Stress Responses: Organization and Impact on the

Immune System

Elizabeth Repasky, Ph.D. Advanced Topics in Immunology

March 24th, 2015

Most Lecture Notes from: Padgett and Glaser Reviews

STRESS RESPONSE in Advanced Immunology?

• Since the 40s and 50s, GC hormones have been prescribed clinically because of their inherent and powerful anti-inflammatory properties..

• 1950 Nobel Prize in Medicine: Edward Kendall, Tadeus Reichstein and Philip Hench for their studies on the hormones of the adrenal cortex.

• Based on their early observations, it has become very clear that GC hormones can regulate a wide variety of immune cell functions.

• Modulate expression of: cytokines, chemoattractants, adhesion-molecules, and impact Immune cell trafficking, lymphocyte proliferation and differentiation, and effector function.

• How do stress hormones mediate this wide spectrum of immunological influence??

• HSPs- Stress Response Proteins: Relationship to immune response?

How does stress influence the immune system?

• Psychoneuroimmunology- PNI • Focuses on the interactions among the central nervous

system (CNS), the endocrine system and the immune system, and the impact that these interactions have on health.

• --a complex network of signals that function in bi-directional communication among these 3 system.

• The hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal medullary (SAM) axes are the two major pathways through which the immune system can be altered.

Immune system can respond to stress

• Lymphocytes, monocytes, or macrophages and granulocytes, exhibit receptors for many neuroendocrine products of the HPA and SAM axes, such as cortisol and catecholamines, which can cause changes in cellular trafficking, proliferation, cytokine secretion, antibody production and cytolysis.

Ecoimmunology- a new discipline related to PNI

• The study of environmental influences on immune function

• Involves the fields of ecology and evolutionary biology.

• Goal-to understand immune function within a broadly integrative, organismal context, but from a more evolutionary perspective,

• Involves the study on interactions and trade-offs between immunity and other life history traits, both within and across individuals, populations and species. (see review by Demas and Carlton, 2015

Goal of Ecoimmunology

• To understand extrinsic and intrinsic factors leading to changes in immune system function and how these changes contribute to disease susceptibility across a wide range of animal species (Usually natural populations, not model systems)

The immune and nervous

systems communicate

with and regulate each

other.

CNS Cytokines STRESSORS

Hypothalamus

CRV AVP

CRV= corticotropin-releasing hormone AVP= arginine vasopressin

ACTH

Adrenal Glands

Glucocorticoids

Immune System (Organs & Cells

Thymus

Immune Cells

Spleen

Lymph Nodes

Bone Marrow

PNS

SNS

Vagus nerve

Paul- Fundamental Immunology

Glaser and Kiecolt-Glaser Nat Rev Immunol. 2005

Stressor

Example: • coricoptrophin-releasing hormone is

secreted from the paraventricular nucleus of the hypothalamus into the hypophyseal portal blood supply and subsequently stimulates the expression of adrenocorticoptrophin hormone ACTH in the anterior pituitary gland,

• ACTH then circulates in the bloodstream to the adrenal glands where it induces the expression and release of glucocorticoid (GC) hormones.

How immune cells are affected • Lymphocytes, monocytes or macrophages, and

granulocytes, exhibit receptors for many neuroendocrine products of the HPA and SAM axes, such as cortisol and catecholamines, which can cause changes in cellular trafficking, proliferation, cytokine secretion, antibody production and cytolytic activity.

• Example: treatment of peripheral blood leukocytes (PBLs) with with catecholamines in vitro results in the suppression of IL-12 synthesis and an increase in IL-10 production.

• This can cause a shift in the phenotype of CD4 + T-helper cells, from a Th1 profile, which functions in cell-mediated immune activities, to a Th2 profile, which is involved in antibody production.

HPA axis and glucocorticoid hormones

• Hypothalamus receives and monitors information about the environment and coordinates responses through nerves and hormones.

• Vision, smell, hearing, temperature sensation and pain alert the hypothalamus to emergencies or hazards in the environment.

• Emotional portions of the brain also relay information to the hypothalamus. • From this integrative center the brain contols hormone secretion from the

pituitary gland and other tissues, such as the adrenal glands.

• EX. Corticotrophin-releasing hormone is secreted from the paraventricular nucleus of the hypothalamus into the hypophyseal portal blood supply and subsequently stimulate the expression of adrenocorticotrophic hormone (ACTH) in the anterior pituitary gland

• ACTH then circulates in the blood stream to the adrenal glands where it induces the expression and release of GC hormones.

