Gliadin, Intestinal Permeability, and Celiac Disease: From Innate Immunity to Autoimmunity

Gliadin, Intestinal Permeability, and Celiac Disease: From Innate Immunity to Autoimmunity: Alessio Fasano, M.D. Mucosal Biology Research Center University of Maryland School of Medicine

Definition
Celiac disease is an autoimmune condition
Occurs in genetically susceptible individuals
DQ2 and/or DQ8 positive HLA haplotype is necessary but not sufficient
A unique autoimmune disorder because:
both the environmental trigger (gluten) and the autoantigen (tissue Transglutaminase) are known
elimination of the environmental trigger leads to a complete resolution of the disease

Normal small bowel Celiac disease Gluten Gluten-free diet

Pathogenesis
+
=

Several genes are involved
The most consistent genetic component depends on the presence of HLA-DQ (DQ2 and / or DQ8) genes
Other genes (not yet identified) account for 60 % of the inherited component of the disease
HLA-DQ2 and / or DQ8 genes are necessary (No DQ2/8, no Celiac Disease!) but not sufficient for the development of the disease
Genetics HLA ? ? ? ? Gluten Celiac Disease + Genes

Dietary Factors Festucoideae Subfamily Tribe Zizaneae Oryzeae Hordeae Aveneae Festuceaea Chlorideae wild rice rice wheat oat finger millet teff (ragi) rye barley The Grass Family - (GRAMINEAE)

The Celiac Iceberg Symptomatic Celiac Disease Silent Celiac Disease Latent Celiac Disease Genetic susceptibility: - DQ2, DQ8 Positive serology Manifest mucosal lesion Normal Mucosa

The Clinical Manifestations of Celiac Disease are More Heterogeneous Than Previously Appreciated A. Fasano, N Engl J Med 2003;348:2568-70.

What is so Special About Gluten? Gluten (gliadin+glutenin) Gliadin Glutenin

CD Pathogenesis Celiac disease Genetics Gluten NECESSARY CAUSES Gender Infant feeding Infections Others RISK FACTORS Pathogenesis ?

Proposed CD Pathogenesis ?????

The Paracellular Pathway … Tight junctions are a ‘dark horse’ implicated in a host of disease states, ranging from acute injury to chronic inflammation and autoimmune diseases

DISCOVERY OF ZONULIN Coomassie Western blot 1 2 3 4 1: Tissue lysate 2: Sephacryl-S300 3: Q-sepharose 4: Immuoaffinity 1 2 3 4 PURIFICATION PROTOCOL FROM HUMAN INTESTINE

Pillole di Saggezza Pillola #1:

Zonulin Signaling: How Does It Work?

A Way to Activate the Zonulin Innate Immunity PAthway Bacteria-Gut Interaction:

Studies on Cell Lines: Time 00:00 Drago et al Scand J Gastroenterol 2006; 41:408-19.

Double staining immunofluorescence experiments on IEC-6 cells Time 00:05: Zonulin packing GLIADIN CONTROL CASEIN Drago et al Scand J Gastroenterol 2006; 41:408-19. Actin Zonulin Nucleus

Studies on Cell Lines: Time 00:15: Zonulin Release Control Bacteria/gliadin Drago et al Scand J Gastroenterol 2006; 41:408-19.

Studies on Cell Lines: Time 00:20: Zonulin Binding PT-gliadin Caco 2 T84 Control IEC6 Drago et al Scand J Gastroenterol 2006; 41:408-19.

Gliadin Co-Localizes with CXCR3 100X Gliadin FITC Nucleus DAPI CXCR3 TRITC Merge

In Situ IFM of Biopsies from Either Celiac Disease Patients or Healthy Controls Showed Apical Expression of CXCR3 Celiac disease Non-celiac control CXCR3 Actin Nucleus

Proposed Zonulin Mechanism of Action Fasano et al, J Clin Invest 1995;96:710-20; el Asmar et al Gastroenterology 2002;123:1607-15 Following Pathway Activation Resting State Freeze-Fracture Zonulin signaling

Celiac Disease Pathogenesis: The Continuous Stimulation by Gluten is Necessary to Perpetuate the Autoimmune Process Intestinal Lumen A. Fasano and T. Shea Donohue, Nature Clin Pract Gastroenterol Hepatol 2005;2:416-22 PRR AGA EMA TTG MyD88 dependent Submucosa Bacteria

