reunion of the family in Belgium First complaints of 76 - April: 46 diagnosis of CD, acute onset - November: 76 appendicitis, no diagnosis CD Diagnosis of CD for August: 80 diagnosis of CD May: 82 diagnosis of CD, acute onset December: 79 diagnosis of CD Joossens M et al. Inflamm Bowel Dis 2007

35 Diet and intestinal immunity: role of aryl hydrocarbon receptor Highly conserved, ligand inducible transcription factor Intestinal immune functions dependent on dietary AhR ligands AhR -/- mice C. rodentium Cruciferous vegetables AhR ligands Tilg H New Engl J Med 2012; Kiss EA Science 2011; Li Y Cell 2011

36 Omega-3 fatty acids (Fish Oil): maintenance of remission in UC Turner D et al Cochrane systematic Review 2011

37 Omega-3 fatty acids (Fish Oil): maintenance of remission in CD Feagan B et al Jama 2008 Turner D et al Cochrane systematic Review 2011

38 Interim conclusion: diet and specific foods Western diet seems noxious for normal gut homeostasis and microbial composition Change to more fibers, fruits, vegetables, less fat and animal protein intake More research needed if specific diets may have therapeutic effect in IBD? Prevention of onset in relatives of multiple affected families? Influence on disease course in early IBD?

39 How to change gut microbial composition? Diet and specific foods Antibiotics Pre-and probiotics Faecal bacteriotherapy: place in IBD?

40 Antibiotics: induction of remission in active CD Khan KJ et al Am J Gastroenterol 2011; 106:

41 Rifaximin in mild CD (n= weeks) 70 CDAI <150 p= CDAI <150 % placebo 400 mg bid 800 mg bid 1200 mg bid Prantera C et al Gastroenterology 2012; 142:

42 Antibiotics: induction of remission in active UC Khan KJ et al Am J Gastroenterol 2011; 106:

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44 0 Steroid-refractory and 64 steroid-dependent C patients enrolled: moxicillin 500 mg, tetracycline 500 mg and etronidazole 250 mg three-times-daily by mouth, or two weeks, as well as stable conventional treatment ith mesalazine, prednisolone, azathioprine, MP teroids were gradually tapered after week 8

45 Effects of ATB combination therapy on clinical activity scores 60.9% steroid-dependent and 53.3% steroid-refractory patients stopped steroids

46 Effects of ATB combination therapy on endoscopic activity scores 46.7% 32.8% 53.3% 35.9% Mucosal healing: mucosal subscore 0-1

47 Interim conclusion: Antibiotics Small studies Heterogeneity in class of antibiotics used Endpoints not always well defined Future studies include culture-independent analyses of changes in microbiota to explore what effects these interventions have on gut flora Focus trials on patients with specific bacteria only? (anti-map antibiotics in MAP+ patients only?, )

48 How to change gut microbial composition? Diet and specific foods Antibiotics Pre-and probiotics Faecal bacteriotherapy: place in IBD?

49 Definitions Probiotics Living non-pathogenic micro-organisms which, when ingested, exert a positive influence on host health or physiology Bifidobacteria, Lactobacilli, E coli Nissle, VSL#3 Prebiotics Nondigestible food ingredients that beneficially affect host by selectively stimulating growth and/or activity of one or number of bacteria, thus improving host health Fructo-oligosaccharides (FOS), inulin, psyllium Synbiotics

50 Special Genetic and Phenotypic Characteristics of E. coli strain Nissle 1917 (EcN) Fitness factors 7 different iron uptake systems Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ 2 Small cryptic plasmids mch mcm cbt fim chromosome ent foc ybt chu rfb csg aer cit kps fla 3 kb 5 kb Antagonistic activity 2 Microcins (M, H47) Adhesion 3 types of Fimbria (F1A, F1C, Curli) Flagella of H1 type without antibiotic resistance genes, not transmissible Special LPS of O6 type (truncated O6 side-chain) K5 capsule Serotype O6 : K5 : H1 O6 serotype : K5 : H1

51 Interactions of Probiotics within the Gut and with the Gut Mucosa Probiotic (e.g. E. coli Nissle 1917) Host cell signaling direct antagonistic Activity Inhibition of Growth / Killing of pathogenic Bacteria and Yeasts Augmentation of Colonization Resistance Biofilm formation, Mucin production, Tight Junction Sealing Immunomodulation, e.g. enhanced IL-10-Level, Defensin Synthesis inflammatory Effects (Inhibition of IL-5, IL-6, IFN-γ, TNF -Stimulation of IL-8 Synthesis) Invasion of Gut Epithelia by Salmonella, EIEC, AIEC, Shigella, Yersinia, Listeria, Candida Darmepithel Intestinal Epithelium

52 120 UC patients Aliment Pharmacol Ther Oct;11(5):853-8.

