The Role of Gut Permeability In

Autoimmunity: Lesson Learned from

Type 1 Diabetes and Celiac Disease
ISPAD 2014
September 3rd 6th, 2014 Toronto, Canada
Alessio Fasano, M.D.
Mucosal Immunology and Biology Research Center
And Center for Celiac Research
Massachusetts General Hospital for Children
Boston MA U.S.A.

Lecture Objectives
Genes + Environment = Autoimmunity
Not So Fast!
Microbiome

Environmental
Factors

Clinic Outcome

The Holy Trinity of the

Autoimmune Mechanisms
in Celiac Disease

Fasano A; Scientific American Aug. 2009

The Leaky Gut Theory:

Facts and Fantasies

Intestine: Interesting Facts

~20 ft long

~3,000 sf!!!

The Paracellular Pathway and its Regulation

PURIFICATION PROTOCOL FROM HUMAN INTESTINE

1 2 3 4

1 2 3 4

1: Tissue lysate
2: Sephacryl-S300
3: Q-sepharose
4: Immuoaffinity
Coomassie

UP

DOWN

2/7 multiple genes

EGF

EGFR

EGFR

Western blot

Tight junctions are a dark horse

implicated in a host of disease
states, ranging from acute injury to
chronic inflammation and
autoimmune diseases

2/5
2

RAC1

MYO9B

-p

2
ROCK1

2/5

Fasano A. et al Lancet 2000;355:1518-1519.Wang W et al J Cell Sci 2000;24:4435-4440

NEWS #1:
Characterization of Zonulin And
Its Signaling

Is impaired intestinal barrier a cause

of disease or an epiphenomenon?
Multiple Sclerosis, Stroke,
Stroke
Schizophrenia.
Asthma, COPD, ARDS
Dilatative cardiomyopathy,
Ischemia-reperfusion
Systemic sclerosis
Transplant Rejection
Type 1 Diabetes,
Autoimmune thyroiditis
Celiac Disease, PBC, IBD,
IBD. IBS
Glomerulosclerosis,
Acute Renal Failure
Rheumatoid Arthritis
Tumors/Metastatic diseases

Serum Zonulin Levels and Their Correlation

With Intestinal Permeability In
Celiac Disease and Type 1 Diabetes
Celiac Disease

Type 1 Diabetes

(ng/mg protein)
zonulin (ng/mg
Serum
protein)
Serum
zonulin

4.0
4

N=339

N=89

N=97

0.12

3.5

0.1

3
3.0
LA/MA

Zonulin-LA/MA

2.5

2
2.0
1.5

0.08
0.06

0.04
multiple R=0.36;
intercept p=1.71E10; X variable 1
p=0.0004

1
1.0

0.02

0.5

00

0
T1D
T1D

T1D
Relatives
T1D
Relatives

Controls
controls

2
3
Zonulin (ng/mg protein)

Sapone et al Diabetes 2006; 55:1443-9

The Two Strongest Stimuli

for Zonulin Release
Small intestinal Bacterial
Overgrowth (SIBO)

Exposure to Gluten

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. 2011 Jan;91(1):151-75

Prove of Concept on Role of ZonulinRegulated Intestinal Permeability in

Autoimmune Pathogenesis
Environment

Genetics

Mucosal Barrier

Autoimmunity

NO

Only CD

AT1001, the Zonulin Inhibitor

H
CH

H
N
C

HO

C
O

O
H2
C

O
C
O

CH

C
C
H2

HN

NH2

O
C

H3C

CH
CH

NH

CH3

C
O
O

H2
C

CH3

CH

CH

NH

CH3

C
CH
HN
H

C
CH

CH3
CH
CH3

NH
C
CH

H2N

C32H55N9O10
Exact Mass: 725.41
Mol. Wt.: 725.83
m/e: 725.41 (100.0%), 726.41 (35.6%), 727.41 (9.2%), 726.40 (3.3%)
C, 52.95; H, 7.64; N, 17.37; O, 22.04

Di Pierro et al, J Biol Chem 2001;276:19160-5.

