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 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