1) bacterial adhesion, 2) internal signaling, 3) signal transmission to the effector cells, 4) zonulin release on the luminal side 5) zonulin binds to its receptor, 6) cascade of events ending 7) by the TJ opening, leading to 8) water and solutes paracellular passage and 9- “wash out” effect, 10) and paracellular passage of food antigens and macromolecules, 11) GALT stimulation and subsequent undesired effects H 2 O 5 6 8 7 11 10 9 H 2 O Lymphocytes Food Antigens Macromolecules Zonulin Bacteria Zonulin 1 4 2 Zonulin Enterocytes 3 3 El Asmar et al J Gastroenterology 2002;123:1607-1615 Zonulin System – Innate Immunity

Innate Immune Response (Early) Adaptive Immune Response (Late) Microbial insult GOAL: Eliminate “danger” Cytokines Costimulatory Molecules Abs/CMI Cytokines Cross Talk Between Innate and Adaptive Immunity Pathways

Novel Paradigm for Autoimmunity
A third key element necessary to develop autoimmunity is the loss of the mucosal barrier function, so allowing the interplay between genes and environment.
Autoimmune diseases involve a miscommunication between innate and adaptive immunity Autoimmunity Genetics Environment Mucosal Barrier Autoimmunity

Autoimmune diseases involve a miscommunication between innate and adaptive immunity.
Molecular mimicry or bystander effects alone may not entirely explain the complex events involved in the pathogenesis of autoimmune diseases. Rather, the continuous stimulation by non-self antigens (environmental triggers) appears necessary to perpetuate the process. This concept implies that the autoimmune response can be theoretically stopped and perhaps reversed if the interplay between autoimmune predisposing genes and trigger(s) is prevented or eliminated.
In addition to genetic predisposition and the exposure to the triggering non-self antigen, the third key element necessary to develop autoimmunity is the loss of the protective function of mucosal barriers that interface with the environment (mainly the GI and lung mucosa).
Alternative Hypothesis for the Pathogenesis of Autoimmune Diseases

Tight Junction dysfunction / injury
BACTERIAL TOXINS
Bacteriodes fragilis (metalloprotease toxin)
Clostridium difficile (toxins A, B)
Clostridium perfrigens (enterotoxin)
E. coli (cytotoxic necrotizing factor 1)
Helicobacter pylori (vacuolating toxin)
Listeria monocytogenes (internalin)
Vibrio cholerae (zonula occludens toxin)
INFECTIONS:
E.Coli
Rotavirus
Salmonella Ty.
HIV
AUTOIMMUNE DISORDERS:
- Celiac disease - Multiple Sclerosis
- Inflammatory bowel diseases - Diabetes Mellitus
Ankylosing spondylitis - IgA nephropathy
Fasano, A. Pathological and therapeutic implications of macromolecules passage through the tight junction
in Cereijido M, Anderson J, eds. Tight Junctions. CRC Press,2001: 697-722
TJ Dysfunction in Disease Pathology
DRUGS:
alcohol
NSAID
Tacrolimus
Ischemia / Reperfusion Injury

0 2.5 5 7.5 10 12.5 15 zonulin ng/mg serum protein HC CD AIH PBC T1D APS-1 MS Clemente et al. Gastroenterology 2002;122 :A15 0.2 Cut-off 3.4 2.1 3.0 1.3 0.3 0.5 * p < 0.0001 * * * * Serum Zonulin in Autoimmune Disorders

Potential Therapeutic Applications

Therapeutic Applications
Close Sesame!
Zonulin Pipeline
Multiple Sclerosis , Stroke
Asthma , COPD, ARDS
Dilatative cardiomyopathy , Ischemia-reperfusion
Systemic sclerosis, Transplant Rejection
Type 1 Diabetes , Autoimmune thyroiditis
Celiac Disease , Primary Biliary Cirrhosis, Inflammatory Bowel Disease.
Glomerulosclerosis , Acute Renal Failiure
Rheumatoid Arthritis
Metastatic Disease
2. Drug and Vaccine Delivery Open Sesame!
Oral Delivery
BBB Delivery
Nasal Delivery
Skin Delivery
~150 patents surrounding Zonulin, its receptor, and various synthetic / natural peptide modulators of Zonulin receptor & TJ function. 3. Diagnostics
Ongoing research
Preliminary data generated
Potential areas of application

AT-1001, the Zonulin Inhibitor Di Pierro et al, J Biol Chem 2001;276:19160-5.

