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Asthmatic Phenotypes in the PhysGen Consomic Rats UW-MCW Collaboration: Project Update

Asthmatic Phenotypes in the PhysGen Consomic Rats UW-MCW Collaboration: Project Update. L.A. Rosenthal, R.L. Sorkness, and R.F. Lemanske, Jr. Morris Institute for Respiratory Research. Genetic Factors. Environmental Factors. Atopy (Cytokine Dysregulation). AGE. Respiratory Viruses

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Asthmatic Phenotypes in the PhysGen Consomic Rats UW-MCW Collaboration: Project Update

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  1. Asthmatic Phenotypes in the PhysGen Consomic Rats UW-MCW Collaboration: Project Update L.A. Rosenthal, R.L. Sorkness, and R.F. Lemanske, Jr. Morris Institute for Respiratory Research

  2. Genetic Factors Environmental Factors Atopy (Cytokine Dysregulation) AGE Respiratory Viruses Allergens Asthma Phenotype

  3. Post-bronchiolitis Asthma-like Phenotype aerosolized parainfluenza type 1 (Sendai) virus weanling rats (3-4 weeks old) Brown Norway (BN) Fischer 344 (F344) high IgE producer “atopic” (Th2) low IgE producer “nonatopic” (Th1) 8-10 weeks postinfection Abnormal Airways Normal Airways • episodic, reversible airway obstruction • chronic airway inflammation • airway hyperresponsiveness to methacholine • airway wall remodeling Model developed in collaboration with W.L. Castleman (U. of Florida)

  4. Genetic Factor Environmental Factor AGE Rat Strain Sendai Virus Asthmatic Phenotype

  5. Development of the Post-bronchiolitis Asthma-like Phenotype in Brown Norway Rats: Host Response(Genetic Factor) to Sendai Virus(Environmental Factor) During Early Life (Developmental Factor) Drives Process

  6. Acute Phase Chronic Phase BN and F344: Bronchiolitis BN: YES F344: NO Dysregulation of innate immune response? d6-8 >Week 4 Sendai Virus Infection Virus shedding Abnormal resolution of acute inflammatory changes? • Development of asthma-like phenotype • episodic, reversible airway obstruction • chronic airway inflammation • airway hyperresponsiveness • airway wall remodeling d2 Viral replication d10-14 Resolution of acute inflammatory changes

  7. Development of Asthma-like Phenotype: Importance of IFN-g During Acute Phase • IFN-g in bronchoalveolar lavage fluid during acute Sendai virus infection in weanling rats: F344 > BN • Aerosolized IFN-g prevents the development of post-bronchiolitis chronic airway dysfunction in BN rats.

  8. Post-bronchiolitis Asthma-like Phenotype aerosolized parainfluenza type 1 (Sendai) virus IFN-g weanling rats (3-4 weeks old) Brown Norway (BN) Fischer 344 (F344) high IgE producer “atopic” (Th2) low IgE producer “nonatopic” (Th1) 8-10 weeks postinfection Normal Airways Normal Airways Sorkness et al. 1999. AJRCCM 160:705

  9. PhysGen Consomic Rats • Parental strains for consomic rats: • Dahl salt sensitive (SS; hypertension model) • Fawn hooded hypertensive (FHH; hypertension and end-stage renal disease model) • Brown Norway (BN; control strain: normotensive) • Useful for studying the role of genetic factors in susceptibility/resistance to the development of the post-bronchiolitis asthma-like phenotype?

