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

INFLAMMATIONACTH

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

Co

ntr

ol

CR

H 1

0-7 M

Ctrl CRH Ctrl CRH

p50 p65

NF-B

n.s.

Ctrl

LPS 10

g/m

lCRH 1

0-7 M

com

petit

or

Mut

ant c

ompe

titor

CRH/hC

RH 10

-6 M

n.s.

NF-B

IB

CO

NT

RO

L

CR

H 1

5min

CR

H 3

0min

Concentration-dependent effects of CRH on MAPK activation in human leukocytes

p38 (5 min) p42/44 (5 min) JNK (30 min)

Con

trol

CR

H (1

0-9M

)C

RH

(10

-8M

)C

RH

(10

-7M

)

TN

F (1

0ng/

ml)

CR

H (1

0-6M

)

Con

trol

CR

H (1

0-9M

)C

RH

(10

-8M

)C

RH

(10

-7M

)

TN

F (1

0ng/

ml)

CR

H (1

0-6M

)

Con

trol

CR

H (1

0-9M

)C

RH

(10

-8M

)C

RH

(10

-7M

)

EG

F (1

0ng/

ml )

CR

H (1

0-6M

)

Is there an important physiological role

for peripheral CRH itself in the regulation

of the inflammatory process ?

The Crh-deficient animal model

Normal lifespanNormal weightNormal food intake

Blunted/absent circadianrhythmicity

Low basal corticosterone

Impaired ACTH and corticosterone responsesto multiple stressors

CRH

ACTH

GC

Muglia et al. Nature, 1995

Sources of peripheral CRH

ACTH

leukocytes

Nerve fibers?

CRHHypothalamus

Pituitary

Adrenals

Inflammatory sites

glucocorticoid

EPINEPHRINE CRH

INFLAMMATION

GLUCOCORTICOID

1 4 8 16 24 300

600

1200

1800

2400IL

-6 (

pg

/mL

)

Time (hr)

Crh+/+ turpentine

Crh-/- turpentine

CYTOKINE RESPONSE TO TURPENTINE

1 4 8 16 24 30 480

100

200

300

400

500

600

AC

TH

(p

g/m

L)

Time (hr)

*

*

Crh +/+ salineCrh+/+ turpentine Crh -/- salineCrh-/- turpentine

1 4 8 16 24 30 480

15

30

45

60

Co

rtic

ost

ero

ne

(ug

/dL

)Time (hr)

HORMONAL RESPONSES TO TURPENTINE

1 2 3 4 5

0.05

0.10

0.15

0.20

0.25

0.30

**

*

*

* #

* #

Fo

od

in

take

(g

r)/m

ou

se w

eig

ht

(gr)

Days following the injection

1 2 3 4 5 6

92

96

100

104

108 *

*

##

#

*

Bo

dy

wei

gh

t (%

of

con

tro

l)

Day following the injection

Crh +/+ salineCrh+/+ turpentine Crh -/- salineCrh-/- 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

0

150

300

450

600

750

900

AC

TH

(p

g/m

l)

0

20

40

60

80

Co

rtic

ost

ero

ne

(g

/dl)

*

$*

* *

Crh+/+

Saline

Crh-/-

SalineLPS LPS

Crh+/+

Saline

Crh-/-

SalineLPS LPS

0

100

200

300

350

AC

TH

(p

g/m

l)

0

10

20

30

40

50

60

Co

rtic

ost

ero

ne

(g

/dl)

HPA axis response 24 hr after LPS or saline

Crh+/+

Saline

Crh-/-

SalineLPS LPS

Crh+/+

Saline

Crh-/-

SalineLPS LPS

$

*

*

*

*

*: 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

0

8

16

24

32

40

Co

rtic

ost

ero

ne

(g

/dl)

0

75

150

225

300

375

AC

TH

(p

g/m

l)

Vehicle+

LPS

Antalarmin+

LPS

Vehicle+

LPS

Antalarmin+

LPS

* n.s.

