Food allergies: from allergen to intestinal inflammation

Eva Untersmayr

Department of Pathophysiology

and Allergy Research

Medical University of Vienna

For educational purpose only

http://www.uni-ulm.de/biometrie-bumd/viktor/aaa_Themes/logos/

Menschenmenge_2%20.jpg

US National Health and Nutrition

Examination Surveys 2007-2010

(20,686 US participants):

8.96% (7.64%) overall self-reported

food allergy, 6.53% in children

Food allergy: a significant problem

Germany DEGS1 study: Sensitization evidenced by specific IgE: >7000

blood samples 18- 79yrs: overall 25.5% pos. results with 50 allergens

tested

Nwaru BI et al. Allergy 2013

Mc Gowan EC, Keet CA. JACI 2013

EAACI Food allergy & Anaphylaxis Guidelines Group:

Pooled life-time prevalence: self reported food allergy: 17.3%;

Point prevalence: specific IgE: 10,1%, SPT: 2,7%, pos. challenges: 0.9%

Haftenberger M et al. Bundesgesundheitsbl 2013

Average number of hospital discharges/year among children (under 18

years) with diagnosis „food allergy“ in the United States 1998-2006

http://www.cdc.gov/nchs/data/databriefs/db10_fig4.gif

Hospitalisation due to food allergy

Gupta R et al. Thorax 2007

Food allergy hospital admission Overall food allergy admission rates: from 5 to 26/million

Children: 7-fold increase from 16 to 107/million

No data from ER included

Increase of admission rates: 500% since 1990

+ 500%

Economic burden by food allergy

Overall food allergy costs: $24.8 billion/year

$4,184 per child and year

Total costs included $4.3 bill. direct medical costs

$20.5 bill. costs to families

Gupta R et al. JAMA Pediatr 2013

Health care cost for Europeans (EuroPrevall studies in 9 countries):

for food-allergic adults $2,016 mean cost/year

for controls $1,089 mean cost/year

No differences for adults and children in each country

significantly related to disease severity Fox M et al. Eur J Public Health 2013

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?

uri=OJ:L:2011:304:0018:0063:EN:PDF

EU legislation on food allergens

Labelling rules for 14 food allergens:

• Eggs

• Milk

• Fish

• Crustaceans

• Mollusks

• Peanuts

• 10 different tree nuts

• Sesame seeds

• Cereals containing gluten

• Soybeans

• Celery and celeriac

• Mustard

• Lupine

• Sulphites SO2 (>10 mg/kg)

http://www.ies.be/node/1615

More than 170 food ingredients cause IgE-mediated reactions

Campari Caviar

Fish

Milk Snails

Peanuts and tree nuts

Avocado

Banana

Camomille Eggs

Shellfish Reviewed in Burks AW et al. JACI 2012

Characteristics of food allergens

• glycoproteins

• low dose activation of

IL4-producing CD4-T-cells

• low molecular weight (10-70 kDa)

diffusion through epithelia facilitated

• solubility

• stability to heat, acid, proteases

• some are enzyme inhibitors

• abundance in food

http://hometestingblog.testcountry.com/wp-

content/uploads/2010/05/food-allergy-wheel.jpg

Characteristics of food allergens

• glycoproteins

• low dose activation of

IL4-producing CD4-T-cells

• low molecular weight (10-70 kDa)

diffusion through epithelia facilitated

• solubility

• stability to heat, acid, proteases

• some are enzyme inhibitors

• abundance in food

http://www.who.int/foodsafety/publications/biot

ech/en/ec_jan2001.pdf

http://usinfo.state.gov/products/pubs/biotech/

Known allergen or gen

source allergen?