• These hormones affect cardiovascular and renal function and metabolism and act together with the nervous system to adjust our responses to the environment.

HPA axis and Glucocorticoid Secretion

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

HPA axis Activation Induced by Stressors • Stress elicits 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

Jankord et al., 2008 and Sternbergh et al 2001

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

Stimulation of a TH2 System by Glucocorticoids

• Why the TH2 preference? Hypothesis:

– 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 2006

Academic stress? • Marshall et al (see Padgett and Glaser Review

in Handouts) supports several published in vitro findings.

• In medical students taking exams, psychological stress produced a shift in the cytokine balance toward a Th2 profile.

• The data showed decreased synthesis of Th1 cytokines, including IFN-γ and increased production of Th2 cytokines, including IL-10.

• It is believed that this stress-induced decrease of Th1 cytokines results in dysregulation of cell mediated immune responses.

Both major and minor stressful events can have direct adverse effects on a variety of

immunological mechanisms, • To help demonstrate causal relations between psychosocial stressors and

the development of infectious illness, investigators have inoculated subjects with several types of vaccines.

• Vaccine responses demonstrate clinically relevant alterations in an immunological response to challenge under well controlled conditions. Accordingly they act as a proxy for responses to an infectious agent.

• EX. Among medical students taking exams, stress and the degree of social support affect the virus specific antibody and T-cell responses to a hepatitis B vaccine.

• Chronic stress associated with caregiving for a spouse with Alzheimer’s disease was associated with a poorer antibody response to an influenza virus vaccine compared to well matched control subjects.

• Therefore, in individuals who produced delayed, weaker and shorter-lived immune responses to vaccines, it is reasonable to assume these same individuals would also be slower to develop immune responses.

Padgett and Glaser, 2003

Psychological stress can influence immune function, alter the pathophysiology of infection.

• Adults who show poorer responses to vaccines also experience higher rates of clinical illness as well as longer lasting infectious episodes.

• Therefore, from these vaccine studies, it is hypothesized that stress puts individuals at greater risk from more severe illness.

• Cohen et al. (1991) showed that human volunteers who were inoculated with 5 difference strains of respiratory viruses showed a dose dependent relationship between stress and clinical symptoms observed after infection.

Animal studies confirm human findings:

• Stress diminishes vaccine responses • Exacerbates viral and bacterial pathogenesis • Slows wound healing • Alters autoimmune disease • Stress hormones inhibit: • the trafficking of neutrophils, macrophages, antigen

presenting cells, NK cells, T and B cells, • Suppress the production of pro-inflammatory cyokines

and chemokines, • Downregulate the production of cytokines necessary for

the generation of adaptive immune responses and impair effector functions of macrophages, NK cells and lymphocytes.

Padgett and Glaser, 2003

IL-12 TNFa

Glucocorticoids can inhibit innate immunity.

Shown to be suppressed

Paul- Fundamental Immunology

HPA axis and glucocorticoid hormones

• The hypothalamus receives and monitors information about the environment and coordinates responses through nerves and hormone.

• Visual information, smell, hearing, temperature sensation and pain alert the hypothalamus to emergencies or environmental changes which could be harmful.

• Also, the emotional part of the brain also relay information to the hypothalamus.

• From this integrative center, the brain controls hormone secretion from the pituitary gland and other tissues such as the adrenal glands.

GC hormones • Affect cardiovascular and renal function and

metabolism • Act together with the nervous system to adjust

our responses to the environment. • One of the “core stress responses” originally

described by Hans Selye in 1936 • The acute production of GC hormones from

the adrenal cortex stimulates the metabolism of glucose to provide for energy to flee or combat an immediate threat.

• But, when chronically activated, the HPA axis can cause deterioration in general health and worse existing disease.

• Since 1940s and 50s, GC hormones have been prescribed clinically because of their inherent and powerful anti-inflammatory properties

• 1950 nobel prize- given to Edward Kendall, Tadeus Reichstein and Philip Hensch for their studies of the hormones of the adrenal cortex.

• Building on these studies, it became clear that GC hormones can regulate a wide variety of immune-cell functions.

• Moculate cytokine expression, • Chemoattractant expression, • Adhesion-molecular expression • Immune cell trafficking, proliferation,

differenatiation and effector function.

