The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of...

The Sympathetic Nervous System (SNS)

A not so “sympathetic”

regulator of immune

function in autoimmune

disease:RA as an example

Kent State University‡

Loma Linda University¤

Dianne Lorton ‡

Denise Bellinger ¤

3rd International Conference on Clin,

& Cell. Immunol, Sept 23- Oct. 1, 2014

Blood Borne

SignalsSympathetic

Nervous

Neural-Immune Cross-Talk

SignalsTNF, IL-1, IL-6

Bone

Marrow

Thymus

Spleen

BloodGALT

Lymph

Node

Lymphocytes

Macrophages

Dendritic cells

Others

Pathogens DiseaseX

Nervous

System

� Autoimmune Disease

� Chronic inflammatory response

� Production of autoantibodies

� Loss of Tolerance: Imbalance

between autoreactive effector T cells

Rheumatoid Arthritis

(CD4+ Th1 & Th 17) and T reg cells

� Th cell balance regulated by the SNS

� SNS activity is chronically elevated in RA patients

� How this impacts Th cell balance is not known

T ββββ2222

CD4+ ββββ2 (200-750 sites/cell)

CD4+ Th1 clones ββββ2 (250 sites/cell)

SNS Regulates Th Cell Differentiation via β2-AR

Activation of cAMP-PKA Pathway

.L

ACGs

nerve

terminal

B

APCs

αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222

CD4+ Th1 clones ββββ2 (250 sites/cell)

CD4+ Th2 clones (no detectable ββββ2)

CD4+ Treg cell 1?

CD4+ Th17 cells?

ACGs3

β2-AR

G –protein

Adenylate cyclase

Lipid Raft

Gs

ATP cAMP5’ AMP

PKA

AC

1Guereschi et al. Eur J Immunol. 2013 Apr;43(4):1001-12.

β2-AR Shifts Th0 cell →Th2 Differentiation

TNF-α, IL-12

IFN-γ, IL-2

Th0-CellTh1

Th1-Cell

Th2-Cell

APCMicrobial

Viral

β2-ARαααα-AR

Tregs?

Th17?

IL-10 IL-4

Th2-Cell

Th2Th0-Cell

Th2-Cell

APCParasite

Allergen

Guereschi et al., 2013

Hypothesis: Reduce disease severity is due in

part to a β2-AR driven shift in Th1 vs Th2 cell

balance.

Disease Onset

Ag Processing/

Clearance

Effector

Phase

Challenge

Joint

InflammationClearance Phase

increasing severity

ββ22--AR agonistAR agonistss

Inflammation

(AA: Lorton et al., 1998; 2004)

(CIA: Malfait et al., 1999; Härle et al., 2005) AA, Terbutaline D12-28

Day 0Terbutaline (β -AR agonist;

Day 12-28

Experimental Design

Adjuvant-Induced

Arthritis (AA)

CFA (0.3 mg M. butyricum

in 100 µµµµl MO)

Terbutaline (β2-AR agonist;

1.5 mg/ml/day i.p.)

Saline Vehicle

Outcome AssessmentsTh1/2 cell cytokines (ELISAs)

Foot Pad Swelling (data not shown)

X-ray analysis (data not shown)

Day 28

Draining Lymph

Nodes (DLN)

Spleen

PBMCs

100

200

300

400

500

IFN

- γγ γγ(p

g/m

l)Spleen: Failure of a β2-AR agonist to shift

from a Th1 to Th2 cytokine profile

50

100

150

200

250

300

TN

F-α

(pg

/ml)

A. B.

No Change in IL-4; (40-80 pg/ml)

0

VEH TERB

0

10

20

30

40

50

VEH TERB

IL-2

(p

g/m

l)

0

200

400

600

800

1000

VEH TERB

IL-1

0 (

pg

/ml)

*

0

VEH TERB

Anova with Bonferoni post-hoc test, N = 8

C. D.

75

150

225

300

375

450

IFN

- γγ γγ(p

g/m

l)

*

0

50

100

150

200

250

TN

F-α

(pg

/ml)

DLN: β2-AR agonist promotes a Th1

cytokine profileA. B.