Sequence homology,

databank search

Allergological testing

Stability to

digestion/processing

(Animal model)

Simulated gastric fluid experiments

http://de.123rf.com/photo_4177027_chemische-reagenzglas-chemische-experiment-mit-laborglas.html

Food proteins

+

Gastric enzymes

pH2

Digestion-labile food allergens

0 s

ec

5 s

ec

1 m

in

1 h

15 m

in

0 s

ec

5 s

ec

1 m

in

1 h

Untersmayr E et al. FASEB J 2005, Untersmayr E et al. COAI 2006, Untersmayr E et al. Proceedings of the 25th CIA,

2005 . Untersmayr E, Jensen-Jarolim E In: B. Wüthrich, T. Werfel (Eds.), Nahrungsmittel und Allergie 3, 2010 .

Shrimp Wheat Milk Parvalbumin

HR test: passive sens. basophils

Untersmayr E et al. JACI 2005

Codfish extract (μg/mL)

His

tam

ine

re

lea

se

(%

) undigested

dig., pH 2

10.900-times

reduction of IgE

binding capacity

Biological activity of digested codfish

http://www.hausarbeiten.de/faecher/vorschau/188567.html

Pepsin and gastric acid producing cells

Foveolar cells: mucus production

Parietal cells: hydrochloric acid

intrinsic factor

Chief cells: Pepsinogen

G-cells: gastrin secretion

Physiological gastric digestion

Untersmayr E, Jensen-Jarolim E. COAI 2006

Untersmayr E, Jensen-Jarolim E. JACI 2008

Transformation of

pepsinogen into the

mature enzyme pepsin

Protein binding in

substrate cleft and

protein cleavage

Releasing of acidic chyme into the

duodenum

Reviewed in Pali-Schöll I, Jensen-Jarolim E. Allergy 2011

Activation of duodenal (and jejunal)

S-cells secretin release

Stimulation pancreas proteases

trypsin, chymotrypsin,

carboxypeptidases release

Physiological protein digestion

http://en.wikipedia.org/wiki/Duodenum

Low gastric pH is decisive for activation and release of

gastric and pancreas proteases

?

Gastric pH elevation

Physiological protein digestion

http://www.wellmona.com/product_info.php/info/p27_5-wwpads-organ-booster--

magen----besaenftigend---hilft-bei-kraempfen-und-schmerzen.html

Hypoacidity and gastric digestion

Codfish digestion, 1min

Milk digestion

Untersmayr E et al. JACI 2005, Untersmayr E et al. FASEB J 2005

pH2

SDS-Page IgE blot

1h

1m

5s

un

dig

.

pH2

1h

, p

H3

1h

, p

H5

1h

, p

H3

1h

, p

H5

1h

1m

5s

un

dig

.

un

dig

.

pH

1.2

5

pH

2.0

pH

2.5

un

dig

.

pH

1.2

5

pH

2.0

pH

2.5

pH

2.7

5

pH

3.5

pH

4.0

pH

4.5

pH

5.0

pH

2.7

5

pH

3.5

pH

4.0

pH

4.5

pH

5.0

Gastric hypoacidity in health and disease

Pharmacological:

• Antacids & Sucralfate

• H2 receptor blockers

• Proton pump inhibitors

Physiological: Newborns

Atrophic gastritis

http://www.kur-apotheke-wolter.de/info-magen.html

Acid suppression medication – mode of action

Olbe L et al. Nat Rev Drug Discov. 2003

Proton pump

inhibitors

H2-receptor

blockers

Antacids: slight bases

neutralizing gastric acid

Sucralfate: Aluminum

compound strong neg.

charge upon Al3+ release

Murine experiments

digestion-labile food allergens

+

Sucralfate

H2 receptor blockers

Proton pump inhibitors

Rennie®

Base powder

Ma

st

ce

ll t

ryp

tas

e

(ng

/mL

)

*

aa. oral ip. naive acidsuppr.