Celiac Disease NDA Explor. Discssn w/ GI/Coag Dvn End of Phase II Meeting; confirm Fasttrack Single Dose Safety & PK, SDSS 4 wk Mult Dose Safety Study, MDSS File Celiac IND, seek fast track Pivotal #1 Celiac Pivotal #2 Celiac Start Phase II 3 mos. Dose ranging in Celiacs & Diabetics Preclinical ADME, Tox, Biology Pivotal #2 T1D T1D NDA ↓ Intest. Perm? Reformulate Yes No File T1D IND Explor. Discssn w/ Metab/Endo Dvn Addtl PD Studies The Zonulin Clinical Trial Roadmap Pivotal #1 T1D

Zonulin, Gliadin, And Type 1 Diabetes Sapone et al. Diabetes 2006

In Vivo permeability and serum glucose levels in DP rats either untreated or treated with AT-1001 Watts et al PNAS 2005;102:2196-21 Glucose LA/MA LA/MA Ratio 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 LA/MA Ratio No Diabetes – FZI/0 Treated Diabetes ICA + (100%) ICA – (92%)

BB/wor DP Rat Beta Cell Rescue Study

Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Age: 52-54 days Treatment Animal flow Placebo Timeframe T 0 1 2 3 4 5 6 7 14 wks Pre-T 0 AT1001 Placebo 60% T1D (N = 10) (N = 20) (N = 30) AT1001 35% T1D T1D Sapone et al. ADA Meeting 2005: late breakthrough abstract Outcome 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 LA/MA Ratio ICA + (100%)

Diabetes Treatment Study: Treatment of Autoimmune Diabetes with AT-1001 Sapone et al. ADA Meeting 2005: late breakthrough abstract Untreated T1D Treated NO T1D 10X 40X

Insulin staining Glucagon staining A B C D Untreated BBDP rats that developed T1D Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process AT1001-treated BBDP rats that DID NOT develop T1D: No insulitis Islet Immunohistochemistry

Insulin staining Glucagon staining A B C D 40X Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Islet Immunohistochemistry AT1001-treated BBDP rats that DID NOT develop T1D: Islets recovering from insulitis and islet regeneration 10X

Role of Gliadin in Type 1 Diabetes Pathogenesis Zonulin Intestinal Permeability Incidence of T1D BBDP rats on regular diet BBDP rats on GFD BBDP rats on regular diet BBDP rats on GFD BBDR rats on regular diet BBDP rats on regular diet BBDP rats on GFD

Human Studies Sapone et al. Diabetes 2006

Sapone et al. Diabetes 2006 Role of Zonulin in T1D in Humans T1D Relatives T1D Controls 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Serum zonulin (ng/mg protein) n= 339 89 97

Serum Zonulin Levels and Intestinal Permeability in T1D, Their Relatives and Controls Intestinal permeability and serum zonulin levels in T1D patients and their relatives. Both parameters were significantly higher in T1D subjects as compared to their relatives. *p=0.04; **p=0.02 Sapone et al. Diabetes 2006

Serum Zonulin and in vivo Permeability in Children with Type 1 Diabetes After Gluten Challenge, vs. Relatives Fig. 1. Serum zonulin levels in T1D patients (n=16) and their relatives (N=35) before and after a gluten-containing meal. *<0.05 compared to baseline. Fig. 2. Serum LA/MAin T1D patients (n=16) and their relatives (N=35) 1 hour before and 1 hour after a gluten-containing meal. *<0.0002; **<0.0005 compared to before meal LA/MA. ** 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0 1 2 3 4 5 Time (hrs) Serum zonulin (ng/mg protein) T1D relatives Gliadin-containing meal * * * 0.000 0.050 0.100 0.150 0.200 0.250 0.300 1 2 3 4 5 Time (hrs) LA/MA ratio T1D relatives *

PROPOSED MODEL Proposed role of aberrant intestinal permeability in Type 1 diabetes pathogenesis. Non-self antigens are present in the intestinal lumen (1) and cross the tj barriers in subjects with dysregulation of the zonulin system (2-3). Antigen peptides bind to HLA receptors present on the surface of APC (4). In turn, these peptides are presented to T lymphocytes (5). In genetically susceptible individuals, an aberrant immune response (both umoral and cell-mediated) (6) leads to the autoimmune process mainly targeting the Langherans islets with subsequent insulin deficiency typical of type 1 diabetes (7). 5 Submucosa T B GAD, IA, IA-2 Tk P 2 3 4 6 1 APC Intestinal lumen 6 7 7

Acknowledgements
Carlo Catassi
Cinzia D’Agate
Mariarosaria Di Pierro
Sandro Drago
Ramzi El Asmar
Simeon Goldblum
Giuseppe Iacono
James Kaper
Ruiling Lu
Regina Macatangay
Klara Margaretten
Sergio Uzzau
Wenle Wang
Tammy Watts

http://nogf.san-lorenzo.com	21
http://www.slideshare.net	5
http://pdemo.secondbrain.com	1

Gliadin, Intestinal Permeability, and Celiac Disease: From Innate Immunity to Autoimmunity

by Letizia Saturni

Accessibility

Categories

Upload Details

Usage Rights

3 Embeds 27

Statistics

Gliadin, Intestinal Permeability, and Celiac Disease: From Innate Immunity to Autoimmunity Presentation Transcript