Download Poster Anna 2009

Activation of Innate and not Adaptive
Immune system in Gluten Sensitivity
Update: Differential mucosal IL-17 expression in gluten sensitivity
and the autoimmune enteropathy celiac disease
A. Sapone, L. de Magistris, M. Cammarota, M.T. Giuliano, M. De Rosa,
M. Carteni, C. Tolone, A. Papparella, R. Stefanile, G.Mazzarella, M.I. Russo,
P. Esposito, A. Frattino, A. Fasano, G. Riegler
1
ABSTRACT
There are cases of gluten reaction defined as gluten sensitivity (GS) in which neither an allergic (wheat allergy) nor an
autoimmune [celiac disease (CD)] mechanism can be advocated. Recent evidences suggest that early changes in
intestinal permeability (IP) and activation of both innate and adaptive immune responses are involved in CD
pathogenesis. Conversely, no data are currently available on the mechanisms leading to GS. Aim was to investigate the
changes in IP, TJ protein genes expression, and innate and adaptive immune responses in GS. Biopsy samples were
obtained from 28 GS patients, 53 pts with active CD, 16 patients with CD in remission, and 37 healthy controls (age
range: 5 years -50 years). Claudin (CL) 1, CL2, CL3, CL4, ZO-1, and TLR1, TLR2 and TLR4, FOXP3, and TGF-ß
gene expression were performed by Real-time PCR. IP was evaluated by means of the lactulose/mannitol test
(LA/MA). The number of intraepithelial lymphocytes (IELs) was detected with CD3 and TCR-?d immunostaining and
examined by counting the peroxidase stained cells. ELISA analysis of IL6, IL8, TNFa, was conducted on PBMC of all
patients. CL3 and CL4 expressions were significantly increased in GS subjects compared to CD patients (p<0.01). In
GS patients, these changes were associated to a lower IP (0.010±0.008) that inversely correlated to CL4 gene
expression (r= -0.6318; p<0.05) compared to healthy controls (0.018±0.009). Conversely, in CD patients an overexpression of CL2 was observed that was associated to increased IP (0.053±0.048) and that returned to baseline
following implementation of a gluten free diet. Interestingly, immunohistochemical examination of biopsy specimen
showed a lower number of CD3+IELs in GS pts compared to active CD pts (27.25/100 and 36.3/100 enterocytes
respectively) that correlated with MARSH 0-1 lesions, with no changes in TCR γδ IELs. In a subgroup of GS pts,
intestinal TLR1 and TLR2 expression was increased and these changes were associated to an increased production of
cytokines related to the innate but not adaptive immune responses. Compared to CD patients, GS subjects showed
normal IP and activation of the innate but not adaptive immune responses. These changes cause only minimal gut
inflammation, suggesting that in GS lack of adaptive immune response involvement prevent the autoimmune gut insult
typical of CD.
2
Background
The evolution of human race did not plan
for the advent and exposure to wheat
gluten.
The “engineering” of gluten-containing
grains 10,000 years ago represented a
“mistake of evolution” that created the
conditions for human diseases related to
gluten exposure.
3
Background:
Reaction to gluten can involve an allergic (wheat allergy) or an
autoimmune [celiac disease (CD)] mechanism.
However, there are cases of gluten reaction in which neither
mechanism is involved.
These cases are defined as gluten
sensitivity (GS).
Recent evidences suggest that early changes in intestinal
permeability (IP) and activation of the innate immune system
through Toll Like Receptor (TLR) signaling pathway may both be
involved in the early steps of CD pathogenesis.
Conversely, no data are currently available on the role of intestinal
barrier dysfunction in the pathogenesis of GS.
4
Aims:
. To investigate the changes in IP and TJ protein
genes expression in GS patients;
. To establish whether these changes are related
to activation of the innate and/or adaptive
immune response.
5
Materials and Methods:
• Biopsy samples were obtained from 26 GS patients,
30 patients with active CD, 6 patients with CD in
remission, and 14 healthy controls.
• Quantitative gene expression of tight junctions
proteins Claudin (CL) 1, CL2, CL3, CL4, ZO-1,
Occludin and of TLR1, TLR2 and TLR4 was
performed by Real-time PCR.
• IP was evaluated by means of the lactulose/mannitol
test (LA/MA).
• The degree of intestinal mucosal damage was
evaluated according to the Marsh classification.
• The numbers of IELs were examined using CD3 and
TCR-γδ immunostaining.
6
Materials and Methods:
GS inclusions criteria:
• Gastrointestinal or extra-intestinal
symptoms mimicking CD;
• Negative CD serology;
• Negative response for atopy patch test
(APT) to wheat and prick test.
