Peripheral actions of the stress hormone Corticotropin Releasing Hormone (CRH): focus on its immunomodulatory effects
The Hypothalamic-Pituitary Adrenal (HPA) axis Hypothalamus CRH VP - + + Anterior pituitary Glucocorticoid - ACTH Adrenal cortex
Sites of CRH synthesis SPINAL CORD BRAIN IMMUNE SYSTEM GI TRACT T-lymphocyte LUNG PLACENTA
CRH Receptors CRH RECEPTOR 1 (CRHR1): Brain, anterior pituitary, immune system, GI tract, adrenal gland, skin CRH RECEPTOR 2 (CRHR2): Brain, pituitary, immune system, GI tract, adrenal gland, heart, skin, skeletal muscle
CRH AND INFLAMMATION CRH INFLAMMATION ACTH PERIPHERAL CRH GC
Dual effects of CRH on the immune /inflammatory response * suppressive, by central CRH *stimulatory, by peripheral CRH
Signaling pathways mediating the immunomodulatory effects of CRH ??
Control CRH 10-7 M p50 p65 l Ctrl CRH Ctrl CRH NF-B n.s.
Mutant competitor CRH/hCRH 10-6 M competitor LPS10g/ml Ctrl CRH 10-7 M NF-B n.s.
CONTROL CRH 15min CRH 30min IB
CRH (10-6M) CRH (10-6M) CRH (10-6M) CRH (10-7M) CRH (10-7M) CRH (10-7M) CRH (10-8M) CRH (10-8M) CRH (10-8M) EGF (10ng/ml ) TNF (10ng/ml) TNF (10ng/ml) CRH (10-9M) CRH (10-9M) CRH (10-9M) Control Control Control Concentration-dependent effects of CRH on MAPK activation in humanleukocytes p42/44 (5 min) p38 (5 min) JNK (30 min)
Is there an important physiological role for peripheral CRH itself in the regulation of the inflammatory process ?
TheCrh-deficient animal model Normal lifespan Normal weight Normal food intake Blunted/absent circadian rhythmicity Low basal corticosterone Impaired ACTH and corticosterone responses to multiplestressors CRH ACTH GC Muglia et al. Nature, 1995
Inflammatory sites Sources of peripheral CRH leukocytes Hypothalamus CRH Nervefibers ? Pituitary ACTH glucocorticoid Adrenals
GLUCOCORTICOID INFLAMMATION EPINEPHRINE CRH
Crh+/+ turpentine Crh-/- turpentine 2400 1800 IL-6 (pg/mL) 1200 600 0 1 4 8 16 24 30 Time (hr) CYTOKINE RESPONSE TO TURPENTINE
Crh +/+ saline Crh+/+ turpentine 60 Crh -/- saline Crh-/- turpentine 45 30 15 0 HORMONAL RESPONSES TO TURPENTINE 600 * 500 400 ACTH (pg/mL) Corticosterone (ug/dL) 300 * 200 100 0 1 4 8 16 24 30 48 1 4 8 16 24 30 48 Time (hr) Time (hr)
0.30 * 108 * # * * * 0.25 * * 104 * # 0.20 Body weight (% of control) Food intake (gr)/mouse weight (gr) # 100 # # 0.15 * 96 0.10 92 0.05 1 2 3 4 5 6 1 2 3 4 5 Day following the injection Days following the injection METABOLIC RESPONSES TO TURPENTINE Crh +/+ saline Crh+/+ turpentine Crh -/- saline Crh-/- turpentine
SYSTEMIC INFLAMMATION: Is there an important physiological role for peripheral CRH itself on the regulation of the inflammatory process, or its effects are linked to its role in stimulating the release of glucocorticoid ?