• How do GCs mediate such a wide spectrum of influence?

Glucocorticoids Regulate Cytokine Production

• Once a T cell response is engaged, 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

T Cell Differentiation and Glucocorticoid Targets

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

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

Molecular Mecanisms of GC action

• Effects of GC on immunity occur through the glucocorticoid receptor (GR),,,an intracellular receptor that is part of the steroid hormone receptor family of ligand dependent transcription factors.

• Characterized by an N-terminal transactivation domain, and a ligand binding domain that includes the Tau 1 domain, a DNA binding domain, and a ligand binding domain.

• Once bound to ligand, the receptor dissociates from a heat shock protein complex, translocates to the nucleus, dimerizes, binds to DNA and activates transcription.

Mechanism of action for GC hormones • Lipophilic- GC hormones pass through the plasma

membrane of all cells in the body. • If a cell possesses a GC receptor, that cell can be a

target. • Two receptors for GC hormones- GR and the

mineralocorticoid receptor (MR).

• Because corticosterone has a higher affinite for MR than for GR at low lowers they bind preferentially to MR receptors…only at high circulating or tissues leves (i.e during stress) is the GR occupied.

• In immune cells (macrophages and T cells the primary receptor for glucocorticoid homnones on immune function is GR; thus mediated by the GR.

What is GR? • A member of the steroid hormone-receptor superfamily • Structurally it can be divided into three distinct regions. • N-terminal domain that is involved in transactivation • Middle section-DNA binding domain, is involved in DNA binding

mediated byt two zinc fingers • C terminal domain, or ligand binding domain of the GR is

responsible for ligand binding and is also involved in transactivation, dimerization and heat shock protein 90 binding.

• In its unactivated state, the GR is located in the cytoplasm where it is part of an oligomeric complex containing HSP90 which is though to hold the GR in a conformation that is available to incoming cortisol or corticosterone.

• Upon ligand binding, GRs dissociate from this complex and translocate to the nucleus where they bind as a homodimer to target glucocorticoid response elements (GREs) through zinc fingers of the DNA binding domain.

• The bound GR-ligand complexes can then influence gene expression by modulating transcription through several proposed mechanisms.

Structure of the glucocorticoid receptor

Paul- Fundamental Immunology

Breakthrough…. • In 1995, two publications (Schenman et al., Science-v.

270,1995 and Auphan et al., v. 270, 1995) presented data that GC could interfere with NF-kB activity….and showed that GC hormones could transactivate an inhibitor of NF-kB.

• Hypothesis: GC induces the transcription of IkBa which then sequesters NFkB in the cytoplasm and prevents it from translocating to the nucleus and inducing gene activation.

• This is a logical explanation for the broad spectrum of cytokines suppression mediated by GCs.

Paul- Fundamental Immunology

1. Many of the cytokines produced by macrophages and by Th cells are under the control of NF-kB, therefore if GCs could repress activation, then individiual cytokines would not need to possess the putative hormone response elements for GCs--- instead, by inhibiting NF-kB transcriptional activity, multiple cytokines genes could be turned off simulaneously.

Some models…..

Model 2

There is substantial cross talk between NF-kB and the GR that might prevent gene expression. The activated GC receptor has the capability of binding directly to NF-kB and preventing its transmigration to the nucleus. Also, the activated GC receptor has the capability of binding directly to the NFkB as it is attached to its putative kB DNA binding locus.

Glucocorticoid repression of NF-kB activity could be caused by competition between the GR and NF-kB f for limited cofactors, such as CBP (cAMP response element binding protein) and SRC-1 (steroid receptor co-activator-1 In some cells, GR repression of NF-kB can be alleviated by excess co-factor..however, McKay and Cidlowski found that that CBP did not mediate GR repression of NF-kB using a competitive model, but rather that CBP functions as an integrator to enhance the physical interaction between the GR and NF-kB.

3rd Model…..

Sympathetic Nervous system, Adrenal Medulla and catecholamines

• In addition to GC hormones, catecholamines also modulate a range of immune functions, inlcuding

• T cell proliferation • Cytokines and antibody production • Cytolytic activity • Cell trafficking. • Catecholamines often act in in concert

with the HPA axis.

What about catecholamines?

• Treatment of peripheral blood leukocytes with catecholamines in vitro results in the suppression of IL-12 synthesis and an increase in IL-10 production.

