Content uploaded by Niranjan Pandey
Author content
All content in this area was uploaded by Niranjan Pandey on Nov 22, 2021
Content may be subject to copyright.
Peptides
35
(2012)
86–94
Contents
lists
available
at
SciVerse
ScienceDirect
Peptides
j
our
na
l
ho
me
p
age
:
www.elsevier.com/locate/peptides
Larazotide
acetate
regulates
epithelial
tight
junctions
in
vitro
and
in
vivo
Shobha
Gopalakrishnana, Malarvizhi
Duraia,
Kelly
Kitchensa,1,
Amir
P.
Tamiza,
Robert
Somervillea,2,
Mark
Ginskia,3,
Blake
M.
Patersona,4,
Joseph
A.
Murrayb,
Elena
F.
Verduc,
Sefik
S.
Alkana,5,
Niranjan
B.
Pandeya,∗
aALBA
Therapeutics,
650
S.
Exeter,
Suite
1040,
10th
Floor,
Baltimore,
MD
21202,
United
States
bMayo
Clinic,
Department
of
Medicine
and
Immunology,
200
First
Street
SW,
Rochester,
MN
55905,
United
States
cDepartment
of
Medicine,
McMaster
University,
HSC-3N5C,
L8N3Z5,
Canada
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
13
December
2011
Received
in
revised
form
20
February
2012
Accepted
20
February
2012
Available
online
27
February
2012
Keywords:
Celiac
disease
Gliadin
ZO-1
Actin
Tight
junction
Permeability
inducer
a
b
s
t
r
a
c
t
Tight
junctions
(TJs)
control
paracellular
permeability
and
apical-basolateral
polarity
of
epithelial
cells,
and
can
be
regulated
by
exogenous
and
endogenous
stimuli.
Dysregulated
permeability
is
associated
with
pathological
conditions,
such
as
celiac
disease
and
inflammatory
bowel
disease.
Herein
we
studied
the
mechanism
by
which
larazotide
acetate,
an
8-mer
peptide
and
TJ
regulator,
inhibits
the
cellular
changes
elicited
by
gliadin
fragments,
AT-1002,
and
cytokines.
Previously,
we
demonstrated
that
AT-1002,
a
6-
mer
peptide
derived
from
the
Vibrio
cholerae
zonula
occludens
toxin
ZOT,
caused
several
biochemical
changes
in
IEC6
and
Caco-2
cells
resulting
in
decreased
transepithelial
electrical
resistance
(TEER)
and
increased
TJ
permeability.
In
this
study,
larazotide
acetate
inhibited
the
redistribution
and
rearrangement
of
zonula
occludens-1
(ZO-1)
and
actin
caused
by
AT-1002
and
gliadin
fragments
in
Caco-2
and
IEC6
cells.
Functionally,
larazotide
acetate
inhibited
the
AT-1002-induced
TEER
reduction
and
TJ
opening
in
Caco-2
cells.
Additionally,
larazotide
acetate
inhibited
the
translocation
of
a
gliadin
13-mer
peptide,
which
has
been
implicated
in
celiac
disease,
across
Caco-2
cell
monolayers.
Further,
apically
applied
larazotide
acetate
inhibited
the
increase
in
TJ
permeability
elicited
by
basolaterally
applied
cytokines.
Finally,
when
tested
in
vivo
in
gliadin-sensitized
HLA-HCD4/DQ8
double
transgenic
mice,
larazotide
acetate
inhibited
gliadin-induced
macrophage
accumulation
in
the
intestine
and
preserved
normal
TJ
structure.
Taken
together,
our
data
suggest
that
larazotide
acetate
inhibits
changes
elicited
by
AT-1002,
gliadin,
and
cytokines
in
epithelial
cells
and
preserves
TJ
structure
and
function
in
vitro
and
in
vivo.
©
2012
Elsevier
Inc.
All
rights
reserved.
Abbreviations:
TJ,
tight
junction;
TEER,
transepithelial
electrical
resistance;
LY,
lucifer
yellow;
AT-1002,
permeability
inducer,
6-mer
peptide;
AT-1001,
tight
junc-
tion
regulator,
8-mer
peptide
(larazotide
acetate).
∗Corresponding
author.
Present
address:
720
Rutland
Ave.,
617
Traylor
Bldg.,
Systems
Biology
Laboratory,
Department
of
Biomedical
Engineering,
School
of
Medicine,
Johns
Hopkins
University,
Baltimore,
MD
21205,
United
States.
Tel.:
+1
410
614
2951;
fax:
+1
410
614
8796.
E-mail
address:
pandey.niranjan@gmail.com
(N.B.
Pandey).
1Present
address:
MPN1,
Rockville,
MD
20855,
United
States.
2Present
address:
National
Cancer
Institute,
Surgery
Branch,
Building
10
CRC,
Room
3-3825,
9000
Rockville
Pike,
Bethesda,
MD
20892,
United
States.
3Present
address:
5217
Scenic
Drive,
Perry
Hall,
MD
21128,
United
States.
4Present
address:
Department
of
Anesthesia
and
Critical
Care
Medicine,
Johns
Hopkins
University
School
of
Medicine,
600
N.
Wolfe
Street,
Meyer
8-140,
Baltimore,
MD
21287,
United
States.
5Present
address:
Alkan
Consulting
LLC,
Mittlere
Strasse
8,
CH-4056
Basel,
Switzerland.
1.
Introduction
Multicellular
organisms
can
survive
only
if
they
establish
a
dis-
tinct
internal
environment,
separating
“self”
from
the
“non-self”
environment.
The
mucosal
surfaces
of
the
genitourinary,
gastroin-
testinal,
and
respiratory
tracts
have
epithelial
barriers
that
are
“sealed”
by
tight
junctions
(TJs)
[31,40].
TJs
are
located
at
the
apico-
lateral
borders
of
adjoining
epithelial
cells,
regulating
the
patency
of
the
paracellular
space
and
limiting
the
bidirectional
diffusion
of
particles,
water,
and
solutes
across
mucosal
surfaces.
TJs
consist
of
over
50
proteins,
including
the
transmembrane
proteins
junctional
adhesion
molecule
(JAM),
occludin,
and
claudin,
and
the
cytoplas-
mic
scaffolding
proteins
zonula
occludens
(ZO)-1,
ZO-2,
and
ZO-3
[1,16].
In
the
absence
of
inflammation
or
epithelial
disruption,
the
functional
state
of
the
TJ
determines
paracellular
permeability
and
polarity
[33].
TJs
are
highly
dynamic,
opening
and
closing
in
response
to
cytoskeletal
reorganization
that
occurs
upon
exposure
to
external
antigens,
such
as
gliadin
in
celiac
disease
[30],
and
inter-
nal
inflammatory
cytokines,
such
as
TNF-␣
[31].
Increased
exposure
0196-9781/$
–
see
front
matter
©
2012
Elsevier
Inc.
All
rights
reserved.
doi:10.1016/j.peptides.2012.02.015
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
87
of
the
submucosa
to
external
antigens
is
putatively
caused
by
“barrier
dysfunction”
in
the
small
bowel,
and
“intestinal
leak”
is
associated
with
numerous
intestinal
and
extra-intestinal
autoim-
mune
conditions,
including
celiac
disease,
inflammatory
bowel
disease,
type
1
diabetes,
multiple
sclerosis,
primary
biliary
cirrho-
sis,
autoimmune
hepatitis,
and
systemic
sclerosis
[14,15,42].
Celiac
disease,
an
autoimmune
disorder,
is
triggered
by
gluten/gliadin
or
gliadin
fragments
in
individuals
with
the
HLA-
DQ2/DQ8
allele
that
facilitates
presentation
of
gliadin
peptides
to
T
cells
[20].
Gliadin
induces
a
loss
of
barrier
function
and
stimu-
lates
innate
and
adaptive
immune
responses,
followed
by
intestinal
damage
[29].
Gliadin
also
disrupts
TJ
integrity
by
altering
actin
and
ZO-1
distribution
in
intestinal
epithelial
cells
[9,12,30].
