Regulation of Stress Induced Regulation of Stress Induced Cytokine Response by Cytokine Response by

GlucocorticoidsGlucocorticoids

Janine GilkesJanine GilkesBiology 520Biology 520

2/11/092/11/09

HPA axis Activation Induced by StressorsHPA axis Activation Induced by Stressors

Stress illicits an immune response resulting Stress illicits an immune response resulting in bi-directional signaling between the in bi-directional signaling between the immune and central nervous system (CNS)immune and central nervous system (CNS)

Cytokines from the periphery can initiate the Cytokines from the periphery can initiate the cycle by crossing the blood–brain barriercycle by crossing the blood–brain barrier

Immune signaling of the CNS activates the Immune signaling of the CNS activates the hypothalamic-pituitary-adrenal (HPA) axis hypothalamic-pituitary-adrenal (HPA) axis

The HPA axis is responsible for the initiation The HPA axis is responsible for the initiation

of glucocorticoid (GC) stress responses in of glucocorticoid (GC) stress responses in all vertebrate animals. all vertebrate animals.

The principal end products of the HPA axis The principal end products of the HPA axis are glucocorticoid hormonesare glucocorticoid hormones

Jankord et al., 2008 and sternbergh et al 2001http://2to3yearsplan.com/stress-cartoon-work.jpg

Effects of Chronic Stress on HPA axisEffects of Chronic Stress on HPA axis

Temporal prolongation of stress exposure (chronic Temporal prolongation of stress exposure (chronic stress) causes marked enhancement in Basal HPA tone stress) causes marked enhancement in Basal HPA tone as well as stress reactivity.as well as stress reactivity.

Excessive stimulation results in baseline glucocorticoid Excessive stimulation results in baseline glucocorticoid hypersecretion, enhanced CRH and vasopressin hypersecretion, enhanced CRH and vasopressin synthesis, and down regulation of glucocorticoid synthesis, and down regulation of glucocorticoid receptors (GR) in key feedback regions.receptors (GR) in key feedback regions.

Excessive stimulation may also a ect severity of ffExcessive stimulation may also a ect severity of ffinfectious disease through their immunosuppressive infectious disease through their immunosuppressive e ects.ffe ects.ff

Jankord et al., 2008

GlucocorticoidsGlucocorticoids Immunomodulatory vs. Immunosuppressive and anti-Immunomodulatory vs. Immunosuppressive and anti-

inflammatoryinflammatory

Most notable mechanism of action is the inhibition of Most notable mechanism of action is the inhibition of cytokine production and action.cytokine production and action.

Beneficial for short-term survival, but prolonged Beneficial for short-term survival, but prolonged exposure can lead to serious metabolic, immune, and exposure can lead to serious metabolic, immune, and psychological dysfunction,psychological dysfunction,

Termination of the GC response is controlled by various Termination of the GC response is controlled by various feedback inhibition mechanisms feedback inhibition mechanisms

Glucocorticoids are lipophilic molecules and so readily Glucocorticoids are lipophilic molecules and so readily pass through the plasma membrane of all cells in the pass through the plasma membrane of all cells in the body. body.

Padgett et al., 2003 and Almawi et al., 1999

HPA axis and Glucocorticoid SecretionHPA axis and Glucocorticoid Secretion

http://speakingoffaith.publicradio.org/programs/stress/images/stressresponse.jpg

Glucocorticoids Regulate Cytokine ProductionGlucocorticoids Regulate Cytokine Production

Once a T cell immune response has flared up, Once a T cell immune response has flared up, glucocorticoids may now modulate its cytokine production glucocorticoids may now modulate its cytokine production pattern. pattern.

The differentiation of CD4+ T cells into TH1 lymphocytes The differentiation of CD4+ T cells into TH1 lymphocytes drive cellular immunity, while differentiation into TH2 drive cellular immunity, while differentiation into TH2 lymphocytes drive humoral immunitylymphocytes drive humoral immunity

This differentiation depends on the type of antigen This differentiation depends on the type of antigen encountered and the type of cytokines produced during encountered and the type of cytokines produced during antigen presentation. antigen presentation.

Glucocorticoids block IL-12 secretion by monocytes and Glucocorticoids block IL-12 secretion by monocytes and dendritic cells, but promote TH2 development by dendritic cells, but promote TH2 development by enhancing IL-10 secretion by macrophagesenhancing IL-10 secretion by macrophages

Franchimont et al., 2004

Subdivisions of Adaptive ImmunitySubdivisions of Adaptive ImmunityCellular armCellular arm

- Antigen mediated immune response Antigen mediated immune response

- Th1 cells primarily secrete interferon Th1 cells primarily secrete interferon IFNγ, IL-2, and TNF-α, which IFNγ, IL-2, and TNF-α, which promote cell mediated immunitypromote cell mediated immunity

- IL-12 (produced by APCs) is the IL-12 (produced by APCs) is the major inducer of Th1 differentiation major inducer of Th1 differentiation and, hence, cellular immunity.and, hence, cellular immunity.

- by producing IFNby producing IFNγγ, Th1 cells activate , Th1 cells activate CD8+ T cells and macrophagesCD8+ T cells and macrophages

- IL-12 and IFNγ inhibit Th2IL-12 and IFNγ inhibit Th2

Humoral ArmHumoral Arm

- - Primarily functions in the production of Primarily functions in the production of antibodies.antibodies.

- Th2 cells secrete primarily IL-4, IL-10, Th2 cells secrete primarily IL-4, IL-10, and IL-13, which enhance humoral and IL-13, which enhance humoral immunity. immunity.

- IL-4 and IL-10 stimulate the IL-4 and IL-10 stimulate the differentiation of B cells into antibody differentiation of B cells into antibody secreting B cells, and immunoglobulin secreting B cells, and immunoglobulin switching to IgE. switching to IgE.

