INFLAMMATORY BOWEL DISEASES

PRIOR APPLICATION INFORMATION

The instant application claims the benefit of US Provisional Patent Application

61/757,394, filed January 28, 2013.

BACKGROUND OF THE INVENTION

Inflammatory bowel diseases (IBDs), consisting of Crohn's disease (CD) and ulcerative colitis (UC), are characterized by a chronic relapsing and remitting course as a result of intestinal inflammation ¹ . The release of inflammatory mediators, including pro-inflammatory cytokines from immune cells ^2,3 mediate tissue injury and exacerbation of IBD. Accordingly, several therapeutic approaches targeting inflammatory cytokines in IBDs have been investigated ⁴ . Cytokine production is controlled by cholinergic signals along the vagus nerve in different inflammatory conditions including IBDs ^5-7 and a7 nicotinic acetylcholine receptors (a7nAChR) on macrophages and other immune cells mediate this regulation ⁸ . This vagus nerve- dependent and a7nAChR-mediated cholinergic anti-inflammatory pathway represents the efferent arm of an inflammatory reflex that controls immune responses and cytokine production ⁹ . Electrical vagus nerve stimulation suppresses tumor necrosis factor (TNF) and other pro-inflammatory cytokine levels in endotoxemia and colitis ¹⁰ . IBDs are associated with an autonomic imbalance and up to 35% of patients with ulcerative colitis (UC) exhibit autonomic dysfunction with impaired efferent vagus nerve activity ¹¹ . We have previously reported that the vagus nerve has a tonic inhibitory role on acute inflammation in a murine model of colitis resembling UC and CD5. In this context, the absence of the vagus nerve worsened acute colitis through a macrophage- mediated mechanism. Moreover, in line with the role of ci7nAChR in mediating anti-inflammatory cholinergic signals, it has been demonstrated that nicotine, a cholinomimetic, and an a7nAChR agonist ameliorates inflammation in UC patients ¹² . Dendritic cells (DCs) are key cells of the innate immune system that bridge innate with adaptive immune responses. Strategically positioned in the lamina propria in proximity to a number of luminal bacteria and antigenic stimuli, these cells perform a key role in activation of the immune response and generation of gut inflammation via their passage into the spleen and interaction with T cells ¹³ . Human studies revealed that there is a significant increase in the numbers of DCs within the inflamed tissue and the peripheral blood of patients with CD or UC ¹⁴ . Furthermore, DCs depletion in dextran sulfate sodium (DSS)-treated CD11c-DT receptor transgenic mice almost completely inhibited experimental colitis ¹⁵ . Nicotinic, including a7nAChR are expressed by human and mouse DCs ¹⁶ .

The cholinergic anti-inflammatory pathway can be centrally activated by muscarinic acetylcholine receptor (mAChR) ligands or acetylcholinesterase (AChE) inhibitors ¹⁷ ' ¹⁸ . Galantamine (GAL) is a reversible, competitive AChE inhibitor, which crosses the blood-brain barrier, increases brain cholinergic network activity ¹⁹ and is widely used in the treatment of Alzheimer's disease. GAL activates efferent vagus nerve activity ²⁰ and its anti-inflammatory activity has been associated with brain mAChR-mediated activation of the cholinergic anti-inflammatory pathway ¹⁸ . Recent findings have highlighted a key role of the spleen in mediating vagus nerve antiinflammatory signaling during endotoxemia ²¹ . The importance of a vagus nerve - to spleen anti-inflammatory axis in the regulation of intestinal inflammation remains to be determined. To provide insight here we studied whether central activation of the cholinergic anti-inflammatory pathway by the AChE inhibitor GAL or mAChR ligands alters the severity of colitis and the specific role of the spleen and DCs. SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method of treating an individual suffering from an inflammatory bowel disease comprising administering to an individual in need of such treatment an effective amount of a M1 mAChR agonist.

According to a further aspect of the invention, there is provided use of a M1 mAChR agonist for treating an inflammatory bowel disease.

According to another aspect of the invention, there is provided a M1 mAChR agonist for use in a medicament for treating an inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Galantamine (GAL) alleviates the severity of DSS-induced colitis. GAL (6 days, i.p.) treatment was started one day prior to colitis induction. ; A: Disease activity index; B: Macroscopic scores; C: Myeloperoxidase (MPO) activity; D: Serum C-reactive protein (CRP). E: Histological score. F: Appearance of a colon in mice with DSS-induced colitis (control group); G: Appearance of a colon in GAL-treated mice with DSS-induced colitis (Hematoxylin and eosin staining, 10X magnifications). Values are shown as means±SEM, n≥8. ^* P<0.05 as compared to control DSS-treated group. ^# P<0.05 as compared to water (H2O)-treated group.

