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