7
Results:
Clinical and laboratory characteristics of the study subjects
Healthy
CD
GS
29.1 ± 5.1
35.2 ± 3.3
28.9 ± 4.4
20/16
15/36
18/10
Dyspepsia
-Chronic diarrhea
-Abdominal pain
-Weight fluctuation
- Weakness
- Smelling, fatty stool
-Diarrhea
- Abdominal pain
-Weight fluctuation
-Gas
Extra-intestinalSymptoms
None
-Bone or joint pain
- Osteoporosis
- Behavioral changes
- Tingling leg numbness
- Muscle cramps
- Missed menstrual periods
- Infertility
- Recurrent miscarriage
- Delayed growth
- Tooth discoloration
-Unexplained anemia
-Bone or joint pain
-Osteoporosis
- Leg numbness
-Muscle cramps
-Unexplained anemia
- Glossitis
-Thyroiditis
EMA
0%
Positive (92.3%)
Negative
AGA
0%
Positive (53.8%)
Positive (45.5%)
tTG
0%
HLA DQ2/DQ8
28.6%
92.3%)
36.4%)
PRICK/ RAST/ PRIST test
Not tested
Negative
Negative
LA/MA test
Normal (< 0.030)
Altered (> 0.030)
Normal (< 0.030)
Age of diagnosis (mean ± SE)
Sex (F/M)
Intestinal Symptoms
Positive (84.6%)
Negative
8
A
Results :Duodenal IHC
CD3+ IELs
B
C
C
40 X
TCR-γδ IELs
CD3+IELs
Nr TCR-γδ IEL/100 enterocytes
Nr CD3+IELs/100 enterocytes
100
90
80
70
60
50
40
30
20
10
0
Healthy
CD active
GS
CD treated
A= Gluten Sensitive
50
45
B= Controls
40
35
30
25
C= CD Active
20
15
10
5
0
Healthy
CD active
GS
CD treated
9
Results:
Change in Intestinal Permeability
in GS and CD patients
In GS patients IP (0.014±0.015) was similar to that detected in healthy controls (0,019±0,018). Conversely, in
CD the increased expression of CL2 was associated to an increase in IP (0.052±0.048). In CD patients in
remission, IP (0.014±0.004) returned to normal levels.
10
Results:
Intercellular TJ expression CD
CLDN1
CLDN3
CLDN2
0.10
5
1.5
0.04
1.0
% 18S
0.06
% 18S
% 18S
**
4
0.08
3
2
0.5
0.02
1
*
A
G
C
H
A
A
G
OCLN
ZO1
2.5
15
2.0
1.0
1.5
% 18S
% 18S
1.5
% 18S
C
H
G
C
A
A
C
C
H
CLDN4
2.0
A
0
0.0
C
0.00
1.0
10
5
0.5
0.5
0.0
A
G
CA
HC
A
G
CA
0
HC
A
G
A
C
H
C
0.0
CL3 and CL4 expressions were significantly increased in GS subjects compared to CD
patients (p<0.01), while no changes in other TJ protein genes expression were
detected. Conversely, in CD patients an over-expression of CL2 was observed that was
associated to increased IP and that returned to baseline following a gluten free diet.
*,** P <0.01 GS vs Celiac active pts
* r (IP vs CL4)= 0,6318; p=0,05
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Results:
TLR signaling pathway involvement
TLR1
0.02
0.01
A
G
A
A
G
A
C
C
0.00
H
A
G
A
C
H
0.10
0.05
0.00
C
0.00
0.15
C
0.05
0.20
C
0.10
**
0.03
% 18S
*
0.15
TLR4
0.25
H
*
0.20
% 18S
TLR2
0.04
% 18S
0.25
TLR1 and TLR2, but not TLR4 resulted significantly increased in GS *,**(p<0,05)
respect to normal controls, while CD showed only a not significant increase of TLR1
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Results: Cytokines expression in cultured PBMC
8000
700
7000
600
6000
Healthy
5000
4000
3000
500
CD active
400
CD active
GS
300
GS
CD gfd
2000
100
0
0
PT
CD gfd
200
1000
medium
Healthy
medium
BSA
PT
BSA
TNFa
IL6
350000
300000
250000
Healthy
200000
CD active
150000
GS
CD gfd
100000
50000
PBMC released IL6, IL8 and TNFa
in all groups after stimulation with
pt-Gliadin.
0
medium
PT
IL8
BSA
13
Results: regulatory molecules gene expression
TGFB1
FOXP3
6
0.10
% 18S
% 18S
0.08
4
2
0.06
0.04
0.02
0
*
A
G
2.0
1.5
% 18S
0.015
0.010
**
1.0
***
0.5
0.005
A
G
A
C
A
G
A
C
C
H
C
0.0
0.000
H
% 18S
0.020
A
HMGB1
IL17A
0.025
C
C
G
H
A
A
C
H
C
0.00
CD pts showed a decreased expression of FOXP3 that was associated to a
significantly increased of IL 17 compared to normal control and GS pts,
while HMGB1 was significantly decreased in both GS and CD pts.
* P=0,04 CD vs C
**,*** P<0,05 CD and Gs vs C
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Conclusions:
•
Compared to CD patients, GS subjects showed normal IP.
•
Up-regulation of CL4 in GS patients is statistically significant and
inversely correlated to IP.
•
GS patients showed an increase in CD3+ IEL but no tissue damage.
•
The significant over expression of TLR 1 and TLR2 in GS but not in CD
may reflect the activation of the innate immune system in this
condition.
•
•
PBMC derived cytokines expression is similarly elicitated by gliadin in
both CD and GS.
IL 17 activation pathway only in CD may suggest that in GS the
response to gluten exposure is limited to activation of the innate immune
system without involvement of the adaptive immune response.
•
Gluten Sensitivity showing a peculiar TJ proteins pattern, confirms
be different from celiac disease.
to
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