Figure 2: Galantamine effects in DSS-induced colitis are mediated through central mAChRs. Atropine sulfate (AS, a mAChR antagonist that crosses the blood- brain barrier) or atropine methyl nitrate (AMN, a mAChR antagonist that does not cross the blood-brain barrier) (4mg/kg, i.p. for 6 days) was administered as a single daily injection 20 min prior to every galantamine administration (4mg/kg, i.p., for 6 days). A: Macroscopic score; B: Myeloperoxidase (MPO) activity; Values are shown as means+SEM, n>8. ^a P<0.05, compared to non DSS-treated group (H20), ^b P<0.05 as compared to vehicle DSS-treated group, ^C P<0.05 as compared to GAL DSS- treated group.

Figure 3: Central administration of a M1 mAChR agonist or a M2mAChR antagonist alleviates the severity of DSS-induced colitis. McN-A-343 (M1 mAChR agonist) or methoctramine (MTT, M2 mAChR antagonist) (5 ng/kg/day, i.c.v., for 6 days) treatment was started one day before colitis induction. A: Macroscopic score; B: Myeloperoxidase (MPO) activity; Values are shown as means+SEM, n≥8. ^a P<0.05, compared to non DSS-treated group (H20), ^b P<0.05 as compared to vehicle DSS- treated group. Figure 4: Galantamine effects in mice with DSS-induced colitis are mediated through vagus nerve and splenic nerve signaling to the spleen. Vagotomy (VXP) and/or splenectomy (SPX), splenic neurectomy (NRX) and/or splenectomy (SPX) were performed prior to initiating galantamine (4 mg/kg/day, i.p.) treatment and/or colitis induction as described in Material and Methods. *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; A: Macroscopic score; B: Myeloperoxidase (MPO) activity; ^a P<0.05 as compared to sham-vehicle-DSS-treated group, P<0.05 as compared to VXP-DSS-treated group or NRX-DSS treated group, ^C P<0.05 as compared to sham-GAL-DSS-treated group.

Figure 5: Cholinergic treatment with galantamine or McN-A-343 results in increased splenic ACh levels, mediated through vagus nerve and splenic nerve signaling. Vagotomy (VXP) and/or splenic neurectomy (NRX) were performed prior to galantamine (GAL, 4mg/kg/day, i.p. for 6 days) or McN- A-343 (5ng/kg/day, i.c.v., for 6 days) treatment and spenic ACh levels analyzed as described in Material and Methods, 24h after the last injection. Splenic ACh levels were determined as postmortem, Values are shown as means+SEM, n≥8. *P<0.05 as compared to control Sham-DSS-treated group. ^# P<0.05 as compared to the same treatment in sham- operated animals with DSS-induced colitis.

Figure 6: Effects of cholinergic treatment on splenic dendritic cell and T cell cytokine production in the context of colitis. A: Interleukin (IL)-12p40 production from dendritic cells. Splenic dendritic cells were isolated from galantamine (GAL, 4 mg/kg/day, i.p. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX). Dendritic cells were also isolated from groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) and incubated ex vivo with GST-21 (a specific a7nAChR agonist, 100 μΜ). !L12p40 was measured in media at 24h following treatment. ^* P<0.05 as compared to DSS control group, ^# P<0.05, n=8. B: INF-γ production from dendritic/T cell co-cultures. Galantamine (in vivo) and GTS-21 (ex vivo) treatments were performed as described above in A. See Material and Methods for details. ^* P<0.05 as compared to vehicle treatment, ^# P<0.05; data from 3 independent experiments with quadruplicated cultures, mean ± SEM. C: IL-17 production from dendritic T cell co- cultures. Galantamine (in vivo) and GTS-21 (ex vivo) treatments were performed as described in A. See Material and Methods for details. ^* P<0.05 as compared to vehicle treatment, P<0.05; data from 3 independent experiments with quadruplicated cultures.

Figure 7: Effect of galantamine on the development of acute colitis induced by 2,4 dinitrobenzene-sulfonic acid (DNBS)-treatment. Influence of 4 days galantamine treatment (4 mg/kg/day, intraperitoneal (i.p.)) starting 1 day before injection of DNBS. A: Myeloperoxidase (MPO) activity. B: Interleukin (ΙΙ_)-1 β colonic level. A significant decrease of both markers was detected compared to the control group. Pre-treatment with atropine sulfate (AS, 4 mg/kg, i.p.), but not atropine methyl nitrate (AMS, 4 mg/kg, i.p.) abolished the beneficial effect induced by galantamine. ^* vs vehicle group, *vs DNBS galantamine-treated group, P < 0.05, n≥8. The values are shown as mean ± SEM. GAL, AS and AMS do not have any effect on any parameters in control conditions.

Figure 8: Effect of muscarinic 1 agonist (McN-A343: 5ng/kg/day, intracerebroventricular (i.c.v.)) and Methoctramine (MTT: 5ng/kg/day, i.c.v.) on the development of colitis induced by 2,4 dinitrobenzen-sulfonic acid (DNBS)-treatment (5mg). Influence of 4 days of i.c.v. infusion starting 1 day before injection of DNBS. A: Myeloperoxidase (MPO) activity, B: Interleukin (IL)-1 β colonic level. A significant decrease of both markers is detected compared to the vehicle group. ^* P < 0.05 vs ethanol 50% group, ^# P < 0.05 vs DNBS group. The values are shown as mean ± SEM,n>8.

