Gut Permeability, The Microbiome, And Metabolic Disorders Leading To Obesity: How to Connect The Dots

Transcription

1 Gut Permeability, The Microbiome, And Metabolic Disorders Leading To Obesity: How to Connect The Dots Alessio Fasano, M.D. W. Allan Walker Chair in Pediatric Gastroenterology and Nutrition Mucosal Biology and Immunology Research Center And Center for Celiac Research And Treatment Massachusetts General Hospital for Children Harvard Medical School

2 Lecture Objec7ves Genes, Nutri7on and the Gut Mucosa Func7ons: How to Connect the Dots Microbiome Environmental Factors Clinic Outcome

3 We Are Not Born With The Destiny to Develop Chronic Inflammatory Diseases

4 The Yin and Yang Between Tolerance and Immune Response Leading To Chronic Inflammatory Diseases Increased Gut Permeability Human Genome Immune Response Environmental Factors Clinic Outcome Microbiome

5 Several Cells Play a Role in Maintaining The Immune Homeostasis Epithelial cells Intes7nal DCs B cells T cells

6 Loss of Mucosal Immune Homeostasis Chronic Inflammation-Allergy Normal/physiologically controlled permeability Minor barrier defect dietary/microbial Ag influx Increased permeability Massive dietary and microbial antigen influx Defensins Mucus Synthesis & Quality SIgA Mucosal Tolerance Homeostasis Anergy Regulatory DC s Macrophages Tregs IL-10/TGF-β Adapted from P. Brandtzaeg, Beneficial Microbes 2010 Inflammation - Allergy Innate or immunoregulatory defect Vicious circle Proinflammatory Allergic cytokines Break of Tolerance Apoptosis resistant T cells Tissue damage Chronic inflammation Allergy

7 The Paracellular Pathway PURIFICATION PROTOCOL FROM HUMAN INTESTINE : Tissue lysate 2: Sephacryl-S300 3: Q-sepharose Tight junctions are a dark horse implicated in a host of disease states, ranging from acute injury to chronic inflammation and autoimmune diseases 4: Immuoaffinity Coomassie Western blot Fasano A. et al Lancet 2000;355: Wang W et al J Cell Sci 2000;24:

8 Zonulin Characterization and Signaling Zonulin signaling Freeze-Fracture Following Pathway Activation Resting State Fasano et al, J Clin Invest 1995;96:710-20; el Asmar et al Gastroenterology 2002;123:

9 Zonulin Gene Is Located on Chromosome 16 Chromosome 16 contains about 98 million bases, or some 3% of the human genome, encoding for ~1,300 genes. Fasano A. Physiol Rev Jan;91(1):151-75

10 Literature Report On Zonulin Association With CID Disease Model Zonulin Shown to be Involved Celiac Disease Human YES Coli7s/IBD (Crohn s disease) Human YES Coli7s Mouse YES Fe metabolism in heart transplant Human NO Glioma Human YES Glioma Cell YES Irritable bowel syndrome Human YES HIV Human YES Mul7ple sclerosis Mouse YES Necro7zing Enterocoli7s (NEC) Rat YES Nonalcoholic fasy liver disease Human YES Non-Celiac Gluten Sensi7vity Human YES Obesity/Insulin resistance Human YES Post-surgery Sepsis Human YES Post-surgery Sepsis Mouse YES Psoriasis Human NO Sepsis Human YES Type 1 diabetes Human YES Type 2 diabetes Human YES

11 Serum Zonulin Levels and Their Correlation With Intestinal Permeability In Celiac Disease and Type 1 Diabetes Celiac Disease Type 1 Diabetes Zonulin-LA/MA A B C N=339 N=89 N= Serum Serum zonulin (ng/mg protein) T1D T1D T1D Relatives controls Controls LA/MA multiple R=0.36; intercept p=1.71e- 10; X variable 1 p= Zonulin (ng/mg protein) Sapone et al Diabetes 2006; 55:1443-9

12 Environmental Triggers Causing Zonulin Release Gluten Gut Microbiome

13 Mechanisms of Zonulin Release: Gliadin Shan L et al, Science. 2002; 297: Lammer K et al, Immunology. 2011;132: Maiuri et al. Scand J Gastroenterol. 1996; 31: Lammer K et al, Gastroenterology 2008;135: CXCR-3 is a seven-transmembrane G couple protein receptor that is preferentially expressed on activated T lymphocytes and subset of B and NK cells. Three known CXCR3 ligands CXCL-9, -10, -11 are produced at the site of inflammation and elicit migration of pathological Th1 cells. CXCR3 has been implicated as a potential target for impeding T-cell-mediated destruction in autoimmune diseases such as multiple sclerosis and type 1 diabetes Mapping of α-gliadin motifs exerting cytotoxic activity (red), immunomodulatory activity (light green), zonulin release and gut permeating activity (blue) and CXCR3-dependent IL8 release in CD patients (dark green).