Alterations
in
ZO-1
phosphorylation
and
expression
have
also
been
reported
in
response
to
gliadin
and
in
celiac
disease
mucosa,
respectively
[7,30].
Protease-resistant
gliadin
peptides
are
transported,
by
as
yet
unknown
mechanisms,
from
the
lumen
of
the
gut
to
the
lamina
propria,
where
tissue
transglutaminase
catalyzes
the
conversion
of
glutamine
in
certain
peptides
to
glutamate.
Deamidated
ver-
sions
of
the
gliadin
peptides
cause
immune
activation
and
initiate
an
inflammatory
cascade
with
the
production
of
IFN␥and
other
cytokines
[17].
One
of
the
“toxic”
gliadin
peptides,
P31–43
(13-
mer),
also
induces
intestinal
permeability
and
stimulates
cytokine
and
chemokine
production
by
macrophages
in
vitro
[39].
Inflam-
matory
cytokines
have
been
shown
to
enhance
the
permeability
of
the
gut
epithelium
[2,8],
which
could
lead
to
the
translocation
of
more
gliadin.
Currently,
a
gluten-free
diet
is
the
only
management
that
helps
to
alleviate
the
symptoms
of
celiac
disease
[10].
There-
fore,
there
is
a
great
need
for
novel
therapeutic
approaches
that
target
the
earliest
event
at
the
mucosal
surface
to
inhibit
barrier
dysfunction,
and
hence
prevent
immune
activation.
We
have
previously
shown
that
AT-1002,
a
synthetic
peptide
comprising
the
first
6
amino
acids
of
the
active
fragment
of
zonula
occludens
toxin
(ZOT)
from
Vibrio
cholerae,
lowers
transepithelial
electrical
resistance
(TEER)
and
increases
TJ
permeability
and
alters
ZO-1
and
actin
distribution
in
Caco-2
cells
[19].
As
mentioned
pre-
viously,
gliadin
also
elicits
similar
responses
in
intestinal
epithelial
cells.
It
is
also
well
established
that
cytokines
decrease
TEER
and
induce
paracellular
permeability
in
epithelial
cells
[32,45].
We
have
developed
an
8-mer
peptide
and
TJ
regulator
larazotide
acetate
that
has
therapeutic
value
for
celiac
disease
owing
to
its
ability
to
inhibit
early
mucosal
events
that
lead
to
barrier
dysfunction
and
immune
activation
[26].
Furthermore,
ZOT-induced
immune
responses
are
inhibited
by
intranasal
administration
of
larazotide
acetate
[22],
and
oral
larazotide
acetate
reduces
the
incidence
of
type
I
diabetes
in
BB
Wor/DP
rats
[46].
In
this
study,
we
examined
the
mechanism
by
which
larazotide
acetate
inhibits
TJ
opening
caused
by
AT-1002,
gliadin,
and
cytokines.
We
studied
effects
on
TJ
and
cytoskeletal
organization
in
vitro,
and
examined
whether
the
“epithelial
leak”
induced
by
various
stimuli
was
inhibited
by
larazotide
acetate.
Finally,
we
examined
whether
these
in
vitro
effects
of
larazotide
acetate
on
TJs
could
be
translated
in
vivo
to
a
gluten-sensitive
trans-
genic
HLA-HCD4/DQ8
mouse
model
developed
at
the
Mayo
clinic
[5].
In
this
mouse
model,
gluten
sensitization
induces
changes
in
intestinal
permeability
and
innate
immune
cell
infiltration
making
it
an
excellent
model
to
test
potential
therapeutics
for
gluten-
induced
effects
[25,28].
2.
Materials
and
methods
2.1.
Reagents
The
permeability
inducer
AT-1002
and
the
13-mer
gliadin
pep-
tide
AT-4067
were
synthesized
using
F-moc
solid
phase
chemistry
as
described
previously
[19].
The
final
products
were
isolated
as
TFA
salts
in
a
lyophilized
form
(>95%
purity
by
HPLC/MS).
Larazotide
acetate
(AT-1001)
was
synthesized
using
solution
phase
synthesis
[9],
and
the
identity
of
the
compound
was
con-
firmed
by
LC/MS.
The
final
product
was
isolated
as
an
acetate
salt
in
a
lyophilized
form
(>99%
purity
by
HPLC/MS).
Pepsin-trypsin
treated
gliadin
(PTG)
was
prepared
as
described
previously
[39].
Recombinant
interleukin
(rIL)-1,
interferon
(IFN)-
␥,
and
tumor
necrosis
factor-␣
(rTNF-␣)
were
purchased
from
R&D
Systems
(Minneapolis,
MN).
2.2.
Cell
lines
Caco-2
BBE
(brush
border-expressing)
cells
were
obtained
from
American
Type
Culture
Collection
(ATCC,
Manassas,
VA)
and
maintained
in
Dulbecco’s
Modified
eagle
Medium
(DMEM;
Invit-
rogen,
Carlsbad,
CA)
containing
10%
fetal
bovine
serum,
penicillin
(100
units/mL),
streptomycin
(100
g/mL),
and
100
g/mL
human
transferrin.
Cells
were
plated
at
100,000
cells
per
12-well
filter
and
used
at
10–14
days
post-seeding.
Caco-2
cells
were
obtained
from
ATCC
and
maintained
in
DMEM
containing
10%
fetal
bovine
serum,
penicillin
(100
units/mL),
and
streptomycin
(100
g/mL).
IEC6
cells
were
obtained
from
ATCC
and
maintained
in
DMEM
containing
0.1
unit/mL
bovine
insulin,
10%
fetal
bovine
serum,
peni-
cillin
(100
units/mL),
and
streptomycin
(100
g/mL).
Cells
were
seeded
on
8-chamber
slides
at
60,000
cells
per
chamber
for
immunofluorescence
studies.
IEC6
cells
were
used
to
study
gliadin
effects
because
Caco-2
cells
did
not
respond
to
gliadin
in
our
hands.
Although
IEC6
cells
formed
TJs
and
expressed
ZO-1
at
cell-cell
junctions,
their
baseline
perme-
ability
was
quite
high.
Therefore,
these
cells
were
used
mainly
for
ZO-1
imaging
studies
and
not
for
functional
assays.
2.3.
Lucifer
yellow
(LY)
permeability
assays
Details
of
this
method
and
modifications
have
been
described
previously
[4,18].
Briefly,
Caco-2
cells
were
seeded
onto
12-well
TranswellsTM and
grown
for
21–28
days
until
fully
differentiated.
The
apical
and
basolateral
compartments
of
Caco-2
cell
monolay-
ers
were
pre-incubated
in
Hank’s
Balanced
Salt
Solution
(HBSS)
at
37 ◦C
for
30
min.
Treatment
solutions
containing
7.5
mM
LY
with
or
without
AT-1002
(7
mM)
and
different
concentrations
of
lara-
zotide
acetate
in
HBSS
were
added
to
the
apical
compartment
of
each
monolayer
and
incubated
at
37 ◦C,
50
rpm
for
180
min.
At
the
end
of
the
incubation,
samples
were
removed
from
the
basolateral
compartment
and
analyzed
in
a
Tecan
Spectrofluor
fluorescence
plate
reader
at
excitation
and
emission
wavelengths
of
485
nm
and
535
nm,
respectively.
The
increase
in
LY
passage
was
calculated
for
each
treatment
and
is
expressed
relative
to
that
of
untreated
controls.
2.4.
Immunofluorescence
Cells
were
washed
in
serum-free
medium
and
incubated
with
12.5
mM
larazotide
acetate
diluted
in
serum-free
medium
for
30
min
at
37 ◦C.
Larazotide
acetate
was
removed
and
cells
were
incubated
with
PTG
(2.5
mg/mL)
or
AT-1002
(7
mM)
in
the
pres-
ence
or
absence
of
larazotide
acetate
(12.5
mM)
at
37 ◦C
for
60
min
or
180
min,
respectively.
Following
treatment,
cells
were
washed
in
PBS
and
fixed
in
4%
paraformaldehyde
for
15
min
at
room
temper-
ature,
or
in
ice-cold
methanol:acetone
(1:1)
for
7
min.
