The role of cholinergic balance in immune responses: a novel anti

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

fo ld o f ch an g e

3 2 .5 2 1 .5 1 0 .5 0 c tl

20

to r p e d o

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 • • • • • • •

Lab members Eran Nizri Michal Irony-TurSinai Yasmine HamraAmitay Neli Boneva Camille Sicsic Omer Lori Hodaya Hadad 46

• • • • • • • •

Collaborators M. Rosin E. Lavi S. Berrih-Aknin H. Soreq O. Abramsky Ester\ Neuroscience AFM Israeli Ministry of Health chief scientist fund