• This can shift the phenotype of CD4+ T-helper (Th) cells from a Th1 profile, which functions in cell-mediated immune activities, to a Th2 profile, which is involved in antibody production.

Paralleling the increase of GC hormone production from the adrenal cortex, activation of the HPA axis also results in catecholamine production from the adrenal medulla Adrenal medulla: Synthesizes and secretes norepinephrine and epinephrine. In humans, 80% of the catecholamine output of the medulla is epinephrine. Norepinephrine is released from sympathetic nerve fibers in direct proximity of target tissues. If acutely activated these catecholaminergic systems can provide the body with a needed “boost” to deal with an immediate threat—the typical and most obvious effect- a primitive mammalian “fight or flight” reaction, in which there is increased heart rates and increased blood flow to muscles.

Chronic activation of SAM can dysregulate immune function

• Noradrenergic sympathetic nerve fibers fun from the CNS to both primary and secondary lymphoid organs.

• Evidence suggests that sympathetic nerve terminals synapse with neighboring immune cells,

• In this synapses the sympathetic nerves release noradrenalin.

• In addition, once released from the adrenal medullla, epinephrine courses through the circulation and binds to specific adrenergic receptors on immune cells, where they induce essentially the same effects as direct sympathetic nervous simulation.

Norepinephrine Increases Tumor Growth and Metastasis

• Norepinephrine increases VEGF, IL-6, and MMPs in ovarian and pancreatic cancers leading to increase metastasis. (Thakar et al, Nature Medicine 2006; Lutgendorf et al, Clinical Cancer Research 2003; Guo et al, Oncology Reports 2009)

• In pancreatic cancer, NE downregulates MHC class 1 and B7-1 on tumor cells leading to immune escape. (Wang et al, Plos One 2009)

• Exposure to norepinephrine enhances prostate tumor survival by upregulating the expression of MAPK, and inactivating the apoptotic molecule, BAD. (Hassan et al, JCI 2012, Magnon et al Science 2013)

40

Mechanisms of action for catecholamines

• Mediate their effect on target tissues through adrenergic receptors.

• Numerous cells of the immune system (incl lymhocytes and macrophages express adrenoreceptors.

• G-protein coupled receptors: α-and β- • Most important for immune cells appears

to be the β2-adrenergic receptor.

B2-adrenergic receptor • A seven membrane spanning, serpentine receptor embedded in

the plasma membranes of many cell types. • Once bound to ligand, the b2-adrenergic receptor

communicates with the cytoplasm by stimulating the activation of a G-protein complex.

• G-protein- formed from 3 distinct protein subunits- α,β and γ • Inactive form- the three G-protein subunits are bound together

in a heterotrimeric complex, and the G-a subunit is bound to guanosine diphosphate (GDP); when active in binds the triphosphate form- GTP

• When this happens the GTP-bound a subunit loses affinity for the receptor and for the b and g subunits, dissociates from them, and then activates adenylate cyclase. In turn, adenylate cyclase catalyzes the synthesis of cAMP from ATP.

Major cellular effects of the activation of the cAMP cascade

• Stimulation of transcription after phorphorylation of transcription factors by cAMP dependent protein kinase A (PKA).

• One is CREB (cAMP response element binding protein binds to the conserved consensus cAMP response element TGACGTCA present in the promoter regions of responsive genes.

• This way, the expression of numerous immune response genes can be modified during times of stress.

Other physiological pathways for stress

• Endogenous opioids diminish NK cell cytotoxicity,

• Substance P, a neuropeptide is able to reduce inflammatory responses by suppressing IL-16 production by eosinophils.

• Whereas the anti-inflammatory function of GC center on NF-kB inhibition, the GR most likely interferes with other transcriptional regulators. (AP-1 and NF-AT.

Glaser and Kiecolt-Glaser Nat Rev Immunol. 2005

Thermoneutral Temperature

●●●

10 20 30

Basal

Ambient Temperature (°C)

Met

abol

ic R

ate

W/ k

g Standard Room

Temperature

• Eat more • Have higher BP, heart

and breathing rates • Have increased

oxygen consumption

At standard ambient temperature mice:

Guyton & Hall, 2002; Cannon, 2004; Gordon, 2012

Generation of extra body heat through adaptive thermogenesis uses a lot of energy