Glu

cose

(g

/dl)

1 24

50

100

150

200

250

50

100

150

200

Crh+/+

Crh-/-

Plasma glucose levels 1 and 24 hr after LPS or saline

Time (hr) following the injection

SalineLPS

Crh+/+ Crh-/-

0

2000

4000

6000

basal saline LPS basal saline LPS

Pg/

ml

P<0.01

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 11/2, 4 hours and 24 hours post-LPS injection

0

5000

10000

15000

20000

25000

30000

11/2 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

0

1000

2000

3000

4000

5000

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

• AngiogenesisCRH 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 healingCrh-/- 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

05101520253035

Lep

tin

(n

g/m

l)

$

*

*

Crh+/+

Saline

Crh-/-

SalineLPS LPS

25

0

5

10

15

20L

epti

n (

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

0

50

100

150

200

250

*

*

TN

F-

(p

g/m

l/m

illi

on o

f p

late

d c

ells

)

control

$

LPSLPS control

Crh+/+ Crh-/- Crh+/+ Crh-/-

TNF-

-actin

A B

0.0

2.5

5.0

7.5

10.0

IL-1

(p

g/m

l/m

illi

on o

f p

late

d c

ells

)

control LPS control LPS

Crh+/+ Crh-/-

* $

Crh+/+Crh-/-

IL-1

-actin

A B

Crh

HT

WTSPL

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 theregulation of innate immunity?

What is the contribution of the above hormonesin the metabolic changes triggered by inflammation?

Immunomodulatory neuropeptides (*acting through GPCRs)

INFLAMMATIONINFECTION

ObesityDiabetesAtherosclerosisCNS disease

Appetite controlBody weight regulationCachexia of chronic diseases

Cytokines and Related peptides

Jie ZhaoLilian vanVlerkenChristina ChandrasMaria VenihakiJerome Gay

Joseph MajzoubChildren’s Hospital Endocrine Division, Boston

Harris PothoulakisEfi Kokkotou

Beth Israel Hospital, Boston

Yassemi KoutmaniDespina XanthakiChristina ChandrasMaria VenihakiDevelopmental Biology Section, Foundation for Biomedical Research, Academy of Athens (IIBEAA)

Reduced toxin A-associated histologic damage Reduced toxin A-associated histologic damage and inflammation in CRH deficient miceand inflammation in CRH deficient mice

Crh +/+

Crh - / -

+, ++ significantly different (p<0.05, 0.01) from WT toxin A treated mice

Epithelial damage Epithelial damage Congestion & edemaCongestion & edema PMNPMN

His

tolo

gic

scor

es

0.0

0.5

1.0

1.5

2.0

+

+ +

Toxin A modulation of plasma corticosterone Toxin A modulation of plasma corticosterone in CRH deficient micein CRH deficient mice

* significantly different (p<0.05,p<0.001) from their respective Crh +/+ counterparts (4 hours)

0

10

20

30

40

50

Crh +/+ Crh -/- Crh +/+ Crh -/-

Time 0 4 hours

**

pla

sma

cort

icos

tero

ne

(g/

dl) buffer

toxin A

0

20

40

60

80

100

120

140

Inte

stin

al f

lui d

sec

reti

o n (

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 CRH receptor antagonists reduce toxin A-induced ileal fluid secretion 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.

0

0.5

1

1.5

2

2.5

3

3.5

lung thymus peritoneal leukocytes pituitary

mR

NA

exp

ress

ion

(d

ensi

omet

ric

un

its)

Crh+/+ saline

Crh+/+ LPS

Crh-/- saline

Crh-/- LPS

p<0.05

p<0.01

p<0.05

p<0.05

TLR4 expression in Crh+/+ and Crh-/- tissuesfollowing LPS or saline administration

LPS

LPS LBP

LPS

CYTOPLASM

TLR4 MD-2

MyD88

IRAK

TRAF6

JNK p38 ERK1

/2

M A P K

IKK/ß

NIK

IB P P

P NF-B

P P

IB

NUCLEUS

TRANSCRIPTION FACTORS

c-Jun c-Fos CREB Elk1

Transcription Transcription NF-B

P

CD14

80

85

90

95

100

105

110

d - 1 d0 24h 48htime

Wei

ght v

aria

tion

(%

of

d-1)

Crh+/+ controls

Crh+/+ TNBS

Crh-/- controls

Crh-/- TNBS

***

****

A

B

0

1

2

3

4

5

24h 48h

Foo

d in

take

(g) Crh+/+ controls

Crh+/+ TNBS

Crh-/- controls

Crh-/- TNBS

Total p38

Con

trol

TNFα

(10n

g/µl

)

CR

H (1

0-6 M

)

Con

trol

TNFα

(10n

g/µl

)

CR

H (1

0-6 M

)

Phospho-p38

CRH

CYTOPLASM

CRHR

JNK p38 ERK1

/2

M A P K

IKK/ß

NIK

IB P P

P NF-B

P P

IB

NUCLEUS

TRANSCRIPTION FACTORS

c-Jun c-Fos CREB Elk1

Transcription Transcription NF-B

P

G

?

?

CRH

intestine

CRHCRH

CRH

CRH

Inflammatory stimulus

CRH deficient

inflammation

CRH2CRH2

Inflammatorystimulus

CRH IBD?