Figure 9: Effect of McN-A-343 intracerebroventricular ((5ng/kg/day, i.c.v.) infusion on the development of acute colitis induced by dextran sulfate sodium (DSS) 5% treatment in the presence or absence of the vagus nerve (vagotomy: VXP) or the spleen (SPX). Influence of a 6 days i.c.v. infusion starting 1 day before induction of DSS colitis. A: Myeloperoxidase (MPO) activity, B : Interleukin (ll_)-1 β colonic level. A significant decrease of both markers was detected compared to the sham vehicle group. VXP and SPX abolished the beneficial effect of M1 i.c.v. infusion. ^* vs sham vehicle group, ^# P < 0.05. The values are shown as mean ± SEM, n≥8.

Figure 10: I.C.V. infusions of the MlmAChR agonist McN-A-343.

Figure 11 : Central administration of a MlmAchR agonist alleviates the severity of dextran sulfate sodium (DSS)-induced colitis through vagus and splenic nerve signaling to the spleen. Vagotomy (VXP) and/or splenectomy (SPX), splenic neurectomy (NRX) and/or splenectomy (SPX) were performed 10 days prior to initiating McN-A-343 (5 ng/kg/day, i.c.v.) treatment and/or colitis induction as described in Material and Methods. ^* Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; A: Serum amyloid protein (SAP); B: Colonic interferon- gamma (IFN-D); C; Colonic interleukin (IL)-17; D: Colonic IL-12p70; E: Colonic IL-23 and F: Colonic IL-4. Values are shown as means±SEM. Samples were collected on day 5 post-DSS induction; mice per group≥8. ^a P<0.05 as compared to sham-saline- DSS-treated group, ^b P<0.05 as compared to VXP-DSS-treated group or NRX-DSS- treated group respectively, ^C P<0.05 as compared to sham-McN-A-343-DSS-treated group.

Figure 12: Central administration of a Ml mAchR agonist alleviates splenic CD1 1c+ dendritic cells (DCs) interleukin (IL)-12p70 and IL-23 release in the context of dextran sulfate sodium (DSS)-induced colitis through vagus and splenic nerve signaling to the spleen. A: IL-12p70 and B; IL-23 production from splenic CD11c ⁺ DCs. Splenic CD 1 c ⁺ DCs were isolated from McN-A-343 (5 ng/kg/day, i.c.v. for 6 days)-treated groups of colitic mice subjected to sham- operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 5 post-DSS induction incubated ex vivo or not with GTS-21 (a specific a7nAChR agonist, 100 μΜ). IL-12p70 and IL-23 were measured in media at 24hrs following treatments. Values are shown as means±SEM, 3 independent experiments with 4 mice per group. ^a P<0.05 as compared to DSS control group, ^b P<0.05.

Figure 13: Implication of the NF-κΒ pathway in splenic CD 1 c ⁺ dendritic cells (DCs) cytokine release in the context of dextran sulfate sodium (DSS)-induced colitis. Interleukin (IL)-12p70 and IL-23 production from splenic CD11 c ⁺ DCs. Splenic CD1 1 c ⁺ DCs were isolated from McN-A-343 (5 ng/kg/day, i.e. v. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 5 post-DSS induction incubated ex vivo or not with GTS-21 (a specific a7nAChR agonist, 100 μΜ) or with A, B: betulinic acid (a specific NF-KB activator, 10μΜ) or C, D: BAY 1 1-7082 (a specific NF-κΒ inhibitor, 10μΜ). IL-12p70 and IL-23 were measured in media at 24hrs following treatments. Values are shown as meansiSE , 3 independent experiments with 4 mice per group. ^a P<0.05 as compared to DSS control group, ^* P<0.05.

Figure 14: Role of the CD1 1 c ⁺ dendritic cells (DCs) in CD4 ⁺ CD25 ^" T cells priming in the context of dextran sulfate sodium (DSS)-induced colitis. Effect of McN- A343 (in vivo) and GTS-21 (in vitro) treatments on splenic CD1 1 c ⁺ DCs function and sequential CD4 ⁺ CD25 cell activation. Splenic CD1 1 c ⁺ DCs isolated from different colitic group were cultured in for 24h before being co-cultured with CD4 ⁺ T lymphocytes isolated from na ^' ive mice. The level of A: Interferon-gamma (IFN-γ), B: Interleukin (IL)-17 and C: IL-4 were measured in media at 24hrs. D: CD4 ⁺ CD25- T cells proliferation. ^a P<0.05 as compared to DSS control group, ^b P<0.05, n=8, data are representative of 3 independent experiments with quadruplicated cultures, meanlSEM.

