The Role of Aryl Hydrocarbon Receptor (AhR) in the Immune System of Aging Mice

Duy PhamDuy PhamDr. Nancy I. KerkvlietDr. Nancy I. Kerkvliet

Department of Environmental and Department of Environmental and Molecular ToxicologyMolecular Toxicology

Oregon State University Oregon State University HHMI Summer 2007HHMI 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 SignalingThe 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

AhR SignalingThe case of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD)

1. Prevent rejection of tissue and organ transplants

2. Reduce overactive immune responses associated with allergy and asthma

3. Treat autoimmune diseases

Immune System

Clinical applications of chemically induced immune suppression

Natural Role of AhR not yet knownHypothesis

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 parameterApproach

1. Activate AhR KO and WT spleen cells with antigenic stimulation

► Lipopolysaccharide (LPS) + Anti-CD3

►Anti-CD3 + Anti-CD28 + IL-4

CYTOKINESCYTOKINES

T cell2T cell2T cell2B cellB cellB cellT cell1T cell1T cell1

T cell2T cell2

T cell2

B cell

B cell

B cell

T cell1

T cell1

T cell1

Culture 24,48 & 72hrs

IL-4

Examine Immune parameterApproach

2. Cytokines Measurement

T cell2

T cell2

T cell2

B cell

B cell

B cell

T cell1

T cell1

T cell1

Cytokines=

ELISA Antibody-Sandwich

ELISA

Examine Immune ParametersMethods & Instruments

Spectra Max250 Spectrophotometer ALS1155

Examine Immune ParameterExperimental Design-Study 1 (female) & 2 (male)

Young (3-5 months of age) &Old (20-23 months of age)

Cytokines

Study 1- female

LPS @ 10ug/ml + anti-CD3 @ 250ng/ml

IL-2, IL-6, IL-12, IFNγ

Study 2-male

1. LPS @ 10ug/ml + anti-CD3 @ 250ng/ml

2. Anti-CD3 @ 250ng/ml + anti-CD28 @ 1ug/ml + IL-4 @ 50 ng/ml

1. IL-2, IL-6, IL-12, 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-6IFN? - Old Mice

* *

0.0

0.2

0.4

0.6

0.8

1.0

1.2

24 48 72

Hours of Culture

IFN

? (

Ab

sorb

ance

)

Old WT

Old KO

IL-6 Old mice

* * *

0

200

400

600

800

1000

1200

24 48 72

hours

pg

/ml

WT

KO

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

*

0

100

200

300

400

500

600

700

24 48 72

hours

pg/ml

WT

KO

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 NewsGood 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 arthritisRheumatoid 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

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute

Dr. Nancy KerkvlietDr. Nancy Kerkvliet

Dr. Kevin AhernDr. Kevin Ahern

Kerkvliet LabKerkvliet Lab

William Vorachek William Vorachek

Linda SteppanLinda Steppan