Cholinergic anti-inflammatory
signaling through α7 nAChR
Talma Brenner
Department of Neurology
Hadassah University Hospital
Jerusalem, Israel
1
Α 7 nicotinic acetylcholine receptor
• The α 7nAChR an anti-inflammatory target in
macrophages and T-cells (B-cells)
• α 7nAChR presence on immune cells may explain
epidemiological data claiming link between cigarette
smoking and several inflammatory disorders
(ulcerative colitis, sarcoidosis)
• Animal studies show that nicotine is active in
reducing Ab production and in signaling through the
TCR
• Involvement of the immune cholinergic system in
the regulation of autoimmune responses
2
Non-neuronal immune
cholinergic system
• Synthesis of ACh
• Expression of the enzyme
cholineacyltransferase (ChAT)
• Response to cholinergic signals; muscarnic
and nicotinic
• Expression of α7 nicotinic acetylcholine
receptor (AChR)
• Termination of cholinergic signals is
mediated by AChE
3
nAChR – structure and function
• Neuronal nAChR are pentameric extracellular complexes of α
and β subunits that form a ligand gated ion channel. (9 α and 4
β subunits).
4
Neuro-immune interactions
*
The
nervous
system is a major
producer of ACh, the
immune
cholinergic
system can mediate
neuro-immune
interactions
* or serve as an
internal regulator of
immune responses
*
α7nAChR
on
macrophages and Tcells can be
antiinflammatory target
5
Neuroimmune interactions
(The cholinergic anti-inflammatory surveillance)
6
(Ulloa, 2007)
AChE: Termination of cholinergic signaling
A12
G4
G2
7
G1
G2
G1
AChE Inhibitors (AChEI)
* Clinical indications: Alzheimer’s Disease (AD)
and Myasthenia Gravis (MG).
* Inhibitors of AChEI enzymatic activity
(Conventional): Donepezil, Rivastigmine,
Tacrine, Pyridostigmine and Edrophonium.
* Inhibitors of AChE synthesis: EN101, antisense targeted to exon 2 of the AChE
mRNA.
8
Anti-sense directed to ACHE mRNA (EN101)
treatment in experimental MG
9
Brenner et al FASEB J.
Oral EN101 improves decremental response in
EAMG rats
10
Brenner et al FASEB J.
Chronic EN101 Retards EAMG
Progression
Stamina
Disease Severity
.
.
.
(sec
)
Running time
Clinical score
.
.
Be fore
Saline
Me stinon
EN
Before
Mestinon
EN
-
(g
)
Weight change
Body Weight
(% )
-day survival
Survival
Saline
Saline
11
Mestinon
EN
Saline
Mestinon
EN
Suppression of Anti-AChR abs and muscle chemokine/chemokine
receptor by chronic EN 101 in EAMG rats
CXCR3 expression
(muscle)
Anti-rat-AChR abs
*
20
16
*
AU
**
12
8
80
4
Pmole
/ml
0
60
40
Control EAMG EAMG+EN101
**
IP10 expression
(muscle)
20
*
16
0
Saline
Control EAMG
EAMG
+EN101
12
8
4
0
12
Control
EAMG
EAMG+EN101
EN 101 in phase Ib in MG
Mean change in QMG
0
Mean Change
-1
-2
Baseline
-3
-4
-5
-6
-7
3.5h
after
first
dose
3.5h
after
2nd
dose
3.5h
after
3rd
dose
Day 2
Day 3
Day
Day 4
Day 5
Day 6
On EN101
500 µg/kg,
day 2
4 wk after
treatment
13
Argov et al
Our working hypothesis:
Activation of the 7 nAChR has
anti-inflammatory effects
7
nAChR
ACh
7
nAChR
ACh
AChEI
AChE
AChE
AChE
AChE
AChE
AChE
Cholinergic up-regulation by inhibition of
(extra-cellular) AChE can activate
the 7 nAChR
14
Reduction in AChE activity in
the extracellular medium of
proliferating T-cells by AChEI
Relative AchE Activity (O.D. 405nm)
1.0
PHA
PHA+EN101 1µM
0.9
PHA+EN101 2µM
0.8
PHA+edrophonium 0.5µM
PHA+edrophonium 1µM
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
5
10
15
25
Minutes in Substrate
15
30
7 nAChR is specifically
up-regulated during
T-cell activation
7 nAChR involvement in T-cell proliferation
cholinergic up-regulation inhibits T-cell proliferation and can render
activated cells to be more sensitive to nicotinic agonists
16
Nizri et al Neuropharmacology 2006
Attenuation of pro-inflammatory cytokine
production by 7 nAChR activation
17
Nizri et al Neuropharmacology 2006
Expression of α7 nAChR by CD4+ T-cells:
increase by stimulation and inhibition by α7 AS
18
Isolated CD4+ cells stained
with FITC αbgtx
Nizri et al J. Immunol 2009
Cells positive for α-Bgtx are activated
cells in replication
19
Up-regulation of α7nAChR in EAMG mice by
Torpedo-AChR
4
3.5
fold of change
3
2.5
2
1.5
1
0.5
0
ctl
20
torpedo
What is so special about 7 nAChR?