Figure 15: Implication of the interleukin (IL)-12p70 and IL-23 pathways during

CD4 ⁺ CD25 ^" T cells priming by splenic CD1 1c ⁺ dendritic cells (DCs) in the context of dextran sulfate sodium (DSS)-induced colitis. Splenic CD1 1 c ⁺ DCs isolated from different colitic group were cultured in for 24h before being co- cultured with CD4 ⁺ T lymphocytes isolated from naive mice in the presence of absence of A; anti-pi 9 mAb (10ug/ml-1 ) or B: anti-p35 mAb (l Oug/ml ¹ ), C: recombinant (r)IL-12p70 (25ng/mr ¹ ) or D: rlL-23 (25ng/ml ^"1 ) protein. Supernatant were collected after 24h. The level of interferon-gamma (IFN-γ) and IL-17 in the culture supernatant were investigated at 24hrs. *P<0.05, data are representative of 3 independent experiments with quadruplicated cultures, mean±SEM. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

It is demonstrated herein that GAL or McN-A-343 (a MlmAChR agonist) significantly ameliorate disease severity and inhibit inflammation in the context of experimental colitis. This therapeutic efficacy is brain mAChR-dependent and mediated through a functional interaction between the vagus nerve and spleen and inhibition of splenic DCs cytokine production through an a7nAChR mechanism.

The cholinergic anti-inflammatory pathway is an efferent vagus nerve-based mechanism that regulates immune responses and cytokine production through a7- nicotinic-acetylcholine-receptor(a7nAChR) signaling. Decreased efferent vagus nerve activity is observed in inflammatory bowel disease (IBD). The inventors determined whether central activation of this pathway alters inflammation in mice with colitis, the mediating role of a vagus nerve-to spleen circuit and a7nAChR signaling. Two experimental models of colitis were used in C57BL/6 mice. Central activation induced by the acetylcholinesterase inhibitor or muscarinic acetylcholine receptor agonist treatments reduced mucosal inflammation associated with decreased proinflammatory cytokine secretion by splenic dendritic ce!ls(DC), mediated by a7nAChR signaling. DCs isolated from treated-mice showed decreased CD4 ⁺ T cell priming. The anti- inflammatory efficacy was abolished in mice with vagotomy, splenic neurectomy or splenectomy. In conclusion, centra! cholinergic activation of a vagus nerve-to spleen circuit controls intestinal inflammation and is of interest for the development of novel therapeutic strategies in the treatment of IBD. For example, as discussed herein, compounds known for treating for example Alzheimer's Disease such as for example cholinesterase inhibitors such as Donepezil (Aricept), Rivastigmine (Exe!on) and Galantamine (Razadyne).

As will be appreciated by one of skill in the art, the fact that cholinesterase inhibitors that cross the blood-brain barrier can be used to treat intestinal diseases such as Crohn's disease and ulcerative colitis is highly surprising. As will be appreciated by one of skill in the art, it is completely unexpected that a medication for treating an inflammatory bowel disease must cross the blood-brain barrier and is not applied directly to the gut or the digestive tract, for example, intraperitoneally. Furthermore, it is unexpected that an immune cell can be regulated directly in this manner and an opposite effect induced.

The results of this study clearly indicate that central cholinergic activation by

AChE inhibitors or selective mAChR ligands results in a decreased susceptibility to experimental colitis. These protective effects are dependent on vagus nerve and splenic nerve signaling, which is mediated at a cellular level through cholinergic suppression of splenic DC activation.

According to a first aspect of the invention, there is provided a method of treating an individual suffering from an inflammatory bowel disease comprising administering to an individual in need of such treatment an effective amount of a 1 mAChR agonist.

As will be appreciated by one of skill in the art, an "effective amount" will depend on for example the age and general condition of the individual in need of such treatment or patient. However, such amounts can be determined through routine experimentation and/or by analysis of previous administrations of the compounds.

In a preferred embodiment, the 1 mAChR agonist crosses the blood-brain barrier.

The M1 mAChR agonist may be selected from the group consisting of McN-A-

343.TBPB , VU0357017, VU0364572 and VU0029767 and mixtures thereof, !n preferred embodiments, the M1 mAChR agonist is McN-A-343. For example, an "effective amount" of these compounds may be similar to the effective amount for treatment of Alzheimer's Disease, that is, 8-24 mg/day.

As will be appreciated by one of skill in the art, a person in need of such treatment is an individual who suffers from or is suspected of suffering from or at risk of developing an inflammatory bowel disease, for example ulcerative colitis or Crohn's disease.

As will be appreciated by one of skill in the art, the effective amount of lmAChR agonist will accomplish at least one of the following: reduction in severity and/or frequency of associated symptoms, including but by no means limited to abdominal pain, vomiting, diarrhea, rectal bleeding, and intestinal cramps; and longer periods of remission and/or being symptom free.

According to a further aspect of the invention, there is provided use of a Ml mAChR agonist for treating an inflammatory bowel disease.

The inflammatory bowel disease may be ulcerative colitis or Crohn's disease. According to another aspect of the invention, there is provided a Ml mAChR agonist for use in a medicament for treating an inflammatory bowel disease.

