The Role of Nutrition, Gut Microbiome, and Gut Permeability in Immunomodulation: The Celiac Disease and Type 1 Diabetes Paradigms

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Allan Walker Chair in Pediatric Gastroenterology and Nutrition Professor of Pediatrics Harvard

1 The Role of Nutrition, Gut Microbiome, and Gut Permeability in Immunomodulation: The Celiac Disease and Type 1 Diabetes Paradigms Alessio Fasano, M.D. W. Allan Walker Chair in Pediatric Gastroenterology and Nutrition Professor of Pediatrics Harvard Medical School Mucosal Biology and Immunology Research Center And Center for Celiac Research Massachusetts General Hospital for Children

2 Meeting Overview On New Concepts of Autoimmunity: Excessive and Inappropriate Inflammatory Process Associated to a Dysfunction of Barriers Microbiome Environmental Factors Clinic Outcome

3 The Epidemics of Immune-Mediated Diseases In The Western Hemisphere: The Hygiene Hypothesis

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

5 The Yin and Yang Between Tolerance and Immune Response Leading To Chronic Inflammatory Diseases: Lesson Learned From Celiac Disease Increased Gut Permeability Immune Response Human Genome Environmental Factors Clinic Outcome Microbiome

6 Several Cells Play a Role in Maintaining The Immune Homeostasis Epithelial cells Intestinal DCs B cells T cells

7 Excessive and Inappropriate Inflammatory Process Associated to a Dysfunction of Barriers: Loss of Mucosal Immune Homeostasis 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-b 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

The Paracellular Pathway PURIFICATION PROTOCOL FROM HUMAN INTESTINE 1 2 3 4 1 2 3 4 1: Tissue lysate

states, ranging from acute injury to chronic inflammation and autoimmune diseases 4: Immuoaffinity

8 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:

9 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:

DSS Mouse Models to Establish The Link Between

to Inflammation C57Bl/6 wild type mouse HP 1-1 (no

gene HP 1-2 (1 zonulin gene) Transgenic C57Bl/6 mouse

11 DSS Mouse Models to Establish The Link Between Distribution of Haptoglobin Alleles And Susceptibility to Inflammation C57Bl/6 wild type mouse HP 1-1 (no zonulin gene) C57Bl/6 mouse transfected with human HP2 gene HP 1-2 (1 zonulin gene) Transgenic C57Bl/6 mouse engineered by duplicating a chain (a1 a2) HP 2-2 (2 zonulin genes) WB a2 a1

mice are put on Normal drinking water Zonulin transgenic Hp2 mice are phenotypically normal despite

12 How Zonulin-Mediated Increased Ag Trafficking Leads to Chronic Inflammation: Insights From The Zonulin Trangenic Mouse Model 3% DSS Normal H2O Ztm WT Ztm WT Daily Body Weight after 7 days all mice are put on Normal drinking water Zonulin transgenic Hp2 mice are phenotypically normal despite increased small intestinal permeability *p<0.05 Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

DSS DSS DSS Untreated Untreated 3% DSS A C57Bl/6 Zonulin Transgenic Hp2 B C57Bl/6

2000 C Histology Score 40 in vivo - FITC-dextran ** 30 20 10 0 Untreated * * *

Untreated DSS Ztm have increased morbidity (colonic mucosal damage) and mortality

13 DSS DSS DSS Untreated Untreated 3% DSS A C57Bl/6 Zonulin Transgenic Hp2 B C57Bl/6 Zonulin Transgenic Hp2 FITC-Dextran Concentration (ng/ml) C Histology Score 40 in vivo - FITC-dextran ** Untreated * * * **** DSS C57Bl/6 Zonulin transgenic Hp2 C57Bl/6 Zonulin transgenic Hp2 D 0 Untreated DSS Ztm have increased morbidity (colonic mucosal damage) and mortality (70%) following DSS induced colitis Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

The Zonulin Inhibitor AT1001 (Larazotide Acetate) Treatment Ameliorates Increased DSS Induced Morbidity and Mortality in Ztm H H N AT1001 CH HO C O C O N O H 2 C C C O CH C H2 NH 2 HN C O H 3C CH CH

14 The Zonulin Inhibitor AT1001 (Larazotide Acetate) Treatment Ameliorates Increased DSS Induced Morbidity and Mortality in Ztm H H N AT1001 CH HO C O C O N O H 2 C C C O CH C H2 NH 2 HN C O H 3C CH CH NH CH 3 C H 2 C CH 3 O CH CH O NH CH 3 C HN CH CH CH 3 H C CH 3 CH O O C NH H 2N CH H C 32H 55N 9O 10 Exact Mass: Mol. Wt.: m/e: (100.0%), (35.6%), (9.2%), (3.3%) C, 52.95; H, 7.64; N, 17.37; O, AT1001 (larazotide acetate) treatment ameliorates increased DSS induced morbidity and mortality in ztm Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

15 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

16 Literature Report On Zonulin Association With CID Disease Model Zonulin Shown to be Involved Ankylosis spondylitis Human YES Autism Human YES Celiac Disease Human YES Colitis/IBD (Crohn s disease) Human YES Colitis Mouse YES Fe metabolism in heart transplant Human NO Glioma Human YES Glioma Cell YES Irritable bowel syndrome Human YES HIV Human YES Multiple sclerosis Mouse YES Necrotizing Enterocolitis (NEC) Rat YES Nonalcoholic fatty liver disease Human YES Non-Celiac Gluten Sensitivity 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

