Download Wheat Amylase Trypsin Inhibitors as Triggers of

Wheat Amylase Trypsin Inhibitors as Triggers of
Innate Immunity
Detlef Schuppan
Molecular and Translational Medicine, Dept. Medicine I, Univ. of
Mainz, Germany
Division of Gastroenterology and Celiac Center, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, USA
2nd International Expert Meeting on Gluten Sensitivity
Munich Nov.30 – Dec. 2, 2012
HARVARD
MEDICAL
SCHOOL
Food intolerances
1. Lactose or fructose intolerance
2. “Histamine intolerance”
3. Food allergy
4. Celiac disease (gluten: wheat, barley, rye)
Recent – very common
5. Non-celiac “gluten” sensitivity
6. FODMAP intolerance
Frequently associated
6. Irritable bowel syndrome
7. Pathological intestinal microbiota
Mesopotamia
Egypt
Moderner Massenanbau
Hallmarks of celiac disease
• Dietary gluten from wheat, barley, or rye as
trigger of adaptive (T cell) immunity
• Genetic Predisposition (HLA-DQ2 or -DQ8)
• IgA autoantibodies to tissue transglutaminase
• A wheat component that drives innate
immunity
Role of the Innate Immune System in celiac disease – prior work
• Stimulation of biopsies from CD patients with PT gliadin or α2 gliadin p31-43
enhances IL-15 positive cells in the lamina propria
(Maiuri et al, Lancet 2003)
• p31-43 induces MICA on intestinal epithelial cells via IL-15, serving as target
for cytotoxic IELs
(Hue et al, Immunity 2004)
• PT gliadin and different gliadin peptides induce activation & maturation of
monocytes, macrophages & DCs
(Tuckova et al, J Leuk Biol 2002; Palova-
Jelinkova et al, FEBS Lett 2004, J Immunol 2005; Nikulina et al, J Immunol 2004; Cinova et al,
J Clin Immunol 2007; Rakhimova et al, J Clin Immunol 2008)
• Gliadin enhances intestinal permeability and DC activation via MyD88 (CXCR3
on intestinal epithelial cells as gliadin receptor)
(Thomas et al, J Immunol, 2006; Lammert et al, Gastroenterology 2008)
1. LPS contamination not strictly ruled out
? 2. No reproducible identification of a certain (set of) gliadin peptide(s)
3. No plausible receptor identified
The innate immune
response in celiac
disease
„gluten“
PAMPs
IL-15
What about
professional APC ?
Is it really gluten ?
IL-15
central growth
factor for
intraepithelial NK
cells and CTL
Jabri B et al, Gastroenterology 2000
Maiuri L et al, Lancet 2003
Hue S et al, Immunity 2004
Meresse B et al, Immunity 2004
Rakhimova M et al, J Clin Immunol 2008
p31-43 and PT gliadin do not stimulate intestinal epithelial
cells, monocytes, macrophages or DCs
Medium
6000
p31-43 20g/ml
scrambled p31-43 20g/ml
4000
scrambled p31-43 40g/ml
2000
7
U-
93
1
PTH
-2
9
0
HT
IL-8 (pg/ml)
p31-43 40g/ml
Junker et al, J Exp Med 2012
10
ng
/m
l
5000
TN
Fa
5n
g/
m
l
50
0
g/
m
l
LP
S
gl
ia
di
n
LPS is not a contaminant
M
ed
iu
m
g
/m
l
10
ng
/m
l
25
0
0
l
0
g/
m
5
0
iu
m
5
25
M
ed
10
IL-8 (ng/ml)
20
15
LP
S
l
25
ze
in
gl
ia
di
n
g
/m
/m
l
10
ng
/m
l
25
0
LP
S
ze
in
g
gfd
PT
PT
PT
25
0
30
P
T
0
gl
ia
di
n
5
M
ed
iu
m
10
IL-8 (ng/ml)
healthy ctr
PT
IL-8 (ng/ml)
15
IL-8 (pg/ml)
gl
M
ed
PT iadi
n
iu
gl
1
00 m
ia
PT di
n g/
25 m
gl
ia
d i 0 g l
PT n 5
/m
ze 00 l
PT in 1 g/m
z e 00  l
PT in 2 g/m
ze 50 l
in
g
50 /m
l
L P 0 g
S
/
10 ml
ng
/m
l
PT
PT gliadin stimulates monocyte derived DCs from controls and celiac patients
20
15
regular diet
10
w/o proteinase K
+ proteinase K
4000
3000
2000
1000
0
Junker et al, J Exp Med 2012
PT gliadin-induces innate immune responses via TLR4 in
vitro and in vivo
KC
KC(ng/ml)
(ng/ml)
30
C3H/HeOuJ
C3H/HeOuJ
C3H/HeJ
C3H/HeJ
20
10
0
C3H/HeJ mice: TLR4 deficient due to a
spontaneous point mutation
Gliadin mediated innate immune responses in vivo
80
60
40
C57BL/6
MyD88-/-
TNF- (pg/ml)
2000
20
15
10
C57BL/6
Rag1-/-
1000
400
300
fold
x-fold mucosal induction
(mRNA)
20
LPS
oral feeding
LPS
gliadin
PBS
15
10
5
0
KC
TNF-
MCP-1
IL-1
gliadin
zein
Oral LPS is inactivated by
stomach acid and intestinal
alkaline phosphatase, while
the activity in gliadin is not
Junker et al, J Exp Med 2012
8
7
1
8
6
5
2
7
6
4
3
3
4
5
6
zein
gliadin
LPS
0
0
9
0
4
100
9
0
1
2
5
5
200
1
2
3
4
S
S
S
KC (ng/ml)
25
i.p. injection
3000
i.p. injection
The activity is contained in the ω-gliadin fraction
50
293-hTLR4/MD2-CD14
IL-8 (ng/ml)
8000
6000
4000
30
10
8
6
overlapping 20mers
4
2
2000
0
0
M
ed
iu
m
LP
S
P
PT MA
R
ek
 tor
-g
lia
di
n
gl

