Cholinergic anti-inflammatory signaling through α 7 n ACh

Cholinergic anti-inflammatory signaling through α 7 n. ACh. R 1 Talma Brenner Department of Neurology Hadassah University Hospital Jerusalem, Israel

Α 7 nicotinic acetylcholine receptor • The α 7 n. ACh. R an anti-inflammatory target in macrophages and T-cells (B-cells) • α 7 n. ACh. R 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 (Ch. AT) • Response to cholinergic signals; muscarnic and nicotinic • Expression of α 7 nicotinic acetylcholine receptor (ACh. R) • Termination of cholinergic signals is mediated by ACh. E 3

n. ACh. R – structure and function • Neuronal n. ACh. R 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 * α 7 n. ACh. R on macrophages and Tcells can be antiinflammatory target 5

Neuroimmune interactions (The cholinergic anti-inflammatory surveillance) 6 (Ulloa, 2007)

ACh. E: Termination of cholinergic signaling A 12 G 4 G 2 7 G 1 G 2 G 1

ACh. E Inhibitors (ACh. EI) * Clinical indications: Alzheimer’s Disease (AD) and Myasthenia Gravis (MG). * Inhibitors of ACh. EI enzymatic activity (Conventional): Donepezil, Rivastigmine, Tacrine, Pyridostigmine and Edrophonium. * Inhibitors of ACh. E synthesis: EN 101, antisense targeted to exon 2 of the ACh. E m. RNA. 8

Anti-sense directed to ACHE m. RNA (EN 101) treatment in experimental MG 9 Brenner et al FASEB J.

Oral EN 101 improves decremental response in EAMG rats 10 Brenner et al FASEB J.

Chronic EN 101 Retards EAMG Progression 11

Suppression of Anti-ACh. R abs and muscle chemokine/chemokine receptor by chronic EN 101 in EAMG rats CXCR 3 expression (muscle) * Anti-rat-ACh. R abs * Pmole /ml ** AU Control EAMG+EN 101 ** IP 10 expression (muscle) * Control EAMG +EN 101 Control 12 EAMG+EN 101

EN 101 in phase Ib in MG Mean change in QMG Baseline On EN 101 500 µg/kg, day 2 4 wk after treatment 13 Argov et al

Our working hypothesis: Activation of the 7 n. ACh. R has anti-inflammatory effects 7 n. ACh. R ACh ACh. EI ACh. E Cholinergic up-regulation by inhibition of (extra-cellular) ACh. E can activate the 7 n. ACh. R 14

Reduction in ACh. E activity in the extracellular medium of proliferating T-cells by ACh. EI 15 7 n. ACh. R is specifically up-regulated during T-cell activation

7 n. ACh. R 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 n. ACh. R activation 17 Nizri et al Neuropharmacology 2006

Expression of α 7 n. ACh. R by CD 4+ T-cells: increase by stimulation and inhibition by α 7 AS 18 Isolated CD 4+ 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 α 7 n. ACh. R in EAMG mice by Torpedo. ACh. R 20

What is so special about 7 n. ACh. R? CD 3 and 7 n. ACh. Rs coimmunoprecipitate (Razani-Boroujerdi , 2007) 21

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Attenuation of EAE by ACh. EI (rivastigmine) Subcutaneous daily injection Delivery by mini osmotic pumps Treatment control rivastigmine+ Mecamyl. Mean severity 3. 4± 0. 4 1. 7 ± 0. 3 * 2. 9 ± 0. 1 Mean severity 2. 2 ± 0. 4 0. 6 ± 0. 1** Cumulative score# 70. 4 ± 4. 8 32. 4 ± 2. 7 * 50. 8 ± 4. 2 * Cumulative score # 48. 8 ± 5. 1 13. 5 ± 5. 8*** Inhibition of ACh. E activity: Brain 56%, Muscle 42% 23 Nizri et al. J Neuroimmunology, 2008

Histological preservation of the CNS following ACh. EI 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 Th 1 and Th 17 activity by ACh. EI treatment in MOG-specific T-cells 27

ACh. EI treatment affect antigen presentation 28

Direct signaling of α 7 n. ACh. R by nicotine 29

Signaling downstream of 7 n. ACh. R 30

Inhibition of Signaling downstream of 7 n. ACh. R by nicotine 31 Nizri et al 2009

Direct activation of 7 n. ACh. R 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 16 15. 7 0. 02 onset 32 Nizri et al J. Immunology 2009

Improved histophatological parameters under nicotine treatment Demyelination PBS Nicotine 33 Microglial activation Axonal damage

Reduced CD 4 and CD 11 b cells in the CNS of EAE mice treated with nicotine 34

Nicotine treatment was associated with reduced T-cell reactivity 35

Α 7 KO mice develop milder EAE without response to nicotine treatment Disease Mean severity WT 3. 3± 0. 4 Α 7 -/1. 5± 0. 2 Α 7 -/- w nicotine 1. 4± 0. 1 36

Altered T-cell reactivity in α 7 n. ACh. R 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 ACh. E 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). • ACh. EI are prescribed for cognitive dysfunction in AD. Recent evidence pointed to their efficacy in MS patients (Krupp 2004). 42

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Conclusions I The immune cholinergic system can be harnessed for immunomodulation Proposed mechanism involves activation of α 7 n. ACh. R Cholinergic up-regulation by ACh. EI treatment results in reduction of neuroinflammation, amelioration of EAE and improvement of cognitive deficit ACh. EI mediate anti-inflammatory effects; Suppression of T-cell activities, proliferation, pro-inflammatory cytokine production 44

Conclusions II α 7 n. ACh. R is expressed on CD 4+ cell surface. Expression increases following stimulation Nicotine treatment suppressed EAE Nicotine treatment reduced T-cell reactivity, Th 1 and Th 17 activity and implicated a skew towards Th 2 with inhibition of NFkb induced signaling Effects of nicotine are α 7 n. ACh. R dependent Differential responses of α 7 n. ACh. R-/-cells; impairment in antigen presentation and increased T -cell proliferation 45

Thanks • • Lab members Eran Nizri Michal Irony-Tur. Sinai Yasmine Hamra. Amitay 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