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

Clinic Outcome

Environmental Factors

Microbiome

Meeting Overview On New Concepts of Autoimmunity:

Excessive and Inappropriate Inflammatory Process Associated to a Dysfunction of Barriers

The Epidemics of Immune-Mediated Diseases In The

Western Hemisphere: The Hygiene Hypothesis

We Are Not Born With The Destiny to

Develop Chronic Inflammatory Diseases

The Yin and Yang Between Tolerance and Immune

Response Leading To Chronic Inflammatory Diseases:

Lesson Learned From Celiac Disease

Environmental Factors

Human Genome

Clinic Outcome

Increased Gut Permeability

Microbiome

Immune Response

Several Cells Play a Role in Maintaining

The Immune Homeostasis Epithelial cells

Intestinal DCs

B cells

T cells

Adapted from P. Brandtzaeg, Beneficial Microbes 2010

Excessive and Inappropriate Inflammatory Process Associated to a Dysfunction of Barriers:

Loss of Mucosal Immune Homeostasis

Mucosal Tolerance

Homeostasis

Anergy

Proinflammatory

Allergic cytokines

Break of Tolerance

Apoptosis resistant T cells

Tissue damage

Chronic inflammation

Allergy

Inflammation - Allergy

Normal/physiologically

controlled permeability

Minor barrier defect

dietary/microbial Ag influx

Increased

permeability

Massive dietary and

microbial antigen influx

Vicious

circle Regulatory DC’s

Macrophages

Tregs

IL-10/TGF-b

Innate or immuno-

regulatory defect

Defensins

SIgA

Mucus Synthesis & Quality

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

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

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

Fasano A. et al Lancet 2000;355:1518-1519.-

Wang W et al J Cell Sci 2000;24:4435-4440

Zonulin Characterization and Signaling

Fasano et al, J Clin Invest 1995;96:710-20; el Asmar et al Gastroenterology 2002;123:1607-15

Zonulin signaling

Following Pathway Activation

Resting State

Freeze-Fracture

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

a1

a2

ZonulinTransgenic Mice (Ztm)

How Zonulin-Mediated Increased Ag

Trafficking Leads to Chronic Inflammation: Insights From The Zonulin Trangenic Mouse Model

3% DSS Normal H2O

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

Ztm WT Ztm WT

Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

Ztm have increased morbidity (colonic mucosal damage) and mortality (70%) following DSS induced colitis

Un

treate

d

Un

treate

dD

SS

DS

S

DS

S

C57Bl/6 Zonulin Transgenic Hp2

C57Bl/6 Zonulin Transgenic Hp2

A B

C

Untreated DSS 0

10

20

30

40

His

tolo

gy S

co

re

****

*

C57Bl/6 Zonulin transgenic

Hp2

Untreated DSS0

2000

4000

6000

8000

10000

FIT

C-D

extr

an

Co

ncen

trati

on

(n

g/m

l)

in vivo - FITC-dextran

**

**

C57Bl/6

Zonulin transgenic

Hp2

D

3% DSS

Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

AT1001 (larazotide acetate) treatment ameliorates

increased DSS induced morbidity and mortality in ztm

The Zonulin Inhibitor AT1001 (Larazotide Acetate) Treatment Ameliorates

Increased DSS Induced Morbidity and Mortality in Ztm

H2N

CH

C

H

O NH

CH

C

O

HN

CH

C

CH

O

CH3

H

NH

CH

CH2C

O CH

CH3

CH3

NH

CH

C

CH

O

H3C

CH3

HN

CH

CH2C

O CH2

C

NH2

ONC

O

HN

CH

C

H

HO

O

CH3

C32H55N9O10

Exact Mass: 725.41Mol. 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

AT1001

Sturgeon C et al. Ann NY Acad Sci 2017 Apr 19 (Epub ahead print)

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

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

Sapone et al Diabetes 2006; 55:1443-9

T1D RelativesT1D Controls

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Se

rum

zo

nu

lin (

ng/

mg

pro

tein

)

N=339 N=89 N=97

0

0.02

0.04

0.06

0.08

0.1

0.12

0 1 2 3 4Zonulin (ng/mg protein)

LA/M

A

multiple R=0.36; intercept p=1.71E-10; X variable 1 p=0.0004

4

3

2

1

0

Seru

m z

on

ulin

(ng/

mg

pro

tein

)

A B C

T1D Relatives controlsT1D

Serum Zonulin Levels and Their Correlation

With Intestinal Permeability In

Celiac Disease and Type 1 Diabetes

Celiac Disease Type 1 Diabetes Zonulin-LA/MA

Type 1 Diabetes

Evidence for Zonulin-Dependent Increased Intestinal Permeability in the Pathogenesis of Type 1 Diabetes

