Mouse Mesenteric Lymph Node Transcriptome Profile And Allergic Reactions In Response To Common

Food Allergens Treatment

Mainul Husain

University of Guelph, Guelph, Ontario,

Canada

Rationale• Development of novel food or novel food

processing techniques may sometimes cause unwanted changes in the food

• Genetically Modified (GM) foods have health safety concerns, because their altered genetic nature could potentially lead to allergic reactions

• Current methods include IFBC-ILSI and FAO-WHO proposed decision tree approach

• This decision tree approach has several limitations

• Need to develop methods for evaluating the allergenicity of foods

Sensitization and Gene Expression• Three different known food allergens were used

to sensitize Balb/c miceo Allergens used: 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

• cDNA microarray (22.4k ESTs) was used to identify the potential bio-marker genes from mesenteric lymph node for type-1 hypersensitivity response

• Real Time RT-PCR was done, to validate the candidate bio-marker genes

Elicitation• 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 • Ear skin was tape stripped • Antigen solution was applied on left ear and PBS was

applied on the right ear• Ear skin thickness was measured after 0.5, 1, 2, 6 and

24 hr• Blood was collected at 24 hr post challenge after

euthanization • Total IgE, IgG1 and histamine levels were assessed to

confirm the type-1 hypersensitivity response in mice2 Weeks

Day 01st Sensitization of

Mice by I.P. Injection

Day 12nd Sensitization of

Mice by I.P. Injection

Day 16Euthanization of

Mice After 24 Hours Observation

Day 15Challenge of Mice by Ear Stripping And 24 Hours

Observation

Ear Swelling Response To Allergen Challenge

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

• ** - P<0.01

24 Hr. Serum IgG1 Response To Allergen Challenge

• ** - P<0.01

24 Hr. Serum IgE Response To Allergen Challenge

• * - P<0.05

• ** - P<0.01

Plasma Histamine Response to Allergen Challenge

• * - P<0.05

• ** - P<0.01

cDNA Microarray

• cDNA microarray was done to determine the gene expression profile of mice mesenteric lymph node in response to known allergens

• Mouse 22.4K cDNA arrays were used from University Health Network, Toronto

• Microarray data was analyzed by Gene Spring GX 7.2 Software

cDNA Microarray

• 1361 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to BLG sensitization

• 533 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to OVA sensitization

• 488 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to PNA sensitization

Overlapping And Unique Genes from 3 Treatment Groups

BLG Treatment Group (1361 Genes)

OVA Treatment Groups (533 Genes)

PNA Treatment Groups (488 Genes)

92 131

77

988

175169

Gene Ontology (GO) Analysis204 Significant (P<0.05) GO Biological

Processes Enriched with the Genes from BLG Treatment Group

327 Significant (P<0.05) GO Biological Processes Enriched with the Genes

from OVA Treatment Groups

296 Significant (P<0.05) GO Biological Processes Enriched with the Genes

from PNA Treatment Groups

24 30

112

104

139114

Gene Ontology (GO) Biological Processes

GO Biological Processes GO IDBLG Treatment PNA Treatment OVA Treatment

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

Response to stimulus 50896 100 0.005 50 0.002 50 0.011

Muscle development 7517 16 0.033 9 0.021 10 0.014

Cytokine and chemokine mediated signaling pathway

19221 10 0.000 5 0.006 4 0.038

Acute inflammatory response 2526 9 0.001 5 0.004 6 0.001

Humoral immune response 6959 8 0.001 5 0.002 6 0.001

Pathways Identified In Response to Known Allergen Treatment

• Complement Pathway

• IL-7 Signal Transduction

• Fc epsilon RI signaling pathway

• Natural killer cell mediated cytotoxicity

• T cell receptor signaling pathway

• B cell receptor signaling pathway

T-Cell Receptor Signaling Pathway

IL-7 Signal Transduction Pathway

Real-Time RT-PCR

Genes:

• Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4

• Igj - Immunoglobulin Joining Chain

• Timp1 - Tissue Inhibitor of Metalloproteinase 1

• Cd79a - CD79A Antigen (Immunoglobulin Associated Alpha)

• Itgb1 - Integrin Beta 1 (Fibronectin Receptor Beta)

Real-Time RT-PCR

Genes:

• Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4

• Igj - Immunoglobulin Joining Chain

• Timp1 - Tissue Inhibitor of Metalloproteinase 1

• Pex13 - Peroxisomal Biogenesis Factor 13

• Syt4 - Synaptotagmin IV

Real-Time RT-PCR

Genes:

• Timp1 - Tissue Inhibitor of Metalloproteinase 1

• Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4

• Igj - Immunoglobulin Joining Chain

Conclusion

• Genes differentially expressed in response to the treatment of known food allergens, may be used as candidate bio-marker genes for the assessment of potential allergenicity of GM food allergy

• Balb/c mice showed strong allergic response to the known food allergen treatment

• Balb/c mice can be used as a model animal to analyze the gene expression profile in response to known food allergens treatment