animals

oral naive

Untersmayr E et al. JACI 2003

Schöll I et al. Am J Clin Nutr 2005

Schöll I et al. FASEB J 2007

Brunner R et al. Clin Exp Allergy. 2007

Untersmayr E et al. Mech Ageing Dev. 2008

Diesner SC et al. Immunol Lett. 2008

Brunner R et al. Allergy. 2009

Riemer AB et al. Clin Exp Allergy 2010

Pali-Schöll I et al. Clin Exp Allergy. 2010

Untersmayr et al. PLoS One 2010

Diesner SC et al. Immunol Lett 2012

Krishnamurthy D et al. Ms submitted

Diesner SC et al. Ms submitted

Microscopic analysis of gastro-intestinal mucosa

Untersmayr E et al. JACI 2003

Untersmayr et al. IAAI 2004

Diesner S et al. Imm Lett 2012

Pali-Schöll I et al. Exp Toxicol Pathol. 2008

Codfish + sucralfate

PBS

Eosinophils

Mast cells

Intestinal architecture

Human studies

http://www.telegraph.co.uk/news/yourview/2446086/Are-

we-too-dependent-on-medication.html

Untersmayr E et al. FASEB J 2005

Schöll et al. Am J Clin Nutr 2005

Bakos N et al. Imm Letters 2006

Observational study: patients with dyspeptic

disorders treated with anti-ulcer medication

Allergologic evaluation of elderly

http://ucfa.nl/ucfa-research/therapy/

Specif. IgE testing SPTs DBPCFC

Untersmayr E et al. JACI 2007

Undig. Dig. pH 2 Dig. pH 3

Changed reactivity profile in allergic patients

Pat.1 Pat.2 Pat.3 Pat.1 Pat.2 Pat.3

LO

AE

L

(c

um

ula

ted

do

se

; m

g)

Non-digestion

reduces

threshold levels

Digestion significantly

reduces skin reactivity

Untersmayr E, Jensen-Jarolim E. JACI 2008

The role of gastric digestion in food allergy

Population based health register

study: Association between maternal

anti-ulcer intake and childhood asthma Dehlink E et al. Clin Exp Allergy 2009

Questionnaire study: correlation

between increasing prevalence of

childhood food allergy anti-ulcer

medication Karen DeMuth et al. Allergy and Asthma

Proceedings 2013

Insurance data base analysis

(14.172 children between 0-18 ys):

acid suppression associated with a

higher food allergy risk Trikha A et al. Pediatric Allergy Immunol 2013

Food allergy model in BALB/c mice

normal Antiacid

Oral immunizations with digestion-labile allergens under

concomitant gastric acid suppression

1st

immunization

2nd

immunization

3rd

immunization

Read-out

experiments

days Antiacids Antiacids Antiacids

Food allergy against OVA proteins

ip. anti-

acid

oral naive

ip.