Cells
were
washed
in
PBS,
permeabilized
in
PBS
containing
0.5%
Triton
X-
100
for
5
min
at
room
temperature
(for
paraformaldehyde
fixed
cells),
and
blocked
in
PBS
containing
2%
goat
serum
for
30
min
at
room
temperature.
Actin
and
ZO-1
were
detected
using
Alexa
88
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
Fluor555-phalloidin
(1:20)
and
FITC-conjugated
anti-ZO-1
anti-
body
(1:200;
Invitrogen),
respectively.
Slides
were
washed
and
mounted
in
Vectashield
containing
DAPI.
Images
were
collected
on
a
Nikon-TE2000
fluorescence
microscope
using
a
40×
objec-
tive
and
quantified
using
Adobe
Photoshop,
as
described
previously
[19].
Briefly,
the
background
level
was
adjusted
using
the
threshold
function.
Then
the
brightness
was
adjusted
to
bring
all
back-
ground
pixels
to
the
threshold
value.
Pixels
at
cell-cell
junctions
were
highlighted
using
the
Magic
wand
and
select/inverse
func-
tions.
The
mean
pixel
intensity
value,
total
number
of
pixels,
and
number
of
threshold
pixels
were
obtained.
Total
junctional
fluo-
rescence
intensity
=
(total
pixels
×
mean
pixel
value)
−
(threshold
pixels
×
threshold)
was
calculated
for
each
sample.
A
total
of
40
cells
were
quantified
per
treatment
per
experiment.
The
settings
were
kept
the
same
for
all
groups.
Two
independent
experiments
were
performed.
Data
are
expressed
as
mean
(SD),
and
a
p
value
of
<0.05
was
considered
significant.
2.5.
F-actin
flow
cytometry
Caco-2
BBE
cells
were
treated
apically
with
AT-1002
(7
mM)
in
the
presence
or
absence
of
larazotide
acetate
(12.5
mM)
for
3
h
at
37 ◦C.
Following
treatment,
cells
were
detached
from
fil-
ters
using
trypsin.
Detached
cells
were
washed
in
PBS,
fixed
in
PBS
containing
4%
paraformaldehyde
for
15
min
at
room
tempera-
ture,
permeabilized
in
PBS
containing
0.5%
Triton
X-100
for
5
min
at
room
temperature,
and
blocked
in
PBS
containing
2%
goat
serum
for
30
min
at
room
temperature.
Cells
were
incubated
with
Alexa
Fluor555-phalloidin
for
1
h
at
room
temperature,
washed
in
PBS,
and
10,000
cells
from
each
sample
were
analyzed
by
flow
cytome-
try
using
FACSCAN
(Becton
Dickinson,
San
Diego,
CA).
2.6.
Gliadin
peptide
translocation
Caco-2
cells
were
seeded
at
100,000
cells/cm2on
12-well
Transwell®filters.
At
21–28
days
post-seeding,
cells
were
treated
with
FITC-labeled
gliadin
13-mer
fragment
(AT-4067;
1
mM)
in
the
presence
or
absence
of
larazotide
acetate
(100
M)
for
3
h
at
37 ◦C,
95%
relative
humidity,
50
rpm,
and
5%
CO2.
At
t
=
3
h,
samples
were
removed
from
the
receiver
compartment
and
analyzed
in
a
Tecan
Spectrofluor
fluorescence
plate
reader
at
excitation
and
emission
wavelengths
of
485
nm
and
535
nm,
respectively.
Gliadin
perme-
ability
was
calculated
for
each
treatment
and
expressed
relative
to
untreated
control
conditions.
2.7.
Cytokine
treatment
and
permeability
assay
Caco-2
cells
were
seeded
at
60,000
cells/cm2in
24-well
Transwell®plates,
and
maintained
in
culture
medium
composed
of
Dulbecco’s
Modified
Eagle’s
Medium,
10%
fetal
bovine
serum,
1%
non-essential
amino
acids,
and
2%
penicillin-streptomycin.
Medium
was
changed
twice
a
week
with
0.2
mL
and
1.0
mL
for
apical
and
basolateral
sides,
respectively.
Assays
were
performed
21–24
days
post-seeding.
On
the
day
of
the
experiment,
Caco-2
cells
were
incubated
in
complete
RPMI
(RPMI
1640
media
con-
taining
1%
penicillin-streptomycin,
1%
HEPES,
1%
sodium
pyruvate,
1.8
L
of
2-mercaptoethanol,
and
5%
human
AB
serum)
for
2
h
at
37 ◦C,
in
5%
CO2.
After
incubation,
Caco-2
cells
were
treated
baso-
laterally
with
1
mL
of
complete
RPMI
containing
50
ng/mL
each
of
TNF-␣,
IFN␥,
and
IL-1
(R&D
Systems)
for
72
h
at
37 ◦C
in
5%
CO2.
For
larazotide
acetate
treatment,
medium
was
replaced
with
differ-
ent
doses
of
larazotide
acetate
on
the
apical
side
after
48
h.
At
the
end
of
the
experiment,
LY
flux
was
measured
as
follows:
medium
was
replaced
with
LY
(7.5
mM)
on
the
apical
side
and
cells
were
incubated
for
1
h
at
37 ◦C,
50
rpm.
Samples
(100
L)
were
collected
from
the
basolateral
side
and
the
amount
of
LY
was
quantified
by
measuring
fluorescence
at
excitation
and
emission
wavelengths
of
485
nm
and
535
nm,
respectively.
2.8.
Treatment
of
HLA-HCD4/DQ8
mice
with
larazotide
acetate
Cohorts
of
HLA-HCD4/DQ8
mice
(n
=
10
each)
were
sensitized
(i.p.)
with
500
g
of
gliadin
(Sigma-Aldrich,
Oakville,
Ontario,
Canada)
dissolved
in
0.02
mM
acetic
acid
in
50
g
of
Complete
Freund’s
Adjuvant
(CFA;
Sigma-Aldrich);
thereafter,
mice
were
gav-
aged
with
gliadin
(2
mg/mouse),
+/−
treatment,
2×/week
for
7
weeks.
Group
1
received
larazotide
acetate
(250
g/mouse)
and
gliadin,
Group
2
received
AT-1002
(250
g/mouse)
and
gliadin,
and
Group
3
was
gavaged
with
gliadin
only.
A
group
of
non-sensitized
controls
(CFA,
i.p.
only)
was
gavaged
with
rice.
Twenty-four
hours
after
the
last
gavage,
small
intestinal
tissue
was
mounted
in
Uss-
ing
chambers
for
the
measurement
of
electrical
parameters
(Isc,
conductance)
and
macromolecule
transport
(horseradish
perox-
idase
[HRP]
flux).
Tissue
was
processed
for
macrophage
counts
by
immunohistochemistry
using
F4/80
antibody
specific
for
a
macrophage-restricted
cell
surface
glycoprotein.
2.9.
Immunohistochemistry
for
macrophages
Jejunal
sections
obtained
from
HLA-HCD4/DQ8
mice
were
immunostained
for
macrophages
using
a
technique
previously
described
[44].
Briefly,
immunostaining
was
performed
on
paraffin
sections
using
a
monoclonal
antibody
specific
for
the
F4/80
antigen.
The
rat
anti-mouse
F4/80
antibody
(1:200;
Serotec,
Oxford,
UK)
was
followed
by
biotinylated
polyclonal
goat
anti-rat
antibody
(1:200;
Cederlane
Laboratories,
Hornby,
BC,
Canada)
and
then
strepta-
vidin/horseradish
peroxidase
(1:300;
Dakocytomation).
Antibodies
were
visualized
by
diaminobenzidine
and
counterstaining
with
Mayer’s
hematoxylin.
Negative
controls
were
performed
in
the
absence
of
primary
antibody.
F4/80-positive
cells
were
quantified
in
5
villi,
in
2
different
sections
per
mouse
(averaged).
Data
were
analyzed
using
two-way
ANOVA,
and
expressed
as
mean
(SD).
A
p
value
<0.05
was
considered
significant.
2.10.