In line with previous findings indicating a role for brain mAChRs in the central activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway ¹⁷ exogenous GAL failed to reduce the severity of colitis in mice with pharmacological blockage (by AS) of central mAChRs. Furthermore, the therapeutic efficacy of McN-A- 343 and MTT further highlighted exploiting central cholinergic activation by MlmAChR agonists or M2mAChR antagonists as novel approaches to alleviate colitis. While it is known that peripheral cholinergic activation may have some effect on colitis ³⁵ , in this study the inventors found no evidence for a significant role of peripheral mAChRs in mediating protective cholinergic effects in the context of this experimental plan. The findings that the effects of GAL and McN-A-343 are mediated through activation of the vagus nerve-based cholinergic anti-inflammatory pathway are consistent with experimental evidence pointing to a protective role of the vagus nerve against acute colitis in animal models ⁵ - ^{36 37} . However, previously, no relation between the pharmacological vagus nerve stimulation and colitis has been made. The disease- alleviating and anti-inf!ammatory effects of both GAL and McN-A-343 treatments were abrogated in mice with VXP, NRX or mice with splenectomy. These results are in line with data demonstrating that vagus nerve stimulation fails to protect against septic shock in rats subjected to common celiac branch vagotomy, splenectomy or NRX ^{2 ,38} . Both therapeutics (GAL and McN-A-343) increased splenic ACh levels in mice and this effect was not observed in mice with VXP and NRX, indicating that the protective cholinergic effect is dependent on an interaction between the vagus nerve and spleen. These results are consistent with recent observations that splenic ACh is released after vagus nerve activation, which can result in a paracrine effect on antigen presenting cells and anti-inflammatory effect during endotoxemia ²¹ . Thus, the results identify a vagus nerve - to spleen axis as an important mediator of the central cholinergic regulation of colitis severity. The importance of the findings was additionally substantiated by the similar profile of results in a DNBS-model; this model closely resembles CD, which broadens the therapeutic implications of cholinergic modalities in the context of IBD.

As will be appreciated by one of skill in the art, much of the recent progress in the understanding of immunity has been achieved by the study of experimental animal models of intestinal inflammation. These models are valuable tools for studying many important disease aspects that are difficult to address in humans. The most widely used and characterized experimental model of UC is the DSS-induced colitis, which was developed by administration of DSS in the drinking water. DSS induces a very reproducible acute colitis characterized by superficial inflammation and infiltrations. Regarding CD, the most widely used and characterized experimental model of CD is the DNBS-induced colitis, which was developed by the intramural administration of 50% ethanol (barrier breaker) containing DNBS. DNBS is believed to haptenize colonic autologous or microbiota protein rendering them immunogenic to the host immune system. This model induces a very reproducible acute colitis characterized by ulceration and infiltration.

Although previous observations clearly implicate macrophages ^5,8 and T cells ³⁹ in the context of the cholinergic anti-inflammatory pathway, it is not clear whether changes in DCs response play a role in regulating gut inflammation by cholinergic signaling. The data demonstrate that activated vagus nerve signaling affects DCs cytokine production, which subsequently plays an important role in regulation of gut inflammation through the CD4 ⁺ T cell population. As a Th1-17 type immune response have been characterized in IBD and as DCs play a crucial role in priming the immune response towards Th1 and Th-17 the inventors examined whether both treatments may have an influence on T cell priming and production of lNF-γ and iL-17. The results demonstrate that DCs isolated from cholinergic drug-treated colitic mice have significantly reduced ability to stimulate naive CD4 ⁺ T cells in vitro to produce IFN-γ and IL-17 as compared to the DCs isolated from colitic non-treated mice. This observation significantly substantiates previous studies related to the importance of DCs in the context of experimental colitis ⁴⁰ and demonstrating amelioration of DSS- induced colitis by depletion of DCs after administration of diphtheria toxin in CD11c- DT receptor transgenic mice ¹⁵ . This is in agreement with clinical data demonstrating that IBD is characterized by increased expression of innate (IL-12p40, IL-6) and adaptive (lNF-γ, IL-17) pro-inflammatory cytokines ⁴¹ , and that polarization is mainly controlled by the IL-12 cytokine family produced by activated DCs ⁴² . Although it is theoretically possible that mesenteric lymphoid DCs may also be involved in mediating cholinergic effects in the context of colitis, the findings clearly point to a pivotal roie of splenic DCs in this regulation.

The inventors have further identified the mediating role of the a7AChR on DCs; a specific a7AChR agonist down-regulated ex vivo IL-12p40 release. This corroborates data demonstrating that in vitro immature spleen DCs that mature in a nicotinic environment manifest lower endocytic and phagocytic activities ⁴³ . In vitro mature spleen DCs that are exposed to nicotine produce decreased levels of IL-12 and displayed reduced ability to induce T-Cell responses ⁴⁴ . Moreover, the in vitro data do not support the possibility that galantamine may act as an allosteric modulator on the a7AChR on DCs to directly affect their activation. As will be appreciated by one of skill in the art, this goes along with the surprising fact developed above, as normally a direct effect on this type of cell should have been seen, but in this case no effect was observed.