80 900 750 60 600 40 450 300 20 150 0 0 Saline Saline LPS LPS Saline Saline LPS LPS Crh+/+ Crh+/+ Crh-/- Crh-/- HPA axis response 1 hr after LPS or saline * * * ACTH (pg/ml) * $ Corticosterone (g/dl) *: P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment
350 60 300 50 40 200 30 20 100 10 0 0 Saline Saline LPS LPS Saline Saline LPS LPS Crh+/+ Crh+/+ Crh-/- Crh-/- HPA axis response 24 hr after LPS or saline * * * * $ ACTH (pg/ml) Corticosterone (g/dl) *: P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment
375 40 300 32 225 24 150 16 75 8 0 0 Effect of pre-treatment with antalarmin on the HPA axis response 24 hr after LPS n.s. Corticosterone (g/dl) ACTH (pg/ml) * Vehicle + LPS Antalarmin + LPS Vehicle + LPS Antalarmin + LPS
250 200 150 100 50 200 150 100 50 Plasma glucose levels 1 and 24 hr after LPS or saline Crh+/+ Glucose (g/dl) Saline LPS Crh-/- 1 24 Time (hr) following the injection
Plasma epinephrine at basal levels, and 24 hours post-LPS or –saline injection P<0.01 P<0.01 6000 Pg/ml 4000 2000 0 basal saline LPS basal saline LPS Crh+/+ Crh-/-
Systemic Bacterial Inflammation ??? CRH CYTOKINES ACTH ? ? GC LEPTIN ? Decreased food intake / Body weight loss-cachexia
30000 P<0.01 * 25000 20000 Pg/ml 15000 * 10000 5000 0 Crh+/+ Crh-/- Crh+/+ Crh-/- Crh+/+ Crh-/- 11/2 hour 24 hours 4 hours Plasma TNFa 11/2, 4 hours and 24 hours post-LPS injection * : statistical significance of p<0.01 from TNFa levels 4 and 24 hours post-LPS injection between animals of the same genotype
5000 4000 3000 2000 1000 0 Plasma IL-6 4 hours and 24 hours post-LPS injection Pg/ml Crh+/+ Crh-/- Crh+/+ Crh-/- 4 hour 24 hour
Systemic Bacterial Inflammation TLRs ??? CRH CYTOKINES ACTH ? ? GC LEPTIN ? Decreased food intake / Body weight loss-cachexia
Increased leptin levels have been reported to exert protective effects during inflammation
CRH and inflammation-associated processes • Angiogenesis CRH receptors are found in endothelial cells CRH causes chemotaxis of endothelial cells in vitro CRH induces NF-kB DNA binding activity in human endothelial cells • Wound healing Crh-/- mice show altered wound healing
HPA axis, LEPTIN, and LPS LPS administration results in * activation of the immune system manifested by increased plasma levels of proinflammatory cytokines, including TNF, IL-1 and IL-6, *activation of the HPA axis, and * induction of the ob gene, most likely by the action of cytokines on adipocytes.
35 30 25 20 15 10 5 0 Saline LPS Saline LPS Crh+/+ Crh-/- Plasma leptin 24 hr after LPS or saline * Leptin (ng/ml) * $ *: P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment
25 20 15 10 5 0 Effect of pre-treatment with antalarmin on leptin secretion 24 hrs after LPS * Leptin (ng/ml) Vehicle + LPS Antalarmin + LPS * P=0.05
$ 250 * 200 150 TNF- TNF- (pg/ml/million of plated cells) * 100 -actin 50 0 control LPS control LPS Crh+/+ Crh-/- Crh-/- Crh+/+ A B
$ * 10.0 7.5 IL-1 IL-1 (pg/ml/million of plated cells) 5.0 2.5 0.0 control LPS control LPS Crh+/+ Crh-/- Crh+/+ Crh-/- A B -actin
WT SPL +LPS WT SPL HT CRHKO Crh
Regulation of systemic inflammation by CRH and glucocortioid What are the target organs / cells? Is there a role for CRH and/or glucocorticoid on the regulation of innate immunity? What is the contribution of the above hormones in the metabolic changes triggered by inflammation?
Immunomodulatory neuropeptides (*acting through GPCRs) Cytokines and Related peptides Appetite control Body weight regulation Cachexia of chronic diseases INFLAMMATION INFECTION Obesity Diabetes Atherosclerosis CNS disease
Jie Zhao Lilian vanVlerken Christina Chandras Maria Venihaki Jerome Gay Joseph Majzoub Children’s Hospital Endocrine Division, Boston Harris Pothoulakis Efi Kokkotou Beth Israel Hospital, Boston Yassemi Koutmani Despina Xanthaki Christina Chandras Maria Venihaki Developmental Biology Section, Foundation for Biomedical Research, Academy of Athens (IIBEAA)
2.0 1.5 1.0 + 0.5 + + 0.0 Reduced toxin A-associated histologic damage and inflammation in CRH deficient mice Epithelial damage Congestion & edema PMN Crh +/+ Crh - / - Histologic scores +, ++ significantly different (p<0.05, 0.01) from WT toxin A treated mice
50 40 30 20 10 0 Toxin A modulation of plasma corticosterone in CRH deficient mice buffer toxin A plasma corticosterone (mg/dl) * * Crh -/- Crh +/+ Crh -/- Crh +/+ Time 0 4 hours * significantly different (p<0.05,p<0.001) from their respective Crh +/+counterparts (4 hours)
140 120 100 80 60 40 20 0 CRH receptor antagonists reduce toxin A-induced ileal fluid secretion * + + Intestinal fluid secretion (mg/cm) + + Control TxA TxA + antalarmin TxA + a-helical CRH * significantly different (p<0.05) from controls ; ++ significantly different (p<0.01) from Tx A
LEPTIN and HPA axis Leptin, the product of the ob gene, interacts reciprocally with the hypothalamic-pituitary adrenal (HPA) axis: Glucocorticoid, stimulates the expression of the ob gene in adipocytes. Administration of leptin increases CRH and ACTH secretion, while it decreases glucocorticoid release in rodents.
TLR4 expression in Crh+/+ and Crh-/- tissues following LPS or saline administration Crh+/+ saline 3.5 p<0.05 Crh+/+ LPS Crh-/- saline 3 Crh-/- LPS 2.5 p<0.05 2 p<0.01 mRNA expression (densiometric units) 1.5 p<0.05 1 0.5 0 lung thymus peritoneal leukocytes pituitary