0

VEH TERB

No Change in IL-4 (40 -80 pg/ml)

0

VEH TERB

0

10

20

30

40

50

60

70

VEH TERB

IL-2

(p

g/m

l)

T

0

50

100

150

200

250

VEH TERB

IL-1

0 (

pg

/ml)

C. D.

Anova with Bonferoni post-hoc test, N = 8; *P<0.05

0

20

40

60

80

100

120

140

VEH TERB

TN

F-α

(pg

/ml)

0

100

200

300

400

500

600

VEH TERB

IFN

- γγ γγ(p

g/m

l)PBMC: Failure of a β2-AR agonist to shift

Th1 cytokine profilesA. B.

VEH TERB

0

20

40

60

80

100

120

140

VEH TERB

IL-2

(p

g/m

l)

0

10

20

30

40

50

VEH TERB

IL-1

0 (

pg

/ml)

VEH TERB

Anova with Bonferoni post-hoc test, N = 8; *P<0.05No Change in IL-4 (40 -80 pg/ml)Wahle et al., 2006. Failure of catecholamines to shift T-cell cytokine responses toward

a Th2 profile in patients with rheumatoid arthritis. Arthritis Res. Ther., 8(5):R138.

C. D.

Conclusions� Different responses in each tissue examined: animal

models critical for understanding RA

� Stimulating β2-ARs after disease onset fails to

inhibit Th1 cell driving cytokines

� Spleen: β2-AR agonists produced no change IFN-γ, IL-2,

α� Spleen: β2-AR agonists produced no change IFN-γ, IL-2,

IL-4 or TNF-α, and increased IL-10 (source ?)

� DLN stimulating β2-ARs promotes IFN-γ & IL-2, no

change in IL-4, IL-10, TNF-α

� β2-AR stimulation under normal circumstances

inhibits IFN-γ and IL-2 production via cAMP-PKA

� These findings indicate abnormal β2-AR functions

Conclusions

� In spleen cells, the inability of terbutaline to reduce

IFN-γ and IL-2 could be easily explained by the

well-known down-regulation and desensitization of

β2-AR with repeated stimulation.

� Subsequent, cAMP assays and receptor binding � Subsequent, cAMP assays and receptor binding

experiments, confirmed this hypothesis (Lorton et

al., Clin Dev Immunol., 2013)

� However, the terbutaline-induced increase in IFN-γand IL-2 were intriguing. � not explained by

canonical signaling of β2-AR

Does Altered β2-AR Coupling to Second Messengers

Occur in DLNs in AA: cAMP-PKA to ERK1/2?

β-Arrestin-dependent, G Protein-

independent ERK1/2 Activation by the β2

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 2, pp. 1261–1273, January 13, 2006

© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Adrenergic Receptor*

Received for publication, June 16, 2005, and in revised form, November 2, 2005 Published,

JBC Papers in Press, November 9, 2005, DOI 10.1074/jbc.M506576200

Sudha K. Shenoy‡1,Matthew T. Drake‡2, Christopher D. Nelson‡, Daniel A. Houtz‡,

Kunhong Xiao‡, Srinivasan Madabushi§, Eric Reiter‡¶, Richard T. Premont‡, Olivier

Lichtarge§, and Robert J. Lefkowitz‡3

GRK5/6

P3

ACGs3P 3

PKA 6b

5

Altered Receptor Signaling in the DLN?

.L

nerve terminal

x

.L

ACGs3

ATP cAMP

L

nerve terminal

.

Gene Regulation

ERK1/2

MAPK

PP

ββββ-Arrestin

ATP cAMP

NoncanonicalCanonical

Gene Regulation

PKA

Transcription Factors Transcription Factors

CFA (0.3 mg M. butyricum

in 100 µµµµl MO)Mineral Oil (MO)

M. Butyricium (in saline;

SMB)

Saline

Day 1

Terbutaline (β -AR agonist;

Day 12-28

Hypothesis: Terbutaline induces a shift in β2-ARs

signaling from cAMP-PKA to ERK 1/2 in the DLN

Adjuvant-Induced

Arthritis (AA)

Saline Terbutaline (β2-AR agonist;

1.5 mg/ml/day i.p.)

Saline Vehicle

Outcome AssessmentsDLN: ββββ2-AR Western Blots

(antibodies to detect β2-ARs,

and β2-ARs phosphorylated by

PKA and or GRK)

Day 21 or 28

Draining Lymph

Nodes (DLN)

Unchanged DLN β2-AR Density Late Disease

0.4

0.6

0.8

1.0

No

rma

lize

d β

2-A

RT

(O.D

.)