ora

l a

ac

id

naiv

e

ip. anti-

acid

oral naive

Diesner SC et al. Immunol Lett 2008

Allergen factors: environmental protein nitration

http://www.kinderwelt.org/grafik/abgas_1.jpg

Chemical modification of airborne

proteins by NO2 and O3

Nitrated Bet v1 in urban dust samples

Conformational change in nitrated proteins

Enhanced allergenic potential of nitrated

Bet v1 for inducing IgE-mediated type I

allergy

Franze T et al. Environ Sci Technol 2005

Souza JM et al. Arch Biochem Biophys 2000

Gruijthuijsen YK et al. IAAI 2006

Shiraiwa M et al. Nature Chem 2011

Endogenous protein nitration

- Gastrointestinal environment Nitrous acid is formed only at low pH

acidic gastric milieu required for nitration

Nitrated proteins cleaved slower by

chymotrypsin than untreated proteins

Pannala AS et al. Free Radic Biol Med 2006

Greenacre SA et al. Free Radic Res 2001 http://bodybymitch.com/page/2/

- Endogenous protein nitration

during inflammation

Sakaguchi AA et al. Free Redic Biol Med 1999

Helicobacter pylori gastritis or inflammatory

bowel disease

Extent of protein nitration was considered

as a maker of oxidative stress in

inflammatory responses Yeo WS et al. BMB reports 2008

Immunization regimen

untreated OVA

sham-nitrated

OVA

nitrated OVA

untreated OVA

sham-nitrated

OVA

nitrated OVA

Induction of OVA specific IgE

untreated

OVA

sham

OVA

nitrated

OVA

naiv

e

ip. ip. ip. anti-

acid anti-

acid

oral oral oral anti-

acid

Allergenicity of nitrated OVA

untreated

OVA sham

OVA

nitrated

OVA

ip. ip. ip. anti-

acid anti-

acid

oral oral oral anti-

acid

untreated OVA

nitrated OVA

nitrated Bet v1

na

ive

SGF assays with OVA preparations untreated

OVA

sham-nitrated

OVA

nitrated

OVA

Characteristics of nitrated OVA

Y:10, F: 20 L:32

Pepsin interaction partners: phenylalanine, tyrosine and

leucine

10 tyrosine residues: 21% nitration grade

MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKD

STRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQIYKPND

VYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARE

LINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDE

DTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGT

MSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMK

MEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEIN

EAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCV

SP

Y107 nitration degree:1

Y112 nitration degree: 0.11

Y282 nitration degree: 0.14

OVA epitopes and IgE recognition

B-cell epitope T-cell epitope

Mine Y et al. BBRC 2009

Mine Y et al. J Agric Food Chem 2008

Mine Y et al. BBA 2007

Nitrated Y107:

B-cell and T-cell epitope

IgE epitope recognized exclusively after oral immunization

MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKD

STRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQIYKPND

VYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARE

LINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDE

DTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGT

MSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMK

MEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEIN

EAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCV

SP

Protein nitration in food allergy

Enhanced triggering capacity of nitrated OVA

proteins in allergic animals

Differences in administration routes in experimental

models of food allergy

Reduced de novo sensitization capacity of nitrated OVA

via the oral route due to enhanced digestibility

underlining the importance of gastric digestion in food

allergy and/or changes in antibody epitopes

Untersmayr E et al. PLoS One 2010

Stucture impacts on food uptake

Reviewed in: Reitsma M et al. Mol Nutr Food Res 2014

Non-sensitized individuals: protein uptake via M-cells and enterocytes

~2% intact proteins reach intestinal lymph nodes and portal circulation Warshaw AL et al. Gastroenterology 1974

Food protein uptake in allergy

Reviewed in Reitsma M et al. Mol Nutr Food Res 2014

sensitized individuals: paracellular route involvement of Mast cells

transcellular route via enterocytes

Intestinal microbiota and allergy

http://www.gutmicrobiotawatch.org/gut-microbiota-info/

Differences in gut microbiota

between atopic and healthy

persons

Reviewed in: Gigante G et al. Dig Dis 2011

Infants with food allergy reveal imbalance between beneficial and

potentially harmful bacteria Reviewed in: Compare D et al. Eur Rev Med Pharmacol Sci 2013

Bacterial strain specific Fox p3+ Treg induction in vivo protects against

airway inflammation and oral IgE induction in mouse allergy models Lyons A et al. Clin Exp Allergy 2010

Germ-free mice: immunodeficient,

without colonization no oral

tolerance development, decreased

intestinal surface, decreased cell

renewal, thinner mucus layer Reviewed in: Natividad JMM et al. Pharmacol Res 2013

Contributors to food allergic gastrointestinal inflammation

• Route of allergen entry

• Microbiota composition

• Inflammatory mediators and

signaling molecules

http://www.coreonehealth.com/gastrointestinal

Intestinal food allergic response gut microbiota

Mast cells

Th2

Lamichhane A et al. J Gastroenterol Hepatol 2013

Recruitment of

inflammatory cells by

mediators

Sphingolipids

• Large family of lipids

• Ubiquitary expressed

• Constituent of cell membranes

and lipid rafts on the outer

leaflet of the plasma

membrane

General chemical structure

of Sphingolipids

Pierce SK. Nature Reviews Immunology 2002

• S1P is produced in various

cell types and impacts on

apoptosis, cell migration,

cell growth, immune cell

activation

• Exogenous introduction of

SM via food de novo

sythesis

• Endogenous production

(S1P)