46

Difference between TT and ST

Macrophages 47

∆HEAT ↑UCP1

Cold Stress

The uncoupling protein in mitochondria drives adaptive thermogenesis

Cold Stress Induces Norepinephrine Production to Facilitate Heat Production

Nguyen et al, Nature 2011

UCP1 Mediates Adaptive Thermogenesis by Uncoupling Oxidative Respiration

48

ATP Synthase

Outer Mitochondrial Space

Inner Mitochondrial Space

H+ H+

H+ H+ H+ Complex I Complex III Complex IV

H+ H+ H+

HEAT

H+

H+

H+

+O2-+2H+ Complex II

2e-

H+ H+

H+

ADP+ppi ATP

H+ UCP1

HEAT

Stress and The Heat Shock Protein Systems: Chaperones and Antigen Processing

In the beginning

HSPs and Drosophila

Protection from

teratogenesis

• Stress proteins are highly conserved throughout all living organisms

• Differentially compartmentalized and functionally regulated

• Constitutively expressed and can be induced under certain stress conditions

• Heat shock proteins (HSPs) and glucose regulated proteins (GRPs) are two major groups

• Act as molecular chaperones in protein maturation and function regulation

Stress Proteins

Endoplasmic Reticulum (ER) grp78(Bip) grp94(gp96) grp170

Cytoplasm, Nucleus, Mitochondria hsp27 hsp40 hsp60 hsp70 hsp90 hsp110

heat oxidizing agents cytoplasmic & mitochondrial damage Heavy Metals

HSF

hsps

Inhibitors of glycosylation;

hypoxia;calcium ionophores

reductive stress ER damage

Stress Proteins and Stress Response

Mammalian stress proteins: heat shock proteins & glucose (hypoxia) regulated

proteins

hsp110 hsp90 hsp70 hsp60

hsp28 Heat Shock Bacteria to

Man: Cold Spring Harbor Press, 1982

grp170

grp94/gp96

grp78

C Heat C Hypoxia

Sciandra et al.PNAS 1984; Shen et al PNAS

1987

How do HSPs protect the cell?

• Inhibition of protein aggregation that arises as a result of protein unfolding during physical (e.g. heat) or pathological (e.g. neurodegenerative diseases) stress: HOLDING

• Refolding the denatured substrate proteins using multi-component protein machines: FOLDING

• Directing damaged substrate proteins to

degradation pathways.

Hsp110 and grp170 are highly effective in chaperoning large protein substrates: inhibition of

heat induced Luciferase aggregation

Heat shock at 43οC (min)

0 5 10 15 20 25 30

% A

ggre

gatio

n

0

20

40

60

80

100

LucLuc + OVALuc + hsc70Luc + grp94/gp96Luc + hsp110Luc + grp170

Subjeck Lab

FOLDING • Once the denatured protein is bound by the

hsp (molecular chaperone), it can then be refolded with the assistance of other chaperones.

• For example: refolding of denatured Luciferase and Other Reporter Proteins

• Holding, Folding, and Degradation activities are essential at all times in cells, during stress or in natural cellular processes

• Holding, folding, assembly and translocation of newly synthesized proteins and multiprotein ensembles, targeting to cellular organelles, membranes, secratory pathway, etc.

Stress Functions and Non-Stress Functions Are Related

Stress Proteins as Danger Signals

• HSPs and GRPs are released from damaged and dying cells.

• These stress proteins will be carry with them peptide or protein elements that are related to the cell damage, e.g. mutated proteins, viral proteins, etc.

• These stress proteins may therefore act as a ‘natural’ vaccine in-situ.

• Stress Proteins are one group of a broader set of natural ‘Danger Signals’ that are released by damaged cells and alert the immune system by binding to receptors on antigen presenting cells and stimulating immune responses.

Chaperones, stress and evolution?

• Forces that govern protein folding may exert a profound effect on how genotypes are translated into phenotypes.

• HSPs promote the correct folding and maturation of proteins in the cell.

• Hsp90-abundant and hihgly specialized chaperone that that works on “metastable” signal transducers that are key regulators of a broad spectrum of biological processes.

• The folding of “clients” of Hsp90 are very sensitive to changes in the external and internal environment of the cell.

See details in Lindquist, 2010

Figure 1. Examples of developmental abnormalities associated with Hsp90 deficits in Drosophila. (A) Thickened wing veins,(B) transformed second leg with an ectopic sex comb, (C) deformed eye with an extraantenna, (D) disorganized abdominal tergites,(E) notched wings, (F) extraneous tissuegrowing out of tracheal pit.

From Lindquist, 2010