Electron
microscope
(EM)
analysis
of
TJ
Additional
jejunal
sections
from
HLA-HCD4/DQ8
mice
were
obtained
and
immediately
fixed
in
2.5%
glutaraldehyde
in
0.1
mol/L
sodium
cacodylate
buffer
(pH
7.4)
for
2
h
at
room
tempera-
ture,
transferred
to
sodium
cacodylate
buffer,
and
stored
at
4◦C
overnight.
Tissues
were
washed
3
times
in
0.05
mol/L
Tris
buffer
and
then
incubated
for
30
min
in
5
mg
of
3,3-diaminobenzadine
tetrahydrochlorine
(Sigma
Chemicals,
St
Louis,
MO)
in
10
mL
of
0.05
mol/L
Tris
buffer
and
0.01%
hydrogen
peroxide.
Tissues
were
subsequently
processed
for
electron
microscopy,
and
photomicro-
graphs
prepared.
The
fraction
of
TJ
with
altered
structures
was
calculated
as
total
open
TJ
divided
by
total
number
of
TJ
evaluated
in
20
fields
per
mouse
in
a
blinded
manner
(3–20
tight
junctions/field,
60–400
tight
junctions/mouse,
4
mice/group).
A
field
is
defined
as
one
square
in
the
EM
grid,
measuring
8100
mm2.
Data
were
ana-
lyzed
using
two-way
ANOVA,
and
expressed
as
mean
(SD).
A
p
value
<0.05
was
considered
significant.
2.11.
Statistics
Data
are
expressed
as
mean
(SD)
or
mean
(SE),
as
indicated
in
the
figure
legends.
Statistical
analysis
was
performed
with
the
t-test
or
ANOVA
using
Microsoft
Excel.
A
p-value
<0.05
was
considered
significant.
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
89
Fig.
1.
Larazotide
acetate
inhibits
AT-1002-induced
tight
junction
permeability
in
Caco-2
cells.
(A)
Mean
permeability
of
lucifer
yellow
(LY)
across
Caco-2
cell
mono-
layers
was
measured
following
exposure
to
AT-1002
in
the
presence
or
absence
of
a
series
of
concentrations
of
larazotide
acetate
(LA)
(n
=
3
experiments
with
3
wells
per
group
per
experiment).
Data
are
expressed
as
mean
(SE).
*p
<
0.05
vs.
AT-1002
alone.
3.
Results
3.1.
Effects
of
larazotide
acetate
on
AT-1002-induced
TJ
dysfunction
in
Caco-2
cells
We
have
previously
shown
that
AT-1002
induces
TJ
barrier
dysfunction
in
human-derived
Caco-2
cells
[19].
To
determine
the
effects
of
larazotide
acetate
on
AT-1002-induced
barrier
dys-
function,
we
measured
the
mean
LY
passage
across
Caco-2
cell
monolayers
following
exposure
to
AT-1002
in
the
presence
or
absence
of
larazotide
acetate
(Fig.
1).
AT-1002
caused
a
substantial
increase
in
LY
passage,
which
was
inhibited
by
larazotide
acetate
in
a
dose-dependent
manner.
Larazotide
acetate
at
15
and
12.5
mM
significantly
inhibited
the
AT-1002-induced
increase
in
LY
passage
by
71
and
38%,
respectively
(p
<
0.05).
3.2.
Effects
of
larazotide
acetate
on
AT-1002-induced
ZO-1
redistribution
and
actin
rearrangement
in
Caco-2
cells
AT-1002
induces
the
redistribution
of
ZO-1,
an
important
marker
of
TJ
integrity
[19].
We
sought
to
determine
whether
lara-
zotide
acetate
inhibited
these
changes.
To
this
end,
Caco-2
BBe
cells
were
treated
with
AT-1002
in
the
presence
or
absence
of
larazotide
acetate,
and
ZO-1
distribution
was
examined
by
immunofluores-
cence.
A
z-series
of
images
was
collected
and
projected
onto
a
single
plane
to
avoid
missing
fluorescence
from
out-of-focus
planes,
and
ZO-1
junctional
fluorescence
intensity
was
quantified.
As
shown
in
Fig.
2A,
untreated
cells
and
cells
treated
with
larazotide
acetate
alone
exhibited
a
smooth
distribution
of
ZO-1
at
cell-cell
junc-
tions.
AT-1002
treatment
caused
extensive
redistribution
of
ZO-1
away
from
cell
junctions,
which
was
inhibited
by
larazotide
acetate.
Quantification
of
total
junctional
fluorescence
intensity
revealed
that
AT-1002
elicited
a
60%
decrease
in
junctional
ZO-1.
However,
co-incubation
of
AT-1002
with
larazotide
acetate
resulted
in
less
than
a
10%
decrease
in
junctional
distribution
of
ZO-1
(p
<
0.05)
(Fig.
2A),
indicating
that
larazotide
acetate
inhibited
AT-1002-
induced
ZO-1
redistribution.
TJ
are
associated
with
and
stabilized
by
the
cortical
actin
cytoskeleton,
and
AT-1002
causes
actin
rearrangement
[19].
To
examine
the
effect
of
larazotide
acetate
on
AT-1002-induced
actin
rearrangement,
Caco-2
BBe
cells
were
treated
with
AT-1002
for
1
h
in
the
presence
or
absence
of
larazotide
acetate,
and
filamen-
tous
actin
was
visualized
by
immunofluorescence
using
fluorescent
phalloidin.
A
z-series
of
images
was
acquired
and
combined
into
a
projection
and
fluorescence
intensity
was
quantified.
Untreated
Caco-2
BBE
cells
exhibited
a
robust
network
of
actin
filaments
(Fig.
2B).
Exposure
to
AT-1002
caused
dissolution
of
the
actin
network,
which
was
prevented
by
larazotide
acetate.
Actin
fluo-
rescence
was
quantified
by
flow
cytometry
(10,000
cells/sample).
AT-1002
decreased
the
mean
F-actin
fluorescence
intensity
to
61%
that
of
control
levels,
whereas
larazotide
acetate
significantly
inhibited
the
AT-1002-induced
loss
of
F-actin
(p
<
0.05)
(Fig.
2B).
3.3.
Effects
of
larazotide
acetate
on
gliadin-induced
changes
in
IEC6
cells
Celiac
disease
is
triggered
by
gluten/gliadin
or
gliadin
pep-
tides,
which
disrupt
TJ
structure
and
function.
Gliadin
causes
actin
rearrangement
and
ZO-1
redistribution.
To
determine
if
larazotide
acetate
could
inhibit
the
effects
of
gliadin,
rat
intestinal
epithelial
(IEC6)
cells
were
treated
with
PTG,
a
source
of
gliadin
fragments
generated
by
digesting
gliadin
with
pepsin
and
trypsin,
diluted
in
serum-free
medium
in
the
presence
or
absence
of
larazotide
acetate
for
1
h,
and
ZO-1
was
visualized
by
immunofluorescence.
In
untreated
IEC6
cells,
ZO-1
exhibited
a
smooth,
uninterrupted
distri-
bution
at
cell-cell
junctions;
PTG
treatment
caused
a
redistribution
of
ZO-1
away
from
cell
junctions,
and
junctional
fluorescence
inten-
sity
decreased
by
75%
(Fig.
3A).
PTG-induced
ZO-1
redistribution
was
inhibited
by
larazotide
acetate
and
the
junctional
ZO-1
fluo-
rescence
intensity
was
maintained
at
near
control
levels
(Fig.
3A).
We
also
examined
the
effect
of
larazotide
acetate
on
the
actin
cytoskeleton
in
PTG-treated
IEC6
cells.
PTG
caused
actin
network
disassembly;
in
cells
treated
with
PTG
and
larazotide
acetate,
the
actin
network
was
protected
from
disassembly.
Furthermore,
in
the
presence
of
larazotide
acetate
the
junctional
distribution
of
actin
appeared
more
robust.
These
data
suggest
that
larazotide
acetate
prevented
the
PTG-induced
disassembly
of
the
actin
cytoskeleton
(Fig.
3B).
3.4.
Effect
of
larazotide
acetate
on
gliadin
translocation
Gliadin
peptides
are
thought
to
translocate
across
the
epithelial
monolayer
in
the
gut
[6,35].