Glucose

LA/MA

LA

/MA

Ratio

0

50

100

150

200

250

300

30 37 44 51 58 65 72

Age (days)

Seru

m g

luco

se (

mg

/dl)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 No Diabetes – Larazotide Treated

ICA + (8%)

Watts et al PNAS 2005;102:2916-21

0

50

100

150

200

250

300

30 37 44 51 58 65 72

Age (days)

Seru

m g

luco

se (

mg

/dl)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

LA

/MA

Ratio

Diabetes - Untreated

ICA + (100%)

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

Larazotide-treated BBDP rats that DID NOT develop T1D:

No insulitis

Islet Immunohistochemistry

Celiac Disease

Larazotide Acetate

Larazotide Acetate Consistently Reduced Gastro-

Intestinal Symptoms in 3 Gluten-Challenge Clinical Trials

0

0,2

0,4

0,6

0,8

1

1,2

CLIN1001-004 Study (Baseline to Day 14, pooled active)

-0,2

-0,1

0

0,1

0,2

0,3

0,4

0,5

0,6

CLIN1001-006 Study (Baseline to LDBTP, 1 mg TID)

0%

10%

20%

30%

40%

50%

60%

70%

80%

CLIN1001-002 Study (Baseline to Day 3, 12 mg QD)

% o

f p

ati

en

ts w

ith

sym

pto

ms

Ch

an

ge i

n G

SR

S d

om

ain

s

Ch

an

ge i

n G

SR

S d

om

ain

s

Placebo Active

LDBTP, Last Double-Blind Treatment Period Visit

Environmental Triggers Causing Zonulin Release

Gluten Gut Microbiome

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)

Dysbiosis is one of the

key factors causing

zonulin release and

subsequent loss of

barrier function

The changing face of gut microbes

•The human gut harbors 1011-1012 bacteria per gram

colonic content (>1014 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 (400-500 per person)

•The human gut harbors 1011-1012 bacteria per gram colonic content (>1014

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 (400-500 per person)

Adapted from http://biology-medicine.blogspot.com/2012/09/scientific-classification-of-organisms.html

Is Microbiome Science Ready For Primetime Clinical Applicability?

Classification of Organisms

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae

Anaerotruncus

Anaerotruncus_sp

Domain

Kingdom

Phylum

Class

Order

Family

Genus

Species

Milky Way

Solar System

Earth

Europe

Spain

Balearic Islands

Mallorca

Universe Bacteria

Why The First 1000 Days Of Life Are

Instrumental For Clinical Destiny

• 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 butyrate-

producing Firmicutes.

Metabolome Analysis In At-Risk Infants Developing CD

CD

T1D

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.

• 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.

Immune Response

RORgt Th17

FOXP3 • Important for differentiation and function of Treg cells.

Delta2

RORgt

Different Foxp3 FL/D2 Ratio Between Gut Mucosa and PBMC

Serena G et al, Clin Exp Immunol. 2017

CD

HC

FOXP3 D2

CDA HC

D2 FL

D2 FL

= /

CDA HC

D2 FL = D2

FL

Increased Expression of FOXP3 D2 is Correlated With Increased Expression of RORGT and

IL17A in CD Patients

CDA HC VS

IFNg

Gut pro-inflammatory

microenvironment Microbiome composition

Serena G et al, Clin Exp Immunol. 2017

IFNg+Butyrate Combination Leads to FOXP3 Isoforms Equilibrium in PBMC From HC BUT NOT CD subjects

D2 FL

D2 FL

B+I HC

CD HC CD

R= -0.9

P<0.005

Serena G et al, Clin Exp Immunol. 2017

D2 FL

D2 FL

B+I

Working Hypothesis Linking Gut Microbiome Composition, Metabolomic Profile, and Immune Functions Leading To CD

Serena G et al, Clin Exp Immunol. 2017

Activated

inflammatory

cells release

cytokines that

cause local

inflammation

responsible for

the GI symptoms

Working Hypothesis: The Gut Microbiome/Permeability

Link in the Pathogenesis of Autoimmune Diseases

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

Dysbiosis causes changes

in metabolic pathways

affecting key mucosal

biology functions

Diet shapes microbiota

composition that can

lead to dysbiosis

Celiac Disease Genomic Environmental Microbiome and Metabolomic Study

www.CDGEMM.org

Modeling the Effects of Early Childhood

Microbiome Composition On Entire Lifespan

Microbiome establishment

and modification

First 1000 days of life

Acknowledgments

The MIBRC Crew NIH DK078699

NIH DK048373

NIH DK104344