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Cholinergic anti-inflammatory signaling

through α7 nAChR

Talma Brenner Department of Neurology

Hadassah University HospitalJerusalem, Israel

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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

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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

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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).

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Neuro-immune interactionsNeuro-immune interactions

** The nervous The nervous system is a major system is a major producer of ACh, the producer of ACh, the immune cholinergic immune cholinergic system can mediate system can mediate neuro-immune neuro-immune interactions interactions

** or serve as an or serve as an internal regulator of internal regulator of immune responsesimmune responses

** αα7nAChR on 7nAChR on macrophages and T-macrophages and T-cells can be anti-cells can be anti-inflammatory targetinflammatory target

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Neuroimmune interactionsNeuroimmune interactions((The cholinergic anti-inflammatory The cholinergic anti-inflammatory

surveillance)surveillance)

(Ulloa, 2007)

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AChE: Termination of cholinergic signalingAChE: Termination of cholinergic signaling

G4

G2 G1

A12 G2 G1

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AChE Inhibitors (AChEI)AChE Inhibitors (AChEI)

* Clinical indications: Alzheimer’s * Clinical indications: Alzheimer’s Disease (AD) and Myasthenia Gravis Disease (AD) and Myasthenia Gravis (MG).(MG).

* Inhibitors of AChEI enzymatic activity * Inhibitors of AChEI enzymatic activity (Conventional): Donepezil, (Conventional): Donepezil, Rivastigmine, Tacrine, Pyridostigmine Rivastigmine, Tacrine, Pyridostigmine and Edrophonium.and Edrophonium.

* Inhibitors of AChE synthesis: EN101, * Inhibitors of AChE synthesis: EN101, anti-sense targeted to exon 2 of the anti-sense targeted to exon 2 of the AChE mRNA.AChE mRNA.

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Anti-sense directed to ACHE mRNA Anti-sense directed to ACHE mRNA (EN101) treatment in experimental MG(EN101) treatment in experimental MG

Brenner et al FASEB J.

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Oral EN101 improves decremental response in EAMG rats

Brenner et al FASEB J.

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Chronic EN101 Retards EAMG Progression

-15

-10

-5

0

5

10

15

Wei

gh

t ch

ang

e

( g )Saline Mestinon EN101

Body Weight

0

0.5

1

1.52

2.5

3

3.5

4

4.5

Cli

nic

al

sco

re

Before Saline Mestinon EN101

Disease Severity

0

50

100

150

200

250

300

350

Ru

nn

ing

tim

e

( sec

)

Before Saline Mestinon EN101

Stamina

0

20

40

60

80

100

30- d

ay s

urv

ival

(%)

Saline Mestinon EN101

Survival

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Suppression of Anti-AChR abs and muscle chemokine/chemokine receptor by chronic EN 101 in

EAMG rats

0

20

40

60

80

Saline

0

4

8

12

16

20

0

4

8

12

16

Anti-rat-AChR abs

CXCR3 expression (muscle)

IP10 expression (muscle)

ControlEAMG EAMG+EN101

ControlEAMG

*

*

**

Pmole/ml

EAMG+EN101

AU

Control EAMG EAMG+EN101

*

**

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EN 101 in phase Ib in MGEN 101 in phase Ib in MG

Baseline

On EN101 500 µg/kg,

day 2

4 wk after treatment

-7

-6

-5

-4

-3

-2

-1

0

3.5hafterfirst

dose

3.5hafter2nddose

3.5hafter3rd

dose

Day 2 Day 3 Day 4 Day 5 Day 6

Day

Mea

n C

han

ge

Mean change in QMG

Argov et al

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Our working hypothesis :

Activation of the 7 nAChR has anti-inflammatory effects

7 nAChR

ACh

Cholinergic up-regulation by inhibition of (extra-cellular) AChE can activate the 7 nAChR

7 nAChR

ACh

AChEAChE

AChE

AChE

AChE

AChE

AChEI

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0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

5 10 15 25 30

Minutes in Substrate

Rel

ativ

e A

chE

Act

ivity

(O

.D. 4

05nm

)

PHA

PHA+EN101 1µM

PHA+EN101 2µM

PHA+edrophonium 0.5µM

PHA+edrophonium 1µM

Reduction in AChE activity inthe extracellular medium of

proliferating T-cells by AChEI

7 nAChR is specifically up-regulated during

T-cell activation

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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

Nizri et al Neuropharmacology 2006

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Attenuation of pro-inflammatory Attenuation of pro-inflammatory cytokine production by cytokine production by 7 nAChR 7 nAChR

activationactivation

Nizri et al Neuropharmacology 2006

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Expression of α7 nAChR by CD4+ T-cells: increase by stimulation and inhibition by α7

AS

Isolated CD4+ cells stained with FITC αbgtx Nizri et al J. Immunol 2009

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Cells positive for α-Bgtx are activated cells in replication

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Up-regulation of α7nAChR in EAMG mice by

Torpedo-AChR

0

0.5

1

1.5

2

2.5

3

3.5

4

ctl torpedo

fold

of

ch

an

ge

2121

What is so special about What is so special about 7 nAChR?7 nAChR? CD3 and 7 nAChRs coimmunoprecipitate

(Razani-Boroujerdi , 2007)

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Treatment control rivastigminerivastigmine+Mecamyl.