- These cytokines also prevent TH1 These cytokines also prevent TH1 cytokine production by inhibiting IL-12cytokine production by inhibiting IL-12

Almawi et al., 1999 and Tausk et al., 2009

Th1 = Pro-Inflammatory ResponseTh2 = Anti-Inflammatory Response

T Cell Differentiation and Glucocorticoid TaretT 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 andcellular immunity.

Glucocorticoids prevent IL-12 action by specifically inhibiting (Stat) 4 phosphorylation

Almawi et al., 2002 Franchimont et al., 2004

Stimulation of a TH2 System by GlucocorticoidsStimulation of a TH2 System by Glucocorticoids

Why the TH2 preference?Why the TH2 preference?

During an immune and inflammatory response, the During an immune and inflammatory response, the activation of the stress system, through induction of a activation of the stress system, through induction of a TH2 shift may protect the organism from systemic TH2 shift may protect the organism from systemic “overshooting” with TH1 pro-inflammatory cytokines. “overshooting” with TH1 pro-inflammatory cytokines.

Elenkov et al, 2006 and calcagni 206

The Immune ResponseThe Immune Response

Tausk et al., 2009

HypothesisHypothesis

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

ELISAELISA

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

Biotinylated

TMB

Cytokine Increase During Stressful SituationsCytokine Increase During Stressful Situations

Entringer et al., 2008

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

Critique: No X-axis label

Cytokine Increase During Stressful SituationsCytokine Increase During Stressful Situations

- N=15 students

- Mitogen culters: PBMC + PHA

- Pre exam: 4 weeks

- Post exam: 2 days

- Cytokine analysis: ELISA

- p < 0.007

Marshall et al., 1998

Critique: No Y-axis label

Figure 1: The effect of exam stress on TH1/TH2 cytokine balance

Glucocorticoids are Released in Response to StressGlucocorticoids are Released in Response to Stress

Furay et al., 2008

- 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

Figure 6: Effects of chronic variable stress on Plasma Corticosterone levels

Critique: X-axis not well defined Result figures punctuated several parts of discussion

Glucocorticoids Inhibit TH1 CytokinesGlucocorticoids Inhibit TH1 Cytokines

Figure 1: Dexamethasone inhibits IL-12 induced INFγ

Franchimont et al., 2000

- 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

Glucocorticoids Inhibit TH1 CytokinesGlucocorticoids Inhibit TH1 Cytokines

Fig 1: Glucocorticoids delay early IL-6 and TNFα expression

Debonera et al., 2003

- 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

Glucocorticoids Mechanism of ActionGlucocorticoids Mechanism of Action

- 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)

Franchimont et al., 2004

Figure 4: Dexamethasone inhibits IL-12 induced phosphorylation of Stat4

Critique: no mention of how long cells were incubated with stat4

Contrasting View of Glucocorticoid RegulationContrasting View of Glucocorticoid Regulation

Wang et al., 2008

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

Glucocorticoids Act DifferentiallyGlucocorticoids Act DifferentiallyFigure 4: Increasing production of GC in thymocytes by de novo synthesis

Qiao et al., 2008

Figure 6: GCs induce apoptosis in Thymocytes

- 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

Summary of Findings: Chronological ProgressionSummary of Findings: Chronological Progression

Human = cortisolRodents = corticosterone

Hypothalamus

Anterior Pituitary

Adrenal Cortex

GCs

CRH

ACTH CAs

EPINEP

Stress

•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

Summary of FindingsSummary of Findings

AuthorsAuthors Summary of figures in relation to hypothesisSummary of figures in relation to hypothesis Support Support

Entringer et al. 2008Entringer et al. 2008 TH2 dominant cytokines are released in response to TH2 dominant cytokines are released in response to stress stimulusstress stimulus

YesYes

Marshall et al., 1998Marshall et al., 1998 The ratio of INFThe ratio of INFγγ:IL-10 is downregulated in response to :IL-10 is downregulated in response to stress stimulusstress stimulus

YesYes

Furay et al., 2008Furay et al., 2008 Chronic stress causes increased Corticosterone release Chronic stress causes increased Corticosterone release into the bloodinto the blood

YesYes

Franchimont et al., Franchimont et al., 20002000

Dexamethasone inhibits TH1 cytokine expression by Dexamethasone inhibits TH1 cytokine expression by inhibititng IL-12 expressioninhibititng IL-12 expression

YesYes

Debonera et al., 2003Debonera et al., 2003 Dexamethasone delays THFDexamethasone delays THFαα and IL-6 expression and IL-6 expression PartiallyPartially

Franchimont et al., 2004Franchimont et al., 2004 Glucocorticoids inhibit TH1 cytokine expression by Glucocorticoids inhibit TH1 cytokine expression by inhibiting Stat4 phosphorylationinhibiting Stat4 phosphorylation

YesYes

Wang et al., 2008Wang et al., 2008 Dexamethasone has no effect on early stage cytokine Dexamethasone has no effect on early stage cytokine responseresponse

NoNo

Qiao et al., 2008Qiao et al., 2008 Thymus derived Glucocorticoids induce apoptosis in Thymus derived Glucocorticoids induce apoptosis in Thymocytes. Thymocytes.

NoNo

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?

Collect peritoneal mast cells from rats.Collect peritoneal mast cells from rats.

Stimulate with ovalbumin Stimulate with ovalbumin (Lennon et al., 2009)(Lennon et al., 2009)

Measure serum titers of IgE OVA-specific antibodies by ELISAMeasure 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.)

Take Home MessagesTake Home MessagesStress 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

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THE END!THE END!

QUESTIONS ANYONE?QUESTIONS ANYONE?

Stress Reduction TechniqueStress Reduction Technique