It should be acknowledged that apart from the spleen the vagus nerve targets the proximal colon. Therefore, it is theoretically possible that alterations in the inflammatory state in the vagus nerve innervated proximal colon affect the enteric nervous system activity in the remainder of the colon ⁴⁵ , which in turn may result in a reduction in neurotransmitters release and suppression of inflammation. This may also account for the effect of vagus nerve signaling on colitis observed in this and other studies ⁵³⁶ . Furthermore, the fact that inventors did not observe significant effects of GAL or McN-A-343 on IL- 0 levels suggests that alterations in antiinflammatory cytokine levels do not play a rote in mediating the beneficial effects of cholinergic modalities on colitis severity. This confirms previous data on sepsis and colitis ^8,24 suggesting a minor role of IL-10. In addition, cholinergic treatments did not alter the stool consistency in control mice, indirectly pointing to a lack of significant effect on gut physiology.

The inventors findings prompt close consideration of the relationship between brain cholinergic activation and disease activity in patients with IBD and have important clinical relevance. IBD patients might be selected for novel treatment strategies including: 1) Centrally-acting AChE inhibitors, which are already clinically approved for the treatment of Alzheimer's disease; 2) highly selective muscarinic receptor agonists or antagonists ⁷ , which are being actively developed for the treatment of Alzheimer's disease and other neurodegenerative disorders, as discussed above; and 3) electrical vagus nerve stimulation, which is now being used for the treatment of refractory depression and epilepsy ⁵⁰ .

Mucosal inflammation in IBD is accompanied by autonomic nervous system dysfunction and a decreased vagus nerve activity ⁵⁸ . It is recognized that this autonomic dysfunction plays an important role in motility and immune response dysregulation during IBD ⁵⁹ > ⁶⁰ . Over the last decade it has been demonstrated that the vagus nerve-based CAIP has a major anti-inflammatory function in different inflammatory conditions including colitis ⁹ ' °· ⁵ While the spleen plays an important mediating role in the cholinergic anti-inflammatory pathway ²¹ ' ⁵⁶ · ⁵⁷ , at certain experimental conditions of intestinal inflammation, this physiological mechanism does not require neural signalling to spleen ⁶¹ . Here it is shown that central muscarinic cholinergic activation results in a decreased susceptibility to experimental colitis; this effect is vagus nerve- and splenic nerve-dependent and mediated at a cellular level by a specific interaction between DCs and CD4 ⁺ CD25" T cells.

The DSS-induced colitis is a widely used model of ulcerative colitis ²³ . Depending on the concentration, the duration, and frequency of DSS administration, animals may develop acute or chronic colitis or even colitis-induced dysplastic lesions. The innate and the adaptive immune response are shown to be an important component in colitis pathogenesis in this model. Recently the inventors demonstrated a role of a CNS pharmacological mAChR-dependent stimulation of the CAIP in alleviating the disease seventy and suppressing colonic proinflammatory cytokine (IL- 6, IL- β and TNF-a) levels in experimental DSS-induced colitis ⁵⁴ . This effect was mediated through the vagus and splenic nerve, and the spleen, via the modulation of proinflammatory cytokines released by splenic dendritic cells ⁵⁴ . Here, after confirming the beneficial effect of central cholinergic activation by a MlmAChR agonist in colitis, the inventors demonstrate that this effect is accompanied by a downregulation of IFN- Y and IL-17 levels in colonic tissues. In the context of colitis, Th2 can downregulate the Th1 cytokines release and IL-4 is known to decrease the production of IL-12 in the context of T cell priming ⁶² . However, in this study the inventors did not observe any significant effects of the McN-A-343 infusion on colonic level of IL-4, suggesting that alterations in IL-4 levels do not play a role in mediating the beneficial effects of cholinergic modalities on colitis severity. This observation is in line with previous data demonstrating the minor role of vagotomy on !L-4 in colitis ⁶³ .

Although previous observations clearly implicate dendritic cells ^{54, 6} and T cells ⁵⁷ in the context of the cholinergic anti-inflammatory pathway, it was not clear whether alterations in antigen presenting cell response and T cell priming play any mediating role in cholinergic regulation of gut inflammation. We have recently demonstrated an increase of splenic IL-12p40 after pharmacological stimulation of the vagus nerve ⁵⁴ . The fact that the p40 subunit is commonly shared by IL-12p70 and IL-23 ⁶⁵ - ⁶⁶ prompted the inventors to study IL-12p70 and IL-23 levels. Alleviated inflammation in DSS-induced colitis was accompanied by reduced level of IL-12p70 and IL-23 in the colon and from splenic DCs isolated from colitic mice treated with McN-A-343. Conversely, the inventors also observed that colon and DCs isolated from group without vagus and splenic nerve produced significantly more IL-12p70 and IL-23 and that the beneficial effect of the MCN-A-343 treatment was abolished in the absence of the vagus and splenic nerves. Moreover, in vitro stimulation with a specific a7nAChR agonist decreased IL-12p70 and IL-23 production of DCs isolated from both groups. This is in accordance with data demonstrating a downregulation of IL-12p70 and IL-23 in PMN treated with nicotine ⁶⁷ .

