The role of estrogen within the Neuroendocrine system and immune system

1. The role of estrogen within theNeuroendocrine system and immune system Monica Delgado Bio 520 Winter 2009

2. Neuroendocrine System and Immune System:Hypothalamic-Pituitary-Adrenal Axis www.nature.com/.../n6/fig_tab/nm0606-612_F1.html

3. Estrogen • Estrogens are a group of steroid compounds • Like all steroid hormones, estrogens readily diffuse across the cell membrane; inside the cell, where they interact with estrogen receptors (ERs) • Produced mainly by developing follicles in the ovaries • Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) stimulate the production of estrogen in the ovaries • Present in both men and women • Usually present higher in women • Estrogen receptor (ER) are activated by 17-β-estradiol (estrogen) • Two types of ERs exist: ER (intracellular receptor) and the estrogen G protein coupled receptor (GPR30) Barnard, A., et al Neuroimmunomodulation (2008)

4. Background: Estrogen and Autoimmunity • Estrogens have a degenerative effect on the primary lymphoid organs, affecting the development, maturation and function of the immune system • Nearly 79% of autoimmune disease patients in the USA are women • ERs are present on bone marrow (BM) stromal cells and resident multipotential stem cells • Today’s focus looks at the one of the primary target s of estrogen; the thymus (which undergoes phenotypic alterations when exposed to estrogen) • However, normal thymic development has also been shown to be dependent on the estradiol/ERα signaling pathway • Estrogen treatment has been shown to activate phagocytic activity and enhance Fc-γR expression on splenic-macrophages • Estradiol drastically reduces the lineage-negative Flt3+ Sca-1+ cKit+ population in the BM (a population that contains thymic homing progenators) • Lin- Sca-1+ cKit+ (LSK) cells are the principal circulating progenitors with T lineage potential Barnard, A., et al Neuroimmunomodulation (2008) Kletcha, A,, et al Journal of Endocrinology (2006)

5. Estrogen Receptor Expression in the BM and Thymus During Development Barnard, A., et al Neuroimmunomodulation (2008)

6. Thymus Structure and Function • In adults, T-cell precursors enter the thymus at the cortico-medullary junction • Uncommited progenators include CD4- CD8- double negative 1 (DN1) cells, in region 2 cells differentiate into the DN2 stage and undergo a proliferative clonal expansion (loss of NK cell potential) • T-cell lineage commitment and the onset of T-cell receptor β-chain rearrangement occurs in DN3 stage (region 3) • Transition from DN to CD4+ CD8+ DP occurs in region 4 • DP cells then migrate back through the cortex and into the medulla Nature Reviews Immunology 4, 278-289 (April 2004)

7. Hypothesis • High levels of estrogens contribute to the development of autoimmunity as a result of degenerative effects on the primary lymphoid organs

8. Estrogen Induces Thymic Atrophy by Eliminating Early ThymicProgenators and Inhibiting Proliferation of β-Selected ThymocytesZoller and Kersh, 2006 • Significant thymic atrophy began 48h after infection of estradiol • (A) Thymus cellularity declines over the period of 6 days where it reached a minimun of 19% (compared to control) • (B) Preferential reduction of DP thymocytes due to estrodiol

9. Estrogen Induces Thymic Atrophy by Eliminating Early ThymicProgenators and Inhibiting Proliferation of β-Selected ThymocytesZoller and Kersh, 2006 • Age-matched pairs of 6-12wk old male C57BL/6 mice (strain used to show Th1 responses) were injected daily with vehicle only or 17-β-estradiol for 1-6 days • (A) While total BM cellularity was not affected, after 24h there was a greater than 60% reduction in the percentage of Flt3+ LSK cells in estradiol treated mice • (B) Further reduction seen at 48h with decrease in lineage-negative c-Kit+ cells, LSK cells, and Flt3+ LSK cells • Loss of cells in the BM is not likely to contribute significantly to the depletion of DP thymocytes CD117 also called C-kit is a cytokine receptor found on Hematopoietic stem cells % of total bone marrow % of Lin-, c-Kit cells % of bone marrow LSK cells