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55 E. coli Nissle 1917 maintains remission in UC patients

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59 Lactobacilli, bifidobacteria and E. coli Nissle induce pro- and antiinflammatory cytokines in periphal blood mononuclear cells. U. Helwig et al., World J. Gastroenterology 12: (2006). Tested Bacterial Strains: - Bifidobacterium breve Y 8 - Bifidobacterium infantis Y 1 - Bifidobacterium longum Y 10 - Lactobacillus acidophilus MB Lactobacillus casei MB Lactobacillus delbrueckii subspecies bulgaricus MB Lactobacillus plantarum MB 452 VSL#3 - Lactobacillus casei subspecies rhamnosus LGG (ATCC Valio, Finland) - E. coli Strain Nissle 1917 (EcN) InfectoDiarrstop LGG Mutaflor MS/US 10108

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62 Oligofructose-inulin (OF-IN) Detailed description of dysbiosis in CD patients Joossens M et al. Gut Effect of OF-IN intake in healthy subjects Joossens M et al. FEMS Log10 B. adolescents CD patients p= Unaffected relatives Unrelated controls Log10 B. adolescents Baseline p<0.01 OF-IN AIMS: 1. Do we see an increase in fecal B. adolescentis in Crohn s disease patients after OF-IN? 2. Does the change in bacteria lead to a change in disease activity? Joossens M et al; GUT 2011 epub

63 FOS in active Crohn s disease 103 patients randomized: 15 g/day FOS (n=54) or placebo (n=49) - 4 weeks Withdrawals: 26% (FOS) versus 8% (placebo): p = no significant differences in the faecal concentration of bifidobacteria and F prausnitzii between the groups % 45 Response remission FOS Placebo 2,5 2 1,5 IL-10 staining DCs 1 0,5 0 p=0.035 FOS Baseline Week 4 p=ns Placebo Benjamin JL et al GUT 2011;60:923-9

64 Pre-and probiotics Prebiotics?? Probiotics: E.coli nissle efficace alternativa alla mesalazina nel mantenimento della remissione in RCU VSL#3 efficace nel mantenimento della remissione nelle pouchiti

65 How to change gut microbial composition? Diet and specific foods Antibiotics Pre-and probiotics Faecal bacteriotherapy: place in IBD?

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67 Fecal microbiota transplant (FMT) Fecal microbiota transplant is the infusion of a fecal suspension of normal stools into the gastrointestinal tract of another person (patient) to cure a specific disease Healthy gut Diseased gut Aroniadis O & Brandt L. Curr Opin Gastroenterol 2012;28 [epub ahead of print]

68 Fecal microbiota transplantation in C. difficile colitis Borody T & Khoruts A. Nat Rev Gastroenterol Hepatol 2012; 9:88

69 Change of the gut microbiome after fecal transplantation for recurrent C.difficile colitis Microbial diversity in C.difficileinfected patients before and after fecal transplantation Khoruts A et al. J Clin Gastroenterol Hepatol 2010; 44:354 Van Nood E et al. N Engl J Med 2013; 368:

70 Established and potential therapeutic effects of fecal microbiota transplant Gastrointestinal diseases C. difficile infection Inflammatory bowel disease Irritable bowel syndrome Chronic constipation Non-gastrointestinal diseases Obesity Autism Idiopathic thrombocytopenic purpura (UC-associated) Myoclonus dystonia (diarrhea-associated) Parkinson s disease (Chronic constipation-associated) Chronic fatigue syndrome Aroniadis O & Brandt L. Curr Opin Gastroenterol 2012;28 [epub ahead of print]

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72 UC patients (> 18 yrs) with Mayo score > 4 and endoscopic subscore > 1 randomized 1:1, once weekly for 6 weeks, to: - 50 ml of FMT, via enema, from healthy anonymous donors (N 38) - placebo (N 37) Primary outcome at week 7: remission defined as a Mayo score < 2 with an endoscopic subscore= 0

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74 TURN trial

75 Double-blind, placebo-controlled, randomized, phase 2 trial UC pz with a SCCAI > 4 < 11 randomized 1:1 to 2 duodenal infusions, at three weeks apart, of FMT from healthy donors (N 23) or of a suspension of autologous feces (N 25) The primary outcome, at w12, was clinical remission* with > 1-point decrease in the endoscopic score * SCCAI score < 2

76 Primary outcome: SCCAI score < 2 + >1-point decrease of endoscopic score Week 12

77 Major differences between the two trials Gastroenterology 2015 Jul;149(1):15-8

78 Pre-and post faecal bacteriotherapy February 7th, 2012 March 30th, 2012

79 Concluding perspectives: dysbiosis and therapeutic potential in IBD? IBD microbiota characterized by: Reduced diversity Increased mucosal bacteria (AIEC) Altered composition ( dysbiosis signature ) Reduction in production of butyrate and other SCFAs Increase in mucin degradation

80 Concluding perspectives: dysbiosis and therapeutic potential in IBD? What components of microbiota are risk factors to develop IBD? need for longitudinal studies in relatives at risk Risk stratification by microbial analysis predictive markers for treatment choice and response? predictive markers for complication risk? New targets for microbiota modulation (including food!)

81 The dominant role of microbes in health and disease Number of cells Number of genes ,000 Human ,000,000 Gut microbes (10X as many) (100X as many) Health: the result of keeping your microbes happy

82 GRAZIE