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

AsthmaPhenotype Genetic Factors Atopy (Cytokine Dysregulation) Environmental Factors Respiratory Viruses AllergensAGE

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

AsthmaticPhenotype Genetic Factor Rat Strain Environmental Factor Sendai Virus AGE

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

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

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

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

PhysGen Consomic Rats Parental strains for 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?Useful for studying the role of genetic factors in susceptibility/resistance to the development of the post-bronchiolitis asthma-like phenotype?

Th1 Th2Th1 Th2 BNF344 SS? FHH? Balance Between Th1- and Th2-type Cytokines “atopic”“non-atopic” Th1-type: IFN- , IL-12; Th2-type: IL-4, IL-5, IL-13

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

A B C D E F Con A 24 h Measure Cytokine Levels in Supernatant Fluids Screening Assay for Th1- and Th2-type Cytokine Production by Spleen Cells

IFN-  Production by Con A-stimulated Spleen Cells

IL-4 Production by Con A-stimulated Spleen Cells

IFN-  /IL-4 Ratio for Con A-stimulated Spleen Cells

IL-13 Production by Con A-stimulated Spleen Cells

IFN-  /IL-13 Ratio for Con A-stimulated Spleen Cells

Th1 Th2Th1 Th2 BNF344 SS FHH Balance Between Th1- and Th2-type Cytokines “atopic”“non-atopic” Th1-type: IFN- , IL-12; Th2-type: IL-4, IL-5, IL-13

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

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

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.

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

A B C D E F Con A 24 h Measure Cytokine Levels in Supernatant Fluids Screening Assay for Th1- and Th2-type Cytokine Production by Spleen Cells

IFN-  Production by Con A-stimulated Spleen Cells

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

SS.BN20 SS.BN20 - Histocompatible with BN? - Histoincompatible with SS? Chr. 20: Major Histocompatibility Complex

A B C D E F h Measure IL-2 and IFN-  Levels in Supernatant Fluids Two-way Mixed Lymphocyte Cultures Strain A Strain B

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

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

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

PhysGen Consomic Rat Development Timeline SS.BN12 SS.BN12 FHH.BN1 (IL-4R  ) FHH.BN1 (IL-4R  ) SS.BN6 (Immunoglobulin) SS.BN6 (Immunoglobulin) SS.BN7 (IFN-  ) SS.BN7 (IFN-  ) SS.BN8 SS.BN8 Fischer CDF(F-344)CrlBR Fischer CDF(F-344)CrlBR BN/SsNHsd/Mcw BN/SsNHsd/Mcw

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

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