The Role of Aryl Hydrocarbon Receptor AhR in the Immune System

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 Alters immune system upon ligand binding bHLH structural motif of the AhR protein http://en.wikipedia.org/wiki/Basic-helix-loop-helix 3. 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 Bcell T cell 1 T cell 1 T cell 1 CYTOKINES IL-4 T2 cell T2 cell T2 cell Examine Immune parameter Approach 2. Cytokines Measurement Bc ll e Tc 1 ell Bc ll e Bc ll e Tc 1 ell Tc 1 ell T cell 2 T cell 2 T cell 2 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) Cytokines Study 1- LPS @ 10ug/ml + anti-CD3 @ female 250ng/ml 1. LPS @ 10ug/ml + anti-CD3 @ 250ng/ml 2. Anti-CD3 @ 250ng/ml + anti-CD28 @ 1ug/ml + IL-4 @ 50 ng/ml IL-2, IL-6, IL-12, IFNγ Study 2male 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 IFN? - Old Mice 1.2 1200 IL-6 IL-6 Old mice 1.0 IFN? (Absorbance) * * pg/ml 1000 * 0.8 800 0.6 600 * * 0.4 400 0.2 Old WT Old KO 200 WT KO 0.0 24 48 Hours of Culture 72 0 24 48 72 hours 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 * 600 500 pg/ml 400 300 200 100 0 24 48 72 WT KO hours Summary Significant changes of cytokines level in old AhR KO relative to old AhR WT IL-2 - IL-5 IL-6 IL-10 ↑ @ 72hrs ↑ @ 24, 48, 72hrs - IL-12 IFN γ ↑ @ 24hrs ↑ @ 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