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Regulation of Stress Induced Cytokine Response by Glucocorticoids

Regulation of Stress Induced Cytokine Response by Glucocorticoids. Janine Gilkes Biology 520 2/11/09. HPA axis Activation Induced by Stressors. Stress illicits an immune response resulting in bi-directional signaling between the immune and central nervous system (CNS)

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Regulation of Stress Induced Cytokine Response by Glucocorticoids

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  1. Regulation of Stress Induced Cytokine Response by Glucocorticoids Janine Gilkes Biology 520 2/11/09

  2. HPA axis Activation Induced by Stressors • Stress illicits an immune response resulting in bi-directional signaling between the immune and central nervous system (CNS) • Cytokines from the periphery can initiate the cycle by crossing the blood–brain barrier • Immune signaling of the CNS activates the hypothalamic-pituitary-adrenal (HPA) axis • The HPA axis is responsible for the initiation of glucocorticoid (GC) stress responses in all vertebrate animals. • The principal end products of the HPA axis are glucocorticoid hormones http://2to3yearsplan.com/stress-cartoon-work.jpg Jankord et al., 2008 and sternbergh et al 2001

  3. Effects of Chronic Stress on HPA axis • Temporal prolongation of stress exposure (chronic stress) causes marked enhancement in Basal HPA tone as well as stress reactivity. • Excessive stimulation results in baseline glucocorticoid hypersecretion, enhanced CRH and vasopressin synthesis, and down regulation of glucocorticoid receptors (GR) in key feedback regions. • Excessive stimulation may also affect severity of infectious disease through their immunosuppressive effects. Jankord et al., 2008

  4. Glucocorticoids • Immunomodulatory vs. Immunosuppressive and anti-inflammatory • Most notable mechanism of action is the inhibition of cytokine production and action. • Beneficial for short-term survival, but prolonged exposure can lead to serious metabolic, immune, and psychological dysfunction, • Termination of the GC response is controlled by various feedback inhibition mechanisms • Glucocorticoids are lipophilic molecules and so readily pass through the plasma membrane of all cells in the body. Padgett et al., 2003 and Almawi et al., 1999

  5. HPA axis and Glucocorticoid Secretion http://speakingoffaith.publicradio.org/programs/stress/images/stressresponse.jpg

  6. Glucocorticoids Regulate Cytokine Production • Once a T cell immune response has flared up, glucocorticoids may now modulate its cytokine production pattern. • The differentiation of CD4+ T cells into TH1 lymphocytes drive cellular immunity, while differentiation into TH2 lymphocytes drive humoral immunity • This differentiation depends on the type of antigen encountered and the type of cytokines produced during antigen presentation. • Glucocorticoids block IL-12 secretion by monocytes and dendritic cells, but promote TH2 development by enhancing IL-10 secretion by macrophages Franchimont et al., 2004

  7. Cellular arm Antigen mediated immune response Th1 cells primarily secrete interferon IFNγ, IL-2, and TNF-α, which promote cell mediated immunity IL-12 (produced by APCs) is the major inducer of Th1 differentiation and, hence, cellular immunity. by producing IFNγ, Th1 cells activate CD8+ T cells and macrophages IL-12 and IFNγ inhibit Th2 Humoral Arm - Primarily functions in the production of antibodies. Th2 cells secrete primarily IL-4, IL-10, and IL-13, which enhance humoral immunity. IL-4 and IL-10 stimulate the differentiation of B cells into antibody secreting B cells, and immunoglobulin switching to IgE. These cytokines also prevent TH1 cytokine production by inhibiting IL-12 Subdivisions of Adaptive Immunity Th2 = Anti-Inflammatory Response Th1 = Pro-Inflammatory Response Almawi et al., 1999 and Tausk et al., 2009

  8. T Cell Differentiation and Glucocorticoid Taret • Antigen-specific activation of T cells ensues upon recognition of processed antigen co-presented with MHC II proteins • Interleukin-12 is critical for Th1 lymphocyte differentiation and secretion of Th1 cytokines such as IFNγ and TNFα • It is the link between humoral and • cellular immunity. • Glucocorticoids prevent IL-12 action by specifically inhibiting (Stat) 4 phosphorylation Franchimont et al., 2004 Almawi et al., 2002

  9. Stimulation of a TH2 System by Glucocorticoids • Why the TH2 preference? • During an immune and inflammatory response, the activation of the stress system, through induction of a TH2 shift may protect the organism from systemic “overshooting” with TH1 pro-inflammatory cytokines. Elenkov et al, 2006 and calcagni 206

  10. The Immune Response Tausk et al., 2009

  11. Hypothesis • Stress induced Glucocorticoids cause a shift of T cell derived cytokine production from TH1 to TH2 patterns.