NF-κΒ, an important transcription factor mediating the proinflammatory cytokine production, is attributed to the initiation and progression of colonic inflammation in mice and humans ^{68, 69} . nAChRs are expressed on antigen presenting ceil including macrophages and DCs ⁷⁰ , and ACh and nicotine inhibit TNF-a and NF- KB production from lipopolysaccharide (LPS)-stimulated human macrophages and splenocytes ^{55, 56} . In the context of polymorphonuclear neutrophil activation, it has also been described that GTS-2 can inhibit phosphorylation and subsequent degradation of I-KB, which is likely to suppress NF-κΒ activation ⁷¹ . As nAChRs are expressed on DC ⁷⁰ , we tested the role of the intracellular NF-κΒ pathway. Increased production of IL-12p70 and IL-23 by DCs in VXP- and NRX mice was significantly inhibited in the presence of the NF-κΒ pathway inhibitor. Moreover, in the presence of NF-KB pathway activator the levels of both cytokines were increased in DCs isolated from stimulated groups. In vitro stimulation with GTS-21 decreased IL-12p70 and IL-23 production of DCs isolated from both group and this effect was abolished in the presence of the NF- Β activator. Together, these findings indicate that deactivation of the NF-κΒ signaling pathway is essential for pharmacological cholinergic stimulation to inhibit DC activation, which is critical in the pathogenesis of colitis in this model. This corroborates data demonstrating that in vitro mature spleen DCs that are exposed to nicotine produce decreased levels of IL-12 and display reduced ability to induce T cell responses ⁷² .

The results further demonstrate that DCs isolated from colitic mice treated with McN-A-343 have significantly reduced ability to stimulate naive CD4+T cells in vitro to produce !FN-γ and IL- 7 as compared to the DCs isolated from colitic non-treated mice. This observation provides direct evidence of the importance of DCs in colitis. It is in agreement with studies in the context of experimental colitis ⁷³ demonstrating amelioration of DSS-induced colitis by depletion of DCs after administration of diphtheria toxin in CD1 1c ^" DT receptor transgenic mice ⁷⁴ .

The clinical relevance of these findings is substantiated by previous data demonstrating that IBD is characterized by increased expression of innate (IL-12, IL- 23) and adaptive (IFN-γ, IL-17) proinflammatory cytokines ⁴¹ , and Th1 , Th 7 polarization is mainly controlled by the IL-12p70 and IL-23 cytokine family produced by activated DCs ⁷⁵ . Increased numbers of MDC8 ⁺ monocytes, which are the precursors of mucosal DC populations, are found in patients with IBD and, hence, anti-TNF treatment results in reduced DC activation ⁷⁶ ' ⁷⁷ . Moreover

in the context of IBD, DCs are increased within the lamina propria and peripheral blood ⁷⁸ and DCs isolated from peripheral blood monocytes are potent immune response stimulators ⁷⁹ .

The inventors also further identify the mediating role of IL-12p70 and IL-23 on

DCs T cell priming. In the absence of an intact vagus or splenic nerve the presence of a specific anti-p35 Ab decreased exclusively l N-γ release from T cells. Conversely, in the presence of anti-p19 Ab only the IL-23 levels were affected. This corroborates clinical and experimental data demonstrating an increase in IL-12p40, IL- 2p70 and IL-23p19 in intestinal tissue ^{80, 81} . A beneficial effect of anti-p40 or p19 mAbs on human and experimental colitis has been proposed ⁸² - ⁸³ . IL- 12 and IL-23 that share the same p40 subunit may play distinct roles in colitis, depending on the mechanism involved. IL-23p19-deficient mice develop severe colitis in IL-10 deficient mice ⁸⁴ and !L-12p35 deficient mice develop a mild colitis ⁸⁵ . These somewhat conflicting observations suggest diverse roles of IL-12 and IL-23 in colitis depending on the model used.

Herein, the role of brain pharmacological activation of the CAIP in controlling T cell priming via DC deactivation and the specific mediating role of IL-12 and IL-23 is highlighted. In addition, these studies show the efficacy of this approach in suppressing proinflammatory cytokine production from DCs through an NF-KB intracellular mechanism and sequential T-cell instigation. The inventors showed the same profile of results in mice with DNBS colitis, indicating a broader applicability of our finding.

It is possible that in addition to the spleen the vagus nerve-based CAIP signals in other organs implicated in the development colonic inflammation ⁸⁶ .

These findings provide new insight into the mechanisms of regulation of mucosal inflammation by the cholinergic anti-inflammatory pathway. In addition to enhancing the understanding of the pathogenesis of colitis, this study provides novel information on vagus-to spleen regulation in the context of immune cell signaling via DC T-cell priming. These findings foster consideration of the relationship between brain cholinergic activation and disease activity in patients with IBD and suggest further development of centrally-acting selective Ml mAChR agonists as novel compounds for treatment of colitis.

