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
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