USING BALB/c MICE AS A MODEL OF FOOD ALLERGY TO STUDY GENE

EXPRESSION PROFILES IN RESPONSE TO COMMON FOOD ALLERGENS

Mainul Husain

Department of Animal & Poultry ScienceUniversity of Guelph

Rationale

• Approximately 2-3% of the adults and 6-8% of the children are affected by food allergy

• Allergy to hen’s egg, cow’s milk, and nuts especially peanuts are the most common

• The only effective way to prevent food allergy is to avoid allergenic foods

• Avoidance of allergenic foods may lead to nutritional deficiency in children with multiple food allergy

Rationale• Food allergy is typically considered as IgE mediated

type-1 hypersensitivity

IL-4IL-13

INF-

IL-12 IL-12IL-4/IL-

10

Allergen Bacteria/Viruses

APC Dendritic Cell

TH2 Cell TH1 Cell

B Cell

IgE

IL-3, IL-4,IL-6, IL-9

Mast Cell

GM-CSFIL-3, IL-5

Allergen

Eosinophil

Inflammatory Mediators

MHC II

• In certain forms of food allergy, for example cow milk allergy in infants, elevated level of Th1 cytokines IL-2 and IFN- are observed

• Such uncertainty over Th1 and Th2 cytokines makes it difficult for accurate diagnosis of food allergy

Rationale

Rationale

• Mice are the widely accepted model to analyze the allergenicity of food proteins

• Only few strains evaluated as models, for example: BALB/c, C3H/HeJ and C57BL/6

• C57BL/6 strain is less sensitive to allergen sensitizations than BALB/c strain

• C3H/HeJ strain does not respond to OVA sensitization

Rationale

• Among other strains, BALB/c has advantages over other strains because of its high IgE response

• It has a tendency to develop IgE response similar to human atopic phenotype

• Responds to several common allergens

• Until now, there is no validated animal models for the assessment of food allergy

Hypothesis• The BALB/c mouse are suitable for

characterizing allergenic food proteins using i.p. sensitization and epicutaneous challenge

• Treatment of BALB/c mice with food allergens will induce gene expression changes that can be identified in mesenteric lymph nodes and spleens

• Genes expressed in response to food allergen treatment in BALB/c mice may be useful as bio-markers to determine the allergenicity of food proteins

Experiment Objectives

1.a. To investigate the effects of sensitization and challenge of food allergens on BALB/c mice over 24 hours time period by measuring the ear swelling response

1.b. To evaluate the potential of BALB/c mice as a model of food allergy by measuring the serum IgG1, IgE and plasma histamine level, and the presence of antigen specific IgEs

Experiment Objectives

2. To investigate the gene expression profile of mesenteric lymph node from BALB/c mice sensitized with 3 known food allergens, using cDNA microarray technology

3. To investigate the gene expression profile of spleen from BALB/c mice treated with egg ovomucoid (OVM), using Affymetrix microarray technology

Experiment 1: Effects of Antigen Treatment And

Validation of BALB/c mice

Animals used:

• Female Balb/c mice (6-8 weeks of age) Allergens used:

• 0.2% Peanut agglutinin (PNA)

• 2% Ovalbumin (OVA) from hen’s egg white

• 2% β-Lactoglobulin (BLG) from cow’s milk Control used:

• Phosphate buffered saline (PBS)

Experiment 1

• Balb/c mice were sensitized for 2 days with 0.2% (PNA) or 2% (BLG & OVA) antigen solution by i.p injection• Mice challenged 2 weeks after 2nd sensitization

• Blood collected 24 hr post challenge after euthanization

• Total IgG1 and IgE levels were assessed to confirm the type-1 hypersensitivity response

• Plasma histamine level measured for systemic anaphylaxis

2 Weeks

Day 01st I.P.

Sensitization

Day 12nd I.P.

Sensitization

Day 16Euthanization

24 H Post Challenge

Day 15Epicutaneous Challenge

by Ear Stripping

Swelling Response0, 0.5, 1, 2, 6 & 24 H

Results: Experiment 1

• SC – Sensitized & Challenged • C Only – Challenged Only

• *** - P<0.001

Results: Experiment 1

SC – Sensitized & Challenged; C – Challenged

* - p<0.02; ** - p<0.01; *** - p<0.002

Results: Experiment 1

SC – Sensitized & Challenged; C – Challenged

* - p<0.06; ** - p<0.05; *** - p<0.01

Results: Experiment 1

SC – Sensitized & Challenged

* - p<0.06; *** - p<0.001

Experiment 1 : Conclusion

• BALB/c mice responded positively to all three allergens as indicated by ear swelling response, serum total IgG1 and IgE, and plasma histamine level

• Ear swelling response can differentiate the potency of different food allergens

• BALB/c mice can be used as a model of food allergy following this sensitization and challenge protocol

Experiment 2: Gene Expression Analysis In Mesenteric Lymph Node (MLN)

• Animals: Female Balb/c mice (6-8 weeks)• Experimental BALB/c mice i.p. sensitized with:

Peanut agglutinin (PNA) – 0.2% solution in PBS Ovalbumin (OVA) from egg white -2% solution in PBS β-Lactoglobulin (BLG) from milk – 2% solution in PBS