We
examined
whether
translocation
of
the
gliadin
13-mer
peptide
AT-4067
(Leu-Gly-Gln-Gln-Gln-
Pro-Phe-Pro-Pro-Gln-Gln-Pro-Tyr)
[13,27]
could
be
inhibited
by
larazotide
acetate.
Thus,
using
the
Transwell
system,
FITC-
labeled
gliadin
13-mer
peptide
fragment
(AT-4067)
and
larazotide
acetate
were
applied
to
the
apical
side
of
Caco-2
cells
at
time
0,
and
the
amount
of
FITC-labeled
gliadin
13-mer
peptide
fragment
in
the
basolateral
compartment
was
measured
3
h
later.
We
found
that
apically
applied
larazotide
acetate
at
concentrations
as
low
as
1
mM
significantly
inhibited
translo-
cation
of
FITC-labeled
gliadin
13-mer
peptide
fragment
into
the
basolateral
side
(p
<
0.05)
(Fig.
4).
We
obtained
similar
results
with
the
FITC-labeled
gliadin
9-mer
and
33-mer
peptides
Pro-Phe-Pro-Gln-Pro-Gln-Leu-Pro-Tyr
and
Leu-Gln-Leu-Gln-
Pro-Phe-Pro-Gln-Pro-Gln-Leu-Pro-Tyr-Pro-Gln-Pro-Gln-Leu-
Pro-Tyr-Pro-Gln-Pro-Gln-Leu-Pro-Tyr-Pro-Gln-Pro-Gln-Pro-Phe,
respectively
[36]
(data
not
shown).
3.5.
Effect
of
larazotide
acetate
on
cytokine-induced
TJ
dysfunction
Gliadin
peptides
cause
immune
activation
and
initiate
an
inflammatory
cascade
with
the
production
of
IFN␥
and
other
cytokines
[20].
It
is
well
established
that
cytokine
treatment
reduces
TEER
and
increases
paracellular
permeability
of
epithelial
cell
monolayers
[32,45].
We
sought
to
determine
whether
lara-
zotide
acetate
could
inhibit
the
cytokine-induced
permeability
of
Caco-2
cells.
As
shown
in
Fig.
5,
basolateral
treatment
of
Caco-2
cells
90
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
Fig.
2.
Larazotide
acetate
(LA)
inhibits
AT-1002-induced
ZO-1
redistribution
and
actin
rearrangement
in
Caco-2
cells.
Caco-2
BBe
cells
grown
on
permeable
membrane
supports
were
treated
apically
with
AT-1002
(7
mM)
in
the
presence
or
absence
of
larazotide
acetate
for
3
h
at
37 ◦C.
(A)
Cells
were
fixed
and
processed
for
immunoflu-
orescence
using
anti-ZO-1
antibodies.
Graph
represents
percent
junctional
fluorescence
intensity
of
ZO-1.
Data
are
expressed
as
mean
(SD)
and
are
representative
of
3
independent
experiments.
*p
<
0.05
vs.
AT-1002.
(B)
Flow
cytometric
analysis
of
F-actin
fluorescence.
Cells
were
fixed
and
stained
for
F-actin
using
fluorescent
phalloidin,
and
10,000
cells/sample
were
analyzed
by
flow
cytometry
for
actin
fluorescence.
Data
are
expressed
as
mean
(SD)
and
are
representative
of
4
independent
experiments.*p
<
0.05
vs.
AT-1002.
with
a
mixture
of
TNF-␣,
IFN-␥,
and
IL-1
(50
ng/mL
each)
caused
a
substantial
increase
in
permeability,
which
was
significantly
inhib-
ited
by
apically
applied
larazotide
acetate
at
3
and
1
mM
(p
<
0.05
vs.
cytokine
treatment
for
both)
(Fig.
5).
3.6.
In
vivo
effect
of
larazotide
acetate
on
intestinal
permeability
in
HLA-HCD4/DQ8
mice
We
tested
the
effects
of
larazotide
acetate
on
gliadin-induced
alterations
in
barrier
function
and
macrophage
recruitment
in
dou-
ble
transgenic
HLA-HCD4/DQ8
mice
[5].
We
found
that
gliadin
sensitization
and
challenge
increased
conductance
(40.1
mS/cm2)
values,
which
is
indicative
of
a
fall
in
tissue
resistance,
compared
to
non-sensitized
controls
(24.4
mS/cm2)
(Fig.
6A).
We
also
found
that
gliadin
sensitization
and
challenge
increased
HRP
flux,
a
mea-
surement
of
transcellular
permeability,
from
19.4
(non-sensitized
controls)
to
86.8
pmol/cm2/h
(Fig.
6B).
Larazotide
acetate
normal-
ized
the
conductance
(26
mS/cm2)
and
moderately
attenuated
the
HRP
flux
(59
pmol/cm2/h).
The
improvement
in
barrier
function
parameters
by
larazotide
acetate
in
gliadin
sensitized-mice
was
associated
with
improved
TJ
structure,
as
assessed
by
electron
microscopy
(Fig.
6D).
Larazotide
acetate
also
reduced
macrophage
counts
in
the
lamina
propria
to
control
levels
(9.5/villi)
(Fig.
6C).
Macrophage
infiltration
is
one
of
the
earliest
responses
to
gliadin
indicating
activation
of
the
innate
immune
system
after
gluten
sen-
sitization
[39].
We
also
tested
effects
of
gliadin
in
conjunction
with
AT-1002
in
this
model.
In
vivo
administration
of
AT-1002
did
not
further
affect
the
gliadin-induced
barrier
abnormalities.
4.
Discussion
“Barrier
dysfunction”
and
“intestinal
leak”
are
associated
with
numerous
intestinal
and
extra-intestinal
autoimmune
conditions,
and
barrier
function
is
a
new
target
for
drug
development,
drug
delivery,
and
adjuvant
development.
In
this
study,
we
examined
the
effects
of
larazotide
acetate,
an
8-mer
peptide
and
TJ
regulator
[10,26],
on
TJ
disruption
caused
by
AT-1002,
gliadin,
and
proinflam-
matory
cytokines.
We
have
previously
shown
that
AT-1002,
a
ZOT-derived
peptide,
disrupts
TJs,
causes
actin
cytoskeleton
rearrangement,
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
91
Fig.
3.
Larazotide
acetate
(LA)
inhibits
PTG-induced
ZO-1
redistribution
and
actin
cytoskeletal
rearrangement
in
IEC6
cells.
IEC6
cells
were
treated
with
PTG
(2.5
mg/mL)
in
the
presence
or
absence
of
larazotide
acetate
(12.5
mM)
for
1
h
at
37 ◦C.
(A)
Cells
were
fixed
and
processed
for
immunofluorescence
using
anti-ZO-1
antibodies.
Projections
from
z-stacks
were
quantified
as
described
in
Section
2.
Graph
represents
percent
junctional
fluorescence
intensity
of
ZO-1.
Data
are
expressed
as
mean
(SD)
and
are
representative
of
2
independent
experiments.
(B)
Cells
were
fixed,
and
F-actin
was
detected
using
fluorescent
phalloidin.
Graph
represents
%
F-actin
fluorescence
intensity.
Data
are
representative
of
2
independent
experiments.
Fig.
4.
Larazotide
acetate
inhibits
gliadin
translocation
across
Caco-2
monolayers.
Caco-2
cells
were
treated
with
FITC-labeled
13-mer
gliadin
fragment
(AT-4067;
1
mM)
in
the
presence
or
absence
of
larazotide
acetate
(LA;
100
M)
for
3
h
at
37 ◦C.
At
t
=
3
h,
samples
were
removed
from
the
receiver
compartment
and
analyzed
in
a
fluorescence
plate
reader
at
excitation
and
emission
wavelengths
of
485
nm
and
535
nm,
respectively.
This
gliadin
passage
was
calculated
for
each
treatment
and
expressed
relative
to
untreated
control
conditions.
Data
are
expressed
as
mean
(SE)
and
are
representative
of
2
independent
experiments
(3
wells
per
group
per
experiment).
*p
<
0.05
vs.
control.