0.4

0.6

0.8

1.0

No

rma

lize

d β

2-A

R

(OD

)

***

***A. D21 B. D28

5Cββββ2-ARDay 28

Saline MO SMB CFA

0.0

0.2

0.4

Saline MO SMB CFA

No

rma

lize

d

(O.D

.)

0.0

0.2

0.4

Saline MO SMB CFA

No

rma

lize

d

(OD

)

P < 0.05; P < 0.01, P<0.001ANOVA; Bonferoni Post-Hoc Test * ***N=4; **

Altered Receptor Signaling in the DLN?

PKA

P3

.L

ACGs3

nerve terminal

xDesensitization

GRK2

P

Conclusions: These findings along with increased IFN-γγγγ indicate

that β2-ARs in DLN are NOT down-regulated or desensitized.

P

Recycled to

membrane Degraded

or

P

ββββ-Arrestin

L

0.0

0.4

0.8

1.2

1.6

2.0

2.4

Saline MO SMB CFA

β2-A

RP

KA:β

2-A

RT

Ra

tio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline MO SMB CFA

β2-A

RP

KA:β

2-A

RT

Ra

tio

***** **

**

β2-AR phosphorylated by PKA and GRK in DLN

A. D21 B. D28

P < 0.05; P < 0.01, P<0.001ANOVA; Bonferoni Post-Hoc Test * ***N=4; **

Saline MO SMB CFA Saline MO SMB CFA

0.0

0.3

0.6

0.9

1.2

1.5

Saline MO SMB CFA

β2-A

RG

RK

:β2-A

RT

Ra

tio

0.0

0.2

0.4

0.6

0.8

Saline MO SMB CFA

β2-A

RG

RK

:β2-A

RT

Ra

tio

***** **

*

*

*****C. D21 D. D28

GRK5/6

P3

ACGs3P 3

PKA 6b

5

Altered Receptor Signaling in the DLN?

L

nerve terminal

xPKA

P3

L

ACGs3

nerve terminal

or

xDesensitization

GRK2

P

Gene Regulation

ERK1/2

MAPK

PP

ββββ-Arrestin

Transcription Factors

P

Recycled to

membrane Degraded

or

P

ββββ-Arrestin

L

Conclusions: These findings coupled with increased IFN-γγγγ,

provide support β2-AR signaling via ERK1/2.

Summary

� Findings support a shift in β2-AR receptor

signaling from cAMP-PKA to ERK1/2 in

DLN

• β2-AR agonist elevated IFN-γ and IL-2• β2-AR agonist elevated IFN-γ and IL-2

• No change in β2-AR density,

• Receptor phosphorylation by PKA increased

PKA (day 21) and GRK phosphorylation (day 21

and 28)

Future Studies

• Are GRK5/6 and ERK 1/2 elevated in DLN cells?

• Can production of IFN-γ be blocked by inhibitors of

ERK1/2 pathway?

• Why the different profiles in the spleen and DLN?• Why the different profiles in the spleen and DLN?

– Inflammatory cytokine levels

– CFA distribution/concentration

• Does the SNS regulate balance between Th17

and Treg cells?

Acknowledgements

Cheri Lubahn, Ph.D.

Jill Schaller Tracy Osredkar

Kent State University

Loma Linda UniversitySchool of Medicine

Denise Bellinger, Ph.D.

Christine Molinaro

Funded by: NIMH, NIAMS, Arizona Disease CRC, Sun Health Research Institute &Sun City West Community Center Fund, LLU Anatomy and Pathology Dept.

SNS Function:

Respond to stress

&

maintain normal

body functions

(homeostasis)

The SNS

integrates the

functions of many

systems required

to mount an

immune response

Bone

T ββββ2222CD4+ ββββ2 (200-750 sites/cell)

CD8+ ββββ2 (500-2500 sites/cell)

CD4+ Th1 clones ββββ (250 sites/cell)

SNS Inhibition of Th1 Cytokines (IFN-γ and IL-2)

to Push Th2 Cell Differentiation Occurs via β2-AR

of cAMP-PKA Pathway

.L

ACGs

nerve

terminal

B

Monocyte/

macrophage

αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222

CD4+ Th1 clones ββββ2 (250 sites/cell)

CD4+ Th2 clones (no detectable ββββ2)

CD4+ Treg cell 1

CD4+ Th17 cells?