  12. ELISA Biotinylated TMB http://www.labmaster.fi/images/figs/imTroponin-1-elisa-test-principle.gif

  13. Cytokine Increase During Stressful Situations Figure 1: Levels of INFγ, IL-4 and IL-10 after PHA stimulation in prenatal stress Prenatal stress defined as “major negative life events that occurred during pregnancy” • - N sample= 62; N control = 28 • - PBMCs from blood isolated via centrifugation • Cell suspension stimulated by 5ug/ml PHA for 24h • Cytokine evaluation via 5 parameter regression formula, likened to ELISA • p < 0.05 Entringer et al., 2008 Critique: No X-axis label

  14. Cytokine Increase During Stressful Situations Figure 1: The effect of exam stress on TH1/TH2 cytokine balance - N=15 students - Mitogen culters: PBMC + PHA - Pre exam: 4 weeks - Post exam: 2 days - Cytokine analysis: ELISA - p < 0.007 Critique: No Y-axis label Marshall et al., 1998

  15. Glucocorticoids are Released in Response to Stress Figure 6: Effects of chronic variable stress on Plasma Corticosterone levels - CVS = rotation stress, warm swim, cold exposure and hypoxia - Stress exposure = 2x daily/2 wks - Day 15 = 30 min restraint stress followed by blood sampling at various intervals - Plasma Corticosterone measured by I Kit Critique: X-axis not well defined Result figures punctuated several parts of discussion Furay et al., 2008

  16. Glucocorticoids Inhibit TH1 Cytokines Figure 1: Dexamethasone inhibits IL-12 induced INFγ - Cells used: NK3.3 at [5x106 /ml] - Incubated with IL-12 [20ng/ml] for 10 hrs - Cell culture sediment collected supernatant containing INF gamma measured by ELISA Critique: IL-12 incubation time not specified on X-axis Franchimont et al., 2000

  17. Glucocorticoids Inhibit TH1 Cytokines Fig 1: Glucocorticoids delay early IL-6 and TNFα expression • - Liver grafts at various time points after transplantation • - DEX [2mg/kg] intraperitonally • IL-6 and TNFα mRNA assessed by RT-PCR • Each lane represents 1 of 3 rats at each time point Critique: None Debonera et al., 2003

  18. Glucocorticoids Mechanism of Action Figure 4: Dexamethasone inhibits IL-12 induced phosphorylation of Stat4 - Cells used: T cells - IL-12 [20 ng/ml] - IL-4 [20 ng/ml] - DEX [1x10-7M] ?? - Treatment -> cell lysis -> immunoprecipitation w/ (stat 4/6) -> immunoblotting (anti-phosphotyrosine and anti stat 4/6) Critique: no mention of how long cells were incubated with stat4 Franchimont et al., 2004

  19. Contrasting View of Glucocorticoid Regulation Figure 5: Effects of high-dose DEX on the release of TNFα and IL-10 after burn injury • N = 130 rats - DEX [5mg/kg] • Rats subjected to 35% of body burn injury - Red blood cells harvested by centrifugation • Plasma concentrations of TNFα and IL-10 - p < 0.05 • determined by ELISA Wang et al., 2008

  20. Figure 6: GCs induce apoptosis in Thymocytes Glucocorticoids Act Differentially Figure 4: Increasing production of GC in thymocytes by de novo synthesis - N=3mice 14 wks old - Thymocytes cocultured for 20h with COS-7 cells cotransfected an expression vector for GR and a GC response element (GRE)-driven reporter luciferase gene - Luciferase activity measured via Luciferase Activity Kit - Control: cells received medium only instead of supernatants from different thymocyte cultures ?? - Cells incubated 48 h and stained with PI and annexin V then measured by FACScan for apoptosis Qiao et al., 2008

  21. Hypothalamus Anterior Pituitary CRH Adrenal Cortex CAs ACTH GCs Summary of Findings: Chronological Progression • Antigen presenting cells secrete cytokines which prefer a TH1 expression • Glucocorticoids inhibit TH1 expression largely by inhibiting the effects of IL-12; thereby, causing a preferential shift to TH2 cytokine patterns • Glucocorticoids exhibit differential effects Stress EPI NEP Human = cortisol Rodents = corticosterone

  22. Summary of Findings

  23. Collect peritoneal mast cells from rats. Stimulate with ovalbumin (Lennon et al., 2009) Measure serum titers of IgE OVA-specific antibodies by ELISA(van den Brandt et al., 2007) Incubate with Corticosterone antagonist Test IgE antibody production with CRA Allergen-Specific IgE Assay (Hitachi Chemical Diagnostics Inc.) Proposed Experiment: Glucocorticoids in Allergy Response Would decreasing the activity of Glucocorticoids result in a decrease of IgE production, and consequently a decrease in Mast Cell response during an allergic response?