Spiegel S et al. Nature Reviews Molecular Cell Biology 2003

Sphingosine-1-Phosphate

S1P

phosphatase

S1P

lyase

Endogenous S1P production

Spiegel S et al. Nature Reviews Molecular Cell Biology 2003

Sphingosine

Mainly in endothelial cells, erytrocytes, platelets, mast cells

sphingosine kinases

(SphK1 and SphK2) S1P

Role of S1P in allergic inflammation

Oskeritzian CA et al. Pharmacol Ther 2007

Sphingolipids and food allergy

S1P-dependent recruiting of

systemically primed CD4+ T-cells

from spleen to intestine

antigen-specific intestinal allergic

hypersensitivity in mice

S1P-mediated migration of mast

cells with specific IgE towards

antigen Kurashima Y et al. J Immunol 2007

Kunisawa J et al. Nutrients 2012

Intestinal tract relatively rich in sphingolipids (~30% of total lipids in

apical membrane of villi, being higher than basolateral) Danielsen EM et al. Mol Membr Biol 20076

S1P increases the transepithelial allergen uptake in vitro

Caco2

21 days Caco2

cultivation

S1P

1 or 5 h S1P

stimulation

OVA transport

for 1 h

OVA

Diesner SC et al. Immunol Letters 2012

Immunization regimen

Food allergic group

Negative control

group

Positive control

Day 0 14 28 42 84 112 140 154

PP

I + o

ral Im

m.

PP

I + o

ral Im

m.

PP

I + o

ral Im

m.

1s

t Tre

atm

en

t

2n

d Tre

atm

en

t

Sensitization Treatment

PIS 1.MIS 2.MIS 3.MIS 4.MIS 5.MIS 6.MIS

Ora

l Ch

alle

ng

e

56 70 98 126

3rd T

rea

tme

nt

7.MIS

Sa

crific

e

8.MIS

168

PP

I + o

ral Im

m.

Ora

l Ch

alle

ng

e

4th T

rea

tme

nt

5th T

rea

tme

nt

9.MIS

6th T

rea

tme

nt

11.MIS10.MIS 12.MIS

Ae

ros

ol C

ha

llen

ge

Ae

ros

ol C

ha

llen

ge

Impact of SphK1 and SphK2 deletion on food allergy

SphK deletion inhibits oral OVA sensitization

IgE

Anaphylactic response is aberrant in SphKs deleted animals

SphK2 deletion enhances systemic mast cells Flow cytometry analysis of spleen cell subpopulations

Mast cells (c-Kit+ FceRI+)

SphK deletion decreases mast cell accumulation in gastric mucosa

Allergen Intestinal epithelial layer

Allergen specific IgE Mast cell

S1P SphK1

SphK2

Sph

Enhanced mediator

release

Proposed mechanism S1P homeostasis influences food allergy development

and effector cell function

Diesner et al. Immunol Letters 2012

Shingolipid homeostasis in the gut

Sphingomyelin,

ceramide Izawa K et al. JACI 2014

http://aokemergencyroom.com/acute-abdominal-pain/

Diverse contributors to food allergy

Digestion

Microbiota

Mediators

Individual factors Allergen factors

Structure

Stability

Genetics,

Epigenetics,..

Modifications

Complex network of contributing factors

Thank you!

Geja Oostingh Tobias Pfaller Albert Duschl Silja Weßler

Kathrin Selzle Yingyi Zhang Ulrich Pöschl

Laboratory for Medical Allergology,

University Hospital Copenhagen

Helle Vestergaard

Hans-Jørgen Malling

Louise Bjerremann Jensen

Michael H. Platzer

Per Stahl Skov

Lars K. Poulsen

Ana Olivera

Sandra Dillahunt

Juan Rivera

Financial acknowledgements:

Austrian Science Fund FWF KLI 284 and P21577

Intramural Research Program of NIAMS, NIH

European Molecular Biology Organization (short-term fellowship ASTF 374-2008)

Susanne Diesner Isabella Pali Erika Jensen-Jarolim Zsolt Szépfalusi Thomas Eiwegger

Barbara Pitzner

Michael Rothballer

Anton Hartmann