17 Serum Serum zonulin (ng/mg protein) LA/MA 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= T1D T1D T1D Relatives controls Controls 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

18 Type 1 Diabetes

19 Serum glucose (mg/dl) Serum glucose (mg/dl) Evidence for Zonulin-Dependent Increased Intestinal Permeability in the Pathogenesis of Type 1 Diabetes 300 Diabetes - Untreated 0.7 No Diabetes Larazotide Treated ICA + (100%) LA/MA Ratio ICA + (8%) LA/MA Ratio Age (days) Glucose LA/MA Age (days) Watts et al PNAS 2005;102:

20 Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Insulin staining Islet Immunohistochemistry Glucagon staining Untreated BBDP rats that developed T1D A B Larazotide-treated BBDP rats that DID NOT develop T1D: No insulitis C D

21 Celiac Disease

22 Larazotide Acetate

23 % of patients with symptoms Change in GSRS domains Change in GSRS domains Larazotide Acetate Consistently Reduced Gastro- Intestinal Symptoms in 3 Gluten-Challenge Clinical Trials 80% CLIN Study (Baseline to Day 3, 12 mg QD) 1,2 CLIN Study (Baseline to Day 14, pooled active) 0,6 CLIN Study (Baseline to LDBTP, 1 mg TID) 70% 1 0,5 60% 50% 0,8 0,4 40% 0,6 0,3 30% 0,4 0,2 20% 10% 0,2 0,1 0% 0 0-0,1-0,2 LDBTP, Last Double-Blind Treatment Period Visit Placebo Active

24 Environmental Triggers Causing Zonulin Release Gluten Gut Microbiome

Dysbiosis is one of the key factors causing zonulin release and subsequent loss of barrier function The Microbiome as Possible Transducer of All

25 Dysbiosis is one of the key factors causing zonulin release and subsequent loss of barrier function 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)

26 The changing face of gut microbes 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)

The human gut harbors 10 11-10 12 bacteria per gram colonic content (>10 14 total bacteria) Total bacteria outnumber human cells 10:1 Total

27 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)

28 Is Microbiome Science Ready For Primetime Clinical Applicability? Classification of Organisms Milky Way Solar System Universe Domain Kingdom Phylum Bacteria Firmicutes Earth Class Clostridia Europe Order Clostridiales Spain Family Ruminococcaceae Balearic Islands Genus Anaerotruncus Mallorca Species Anaerotruncus_sp Adapted from

29 Why The First 1000 Days Of Life Are Instrumental For Clinical Destiny

30 Metabolome Analysis In At-Risk Infants Developing CD CD T1D Infants who developed autoimmune diseases during the time of the study had high levels of lactate combined with low levels of butyrate before the onset of the disease; Just before developing the disease, lactate decreased while butyrate increased; These changes paralleled changes in microbiome composition, with decreased of Lactobacillaceae and increased of butyrateproducing Firmicutes.

31 T REGULATORY CELLS IN CD Minor subpopulation of CD4 + T cells (5-10%). Traditionally described as CD4 + CD25 ++ FOXP3 + Maintenance of self-tolerance and immune homeostasis. Induction of oral tolerance. Immune Response Low frequency of CD4 + CD25 + Treg cells in 1. Systemic Lupus Erythematosus patients 2. Multiple Sclerosis patients 3. in Type 1 diabetes Number of Treg cells is increased in active celiac patients. Suppressive function of Treg cells is significantly impaired in celiac patients.

32 FOXP3 Important for differentiation and function of Treg cells. Delta2 Th17 RORgt RORgt

33 Different Foxp3 FL/D2 Ratio Between Gut Mucosa and PBMC FOXP3 D2 CD HC CDA HC CDA HC FL D2 = / FL D2 = FL FL D2 D2 Increased Expression of FOXP3 D2 is Correlated With Increased Expression of RORGT and IL17A in CD Patients Serena G et al, Clin Exp Immunol. 2017

34 CDA VS HC Gut pro-inflammatory microenvironment Microbiome composition IFNg Serena G et al, Clin Exp Immunol. 2017

35 IFNg+Butyrate Combination Leads to FOXP3 Isoforms Equilibrium in PBMC From HC BUT NOT CD subjects HC B+I FL D2 FL D2 HC CD CD FL D2 B+I FL D2 R= -0.9 P<0.005 Serena G et al, Clin Exp Immunol. 2017

36 Working Hypothesis Linking Gut Microbiome Composition, Metabolomic Profile, and Immune Functions Leading To CD Serena G et al, Clin Exp Immunol. 2017

Working Hypothesis: The Gut Microbiome/Permeability Link in the Pathogenesis of

mucosal biology functions Diet shapes microbiota composition that can lead to

inflammation responsible for the GI symptoms Non-self antigens, including food

presenting cells (APC) present the non-self antigens to other immune cells Activated

37 Working Hypothesis: The Gut Microbiome/Permeability Link in the Pathogenesis of Autoimmune Diseases Dysbiosis causes changes in metabolic pathways affecting key mucosal biology functions Diet shapes microbiota composition that can lead to dysbiosis Activated inflammatory cells release cytokines that cause local inflammation responsible for the GI symptoms Non-self antigens, including food antigens and microorganisms components gain access into the lamina propria Antigen presenting cells (APC) present the non-self antigens to other immune cells Activated inflammatory cells migrate to other districts where they cause local inflammation responsible for systemic symptoms

38 Celiac Disease Genomic Environmental Microbiome and Metabolomic Study

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

40 Acknowledgments NIH DK NIH DK NIH DK The MIBRC Crew

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

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