Fig.
5.
Larazotide
acetate
inhibits
cytokine-induced
tight
junction
permeability
in
Caco-2
cells.
(A)
Caco-2
cells
were
treated
with
a
mixture
of
TNF-␣,
IFN␥,
and
IL-1
for
72
h.
For
larazotide
acetate
treatment,
the
medium
was
replaced
with
different
doses
of
larazotide
acetate
on
the
apical
side
after
48
h.
At
the
end
of
the
experiment,
LY
passage
was
measured.
Data
are
expressed
as
mean
(SE)
and
are
representative
of
3
independent
experiments
(3
wells
per
group
per
experiment).
*p
<
0.05
vs.
control;
#p
<
0.05
vs.
cytokine
treatment.
92
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
Fig.
6.
Larazotide
acetate
inhibits
intestinal
permeability
in
gluten-sensitive
transgenic
mice.
Cohorts
of
HLA-HCD4/DQ8
mice
(n
=
10
each)
were
sensitized
(i.p.)
with
gliadin
(500
g)
and
Complete
Freund’s
Adjuvant
(CFA),
and
thereafter
were
gavaged
with
gliadin
(2
mg/mouse),
+/−
treatment,
2×/week
for
7
weeks.
Group
1
received
larazotide
acetate
(LA;
250
g/mouse)
and
gliadin,
Group
2
received
AT-1002
(250
g/mouse)
and
gliadin,
and
Group
3
was
gavaged
with
gliadin
only.
A
group
of
non-sensitized
controls
(CFA;
i.p.
only)
was
gavaged
with
rice.
At
24
h
after
the
last
gavage,
small
intestinal
tissue
was
mounted
in
Ussing
chambers
for
the
measurement
of
following
parameters:
(A)
conductance
and
(B)
permeability
(horseradish
peroxidase
[HRP]
flux).
(C)
Tissue
was
obtained
for
the
examination
of
macrophage
counts
by
immunohistochemistry
(F4/80)
(*p
<
0.01
vs.
gliadin;
**p
<
0.01
vs.
control; #p
<
0.05
vs.
gliadin
+
AT-1002.
(D)
Electron
micrographs
and
quantitation
showing
the
preservation
of
TJ
structures
by
larazotide
acetate
(*p
<
0.05
vs.
control; #p
<
0.01
vs.
control;
**p
<
0.01
vs.
gliadin).
Data
are
expressed
as
mean
(SD).
phosphorylation
and
redistribution
of
ZO-1,
and
activates
src
and
MAP
kinase
pathways
in
vitro
[19].
AT-1002
also
enhances
the
systemic
exposure
of
antigens,
such
as
salmon
calcitonin,
in
vivo
[38].
In
the
present
study,
AT-1002
increased
paracellular
perme-
ability
and
elicited
actin
rearrangement
and
ZO-1
redistribution
in
Caco-2
cells,
as
shown
previously
[19].
Larazotide
acetate
inhibited
the
AT-1002-induced
increase
in
permeability,
and
this
corre-
lated
with
its
ability
to
inhibit
AT-1002-induced
actin
and
ZO-1
redistribution.
In
celiac
disease,
barrier
function
is
compromised
and
celiac
disease
patients
exhibit
enhanced
intestinal
permeability
and
dis-
rupted
TJs
[24,34].
Barrier
function
is
regulated
by
TJ
proteins
and
the
actin
cytoskeleton.
Agents
that
disrupt
the
actin
cytoskeleton
affect
paracellular
permeability
[21,41].
In
our
study,
PTG
caused
actin
network
disassembly,
which
was
inhibited
by
larazotide
acetate.
This
result
is
consistent
with
that
of
Clemente
et
al.
who
showed
that
larazotide
acetate
inhibits
gliadin-induced
cytoskele-
tal
reorganization
in
IEC-6
cells
[9].
PTG
has
also
been
shown
to
induce
ZO-1
and
occludin
redistribution
in
an
in
vitro
three-
dimensional
model
system
and
intestinal
epithelial
cells
[11,30].
In
accordance
with
these
observations,
PTG
induced
ZO-1
redistribu-
tion
in
IEC-6
cells,
which
was
inhibited
by
larazotide
acetate.
In
celiac
disease,
the
mechanisms
underlying
gliadin
peptide
transport
from
the
gut
lumen
to
the
lamina
propria
are
not
well
understood.
No
agreement
has
been
reached
in
the
literature
about
whether
the
initial
mode
of
gliadin
transport
is
transcellular,
para-
cellular,
or
both.
Secretory
IgA
has
been
shown
to
mediate
intestinal
transport
of
gliadin
33-mer
and
19-mer
peptides
via
the
CD71
transferrin
receptor
[23].
In
the
present
study,
labeled
gliadin
pep-
tides
translocated
from
the
apical
to
the
basolateral
side
of
a
fully
differentiated
Caco-2
monolayer.
Larazotide
acetate
inhibited
the
transport
of
FITC-labeled
gliadin
13-mer
peptide
by
more
than
50%.
Compromised
barrier
function
may
allow
gliadin
to
cross
the
para-
cellular
space.
We
have
also
presented
convincing
evidence
that
larazotide
acetate
is
an
inhibitor
of
paracellular
permeability,
which
is
consistent
with
recent
findings
by
Silva
et
al.
[37].
However,
crosstalk
and
interaction
between
the
paracellular
and
transcellu-
lar
pathways
have
been
demonstrated
[43].
Thus,
larazotide
acetate
may
inhibit
the
transport
of
gliadin
peptide
regardless
of
the
trans-
port
route.
Gliadin
peptides
cause
immune
activation
and
initiate
a
cascade
of
inflammatory
cytokines,
which
have
been
shown
to
enhance
the
permeability
of
the
gut
epithelium
[2,8].
The
enhanced
per-
meability
would
allow
more
gliadin
peptides
to
enter
the
lamina
propria,
which
in
turn
would
lead
to
an
increase
in
inflammatory
cytokine
levels,
and
an
“inflammatory-permeability
loop”
would
be
established.
A
novel
way
to
treat
celiac
disease
would
be
to
break
the
inflammatory-permeability
loop.
Here,
we
show
that
larazotide
acetate
can
break
the
proposed
inflammatory-permeability
loop
by
decreasing
the
transport
of
inflammatory
gliadin
peptides,
and
by
inhibiting
the
increased
permeability
induced
by
cytokines.
Interestingly,
larazotide
acetate
inhibited
transport
of
gliadin
peptides
even
at
0.1
mM,
which
is
100-fold
lower
than
that
required
to
inhibit
the
AT-1002-induced
effects.
It
is
known
that
in
ani-
mal
experiments
using
BB
Wor
rats
[46]
and
IL-10
−/−
mice
[3],
the
effective
in
vivo
dose
of
larazotide
acetate
is
on
the
order
of
0.2–100
M.
In
this
study,
a
single
dose
of
larazotide
acetate
at
approximately
100
M
was
found
to
be
active
in
a
gluten
sensitivity
model.
However,
the
concentration
of
larazotide
acetate
required
to
effectively
inhibit
the
effect
of
cytokines,
gliadin
peptides,
or
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
93
AT-1002
in
vitro
varied
considerably
(from
0.1
to
10
mM).
The
rea-
sons
for
this
discrepancy
are
not
known.
We
have
previously
found
that
intraperitoneal
sensitization
with
gliadin
and
CFA,
followed
by
an
oral
gliadin
challenge
to
sin-
gle
transgenic
HLA-DQ8
mice
increases
neurally
stimulated
ion
transport
[44].
We
have
also
shown
that
gliadin
sensitization
of
HLA-HCD4-DQ8
mice
increases
paracellular
and
transcellular
per-
meability
of
macromolecules
as
assessed
by 51Cr-EDTA
and
HRP
flux,
respectively
[28,37].
In
this
study
we
found
increased
con-
ductance
values
in
gliadin-sensitized
mice,
indicating
a
reduction
in
tissue
resistance
and
suggesting
an
alteration
in
the
paracel-
lular
pathway.
This
change
was
normalized
by
larazotide
acetate
therapy.