Mean severity 3 .4± 0.4 1.7 ± 0.3 * 2.9 ± 0.1

Cumulative score# 70.4 ± 4.8 32.4 ± 2.7 * 50.8 ± 4.2 *

Attenuation of EAE by AChEI Attenuation of EAE by AChEI (rivastigmine)(rivastigmine)

Delivery by mini osmotic pumps

Inhibition of AChE activity: Brain 56%, Muscle 42%

Subcutaneous daily injection

Treatment control rivastigmine

Mean severity 2.2 ± 0.4 0.6 ± 0.1**

Cumulative score# 48.8 ± 5.1 13.5 ± 5.8***

Nizri et al .J Neuroimmunology, 2008

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Histological preservation of Histological preservation of the CNS following AChEI the CNS following AChEI

treatmenttreatment

Nizri et al J. Neuroimmunology, 2008

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Quantification of histopathological Quantification of histopathological parametersparameters

Pathologicalparameter

control (n=6) rivastigmine (n=7)

InflammationDemyelinationAxonal damage

2.3 ± 0.491.8 ± 0.471.8 ± 0.37

0.71 ± 0.45*0.71 ± 0.24*0.57 ± 0.35*

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The inflammatory demyelinating lesions in EAE and MS

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Reduction of Th1 and Th17 activity by Reduction of Th1 and Th17 activity by AChEI treatmentAChEI treatment in MOG-specific T- in MOG-specific T-

cellscells

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AChEI treatment affect antigen AChEI treatment affect antigen

presentationpresentation

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Direct signaling of α7 nAChR by nicotine

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Signaling downstream of Signaling downstream of 7 7 nAChRnAChR

3131

Inhibition of Signaling Inhibition of Signaling downstream of downstream of 7 nAChR7 nAChR by nicotine by nicotine

Nizri et al 2009

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Direct activation of 7 nAChR by nicotine

  control nicotine p

cumulative score 70±8.2 17±7.1* 0.0011

mean severity 3.3±0.4 0.8±0.1* 0.0001

onset 16 15.7 0.02

Nizri et al J. Immunology 2009

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Improved histophatological parameters under nicotine

treatment Demyelination Microglial activation Axonal damage

PBS

Nicotine

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Reduced CD4 and CD11b cells in the CNS of

EAE mice treated with nicotine

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Nicotine treatment was associated with reduced T-cell reactivity

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Disease Mean severity

WT 3.3±0.4Α7-/- 1.5±0.2Α7 -/- w nicotine 1.4±0.1

Α7 KO mice develop milder EAE without response to nicotine treatment

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Altered T-cell reactivity in α7 nAChR KO, lack of response to nicotine and reduction in antigen

presentation

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Does nicotine posses anti-Does nicotine posses anti-inflammatory effectsinflammatory 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).

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History may be revealingHistory may be revealing……

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Inhibition of AChE by Rivastigmine Inhibition of AChE by Rivastigmine reversed cognitive impairment reversed cognitive impairment

associated with EAEassociated with EAE

clinical signs

0 7 14

Assay periodinduction

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EAE is associated with inflammatory infiltrates near the

hippocampus

4242

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).

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Conclusions IConclusions IThe immune cholinergic system can The immune cholinergic system can be harnessed for immunomodulationbe harnessed for immunomodulationProposed mechanism involves Proposed mechanism involves activation of activation of αα7 nAChR7 nAChRCholinergic up-regulation by AChEI Cholinergic up-regulation by AChEI treatment results in reduction of treatment results in reduction of neuroinflammation, amelioration of neuroinflammation, amelioration of EAE and improvement of cognitive EAE and improvement of cognitive deficit deficit AChEI mediate anti-inflammatory AChEI mediate anti-inflammatory effects; Suppression of T-cell effects; Suppression of T-cell activities, proliferation, pro-activities, proliferation, pro-inflammatory cytokine productioninflammatory cytokine production

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Conclusions IIConclusions II

αα7 nAChR is expressed on CD47 nAChR is expressed on CD4++ cell surface. cell surface. Expression increases following stimulationExpression increases following stimulation

Nicotine treatment suppressed EAENicotine treatment suppressed EAE

Nicotine treatment reduced T-cell reactivity, Th1 Nicotine treatment reduced T-cell reactivity, Th1 and Th17 activity and implicated a skew towards and Th17 activity and implicated a skew towards Th2 with inhibition of NFkb induced signalingTh2 with inhibition of NFkb induced signaling

Effects of nicotine are Effects of nicotine are αα7 nAChR dependent 7 nAChR dependent

Differential responses of Differential responses of αα7 nAChR7 nAChR-/--/-cells; cells; impairment in antigen presentation and impairment in antigen presentation and increased T-cell proliferation increased T-cell proliferation

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Thanks Lab members • Eran Nizri• Michal Irony-Tur-

Sinai• Yasmine Hamra-

Amitay• Neli Boneva• Camille Sicsic• Omer Lori• Hodaya Hadad

Collaborators • M. Rosin• E. Lavi• S. Berrih-Aknin• H. Soreq• O. Abramsky

• Ester\ Neuroscience

• AFM• Israeli Ministry of

Health chief scientist fund