14 Proximal Bowel Contamination Activates the Zonulin Pathway CONTROL BACTERIA Zonulin (ng/mg protein) Zonulin Release Changes in Tissue Resistance Uninfected E.Coli 6-1 Salmonella control (non-pathogen) ty phimurium (Close bars: Mucosal Side; Open bars: Serosal Side) Δ- TEER (Ohms.cm 2 ) Uninfected E.Coli 6-1 Salmo nella control (non-pathogen) typhim urium Zonulin Actin Nucleus Changes in Tissue Resistance Is Prevented by the Zonulin Inhibitor At1001 Δ- TEER (Ohms.cm 2 ) Media Control E. Coli 6-1 E. Coli 6-1+AT Time (minutes) El Asmar et al Gastroenterology 2002;123:

15 The Microbiome as Possible Transducer of All Environmental Factors Affecting Onset of CID in Genetically Susceptible Individuals Martin VJ, et al J Pediat 2016 Sept 12, (Epub ahead of print)

16 The human gut harbors bacteria per gram colonic content (>10 14 total bacteria) Total bacteria outnumber human cells 10:1 Total bacterial genes outnumber human genes >150:1 >10,000 different species of bacteria are resident in the human intestinal microbiota ( per person)

17 Is Microbiome Science Ready For Primetime Clinical Applicability? Classification of Organisms Milky Way Solar System Universe Domain Kingdom Phylum Bacteria Firmicutes Earth Class Clostridia North America Order Clostridiales U.S.A. Family Ruminococcaceae North Carolina Genus Anaerotruncus Wilmington Species Anaerotruncus_sp Adapted from

18 The Omics Revolution Microbiome (Who is there): A powerful tool used to analyze microbial communities (commensal, symbiotic, and pathogenic microorganisms) regardless of the ability of member organisms to be cultured in the laboratory Metagenomics/Metatrascriptomic (What language do they speak and what are they talking about): Genomic and trascriptome analysis of microbial DNA extracted directly from communities present in a specific environment, for example the human gut. E Schwager et al. Microbes, 2015;10:

19 Distribu7on of microbes in the gut Present in all parts of the intes7nal tract Increase from esophagus to colon acid produc7on bile mo7lity ileocecal valve Surface-lumen axis: more anaerobes in the outer mucus FISH: bacteria are not in direct contact with the mucosa at least, in healthy subjects (vs. Crohn s disease) The gut microbiota and host health: a new clinical frontier. JR Marchesi et al. Gut 2016;65;

20 Why The First 1000 Days Of Life Are Instrumental For Clinical Destiny Vaginal Delivery Proper Nutrition No infections No Antibiotic treatments C section Inappropriate Nutrition Multiple infections Antibiotic treatments Microbiome Composition Probiotics Prebiotics Symbiotics Balanced Microbiome Dysbiosis Appropriate GALT Maturation + Genetic Predisposition + Inappropriate GALT Maturation Tolerogenic Response to Food Antigens - State of Health Pro-inflammatory Response to Food Antigens - CID

21 Role of Breastmilk Impact of human milk glycobiome on the infant intestinal microbiota Zivkovica AM, et al. PNAS 2011;108:

22 Gut Microbiome Clusters by Diet: Ley et al, Science 2008;320:

23 Microbiota in immunity & inflamma7on Y Belkaid & TW Hand, Cell 2014;157:

24 Low diversity (less modular) gut microbiota in IBD and in obesity 124 Danish & Spanish adults 42 BMI >30 25 IBD S Greenblum et al., PNAS 2012;109:

25 Core microbiome in obese and lean twins: Missouri Adolescent Female Twin Study PJ Turnbaugh et al. Nature 2009;457: with restora7on ager gastric bypass: H Zhang et al. PNAS 2009;106:

26 Intes7nal Trialogue: epithelium, microbiome, immunity, nutri7on N Shulzhenko et al. (Macpherson, McCoy, Fraser-Ligget, Matzinger) Nature Medicine 2011;17:

27 Gut microbiota of elite athletes (black) vs. low BMI (red) and high BMI (green) controls. Clarke SF et al. Gut 2014;63:

28 Gut Dysbiosis

29 Reduced bacterial diversity (dysbiosis): an emerging theme across diseases Microbiota affected by: - Infec3ons - An3bio3cs - Xenobio3cs Diabetes mellitus Obesity Cancers: gastric, colonic Inflammatory bowel diseases Necro3zing enterocoli3s Irritable bowel syndrome, colic Scientific American June 2012 B Schroeder & F Backhed, Nat Med 2016;22(Oct):

30 Correla7on Between Dysbiosis And Human Diseases

31 Role of Gut Microbiota In Metabolic Disorders

32 Why A Leaky Gut Together With Dysbiosis Can Make Us Fat

33 RATIONALE An increase in intestinal permeability is considered to be associated with gut inflammatory tone and development of obesity, fatty liver (typical of obese subjects) and type 2 diabetes

34 Microbes and Host Metabolism Kristine H Allin et al. Eur J Endocrinol 2015;172:R167-R177

35 Factors Influencing Bacterial Fig. 3 Transloca7on (BT) - Endotoxemia

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39 Loss of Mucosal Immune Homeostasis Leading to NAFLD Normal/physiologically controlled permeability Minor barrier defect dietary/microbial Ag influx Increased permeability Massive dietary and microbial antigen influx Microbiome/ Metatranscriptomic Defensins Mucus Synthesis & Quality SIgA Metabolome Human RNA seq Inflammation Mucosal Tolerance Homeostasis Anergy (HEALTH) Regulatory DC s Macrophages Tregs IL-10/TGF-β Innate or immunoregulatory defect Proinflammatory Allergic/Th1 cytokines Vicious circle

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41 Circulating Zonulin, a Marker of Intestinal Permeability, Is Increased in Association with Obesity-Associated Insulin Resistance The correlation between insulin sensitivity and circulating zonulin in participants with normal glucose tolerance (NGT, n = 82) and with glucose intolerance (GI, n = 41).

42 Increased circulating zonulin in children with biopsy-proven nonalcoholic fatty liver disease Zonulin levels for obese children. A: Zonulin levels for obese children with and without nonalcoholic fatty liver disease (NAFLD); B: Zonulin levels for obese children with NAFLD according to severity of steatosis. World J Gastroenterol Dec 7; 20(45):

43 Probiotic With or Without Fiber Controls Fig. Body 3 Fat Mass, Associated With Serum Zonulin, in Overweight and Obese Adults-Randomized Controlled Trial. Correlation between serum zonulin, serum hscrp and trunk fat mass. Evolution of zonulin (a) and hscrp (c), and correlations between zonulin (b) and hscrp (d) with trunk fat mass as changes from baseline to end-of-intervention (6months) in the Per Protocol population. Panels a) and c) display changes from baseline as mean ± 95% confidence intervals at each time point. Placebo n=35 36, LU n=35 36, B420 n=24 25, LU+B420 n= EBioMedicine Oct 26

44 USA Only in.:

45 The Complexity of the Human Body Over the years we came to appreciate the complexity of the human body Only 25,000 genes, 99.5% identical to chimpanzee, cannot explain such complexity and difference with other primates. However, it would be inappropriate to describe the human body without considering the 300,000,000,000 bacteria (collectively defined as microbiome) gladly living inside us and that express ~100 fold more genes that the human genome.

46 The Real Story of Our Genetic Complexity: We Inherit two Parallel Genomes Human Genome: Inherited from both parents, stable, never change in its composition Microbiome: Inherited from the mother, extremely dynamic, changes from individual to individual and in the same individual over time

47 Microbiome (140-fold Human Genome) Dynamic Human Genome (~30,000 genes) Stable Metabonome Jazz Pop Clinical Outcome Classic

48 Modeling the Effects of Early Childhood Microbiome Composition On Entire Lifespan First 1000 days of life Microbiome establishment and modification

49 Acknowledgments NIH DK NIH DK NIH DK The MIBRC Crew