i
1. adi
n
2gl
i
 adi
n
5gl
ia
di
n
IL-8 (pg/ml)
12000
40

5
LP Me
g
Pa lia S diu
m din 10n m
3C 1 g
SK 00 /m
4 mg l
10 /m
m l
g/
m
1 l
10 to 9
to
19 18
to
28 27
to
38 36
to
1 43
to
43
16000
Comparison of the gliadin fractions by
SDS-PAGE showed a minor component
of 15kDa associated only with ω-gliadins
There is a little hidden bird on this foto – find it !
Wheat amylase-trypsin Inhibitors (ATIs) trigger intestinal innate immunity in
macrophages and dendritic cells via TLR4
Junker Y et al, J Exp Med 2012
Oda Y et al, Biochemistry 1997
Characteristics und function of wheat ATIs
•
•
•
•
•
Family of up to 11 similar, small and compact proteins
5(4) intramolecular SS-bonds, resistant to intestinal degradation
Pest control (inhibition of parasite enzymes) Tatham & Shewry, Clin Exp Allergy 2008
Zevallos VF et al, DDW 2012, #1309
Known major allergens of baker‘s asthma
Content paralles that of gluten – association with omega-gliadins
Activity of 2 major wheat ATIs expressed in eukaryotic cells
Activation of monocytes-macrophages
Inactivation by S-S reduction
Activation of both TLR4 pathways
Physical interaction with TLR4
Oral feeding of ATI
(50g/mouse) causes
low level intestinal
inflammation
ATI promotes
adaptive immunity
in human CD
biopsies
Junker et al, J Exp Med 2012
Classification of plants according to their
relative potency to induce innate immunity
IL8 (1 unit= 100 pg)
Units of IL-8/g of flour in U937 cells
300
250
234,18
208,80
200
Wheat
Triticum aestivum
Barley
Hordeum vulgare L.
Rye
Secale cereale
155,27
150
100
High: gluten containing
79,40
71,89
Medium: gluten-free (gluten-poor)
78,89
50
0
Soya
Glycine Max
Quinoa
Chenopodium quinoa
Buckwheat Fagopyrum esculentum
IL8 (1 unit= 100 pg)
25
22,62
20
16,60
Peas
Pisum sativum
Early
Crops
Einkorn<Emmer<Spelt
15
Low: gluten free
10
Rice
Oryza sativa
Millet
Panicum miliaceum
Oats
Avena sativa
Maize
Zea mays L.
Amaranth
Amaranthus caudatus
5
0
1,69
2,42
1,18
1,23
Zevallos VF et al, unpublished
Emmer
Einkorn
Spelt
Modern wheat
Origin of Wheat
Breeding
Crossing
Chromosome sets
Goat wheat
Increase of content of immunogenic gluten epitopes and ATIs
with resistance breeding and higher ploidity
Breeder‘s dilemma
Breeding for high yield and high pest resistance

• Reduction of nutritional value (essential
amino acids, minerals, vitamins)
• Increase in pest resistance proteins/
molecules potentially harmful to man
Morris CE and Sands DC, Nat Biotechnol 2006
Non-celiac „gluten“ sensitivity
„Gluten sensitivity“ without
overt small intestinal damage
or auto-Abs
Estimated prevalence 3-10%
Role of ATIs ?
Studies in IBS, autoimmune-,
metabolic, cardiovascular……
diseases are warranted
Intestinal lumen
Intestinal epithelium
Basement membrane
IEL activation
Lamina propria
PMN attraction/activation
Stimulation
of TLR4
on MΦ & DC
Fuelling inflammation &
autoimmunity in the gut
and at distant sites
Potentiation of already
existing intestinal adaptive
immune activation
ATI
Antigen
MΦ attraction/activation
Intestine and
lymph nodes
Recirculation and
homing to other sites
adipose
↓
SERIAL
KILLERS
Innate Immunity in Celiac Disease

The mechanism by which certain gluten peptides (p31-43)
may elicit innate mechanisms in biopsies ex vivo remains to
be defined

ATIs of gluten containing grains are strong inducers of
innate immunity in DCs > macrophages > monocytes via
TLR4

Ingested ATIs induce low level intestinal inflammation in vivo

ATI content of modern wheat has increased due to
resistance breeding

ATIs of gluten free foods have much less stimulatory activity

Innate immunity to ATIs likely impacts other intestinal and
non-intestinal inflammatory diseases
Research and Clinical Team
Acknowledgements
BIDMC GI:
Donatella Barisani
Melinda Dennis
Tobias Freitag
Yvonne Junker
Ciaran Kelly
Daniel Leffler
Seong-Jun Kim
BWH:
Sebastian Zeissig
BIDMC Immunology:
Ulrich v. Andrian
Cox Terhorst
Svend Rietdijk
BIDMC Proteomics Center:
Towia Liberman
Simon Dillon
MGH:
Atul Bhan
Hans-Chr. Reinecker
Celiac Center Boston
Germany:
Walburga Dieterich
Minna Hietikko
Martin Hils
George Kahaly
Norbert Krauss
Moises Laparra
Ralf Pasternack
Martin Rosenthal
Mareike Roth
Nina Rüssel
Nicole Voltz
Herbert Wieser
Jessy Willim
Victor Zevallos
Stanford:
Grete Sønderstrup
Chaitan Khosla
Support
NIH-NIAID BMBF DFG
German and US Celiac
Sprue Associations
Celiac Center Mainz