  24. Take Home Messages • Stress activates the production of adrenally derived glucocorticoids • Glucocorticoids cause a shift in cytokine production from TH1 to TH2 patterns mainly by inhibiting IL-12 • Glucocorticoids may function differentially depending on type of stress and site of production

  25. References 1. Almawi, W. et al. (1999) An alternate mechanism of glucocorticoid anti-proliferative effect: promotion of a Th2 cytokine-secreting profile. Clinical Transplantation 13, 365-374 2. Chen, X., Murakami, T., Oppenheim, J., Howard, O. (2004) Differential response of murine CD4+CD25+ and CD4+CD25- T cells to dexamethasone-induced cell death. European Journal of Immunology34, 859-692. 3. Calcagni, E. et al. (2006) Stress system activity, innate and T helper cytokines, and susceptibility to immune-related diseases. Ann. N. Y. Acad. Sci 1069, 62-76 4. Daniel, C., Sartory, N. A., Zahn, N., Radeke, H. H., Stein, J. M. (2008) Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. Journal of Pharmacoloy and Experimental Therapeutics324, 23-333. 5. Debonera, F., Krasinkas, A. M., Gelman, A. E., Aldeguer, X., Que, X., Shaked, A., Olthoff, K. M. (2003) Dexamethasone inhibits early regenerative response of rat liver after cold preservation and transplantation. Hepatology38, 1563-15724. 6. Entringer, S., et al. (2008) Influence of Prenatal Psychological Stress on Cytokine Production in Adult Women. 50, 579-587 7. Franchimont, D., Galon, J., Gadina, M., Visconti, R., Zhou, Y., Aringer, M., Frucht, D. M., Chrousos, G. P., O'Shea, J. J. (2000) Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes. Journal of Immunology164, 1768-17745. 8. Franchimont D. (2004) Overview of the Actions of Glucocorticoids in the Immune Response. Ann. N. Y. Acad. Sci. 1024, 124-137 9. Frick, L. R., Rapanelli, M., Bussmann, U. A., Klecha, A. J., Arcos, M. L., Genaro, A. M., Cremaschi, G. A. (2009) Involvement of Thyroid Hormones in the Alterations of T-Cell Immunity and Tumor Progression Induced by Chronic Stress. Biological PsychiatryDOI:10.1016/j.biopsych.2008.12.013 6.

  26. References 10. Furay, R. A., Bruestlt, E. A., Herman, P. J. (2008) The Role of Forebrain Glucocorticoid Receptor in Acute and Chronic Stress. Endocrinology149, 5482-54907. 11. Jankord, H. et al. (208) Limbic Regulation of Hypothalamo-Pituitary-Adrenocortical Function during Acute and Chronic Stress. Ann. N. Y. Acad. Sci. 1146, 64-73 12. Marshall, G., et al. (1998) Cytokine Dysregulation Associated with Exam Stress in Healthy Medical Students. Brain, Behacior and Immunity 12, 297-307 13. Qiao, S. et al. (2008) Age-Related synthesis of Glucocortocoids in Thymus. Experimental Cell Research 314, 3027-3035 14. Sternberg, E. (2001) Neuroendocrine regulation of autoimmune/inflammatory disease. Journal of Endocrinology 169, 429-435 15. Scheu, S., Stetson, D. B., Reinhardt, R. L., Leber, J. H., Mohrs, M., Locksley, R. M. (2006) Activation of the integrated stress response during T helper cell differentiation. Nature Immunology7, 644-651 16. Shibolet, O., Alper, R., Ilan. Y., Weidenfeld, J. (2005) Regulatory role of the pituitary-adrenal axis in experimental colitis: effect of adrenalectomy on the clinical course and the TH1/TH2 immune profile. Inflammatory BowelDiseases11, 1053-10599. 17. Tausk, F., Elenkov, I., Moynihan, J. (2008) Psychoneuroimmunology. Dermatologic Therapy21, 22-3110. 18. van den Brandt, J., Lühder, F., McPherson, K. G., de Graaf, K. L., Tischner, D., Wiehr, S., Herrmann, T., Weissert, R., Gold, R., Reichardt, H. M. (2007) Enhanced glucocorticoid receptor signaling in T cells impacts thymocyte apoptosis and adaptive immune responses. American Journal of Pathology 170, 1041-1053 19. Wang, J. et al. (2008) Time-course changes in nuclear translocation of hepatic glucocorticoid receptor in rats after burn trauma and its pathophysiological significance. Shock 30, 747-752

  27. THE END! QUESTIONS ANYONE?

  28. Stress Reduction Technique

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