10. Regulatory T Cells Mediate Maternal Tolerance to the FetusAluvihare, Kallikourdis, and Betz.Nature Immunology (2004) • Pregnancy constitutes a major challenge to the immune system • Although localized mechanisms contribute to fetal evasion from immune attack, maternal alloreactive lymphocytes persist • In addition to their function in suppressing autoimmune responses, maternal regulatory T cells suppress aggressive allogeneic response directed at the fetus • Positive effects of pregnancy (counter hypothesis) • (A) Regulatory T cells prevent rejection of the fetal allograft • In an allogeneic context, regulatory T cells are necessary for the prevention of a maternal immune response against the fetus Filled circles = females fertilized by C57BL/6 males (Th1 response) Open circles= females fertilzed by BALB/c males (Th2 response) Filled boxes= non pregnant females Allogeneic = sourced from a genetically non-identical member of the same species Syngeneic = Genetically identical or closely related, immunologically compatible

11. GPR30 Contributes to Estrogen-Induced Thymic AtrophyWang et al, Molecular Immunology 2008 • Exposure to pregnancy levels of E2 reduced thymic size and cellularity (A) • Thymic atrophy was attenuated in AERKO mice but not obliterated (B) • E2 induced thymic atrophy was fractionally seen in both ER-α and GPR30-but not in ERβ-deficient mice (B) • Suggest that ERα and GPR30, but not Erβ, played partial roles in E2-induced thymic atrophy • Note: Erα- and Erβ-knockout (AERKO and BERKO) mice Critique: Column B thymic size irregularities were not addressed

12. Estrogen Receptor Specificity for the Estrogen in Ovariectomized MiceLindberg, M. K., et alJournal of Endocrinology (2002) 100 – Dependent effect WT – DERKO / WT X 100 Note: DERKO = mice with inactivated ERα and ERβ The estrogenic response to thymic atrophy were absent or largely reduced in DERKO mice

13. A Potential Role for Estrogen in Experimental Autoimmune EncephalomyelitisOffner, H., and Polanczyk, M. Ann. N.Y. Acad. Sci (2006) • E2 Treatment inhibits TNF-α producing inflammatory cells in CNS of mice with Experimental autoimmune encephalomyelitis (EAE) • Myelin oligodendrocyte glycoprotein (MOG)-35-55-stimulated CNS mononuclear cells from E2-treated C57BL/6J mice showed a large reduction in intracellular staining TNF-α Critique: Increase in T cell was not really addressed

14. Estrogen Regulation of Nitric Oxide and Inducible Nitric Oxide Synthase (iNOS) in Immune Cells: Implications for Immunity, Autoimmune Diseases, and ApoptosisKarpuzoglu, E., Ahmed, S. A. Nitric Oxide (2006) • Estrogen treatment increases nitric oxide levels in the supernatants from Con-A-stimulated splenocytes when compared to that from placebo treated mice • Blocking with Con-A + CTLA-41g fusion protein leads to a decrease in nitric oxide levels • Previous studies by Karpuzoglu’s group suggest that estrogen-induced up-regulation of iNOS/nitric oxide in activated splenocytes is likely to be mediated through IFN-γ Note: Con-A = T cell mitogen CTLA4 (Cytotoxic T-Lymphocyte Antigen 4)

15. Hypothesis stated that: High levels of estrogens contribute to the development of autoimmunity as a result of degenerative effects on the primary lymphoid organs

16. Take Home Message • Elevated estradiol induces loss of T lineage progenators in the BM (Zoller and Kersh 2006) • Regulatory T cells are required for the maternal immune system to tolerate the fetal allograft (Aluvihare, Kallikourdis, and Betz 2004) • Wang et al showed that E2-induced thymic atrophy involves both ERα and GPR30, but not ERβ • Karpuzoglu’s group showed that estrogen treatment in mice markedly upregulates the levels of iNOS mRNA through IFN-γ; Also estrogens are not only involved in regulation of normal immune responses, but also are implicated in many autoimmune diseases • The exact pathway of estrogen-induced thymic degeneration is yet to be elucidated/established

17. Future Studies • Further research should focus on the different and frequently opposite effects exerted by physiologic and pharmacologic doses of estrogens (dose-related effects) • Further studies must be directed to the inflammatory mediators (i.e., cytokines) that seem to alter the peripheral metabolism of sex hormones and complicate the effects of sex hormones on susceptibility to autoimmunity • There are no available data on cytokine measurements on non-immune hyperthyroidsm

18. Thank you Questions?