Transcriptome Profile Of Bal Bc Mouse
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Transcript of Transcriptome Profile Of Bal Bc Mouse
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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
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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
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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
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• 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
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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
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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
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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
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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
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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
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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)
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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
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Results: Experiment 1
• SC – Sensitized & Challenged • C Only – Challenged Only
• *** - P<0.001
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Results: Experiment 1
SC – Sensitized & Challenged; C – Challenged
* - p<0.02; ** - p<0.01; *** - p<0.002
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Results: Experiment 1
SC – Sensitized & Challenged; C – Challenged
* - p<0.06; ** - p<0.05; *** - p<0.01
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Results: Experiment 1
SC – Sensitized & Challenged
* - p<0.06; *** - p<0.001
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Results: Experiment 3 - Biological Network
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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
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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
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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
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Common GO Processes from Experiments 2 and 3
• Immune Response
• Inflammatory Response
• Complement Activation
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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