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

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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

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Immune System

• Recognizes and responds to antigenic substances
  associated with microbial pathogens and
  parasites that can cause disease or allergic
  reactions

Immune response
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Aryl Hydrocarbon Receptor (AhR)

• Cellular protein
• Responses to variety of environmental agents
• ? polycyclic aromatic hydrocarbons
• ? polychlorinated dioxin compound (TCDD)
• ? Many dietary substances
• Alters immune system upon
• ligand binding

bHLH structural motif of the AhR protein
http//en.wikipedia.org/wiki/Basic-helix-loop-hel
ix
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AhR SignalingThe case of 2,3,7,8-tetrachlorodibe
nzo-dioxin (TCDD)
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AhR SignalingThe case of 2,3,7,8-tetrachlorodibe
nzo-dioxin (TCDD)

• TCDD-induced AhR activation
• increases the expression of
• Cytochrome P4501A1 (Cyp1A1)
• Cyp1A2
• Cyp1B1
• IL-2
• and many more

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Immune System
Clinical applications of chemically induced
immune suppression

• Prevent rejection of tissue and organ transplants
• Reduce overactive immune responses associated
  with allergy and asthma
• Treat autoimmune diseases

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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

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Examine Immune parameterApproach

• 1. Activate AhR KO and WT spleen cells with
  antigenic stimulation
• ? Lipopolysaccharide (LPS) Anti-CD3
• ?Anti-CD3 Anti-CD28 IL-4

Culture 24,48 72hrs
CYTOKINES
IL-4
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Examine Immune parameterApproach

• 2. Cytokines Measurement

Cytokines
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Examine Immune ParametersMethods Instruments

• ELISA
• Antibody-Sandwich ELISA

• Spectra Max250
• Spectrophotometer
• ALS1155

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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 _at_ 10ug/ml anti-CD3 _at_ 250ng/ml IL-2, IL-6, IL-12, IFN?
Study 2-male 1. LPS _at_ 10ug/ml anti-CD3 _at_ 250ng/ml 2. Anti-CD3 _at_ 250ng/ml anti-CD28 _at_ 1ug/ml IL-4 _at_ 50 ng/ml 1. IL-2, IL-6, IL-12, IFN? 2. IL-5, IL-10
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Cytokines Measurement Study 2 Male - Results
IFN ? Il-6 levels in culture supernatants from
spleen cells stimulated with LPS and anti-CD3
IFN ?
IL-6
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Cytokines Measurement Study 2 Male Results
cont
IL-5 levels in culture supernatants from spleen
cells stimulated with anti-CD3, anti-C28 IL-4
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Summary
Significant changes of cytokines level in old AhR KO relative to old AhR WT
IL-2 -
IL-5 ? _at_ 72hrs
IL-6 ? _at_ 24, 48, 72hrs
IL-10 -
IL-12 ? _at_ 24hrs
IFN ? ? _at_ 48 and 72hrs
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Good News vs. Bad News

• Increase in pro-inflammatory cytokines in old
  AhR KO (IL6, IL12, IFN ? )
• More inflammatory response
• More susceptible to inflammatory diseases

Rheumatoid arthritis
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Good News vs. Bad News

• Increase in IL-5 production in old AhR KO
• Increase antibody secretion
• More allergic response

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Future Work

• ?Challenge mice with LPS in vivo
• ? Measure the immune response
• Liver damage
• Inflammation
• many more

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Acknowledgements

• Howard Hughes Medical Institute
• Dr. Nancy Kerkvliet
• Dr. Kevin Ahern
• Kerkvliet Lab
• William Vorachek
• Linda Steppan