Transcript Document 634229

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
Atopy
Environmental
Factors
AGE
(Cytokine
Dysregulation)
Respiratory
Viruses
Allergens
Asthma
Phenotype
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
•episodic, reversible airway obstruction
•chronic airway inflammation
•airway hyperresponsiveness to methacholine
•airway wall remodeling
Normal Airways
Model developed in collaboration
with W.L. Castleman (U. of Florida)
Genetic Factor
Rat Strain
Environmental
Factor
AGE
Sendai Virus
Asthmatic
Phenotype
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
Acute Phase
Chronic Phase
BN and F344:
Bronchiolitis
BN: YES
F344: NO
Dysregulation of
innate immune
response?
Sendai
Virus
Infection
d2
Viral replication
d6-8
Virus shedding
Abnormal
resolution of
acute
inflammatory
changes?
d10-14
Resolution of
acute
inflammatory
changes
> Week 4
Development of asthma-like phenotype
•episodic, reversible airway obstruction
•chronic airway inflammation
•airway hyperresponsiveness
•airway wall remodeling
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.
Post-bronchiolitis Asthma-like Phenotype
IFN-g
Brown Norway (BN)
aerosolized
parainfluenza type 1
(Sendai) virus
weanling rats
(3-4 weeks old)
high IgE producer
“atopic” (Th2)
Fischer 344 (F344)
low IgE producer
“nonatopic” (Th1)
8-10 weeks postinfection
Normal Airways
Sorkness et al. 1999. AJRCCM 160:705
Normal Airways
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?
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
Are the Immune Responses of SS and FHH
Rats Th1-biased Like Those of F344 Rats?
Screening Assay for Th1- and Th2-type
Cytokine Production by Spleen Cells
Con A
1
2
3
4
24 h
5
6
7
A
B
8
C
9
D
E
10
F
Measure Cytokine Levels
in Supernatant Fluids
IFN-g Production by Con A-stimulated Spleen Cells
8
IFN-g (ng/ml)
7
6
5
4
3
2
1
0
BN
SS
FHH
IL-4 Production by Con A-stimulated Spleen Cells
16
14
IL-4 (pg/ml)
12
10
8
6
4
2
0
BN
SS
FHH
IFN-g/IL-4 Ratio for Con A-stimulated Spleen Cells
1000
900
IFN-g/ IL-4
800
700
600
500
400
300
200
100
0
BN
SS
FHH
IL-13 (pg/ml)
IL-13 Production by Con A-stimulated Spleen Cells
65
60
55
50
45
40
35
30
25
20
15
10
5
0
BN
SS
FHH
IFN-g/IL-13 Ratio for Con A-stimulated Spleen Cells
700
IFN-g/ IL-13
600
500
400
300
200
100
0
BN
SS
FHH
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
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.
Screening of Consomic Rat Strains
• SS.BN13
• SS.BN16
• SS.BN18
• SS.BN9
• SS.BN20
Screening Assay for Th1- and Th2-type
Cytokine Production by Spleen Cells
Con A
1
2
3
4
24 h
5
6
7
A
B
8
C
9
D
E
10
F
Measure Cytokine Levels
in Supernatant Fluids
IFN-g (ng/ml)
IFN-g Production by Con A-stimulated Spleen Cells
10
1
BN
SS
13
16
18
9
SS.BN
20
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)] .
Chr. 20: Major Histocompatibility Complex
• SS.BN20
- Histocompatible with BN?
- Histoincompatible with SS?
Two-way Mixed Lymphocyte Cultures
Strain A
72 h
1
2
3
4
5
6
7
A
B
8
C
9
D
E
10
F
Strain B
Measure IL-2 and IFN-g Levels
in Supernatant Fluids
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
15
20
25
30
35
IL-2 (pg/ml)
40
45
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
16 18 20 22 24 26 28 30 32 34
IL-2 (pg/ml)
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
0
100
200
300
IFN-g (pg/ml)
400
500
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
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
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)
Funded by:
NIH/NIAID Asthma and Allergic Disease Research
Center Grant AI050500