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Mouse Model of Allergy

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Mouse Model of Allergy

  1. 1. Mouse Mesenteric Lymph Node Transcriptome Profile And Allergic Reactions In Response To Common Food Allergens Treatment Mainul Husain Department of Animal & Poultry Science University of Guelph Ontario, Canada
  2. 2. Rationale <ul><li>Development of novel food or novel food processing techniques may sometimes cause unwanted changes in the food </li></ul><ul><li>Genetically Modified (GM) foods have health safety concerns, because their altered genetic nature could potentially lead to allergic reactions </li></ul><ul><li>Current methods include IFBC-ILSI and FAO-WHO proposed decision tree approach </li></ul><ul><li>This decision tree approach has several limitations </li></ul><ul><li>Need to develop methods for evaluating the allergenicity of foods </li></ul>
  3. 3. Sensitization and Gene Expression <ul><li>Three different known food allergens were used to sensitize Balb/c mice </li></ul><ul><ul><li>Allergens used : </li></ul></ul><ul><ul><li>Peanut agglutinin (PNA) – 0.2% solution in PBS </li></ul></ul><ul><ul><li>Ovalbumin (OVA) from egg white -2% solution in PBS </li></ul></ul><ul><ul><li>β-Lactoglobulin (BLG) from milk – 2% solution in PBS </li></ul></ul><ul><li>cDNA microarray (22.4k ESTs) was used to identify the potential bio-marker genes from mesenteric lymph node for type-1 hypersensitivity response </li></ul><ul><li>Real Time RT-PCR was done, to validate the candidate bio-marker genes </li></ul>
  4. 4. Elicitation <ul><li>Balb/c mice were sensitized for 2 days with 0.2% (PNA) or 2% (BLG & OVA) antigen solution by i.p injection </li></ul><ul><ul><li>Mice challenged 2 weeks after 2 nd sensitization </li></ul></ul><ul><ul><li>Ear skin was tape stripped </li></ul></ul><ul><ul><li>Antigen solution was applied on left ear and PBS was applied on the right ear </li></ul></ul><ul><ul><li>Ear skin thickness was measured after 0.5, 1, 2, 6 and 24 hr </li></ul></ul><ul><ul><li>Blood was collected at 24 hr post challenge after euthanization </li></ul></ul><ul><li>Total IgE, IgG1 and histamine levels were assessed to confirm the type-1 hypersensitivity response in mice </li></ul>2 Weeks Day 0 1st Sensitization of Mice by I.P. Injection Day 1 2nd Sensitization of Mice by I.P. Injection Day 16 Euthanization of Mice After 24 Hours Observation Day 15 Challenge of Mice by Ear Stripping And 24 Hours Observation
  5. 5. Ear Swelling Response To Allergen Challenge <ul><li>SC – Sensitized & Challenged </li></ul><ul><li>C Only – Challenged Only </li></ul><ul><li>** - P<0.01 </li></ul>
  6. 6. 24 Hr. Serum IgG1 Response To Allergen Challenge <ul><li>** - P<0.01 </li></ul>
  7. 7. 24 Hr. Serum IgE Response To Allergen Challenge <ul><li>* - P<0.05 </li></ul><ul><li>** - P<0.01 </li></ul>
  8. 8. Plasma Histamine Response to Allergen Challenge <ul><li>* - P<0.05 </li></ul><ul><li>** - P<0.01 </li></ul>
  9. 9. cDNA Microarray <ul><li>cDNA microarray was done to determine the gene expression profile of mice mesenteric lymph node in response to known allergens </li></ul><ul><li>Mouse 22.4K cDNA arrays were used from University Health Network, Toronto </li></ul><ul><li>Microarray data was analyzed by Gene Spring GX 7.2 Software </li></ul>
  10. 10. cDNA Microarray <ul><li>1361 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to BLG sensitization </li></ul><ul><li>533 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to OVA sensitization </li></ul><ul><li>488 genes were significantly (p<0.05) differentially (±1.5 fold) expressed in response to PNA sensitization </li></ul>
  11. 11. 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 77 988 175 169
  12. 12. Gene Ontology (GO) Analysis 204 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 30 112 104 139 114
  13. 13. Gene Ontology (GO) Biological Processes GO Biological Processes GO ID BLG 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
  14. 14. Pathways Identified In Response to Known Allergen Treatment <ul><li>Complement Pathway </li></ul><ul><li>IL-7 Signal Transduction </li></ul><ul><li>Fc epsilon RI signaling pathway </li></ul><ul><li>Natural killer cell mediated cytotoxicity </li></ul><ul><li>T cell receptor signaling pathway </li></ul><ul><li>B cell receptor signaling pathway </li></ul>
  15. 15. T-Cell Receptor Signaling Pathway
  16. 16. IL-7 Signal Transduction Pathway
  17. 17. Real-Time RT-PCR <ul><li>Genes: </li></ul><ul><ul><li>Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4 </li></ul></ul><ul><ul><li>Igj - Immunoglobulin Joining Chain </li></ul></ul><ul><ul><li>Timp1 - Tissue Inhibitor of Metalloproteinase 1 </li></ul></ul><ul><ul><li>Cd79a - CD79A Antigen (Immunoglobulin Associated Alpha) </li></ul></ul><ul><ul><li>Itgb1 - Integrin Beta 1 (Fibronectin Receptor Beta) </li></ul></ul>
  18. 18. Real-Time RT-PCR <ul><li>Genes: </li></ul><ul><ul><li>Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4 </li></ul></ul><ul><ul><li>Igj - Immunoglobulin Joining Chain </li></ul></ul><ul><ul><li>Timp1 - Tissue Inhibitor of Metalloproteinase 1 </li></ul></ul><ul><ul><li>Pex13 - Peroxisomal Biogenesis Factor 13 </li></ul></ul><ul><ul><li>Syt4 - Synaptotagmin IV </li></ul></ul>
  19. 19. Real-Time RT-PCR <ul><li>Genes: </li></ul><ul><ul><li>Timp1 - Tissue Inhibitor of Metalloproteinase 1 </li></ul></ul><ul><ul><li>Stard4 – StAR Related Lipid Transfer (START) Domain Containing 4 </li></ul></ul><ul><ul><li>Igj - Immunoglobulin Joining Chain </li></ul></ul>
  20. 20. Conclusion <ul><li>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 </li></ul><ul><li>Balb/c mice showed strong allergic response to the known food allergen treatment </li></ul><ul><li>Balb/c mice can be used as a model animal to analyze the gene expression profile in response to known food allergens treatment </li></ul>

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