Peripheral actions of the stress hormone Corticotropin Releasing Hormone (CRH): focus on its immunomodulatory effects

ACTH Glucocorticoid Hypothalamus Adrenal cortex Anterior pituitary CRH The Hypothalamic-Pituitary Adrenal (HPA) axis VP

Sites of CRH synthesis BRAIN IMMUNE SYSTEM T-lymphocyte PLACENTA SPINAL CORD GI TRACT LUNG

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 INFLAMMATION ACTH CRH GC PERIPHERAL CRH

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 ??

l Control CRH M Ctrl CRH Ctrl CRH p50p65 NF-  B n.s.

Ctrl LPS 10  g/ml CRH M competitor Mutant competitor CRH/  hCRH M n.s. NF-  B

IBIB CONTROL CRH 15min CRH 30min

Concentration-dependent effects of CRH on MAPK activation in human leukocytes p38 (5 min) p42/44 (5 min) JNK (30 min) Control CRH (10 -9 M) CRH (10 -8 M) CRH (10 -7 M) TNF  (10ng/ml) CRH (10 -6 M) Control CRH (10 -9 M) CRH (10 -8 M) CRH (10 -7 M) TNF  (10ng/ml) CRH (10 -6 M) Control CRH (10 -9 M) CRH (10 -8 M) CRH (10 -7 M) EGF (10ng/ml ) CRH (10 -6 M)

Is there an important physiological role for peripheral CRH itself in the regulation of the inflammatory process ?

The Crh-deficient animal model Normal lifespan Normal weight Normal food intake Blunted/absent circadian rhythmicity Low basal corticosterone Impaired ACTH and corticosterone responses to multiple stressors CRH ACTH GC Muglia et al. Nature, 1995

Sources of peripheral CRH ACTH leukocytes Nerve fibers ? CRH Hypothalamus Pituitary Adrenals Inflammatory sites glucocorticoid

EPINEPHRINE CRH INFLAMMATION GLUCOCORTICOID

IL-6 (pg/mL) Time (hr) Crh+/+ turpentine Crh-/- turpentine CYTOKINE RESPONSE TO TURPENTINE

ACTH (pg/mL) Time (hr) * * Crh +/+ saline Crh+/+ turpentine Crh -/- saline Crh-/- turpentine Corticosterone (ug/dL) Time (hr) HORMONAL RESPONSES TO TURPENTINE

* * # # # * Body weight (% of control) Day following the injection Crh +/+ saline Crh+/+ turpentine Crh -/- saline Crh-/- turpentine METABOLIC RESPONSES TO 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 ?

HPA axis response 1 hr after LPS or saline * : P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment ACTH (pg/ml) Corticosterone (  g/dl) * $ * * * Crh+/+ Saline Crh-/- SalineLPS Crh+/+ Saline Crh-/- SalineLPS

ACTH (pg/ml) Corticosterone (  g/dl) HPA axis response 24 hr after LPS or saline Crh+/+ Saline Crh-/- SalineLPS Crh+/+ Saline Crh-/- SalineLPS $ * * * * * : P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment

Effect of pre-treatment with antalarmin on the HPA axis response 24 hr after LPS Corticosterone (  g/dl) ACTH (pg/ml) Vehicle + LPS Antalarmin + LPS Vehicle + LPS Antalarmin + LPS * n.s.

Glucose (  g/dl) Crh+/+ Crh-/- Plasma glucose levels 1 and 24 hr after LPS or saline Time (hr) following the injection Saline LPS

Crh+/+Crh-/ basalsalineLPS basalsalineLPS Pg/ml P<0.01 Plasma epinephrine at basal levels, and 24 hours post-LPS or –saline injection

CRH GC Systemic Bacterial Inflammation LEPTIN ACTH CYTOKINES ? ? ??? Decreased food intake / Body weight loss-cachexia ?

Plasma TNF  1 1 / 2, 4 hours and 24 hours post-LPS injection / 2 hour Crh+/+Crh-/-Crh+/+Crh-/- Pg/ml Crh-/-Crh+/+ 4 hours 24 hours P<0.01 * * * : statistical significance of p<0.01 from TNF  levels 4 and 24 hours post-LPS injection between animals of the same genotype

4 hour 24 hour Pg/ml Crh+/+Crh-/- Crh+/+Crh-/- Plasma IL-6 4 hours and 24 hours post-LPS injection

CRH GC Systemic Bacterial Inflammation LEPTIN ACTH CYTOKINES ? ? TLRs ??? 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-  B 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.

Plasma leptin 24 hr after LPS or saline * : P<0.05 between treatments in the same genotype $: P<0.05 between genotypes following the same treatment Leptin (ng/ml) $ * * Crh+/+ Saline Crh-/- SalineLPS

Leptin (ng/ml) Effect of pre-treatment with antalarmin on leptin secretion 24 hrs after LPS Vehicle + LPS Antalarmin + LPS * * P=0.05

Responses of Crh-/- splenocytes to LPS

* * TNF-  (pg/ml/million of plated cells) control $ LPS control Crh+/+ Crh-/- Crh+/+Crh- /- TNF-   - actin AB

IL-1  (pg/ml/million of plated cells) controlLPScontrolLPS Crh+/+Crh-/- * $ Crh+/+Crh-/- IL-1   -actin AB

Crh HT WT SPL WT SPL +LPS CRHKO

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) INFLAMMATION INFECTION Obesity Diabetes Atherosclerosis CNS disease Appetite control Body weight regulation Cachexia of chronic diseases Cytokines and Related peptides

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)

Reduced toxin A-associated histologic damage and inflammation in CRH deficient mice Crh +/+ Crh - / - +, ++ significantly different (p<0.05, 0.01) from WT toxin A treated mice Epithelial damage Congestion & edema PMN Histologic scores

Toxin A modulation of plasma corticosterone in CRH deficient mice * significantly different (p<0.05,p<0.001) from their respective Crh +/+ counterparts (4 hours) Crh +/+ Crh -/- Crh +/+ Crh -/- Time 04 hours * * plasma corticosterone (  g/dl) buffer toxin A

Intestinal fluid secretion (mg/cm) Control TxA TxA + antalarmin * significantly different (p<0.05) from controls ; ++ significantly different (p<0.01) from Tx A * + TxA +  -helical CRH CRH receptor antagonists reduce toxin A-induced ileal fluid secretion

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.

lungthymusperitoneal leukocytes pituitary mRNA expression (densiometric units) Crh +/+ saline Crh+/+ LPS Crh-/- saline Crh -/- LPS p<0.05 p<0.01 p<0.05 TLR4 expression in Crh+/+ and Crh-/- tissues following LPS or saline administration

d - 1d024h48h time Weight variation (% of d-1) Crh +/+ controls Crh +/+ TNBS Crh -/- controls Crh -/- TNBS *** ** A B h48h Food intake (g) Crh +/+ controls Crh +/+ TNBS Crh -/- controls Crh -/- TNBS

Total p38 Control TNFα (10ng/µl) CRH (10 -6 M)Control TNFα (10ng/µl) CRH (10 -6 M) Phospho-p38

CRH intestine CRH Inflammatory stimulus CRH deficient inflammation CRH2 Inflammatory stimulus CRH IBD ?