1. 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

2. Immune System Recognizes and responds to antigenic substances associated with microbial pathogens and parasites that can cause disease or allergic reactions Immune response

3. 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

4. AhR Signaling The case of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD)

5.  TCDD-induced AhR activation  increases the expression of:  Cytochrome P4501A1 (Cyp1A1)  Cyp1A2  Cyp1B1  IL-2  and many more AhR Signaling The case of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD)

7. 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

8. 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

9. 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 CYTOKINES Culture 24,48 & 72hrs IL-4

10. Examine Immune parameter Approach 2. Cytokines Measurement Cytokines=

11.  ELISA  Antibody-Sandwich ELISA Examine Immune Parameters Methods & Instruments  Spectra Max250  Spectrophotometer  ALS1155

12. Examine Immune Parameter Experimental 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

14. 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 * * 0.0 0.2 0.4 0.6 0.8 1.0 1.2 244872 Hours of Culture IFN? (Absorbance) Old WT Old KO IL-6 Old mice * * * 0 200 400 600 800 1000 1200 244872 hours pg/ml WT KO

16. Cytokines Measurement Study # 2 Male – Results cont’ IL-5 levels in culture supernatants from spleen cells stimulated with anti-CD3, anti-C28 & IL-4

17. 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

18. 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

19. Good News vs. Bad News  Increase in IL-5 production in old AhR KO  Increase antibody secretion  More allergic response

21. Future Work ►Challenge mice with LPS in vivo ► Measure the immune response Liver damage Inflammation many more

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