  10. Balance Between Th1- and Th2-type Cytokines Th2 Th1 Th1 Th2 F344 BN SS? FHH? “non-atopic” “atopic” Th1-type: IFN-g, IL-12; Th2-type: IL-4, IL-5, IL-13

  11. Are the Immune Responses of SS and FHH Rats Th1-biased Like Those of F344 Rats?

  12. 1 2 3 4 5 6 7 A B 8 C 9 D E 0 1 F Screening Assay for Th1- and Th2-type Cytokine Production by Spleen Cells Con A 24 h Measure Cytokine Levels in Supernatant Fluids

  13. IFN-g Production by Con A-stimulated Spleen Cells

  14. IL-4 Production by Con A-stimulated Spleen Cells

  15. IFN-g/IL-4 Ratio for Con A-stimulated Spleen Cells

  16. IL-13 Production by Con A-stimulated Spleen Cells

  17. IFN-g/IL-13 Ratio for Con A-stimulated Spleen Cells

  18. Balance Between Th1- and Th2-type Cytokines Th2 Th1 Th1 Th2 F344 BN SS FHH “non-atopic” “atopic” Th1-type: IFN-g, IL-12; Th2-type: IL-4, IL-5, IL-13

  19. Are SS Rats, Like F344 Rats, Resistant to the Development of Post-bronchiolitis Airway Dysfunction?

  20. Post-bronchiolitis Airway Dysfunction in BN, but not SS, Rats

  21. Therefore, in vitro cytokine data and in vivo physiology data suggest that the PhysGen consomic rat strains will be useful tools for studying the role of genetic factors in susceptibility/resistance to the development of the post-bronchiolitis asthma-like phenotype.

  22. Screening of Consomic Rat Strains • SS.BN13 • SS.BN16 • SS.BN18 • SS.BN9 • SS.BN20

  23. 1 2 3 4 5 6 7 A B 8 C 9 D E 0 1 F Screening Assay for Th1- and Th2-type Cytokine Production by Spleen Cells Con A 24 h Measure Cytokine Levels in Supernatant Fluids

  24. IFN-g Production by Con A-stimulated Spleen Cells

  25. Th1 and Th2 Cytokine Production • No strain differences were observed among SS and these five SS.BN strains with regard to Con A-stimulated IFN-g production [or IL-4 or IL-13 production (not shown)] .

  26. Chr. 20: Major Histocompatibility Complex • SS.BN20 • Histocompatible with BN? • Histoincompatiblewith SS?

  27. 1 2 3 4 5 6 7 A B 8 C 9 D E 0 1 F Two-way Mixed Lymphocyte Cultures 72 h Strain A Strain B Measure IL-2 and IFN-g Levels in Supernatant Fluids

  28. IL-2 Production in 2-way Mixed Lymphocyte Cultures: BN and SS.BN20 Splenocytes are Histocompatible BN+BN F344 + F344 SS.BN20+SS.BN20 BN+ F344 F344 +SS.BN20 BN+SS.BN20

  29. IL-2 Production in 2-way Mixed Lymphocyte Cultures: SS and SS.BN20 Splenocytes are Histoincompatible BN+BN SS + SS SS.BN20+SS.BN20 BN+ SS SS +SS.BN20 BN+SS.BN20

  30. IFN-g Production in 2-way Mixed Lymphocyte Cultures: SS and SS.BN20 Splenocytes are Histoincompatible BN+BN SS + SS SS.BN20+SS.BN20 BN+ SS SS +SS.BN20 BN+SS.BN20

  31. Potential Uses of the Histocompatible SS.BN20 Strain for Studies in BN Rats • in vitro • cell mixing studies • in vivo • adoptive cell transfer studies • organ transplant studies

  32. PhysGen Consomic Rat Development Timeline • SS.BN12 • FHH.BN1 (IL-4Ra) • SS.BN6 (Immunoglobulin) • SS.BN7 (IFN-g) • SS.BN8 • Fischer CDF(F-344)CrlBR • BN/SsNHsd/Mcw http://pga.mcw.edu

  33. Future Directions • Continue screening spleens from adult consomic rats. • PhysGen consomic rat strains to be distributed • through Charles River Laboratories. • Availability of weanling consomic rats will significantly increase options with regard to screening. • Level of characterization of weanlings from each consomic strain? Resources that will inform these decisions include: • Rat Genome Database; Rat Genome Sequencing Project • Virtual Comparative Maps • MCW-TIGR microarray expression profiling of rat • consomic panels (lung)

  34. Funded by: NIH/NIAID Asthma and Allergic Disease Research Center Grant AI050500

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