• Control mice i.p. sensitized with PBS

24 h

Day 0First I.P. Sensitization

Day 2Euthanization and Collection of MLN

Day 1Second I.P. Sensitization

cDNA Microarray

• cDNA microarray experiment was conducted to analyze the gene expression in MLN

• Each cDNA array contains 22.4k probes (mouse ESTs)

• Each probe is represented by one single spot

Results: Experiment 2Overlapping & Unique Genes from Allergen Sensitized Groups

BLG Sensitized Group (1361 Genes)

OVA Sensitized Group(533 Genes)

PNA Sensitized Group(488 Genes)

92 131

77

988

175169

Results: Experiment 2 Gene Ontology (GO) Analysis

BLG Sensitized Group 204 Significant (P<0.05) GO

Biological Processes

OVA Sensitized Group 327 Significant (P<0.05) GO

Biological Processes

PNA Sensitized Group 295 Significant (P<0.05) GO

Biological Processes

24 30

112

104

139113

Results: Experiment 2

GO Biological ProcessesBLG Treatment PNA Treatment OVA Treatment

Genes P-Value Genes P-Value Genes P-Value

Response to stimulus 100 0.005 50 0.002 50 0.011

Muscle development 16 0.033 9 0.021 10 0.014

Cytokine and chemokine mediated signaling pathway

10 0.000 5 0.006 4 0.038

Acute inflammatory response 9 0.001 5 0.004 6 0.001

Humoral immune response 8 0.001 5 0.002 6 0.001

Results: Experiment 2 Validation of Microarray Data

R - 0.923p<0.001

Genes from BLG sensitized group () - Stard4, Igj, Timp1, Cd79a and Itgb1Genes from OVA sensitized group () - Stard4, Igj, Timp1, Pex13 and Syt4 Genes from PNA sensitized group () - Stard4, Igj and Timp1

Experiment 2: Conclusion

• Sensitization of BALB/c mice with known food allergens induce change in its gene expression

• Genes differentially expressed in the MLN of BALB/c mice in response to food allergen sensitization are related with food allergy

Experiment 3: Gene Expression Analysis In Spleen

• Animals: Female Balb/c mice (6-8 weeks)

• Allergen: Egg Ovomucoid (OVM; 1 mg/100 µl)

• Control: Amino acid solution (1 mg/100 µl)

• Adjuvant: Cholera toxin (10 µg)

• Sensitization/exposure: Intra-gastric (i.g.) gavage

• Challenge: Intra-peritoneal (i.p.) injection

Week 6I.P. Challenge & Euthenization 40

Min Post Challenge

Week 2Two I.G. Exposure

Week 3Two I.G. Exposure

Week 4Two I.G. Exposure

Week 1Two I.G. Exposure

Week 5Two I.G. Exposure

Results: Experiment 3

• Affymetrix Mouse Genome 430 2.0 arrays were used

• Each array contained 45K probes, representing 34K genes

• A total of 87 genes were differentially expressed

• 28 genes were up-regulated

• 59 genes were down-regulated

Results: Experiment 3 Gene Ontology Biological Process

GO Biological ProcessEnriched

GenesFisher’s Exact

P-Value

Protein metabolism 20 0.009

Response to stress 9 0.008

Defense response 7 0.023

Response to pest, pathogen or parasite 6 0.004

Immune response 6 0.028

Inflammatory response 3 0.029

Complement activation 2 0.011

Humoral immune response 2 0.043

Results: Experiment 3 - Biological Network

Results: Experiment 3 Validation of Genes by Real Time RT-PCR

GenesReal-time RT-PCR Microarray

Fold Change P-Value Fold ChangeP-

Value

Adipsin (Adn) 7.13 0.001 1.71 0.031

Carbonic Anhydrase 3 (Car3) 4.57 0.001 7.56 0.017

Immunoglobulin Heavy Chain 4 (Igh4) 3.66 0.015 3.38 0.001

Interleukin 6 (IL-6) 4.12 0.011 1.78 0.019

Prostaglandin-endoperoxide Synthase 2 (Ptgs2)

21.51 0.001 2.28 0.045

Experiment 3: Conclusion

• Intra-gastric exposure and i.p. challenge of BALB/c mice with egg OVM induced gene expression changes in spleen

• Genes differentially expressed in spleen in response OVM treatment are related with food allergy

Common Genes from Experiments 2 and 3

• Additional 41 similar genes were differentially expressed from experiment 2 and experiment 3

Experiment 2 (cDNA Microarray) 2382 Differentially Expressed

Genes From MLN

Experiment 3 (Affy Microarray) 87 Differentially Expressed

Genes From Spleen

132369 74

Common GO Processes from Experiments 2 and 3

• Immune Response

• Inflammatory Response

• Complement Activation

Overall Conclusion

• BALB/c mice can be used as a model to evaluate the allergenicity of food proteins

• Treatment of BALB/c mice with known food allergens induce changes in the gene expression profile in MLN and spleen

• Genes differentially expressed in response to known food allergen treatment in BALB/c mice may be useful as candidate bio-marker genes for the assessment of allergenicity of food proteins