This
is
in
agreement
with
the
recent
finding
of
Silva
et
al.
that
larazotide
acetate
also
normalizes
paracellular
macromolecule
transport
[37].
We
also
showed
that
increased
HRP
flux,
indica-
tive
of
transcellular
permeability,
was
improved
with
larazotide
acetate,
but
values
were
still
high
compared
to
non-sensitized
con-
trols.
Improvement
in
HRP
flux
by
larazotide
acetate
may
relate
to
overall
enhancement
of
epithelial
homeostasis,
as
it
correlated
with
an
improvement
in
TJ
structure
in
EM
and
a
decrease
in
macrophage
infiltration
in
gliadin-sensitized
and
challenged
mice.
We
expected
that
AT-1002,
a
permeability
enhancing
peptide,
would
augment
gliadin’s
detrimental
effects
in
the
intestine.
In
fact,
in
a
recent
study
AT-1002
was
shown
to
increase
intestinal
perme-
ability
in
vivo
[3].
In
this
study
however,
AT-1002
did
not
augment
the
effect
of
gliadin
in
any
of
the
measures.
Perhaps
AT-1002
cannot
add
to
the
already
extensive
effects
of
gliadin.
Taken
together,
our
data
indicate
that
larazotide
acetate
inhibits
the
cytoskeletal
rearrangement
and
ZO-1
redistribution
caused
by
gliadin
and
AT-1002
in
epithelial
cells.
Larazotide
acetate
inhibits
gliadin
transport
in
vitro
and
preserves
barrier
function
in
vitro
and
in
vivo.
Furthermore,
in
an
in
vivo
gluten
sensitivity
model,
larazotide
acetate
inhibited
gliadin-induced
macrophage
accumu-
lation
in
the
intestine
and
preserved
normal
TJ
structure.
These
data
offer
insights
into
larazotide
acetate’s
mechanisms
of
action
for
the
treatment
of
gluten-induced
enteropathy,
and
provide
support
for
larazotide
acetate
as
a
novel
candidate
with
potential
therapeutic
value
in
the
management
of
celiac
disease.
Acknowledgments
This
work
was
partially
supported
by
grants
from
the
Cana-
dian
Association
of
Gastroenterology
(CAG)/Canadian
Institute
of
Health
Research
(CIHR)
(GN2-114709),
the
Canadian
Celiac
Associ-
ation
New
Investigator
Award
(to
E.
Verdu),
and
ALBA
Therapeutics.
E.
Verdu
holds
a
McMaster
University
Dep.
of
Medicine
Internal
Career
Research
Award.
Joseph
Murray
was
supported
by
NIH
grant
DK
70031.
We
are
grateful
to
Drs.
Alessio
Fasano,
Linda
Arterburn,
and
Francisco
Leon
for
critical
reading
of
the
manuscript.
References
[1]
Aijaz
S,
Balda
MS,
Matter
K.
Tight
junctions:
molecular
architecture
and
func-
tion.
Int
Rev
Cytol
2006;248:261–98.
[2]
Al-Sadi
R,
Ye
D,
Dokladny
K,
Ma
TY.
Mechanism
of
IL-1beta-induced
increase
in
intestinal
epithelial
tight
junction
permeability.
J
Immunol
2008;180:5653–61.
[3]
Arrieta
MC,
Bistritz
L,
Meddings
JB.
Alterations
in
intestinal
permeability.
Gut
2006;55:1512–20.
[4] Artursson
P,
Magnusson
C.
Epithelial
transport
of
drugs
in
cell
culture.
II:
Effect
of
extracellular
calcium
concentration
on
the
paracellular
transport
of
drugs
of
different
lipophilicities
across
monolayers
of
intestinal
epithelial
(Caco-2)
cells.
J
Pharm
Sci
1990;79:595–600.
[5]
Black
KE,
Murray
JA,
David
CS.
HLA-DQ
determines
the
response
to
exogenous
wheat
proteins:
a
model
of
gluten
sensitivity
in
transgenic
knockout
mice.
J
Immunol
2002;169:5595–600.
[6] Bodinier
M,
Legoux
MA,
Pineau
F,
Triballeau
S,
Segain
JP,
Brossard
C,
et
al.
Intestinal
translocation
capabilities
of
wheat
allergens
using
the
Caco-2
cell
line.
J
Agric
Food
Chem
2007;55:4576–83.
[7]
Ciccocioppo
R,
Finamore
A,
Ara
C,
Di
Sabatino
A,
Mengheri
E,
Corazza
GR.
Altered
expression,
localization,
and
phosphorylation
of
epithelial
junctional
proteins
in
celiac
disease.
Am
J
Clin
Pathol
2006;125:502–11.
[8]
Clayburgh
DR,
Shen
L,
Turner
JR.
A
porous
defense:
the
leaky
epithelial
barrier
in
intestinal
disease.
Lab
Invest
2004;84:282–91.
[9]
Clemente
MG,
De
Virgiliis
S,
Kang
JS,
Macatagney
R,
Musu
MP,
Di
Pierro
MR,
et
al.
Early
effects
of
gliadin
on
enterocyte
intracellular
signalling
involved
in
intestinal
barrier
function.
Gut
2003;52:218–23.
[10]
Crespo
Perez
L,
Castillejo
de
Villasante
G,
Cano
Ruiz
A,
Leon
F.
Non-dietary
therapeutic
clinical
trials
in
coeliac
disease.
Eur
J
Intern
Med
2012;23:9–14.
[11]
Dolfini
E,
Roncoroni
L,
Elli
L,
Fumagalli
C,
Colombo
R,
Ramponi
S,
et
al.
Cytoskeleton
reorganization
and
ultrastructural
damage
induced
by
gliadin
in
a
three-dimensional
in
vitro
model.
World
J
Gastroenterol
2005;11:
7597–601.
[12]
Elli
L,
Roncoroni
L,
Doneda
L,
Ciulla
MM,
Colombo
R,
Braidotti
P,
et
al.
Imaging
analysis
of
the
gliadin
direct
effect
on
tight
junctions
in
an
in
vitro
three-
dimensional
Lovo
cell
line
culture
system.
Toxicol
In
Vitro
2011;25:45–50.
[13] Ellis
HJ,
Rosen-Bronson
S,
O’Reilly
N,
Ciclitira
PJ.
Measurement
of
gluten
using
a
monoclonal
antibody
to
a
coeliac
toxic
peptide
of
A-gliadin.
Gut
1998;43:190–5.
[14] Fasano
A,
Shea-Donohue
T.
Mechanisms
of
disease:
the
role
of
intestinal
barrier
function
in
the
pathogenesis
of
gastrointestinal
autoimmune
diseases.
Nat
Clin
Pract
Gastroenterol
Hepatol
2005;2:416–22.
[15]
Fink
MP,
Delude
RL.
Epithelial
barrier
dysfunction:
a
unifying
theme
to
explain
the
pathogenesis
of
multiple
organ
dysfunction
at
the
cellular
level.
Crit
Care
Clin
2005;21:177–96.
[16]
Furuse
M.
Molecular
basis
of
the
core
structure
of
tight
junctions.
Cold
Spring
Harb
Perspect
Biol
2010;2:a002907.
[17]
Garrote
JA,
Gomez-Gonzalez
E,
Bernardo
D,
Arranz
E,
Chirdo
F.
Celiac
disease
pathogenesis:
the
proinflammatory
cytokine
network.
J
Pediatr
Gastroenterol
Nutr
2008;47(Suppl.
1):S27–32.
[18] Ginski
MJ,
Taneja
R,
Polli
JE.
Prediction
of
dissolution-absorption
relationships
from
a
continuous
dissolution/Caco-2
system.
AAPS
Pharm
Sci
1999;1:E3.
[19]
Gopalakrishnan
S,
Pandey
N,
Tamiz
AP,
Vere
J,
Carrasco
R,
Somerville
R,
et
al.
Mechanism
of
action
of
ZOT-derived
peptide
AT-1002,
a
tight
junction
regulator
and
absorption
enhancer.
Int
J
Pharm
2009;365:121–30.
[20]
Kagnoff
MF.
Celiac
disease:
pathogenesis
of
a
model
immunogenetic
disease.