CD3 and 7 nAChRs
coimmunoprecipitate
(Razani-Boroujerdi , 2007)
21
22
Attenuation of EAE by AChEI
(rivastigmine)
Subcutaneous daily injection
Delivery by mini osmotic pumps
Treatment
control
rivastigmine
2.2 ± 0.4
0.6 ± 0.1**
48.8 ± 5.1
13.5 ± 5.8***
Treatment
control
rivastigmine
rivastigmine+
Mecamyl.
Mean severity
3 .4± 0.4
1.7 ± 0.3 *
2.9 ± 0.1
Mean severity
Cumulative score#
70.4 ± 4.8
32.4 ± 2.7 *
50.8 ± 4.2 *
Cumulative score
#
Inhibition of AChE activity: Brain 56%, Muscle 42%
23
Nizri et al .J Neuroimmunology, 2008
Histological preservation of the
CNS following AChEI treatment
24
Nizri et al J. Neuroimmunology, 2008
Quantification of histopathological
parameters
Pathological
parameter
Inflammation
Demyelination
Axonal damage
25
control (n=6)
rivastigmine (n=7)
2.3 ± 0.49
1.8 ± 0.47
1.8 ± 0.37
0.71 ± 0.45*
0.71 ± 0.24*
0.57 ± 0.35*
The inflammatory demyelinating lesions
in EAE and MS
26
Reduction of Th1 and Th17 activity by
AChEI treatment in MOG-specific T-cells
27
AChEI treatment affect antigen
presentation
28
Direct signaling of α7 nAChR by
nicotine
29
Signaling downstream of 7 nAChR
30
Inhibition of Signaling downstream of
7 nAChR by nicotine
31
Nizri et al 2009
Direct activation of 7 nAChR by nicotine
control
p
cumulative
score
70±8.2
17±7.1*
0.0011
mean
severity
3.3±0.4
0.8±0.1*
0.0001
16
15.7
0.02
onset
32
nicotine
Nizri et al J. Immunology 2009
Improved histophatological parameters under nicotine
Demyelination
PBS
Nicotine
33
treatment
Microglial activation
Axonal damage
Reduced CD4 and CD11b cells in the CNS of EAE
mice treated with nicotine
34
Nicotine treatment was associated with reduced T-cell reactivity
35
Α7 KO mice develop milder EAE without response to nicotine treatment
Disease Mean severity
WT
3.3±0.4
Α7-/1.5±0.2
Α7 -/- w nicotine 1.4±0.1
36
Altered T-cell reactivity in α7 nAChR KO, lack of response
to nicotine and reduction in antigen presentation
37
Does nicotine posses antiinflammatory effects?
• Invasive pneumococcal disease is more
prevalent in smokers (Nuorti, 2000).
Nicotine increases intracellular replication
of Legionella (Matsunaga, 2001).
• Smoking AD patients have reduced levels of
Aβ 40 and Aβ 42 (Hellstrom-Lindahl,
2004).
• Smoking protects against UC (Rubin and
Hanauer, 2000). Clinical trials with nicotine
in TDP reduced UC severity (Van Assche,
2005).
38
History may be revealing…
39
Inhibition of AChE by Rivastigmine
reversed cognitive impairment associated
with EAE
Assay period
induction
0
40
clinical signs
7
14
EAE is associated with inflammatory
infiltrates near the hippocampus
41
Cognitive dysfunction in MS
• Most MS patients develop cognitive dysfunction
(memory, attention, information processing and
verbal fluency).
• These patients are more prone to develop
affective disorders (Gilchrist and Creed, 1994).
• Immunmodulatory treatment can slow the cognitive
decline in MS, there’s still a need for symptomatic
treatment (Henze, 2006).
• AChEI are prescribed for cognitive dysfunction in
AD. Recent evidence pointed to their efficacy in
MS patients (Krupp 2004).
42
43
Conclusions I
The immune cholinergic system can be
harnessed for immunomodulation
Proposed mechanism involves activation of
α7 nAChR
Cholinergic up-regulation by AChEI
treatment results in reduction of
neuroinflammation, amelioration of EAE
and improvement of cognitive deficit
AChEI mediate anti-inflammatory effects;
Suppression of T-cell activities,
proliferation, pro-inflammatory cytokine
production
44
Conclusions II
α7 nAChR is expressed on CD4+ cell surface.
Expression increases following stimulation
Nicotine treatment suppressed EAE
Nicotine treatment reduced T-cell reactivity, Th1
and Th17 activity and implicated a skew towards
Th2 with inhibition of NFkb induced signaling
Effects of nicotine are α7 nAChR dependent
Differential responses of α7 nAChR-/-cells;
impairment in antigen presentation and increased
T-cell proliferation
45
Thanks
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Lab members
Eran Nizri
Michal Irony-TurSinai
Yasmine HamraAmitay
Neli Boneva
Camille Sicsic
Omer Lori
Hodaya Hadad
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Collaborators
M. Rosin
E. Lavi
S. Berrih-Aknin
H. Soreq
O. Abramsky
Ester\ Neuroscience
AFM
Israeli Ministry of
Health chief scientist
fund