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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
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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
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� 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
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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
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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
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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
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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)
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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; **
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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
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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
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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.
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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)
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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?
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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.
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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
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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.
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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)
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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
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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
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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
)
![Page 30: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/30.jpg)
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
****** **** *
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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
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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
![Page 33: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/33.jpg)
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
![Page 34: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/34.jpg)
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
![Page 35: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/35.jpg)
-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
![Page 36: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/36.jpg)
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
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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
![Page 38: The Sympathetic Nervous System (SNS) · The Sympathetic Nervous System (SNS) ... Activation of cAMP-PKA Pathway. L Gs AC nerve terminal B APCs ... 1.6 2.0 2.4 Saline MO SMB CFA](https://reader030.vdocuments.us/reader030/viewer/2022021521/5bfa0a3a09d3f254508bbdaf/html5/thumbnails/38.jpg)
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