ACGs3

β2-AR

G –protein

Adenylate cyclase

Lipid Raft

Gs

ATP cAMP5’ AMP

PKA

AC

1Guereschi et al. Eur J Immunol. 2013 Apr;43(4):1001-12.

The SNS

integrates the

functions of many

systems required

to mount an

immune response

Function:

Bone

Function:

Respond to stress

&

maintain normal

body functions

(homeostasis)

(allostasis- a new

“adaptive”

normal)

Reciprocal Immune System to

SNS Communication in

RA

n

Circulating

TNF-αααα. IL-1,

IL-6 RVLM

Hypothalamus

Cerebral Cortex

(Insula, limbic)

Stress

� Mechanism for

emotional distress to

impact health & disease n

Spleen

Pre-ganglionic

nerves

Joints

Lymph nodesimpact health & disease

� ~ 80% of patients

associate disease onset

with a severe emotional

life stressor (Trigger?)

� Stroke Victims: no RA in

paralyzed limbs (↓↓↓↓ vs ↑↑↑↑SNS nerve activity)

SNS

Saline Rx

0

500

1000

1500

2000

2500

3000

3500

4000

4500 4497Bmax =

Kd = 12.08

CP

M

0 1000 2000 3000 4000 50000

100

200

300

400Scatchard

Bound

Bo

un

d/F

ree

SMB Rx

0

1000

2000 Bmax =Kd =

2023

8.216

cp

m

0 500 1000 1500 2000 25000

50

100

150

200

250

Scatchard

Bound

Bo

un

d/F

ree

Splenocyte β2-AR Receptor Binding in Arthritic

Rats: Saturation Curves

0 25 50 75 100 1250

[I125

]CYP (pM)

0 25 50 75 100 1250

[I125

]CYP (pM)

mineral oil Rx

0 25 50 75 100 1250

1000

2000

3000

4000

5000 5405Bmax =Kd = 25.82

[I125

]CYP (pM)

cp

m

0 1000 2000 3000 4000 5000 60000

100

200

300

Scatchard

Bound (cpm)

Bo

un

d/F

ree

CFA Rx

0 25 50 75 100 1250

500

1000

1500

2000

2500

3000

3500Bmax =Kd =

382823.41

[I125] CYP (pM)

cp

m

0 1000 2000 3000 4000 50000

100

200

Scatchard

Bound (cpm)

Bo

un

d/F

ree

SNS-IS Cross-Talk Pathology in RA:

Reduced Spleen β2-AR Density Late Disease

0.6

1.0

No

rma

lize

d β

2-A

RT

(O.D

.)

0.4

0.6

0.8

1.0

No

rma

lize

d β

2-A

RT

(O.D

)

**

* *** * **A. D21 B. D28

Lorton et al., (2013) Clin Dev Immunol.;2013:764395P < 0.05; P < 0.01, P<0.001

ANOVA; Bonferoni Post-Hoc Test

* ***N=4; **

-0.2

0.2

Saline MO SMB CFA

No

rma

lize

d

0.0

0.2

Saline MO SMB CFA

No

rma

lize

d

(O.D

)

β2-AR Phosphorylation Patterns in the

Spleen

0.0

0.4

0.8

1.2

1.6

Saline MO SMB CFA

β2

-AR

PK

A:β

2-A

RT

Ra

tio

0.0

0.4

0.8

1.2

1.6

Saline MO SMB CFA

β2-A

RP

KA:β

2-A

RT

Ra

tio

**** * *** *

P < 0.05; P < 0.01, P<0.001

ANOVA; Bonferoni Post-Hoc Test

* ***N=4; **

Saline MO SMB CFA

0.0

0.2

0.4

0.6

0.8

1.0

Saline MO SMB CFA

β2-A

RG

RK

:β2-A

RT

Ra

tio

Saline MO SMB CFA

0.0

0.4

0.8

1.2

1.6

2.0

2.4

Saline MO SMB CFA

β2-A

RG

RK

:β2-A

RT

Ra

tio

****** **** *

Hypothesis: Chronic high SNS activity in RA induces

β2-AR down regulation and desensitization

aCFA/ICA (vehicle)

