Transcript Aging Study in mice

The Role of Aryl Hydrocarbon Receptor (AhR) in the
Immune System of Aging Mice
Duy Pham
Dr. Nancy I. Kerkvliet
Department of Environmental and
Molecular Toxicology
Oregon State University
HHMI Summer 2007
Immune System
Recognizes and
responds to antigenic
substances associated
with microbial
pathogens and parasites
that can cause disease or
allergic reactions
Immune response
Aryl Hydrocarbon Receptor (AhR)
1.
Cellular protein
2.
Responses to variety of
environmental agents:
► polycyclic aromatic
hydrocarbons
► polychlorinated dioxin
compound (TCDD)
► Many dietary substances
3.
Alters immune system upon
ligand binding
bHLH structural motif of the AhR protein
http://en.wikipedia.org/wiki/Basic-helix-loop-helix
AhR Signaling
The case of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD)
AhR Signaling
The case of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD)
 TCDD-induced AhR activation
 increases the expression of:
 Cytochrome P4501A1
(Cyp1A1)
 Cyp1A2
 Cyp1B1
 IL-2
 and many more
Immune System
Clinical applications of chemically induced immune suppression
1.
2.
3.
Prevent rejection of tissue and organ transplants
Reduce overactive immune responses associated
with allergy and asthma
Treat autoimmune diseases
Natural Role of AhR not yet known
Hypothesis
The AhR responds to endogenous or exogenous (eg.
dietary) ligands that leads to down-regulation of the
immunity and inflammatory response over a lifetime.
Therefore, older AhR KO mice would be expected to show
enhanced immune response and inflammatory response
compared to AhR WT mice
Examine Immune parameter
Approach
1. Activate AhR KO and WT spleen cells with antigenic stimulation
► Lipopolysaccharide (LPS) + Anti-CD3
►Anti-CD3 + Anti-CD28 + IL-4
Bcell
Culture
24,48 &
72hrs
Bcell
T1 cell
Bcell
T1 cell
T1 cell
IL-4
T2 cell
T2 cell
T2 cell
CYTOKINES
Examine Immune parameter
Approach
2. Cytokines Measurement
Bcell
Bcell
T1 cell
Bcell
T1 cell
T1 cell
T2 cell
T2 cell
T2 cell
Cytokines=
Examine Immune Parameters
Methods & Instruments
 ELISA

Antibody-Sandwich
ELISA
 Spectra Max250

Spectrophotometer

ALS1155
Examine Immune Parameter
Experimental Design-Study 1 (female) & 2 (male)
Young (3-5 months of age) &
Old (20-23 months of age)
Study 1- LPS @ 10ug/ml + anti-CD3 @
female 250ng/ml
Study 2male
1. LPS @ 10ug/ml + anti-CD3 @
250ng/ml
2. Anti-CD3 @ 250ng/ml + anti-CD28
@ 1ug/ml + IL-4 @ 50 ng/ml
Cytokines
IL-2, IL-6, IL-12,
IFNγ
1. IL-2, IL-6, IL12, IFNγ
2. IL-5, IL-10
Cytokines Measurement
Study # 2 Male - Results
IFN γ & Il-6 levels in culture supernatants from spleen cells
stimulated with LPS and anti-CD3
IFN γ
IL-6
IFN? - Old Mice
IL-6 Old mice
1.2
1200
*
*
1000
*
*
*
800
0.8
pg/ml
IFN? (Absorbance)
1.0
0.6
600
400
0.4
0.2
Old WT
200
WT
KO
Old KO
0
0.0
24
48
Hours of Culture
72
24
48
hours
72
Cytokines Measurement
Study # 2 Male – Results cont’
IL-5 levels in culture supernatants from spleen cells stimulated
with anti-CD3, anti-C28 & IL-4
IL5-old mice
700
*
600
pg/ml
500
400
300
200
WT
100
KO
0
24
48
hours
72
Summary
Significant changes of cytokines level in
old AhR KO relative to old AhR WT
IL-2
-
IL-5
↑ @ 72hrs
IL-6
↑ @ 24, 48, 72hrs
IL-10
-
IL-12
↑ @ 24hrs
IFN γ
↑ @ 48 and 72hrs
Good News vs. Bad News

Increase in pro-inflammatory
cytokines in old AhR KO (IL6, IL12,
IFN γ )


More inflammatory response
More susceptible to inflammatory
diseases

Rheumatoid arthritis
Good News vs. Bad News

Increase in IL-5 production
in old AhR KO
 Increase antibody secretion
 More allergic response
Future Work
►Challenge mice with LPS in vivo
► Measure the immune response
Liver damage
Inflammation
many more
Acknowledgements
Howard Hughes Medical Institute
Dr. Nancy Kerkvliet
Dr. Kevin Ahern
Kerkvliet Lab
William Vorachek
Linda Steppan