J
Clin
Invest
2007;117:41–9.
[21]
Madara
JL,
Barenberg
D,
Carlson
S.
Effects
of
cytochalasin
D
on
occluding
junc-
tions
of
intestinal
absorptive
cells:
further
evidence
that
the
cytoskeleton
may
influence
paracellular
permeability
and
junctional
charge
selectivity.
J
Cell
Biol
1986;102:2125–36.
[22]
Marinaro
M,
Fasano
A,
De
Magistris
MT.
Zonula
occludens
toxin
acts
as
an
adjuvant
through
different
mucosal
routes
and
induces
protective
immune
responses.
Infect
Immun
2003;71:1897–902.
[23]
Matysiak-Budnik
T,
Moura
IC,
Arcos-Fajardo
M,
Lebreton
C,
Menard
S,
Candalh
C,
et
al.
Secretory
IgA
mediates
retrotranscytosis
of
intact
gliadin
peptides
via
the
transferrin
receptor
in
celiac
disease.
J
Exp
Med
2008;205:143–54.
[24] Montalto
M,
Cuoco
L,
Ricci
R,
Maggiano
N,
Vecchio
FM,
Gasbarrini
G.
Immuno-
histochemical
analysis
of
ZO-1
in
the
duodenal
mucosa
of
patients
with
untreated
and
treated
celiac
disease.
Digestion
2002;65:227–33.
[25] Natividad
JM,
Huang
X,
Slack
E,
Jury
J,
Sanz
Y,
David
C,
et
al.
Host
responses
to
intestinal
microbial
antigens
in
gluten-sensitive
mice.
PLoS
One
2009;4:e6472.
[26]
Paterson
BM,
Lammers
KM,
Arrieta
MC,
Fasano
A,
Meddings
JB.
The
safety,
tolerance,
pharmacokinetic
and
pharmacodynamic
effects
of
single
doses
of
AT-
1001
in
coeliac
disease
subjects:
a
proof
of
concept
study.
Aliment
Pharmacol
Ther
2007;26:757–66.
[27]
Picarelli
A,
Di
Tola
M,
Sabbatella
L,
Anania
MC,
Di
Cello
T,
Greco
R,
et
al.
31–43
amino
acid
sequence
of
the
alpha-gliadin
induces
anti-endomysial
antibody
production
during
in
vitro
challenge.
Scand
J
Gastroenterol
1999;34:1099–102.
[28]
Pinier
M,
Verdu
EF,
Nasser-Eddine
M,
David
CS,
Vezina
A,
Rivard
N,
et
al.
Poly-
meric
binders
suppress
gliadin-induced
toxicity
in
the
intestinal
epithelium.
Gastroenterology
2009;136:288–98.
[29] Rubio-Tapia
A,
Murray
JA.
Celiac
disease.
Curr
Opin
Gastroenterol
2010;26:116–22.
[30]
Sander
GR,
Cummins
AG,
Henshall
T,
Powell
BC.
Rapid
disruption
of
intesti-
nal
barrier
function
by
gliadin
involves
altered
expression
of
apical
junctional
proteins.
FEBS
Lett
2005;579:4851–5.
[31]
Sawada
N,
Murata
M,
Kikuchi
K,
Osanai
M,
Tobioka
H,
Kojima
T,
et
al.
Tight
junctions
and
human
diseases.
Med
Electron
Microsc
2003;36:147–56.
[32]
Schmitz
H,
Fromm
M,
Bentzel
CJ,
Scholz
P,
Detjen
K,
Mankertz
J,
et
al.
Tumor
necrosis
factor-alpha
(TNFalpha)
regulates
the
epithelial
barrier
in
the
human
intestinal
cell
line
HT-29/B6.
J
Cell
Sci
1999;112(Pt
1):137–46.
[33]
Schneeberger
EE,
Lynch
RD.
The
tight
junction:
a
multifunctional
complex.
Am
J
Physiol
Cell
Physiol
2004;286:C1213–28.
[34]
Schulzke
JD,
Bentzel
CJ,
Schulzke
I,
Riecken
EO,
Fromm
M.
Epithelial
tight
junc-
tion
structure
in
the
jejunum
of
children
with
acute
and
treated
celiac
sprue.
Pediatr
Res
1998;43:435–41.
[35]
Schumann
M,
Richter
JF,
Wedell
I,
Moos
V,
Zimmermann-Kordmann
M,
Schneider
T,
et
al.
Mechanisms
of
epithelial
translocation
of
the
alpha(2)-
gliadin-33mer
in
coeliac
sprue.
Gut
2008;57:747–54.
[36]
Shan
L,
Molberg
O,
Parrot
I,
Hausch
F,
Filiz
F,
Gray
GM,
et
al.
Structural
basis
for
gluten
intolerance
in
celiac
sprue.
Science
2002;297:2275–9.
[37] Silva
MA,
Jury
J,
Sanz
Y,
Wiepjes
M,
Huang
X,
Murray
JA,
et
al.
Increased
bac-
terial
translocation
in
gluten-sensitive
mice
is
independent
of
small
intestinal
paracellular
permeability
defect.
Dig
Dis
Sci
2012.
94
S.
Gopalakrishnan
et
al.
/
Peptides
35
(2012)
86–94
[38]
Song
KH,
Fasano
A,
Eddington
ND.
Enhanced
nasal
absorption
of
hydrophilic
markers
after
dosing
with
AT1002,
a
tight
junction
modulator.
Eur
J
Pharm
Biopharm
2008;69:231–7.
[39]
Thomas
KE,
Sapone
A,
Fasano
A,
Vogel
SN.
Gliadin
stimulation
of
murine
macrophage
inflammatory
gene
expression
and
intestinal
permeability
are
MyD88-dependent:
role
of
the
innate
immune
response
in
Celiac
disease.
J
Immunol
2006;176:2512–21.
[40] Tsukita
S,
Furuse
M,
Itoh
M.
Multifunctional
strands
in
tight
junctions.
Nat
Rev
Mol
Cell
Biol
2001;2:285–93.
[41] Turner
JR,
Rill
BK,
Carlson
SL,
Carnes
D,
Kerner
R,
Mrsny
RJ,
et
al.
Physiological
regulation
of
epithelial
tight
junctions
is
associated
with
myosin
light-chain
phosphorylation.
Am
J
Physiol
1997;273:C1378–85.
[42]
Ukabam
SO,
Clamp
JR,
Cooper
BT.
Abnormal
small
intestinal
permeability
to
sugars
in
patients
with
Crohn’s
disease
of
the
terminal
ileum
and
colon.
Diges-
tion
1983;27:70–4.
[43]
Van
Driessche
W,
Kreindler
JL,
Malik
AB,
Margulies
S,
Lewis
SA,
Kim
KJ.
Inter-
relations/cross
talk
between
transcellular
transport
function
and
paracellular
tight
junctional
properties
in
lung
epithelial
and
endothelial
barriers.
Am
J
Physiol
Lung
Cell
Mol
Physiol
2007;293:L520–4.
[44]
Verdu
EF,
Huang
X,
Natividad
J,
Lu
J,
Blennerhassett
PA,
David
CS,
et
al.
Gliadin-dependent
neuromuscular
and
epithelial
secretory
responses
in
gluten-sensitive
HLA-DQ8
transgenic
mice.
Am
J
Physiol
Gastrointest
Liver
Physiol
2008;294:G217–25.
[45]
Wang
F,
Schwarz
BT,
Graham
WV,
Wang
Y,
Su
L,
Clayburgh
DR,
et
al.
IFN-
gamma-induced
TNFR2
expression
is
required
for
TNF-dependent
intestinal
epithelial
barrier
dysfunction.
Gastroenterology
2006;131:1153–63.
[46] Watts
T,
Berti
I,
Sapone
A,
Gerarduzzi
T,
Not
T,
Zielke
R,
et
al.
Role
of
the
intestinal
tight
junction
modulator
zonulin
in
the
pathogenesis
of
type
I
diabetes
in
BB
diabetic-prone
rats.
Proc
Natl
Acad
Sci
U
S
A
2005;102:2916–21.