Autoantigen: HSP 65

Day 1a0.3 mg Mycobacterium butyricum

in 0.1 ml sterile mineral oil

Day 28

Day 1

cAMP assay

ββββ2-AR Receptor Binding Assays

ββββ2-AR Western Blots

using antibodies to detect

phosphorylated receptor

Day 21 or 28Harvest Spleen & DLN cells

AA

Day 28

Day 14

Day 28

Veh AA

Adjuvant-Induced Arthritis Rat Model

Day 1

Day 14

Day 1 Veh AA

Day 28

A B C

D

E

Day 28CFA (0.3 mg M. butyricum in

100 µµµµl MO)

0 3 7 12 21 28 Days

Autoreactive

T cells in

DLNs

Disease

InductionDisease

Onset

Autoreactive

T cells in

spleen

Chronic

Disease

Peak

Disease

F

SNS-IS Cross Talk in the Spleen?

ß2-AR & Signaling via the Canonical Pathway:

cAMP

.L

ACGs

nerve

terminal

P3

.L

ACGs

3

nerve terminal

xP

L

��� � ��

3

G –protein

Adenylate cyclase

Lipid Raft

Gs

ATP cAMP5’ AMP

PKAAC

Hypothesis: Chronic high SNS activity in RA induces β2-AR

down regulation and desensitization in splenocytes

PKA

P

P

Dephosphorylated

Recycled to membrane

Transported

to lysosome

Degraded

or

xDesensitization

GRK2

P

P

CFA (0.3 mg M. butyricum

in 100 µµµµl MO)Mineral Oil (MO)

M. Butyricium (in saline;

SMB)

Saline

Day 1

Terbutaline (β -AR agonist;

Day 12-28

Hypothesis: Chronic high SNS activity in RA induces

β2-AR down regulation and desensitization in the spleen

Saline Terbutaline (β2-AR agonist;

1.5 mg/ml/day i.p.)

Saline Vehicle

Outcome AssessmentsSpleen: cAMP assay (spleen)

ββββ2-AR Binding Assays

DLN: ββββ2-AR Western Blots

(antibodies to detect β2-ARs)

Day 21 or 28

Spleen

-6cell

s)

250

300

350

NoRx

Forskolin

Isoproteronol* * *

SNS-IS Cross-Talk Pathology in RA:

β2-AR Agonist Fails to Induce cAMP in SplenocytescA

MP

(fM

ol/

2 x

10

-

Non-AA AA

0

50

100

150

200

250 Isoproteronol

*

Anova with Bonferoni post-hoc test,

Day 28; N = 8; *P<0.05Lorton et al., (2013) Clin Dev Immunol.;2013:764395

SNS-IS Cross-Talk Pathology in RA:

Splenocyte β2-AR have Reduced Agonist

Affinity and Density

20

25

Kd

(I

CY

P (

pM

))

# # 1000

*

#

A. B.

Mineral Oil

Saline SMB CFA0

5

10

15

Kd

(I

CY

P (

pM

))

Saline Mineral Oil

SMB CFA

0

250

500

750

Bm

ax (

sit

es/c

ell)

*

P < 0.05; P < 0.01

ANOVA; Bonferoni Post-Hoc Test

* #N=6; Lorton et al., (2013) Clin Dev Immunol.;2013:764395

CFA (0.3 mg M. butyricum

in 100 µµµµl MO)Mineral Oil (MO)

M. Butyricium (in saline;

SMB)

Saline

Day 1

Terbutaline (b2-AR agonist;

Day 12-28

Hypothesis: Chronic high SNS activity in RA induces

β2-AR down regulation and desensitization in the spleen

Saline Terbutaline (b2-AR agonist;

1.5 mg/ml/day i.p.)

Saline Vehicle

Outcome AssessmentsSpleen: cAMP assay (spleen)

ββββ2-AR Binding Assays

DLN: ββββ2-AR Western Blots

(antibodies to detect β2-ARs)

Day 21 or 28

Draining Lymph

Nodes (DLN)Spleen

Altered Receptor Signaling in the DLN?

GRK5/6

P3

.L

ACGs3

nerve terminal

P 3

PKA6b

��� �

�� ��

PKA

P3

.L

ACGs3

nerve terminal

xDesensitization

↑↑↑↑↑↑↑↑↑↑↑↑ activity

��� �

�� ��

GRK2

P

Transcription Factors

Gene Regulation

ERK1/2

MAPK

5

PP

P

Dephosphorylated

Recycled to membrane Transported to lysosome

Degraded

or

Canonical

P

ββββ-Arrestin

L