News #2:
Prove of Concept of
the Role of Zonulin in
Autoimmune Pathogenesis

Type 1 Diabetes

Evidence for Zonulin-Dependent Increased

Intestinal Permeability in the
Pathogenesis of Type 1 Diabetes

250

300

0.7

0.6

250

0.6

0.5
0.4

150
0.3
100

0.2

50

0.1

0
30

37

44

51

58

Age (days)

65

72

0.5

200

0.4

ICA + (8%)

150

0.3
100

0.2

50

LA/MA Ratio

ICA + (100%)

200

No Diabetes Larazotide Treated

0.7

Serum glucose (mg/dl)

Diabetes - Untreated

LA/MA Ratio

Serum glucose (mg/dl)

300

0.1

0
30

Glucose
LA/MA

37

44

51

58

65

72

Age (days)

Watts et al PNAS 2005;102:2916-21

Blocking the Zonulin-Dependent Increased Intestinal

Permeability Aborts The Autoimmune Process
Islet Immunohistochemistry
Glucagon staining

Insulin staining

Untreated BBDP rats

that developed T1D

Larazotide-treated
BBDP rats that DID
NOT develop T1D:
No insulitis

Effect of GFD in BBWor T1D rats

Visser J.T.J. et al Diabetologia 2010 (on line ahead print)

News #2:
Prove of Concept of
the Role of Zonulin in
Autoimmune Pathogenesis

Celiac Disease

Celiac Disease PoC Data Phase 1b CLIN1001-002

Larazotide Acetate Blocks Acute Gluten-induced
Permeability & Immune Activation
Phase Ib Gastrointestinal Signs & Symptoms

(*p=0.04)

Placebo

100%

*0.017

80%
Placebo

60%

AT-1001

40%
20%

n=7

Dimension

Larazotide Acetate
1.00
n = 14

0.75

Day 1

Day 2

Day 3

Day 7

Serum IFN-g

ng
iti
om

N
au
se
a

Fl

at
ul
en
ce

rrh
ea
D
ia

P
in
al

bd
om

(**p=0.07)

1.25

ai
n
C
on
st
ip
at
io
n

0%

1.50

GI Symptoms

* 0.018

ym
pt
om
s

1.75

Blind Gluten Challenge, 2.5gm

120%

G
IS

La/Ma (day x) / La/Ma (day 1)

2.00

% (Placebo n=7; AT-1001 n=14)

Intestinal Permeability

How Zonulin Is Released and

How Does It Works

Gluten Causes Leaky Gut and

Inflammation by Releasing Zonulin

Brain Inflammation

NEWS #3:
We Are Not Born With The Destiny
to Develop Autoimmunity

Autoimmunity Epidemics
Diabetes

Which Factors are Driving This

Autoimmunity Epidemics?
Nutrition

Microbiome
Composition
Maturation
GALT

Genetic
+ Predisposition

Immune-Mediated
Diseases

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.

The Real Story of Our Genetic Complexity:

We Inherit two Parallel Genomes
Microbiome:
Human Genome:
Inherited from both parents, stable,
never change in its composition

Inherited from the mother, extremely

dynamic, changes from individual to
individual and in the same
individual over time

Microbiome

Human Genome

(140-fold Human Genome)

(~25,000 genes)

Dynamic

Stable

Metabonome

Jazz

Pop

Clinical
Outcome

Classic

1H-NMR metabolomics analysis.

Celiac Disease

A. PCA analysis of metabolomic profiles (A).

Relative concentration of major metabolites
measured from stool NMR profiles in
selected subjects over the 24-month study
period (BF). B. Succinate; C. Acetate; D.
Lactate; E. Butyrate and F. Propionate. The
chemical shift (ppm) value of each
metabolite is indicated above each panel.

T1D

The Best Way To Predict The

Future Is To Create It

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

Acknowledgments

NIH DK078699
NIH DK048373

The MIBRC Crew