Results

Centrally-acting acetylcholinesterase inhibitor treatment ameliorates the severity of colitis. Previous findings have characterized GAL as a central activator of the cholinergic anti-inflammatory pathway ^{20 18} ' ³⁰ . Daily administration of GAL (1-4 mg/kg/day, i.p.), starting one day before disease induction, dose-dependently reduced the severity of colitis (Figure 1A, B, C, D). GAL-treated mice with colitis showed a significantly lower DAI for the last 2 days as compared to saline-treated controls with the disease (Figure A). This drug effect was dose-dependent and the highest reduction was achieved with a dose of 4mg/kg/day, which has been previously shown to inhibit mouse brain AChE activity by 43% ³⁰ . The decreased severity of colitis in GAL (4 mg/kg/day)-treated mice as compared to saline-treated controls was also further demonstrated by the 3.2-fold decrease in the macroscopic damage score, in the 3.4-fold decrease in MPO activity and in the 1 .25-fold decrease in serum CRP levels (Figure 1 B, C, D). GAL (4 mg/kg/day) treatment of mice with DSS-induced colitis also significantly lowered the histological manifestation of the disease (Figure 1 E, F, G) and inflammation (Table 1). IL-Ι β levels in GAL-treated mice were 4-fold lower as compared to the DSS-saline treated group (Table 1). Accordingly, IL-6 and TNF-α levels were 2- and 2.3-fold lower respectively (Table 1 ). No significant changes were detected for IL-10 (Table 1).

To characterize the specificity of brain AChE inhibition in preventing the development of colitis, we utilized Huperzine-A, a structurally distinct, highly selective, centrally-acting AChE inhibitor. Huperzine-A administration (0.4 mg/kg/day, i.p.) significantly reduced the disease severity and inflammation in mice with DSS-colitis (Table 4). In addition to their efficacy in DSS colitis, GAL and Huperzine-A treatments decreased the severity of DNBS colitis. Taken together, these findings indicate that centrally-acting AChE inhibitor treatments result in disease-alleviating and counter- inflammatory effects in two experimental models of colitis.

Central muscarinic receptors are essential for alleviation of colitis and inhibition of colonic inflammation by an acetylcholinesterase inhibitor. The inventors next studied whether central mAChRs, which critically mediate brain cholinergic pathways and vagus nerve activation ¹⁸ , are required for the anti- inflammatory effect of GAL. Pre-treatment with AS, a mAChR antagonist that penetrates the BBB, abrogated the suppressive GAL effect on colitis development (Figure 2A, B) and inflammation (Table 1 ). In contrast, pre-treatment with AMN, a mAChR antagonist that does not cross the BBB, failed to alter the beneficial effects of GAL on colitis (Figure 2 and Table 1 ). Neither AMN nor AS alone significantly modified the expression of colitis. In the context of DNBS colitis, results were similar for MPO activity (Figure 7). However AS and AMN and treatments resulted in a slight, but statistically significant decrease in IL-Ι β levels (Figure 7). These results indicate that the anti-infiammatory effects of GAL in the context of colitis are mediated through a brain mAChR-dependent mechanism. Although the presence of mAChRs has been reported on immune cells ¹⁶ , our data indicate that direct modulation of peripheral mAChRs has no significant effect on the magnitude of colonic inflammation (Table 1).

Central administration of mAChR ligands attenuates the severity of colitis and inhibits colonic inflammation. As central mAChRs are important in mediating the effects of GAL, the inventors further assessed the regulatory involvement of these receptors in the context of colitis. Previously, a role for a M1 mAChR activation and M2mAChR inhibition in the regulation of inflammation during endotoxemia has been described ¹⁷ . Accordingly, we performed experiments with mice subjected to i.c.v. infusions of specific mAChR ligands. Treatment with the MlmAChR agonist (McN-A- 343; 5 ng/kg/day) starting one day before induction of DSS-colitis decreased the disease severity (Figure 3A, B) and serum CRP levels and colonic tissue proinflammatory cytokine levels (Table 2).

Then, we examined the anti-inflammatory efficacy of an alternative approach of central cholinergic activation. Central ACh release is negatively regulated by the presynaptic M2mAChR autoreceptor ³¹ ' ³² . Therefore, the inventors studied the efficacy of i.c.v. infusion of the M2mAChR antagonist methoctramine (MTT, 5 ng/kg/day). MTT administration, initiated one day before induction of colitis resulted in comparable significant decrease of colitis severity (Figure 3) and inflammation (Table 2). The inventors also demonstrated the beneficial effect of both treatments in the context of DNBS colitis (Figure 8). These findings highlight the specific regulatory role of central mAChRs in suppressing colitis pathogenesis and are in line with the previously reported anti-inflammatory effects of these two drugs in the context of endotoxemia ¹⁷ .

Central cholinergic disease-alleviating and anti-inflammatory efficacy is mediated through vagus and splenic nerve signaling and the release of ACh in the spleen. To study whether the beneficial effects of central cholinergic activation in the context of colitis depend on the vagus nerve-to spleen axis the inventors performed a series of experiments with mice subjected to vagotomy (VXP), splenic neurectomy (NRX) and/or splenectomy (SPX). VXP significantly exacerbated the severity of colitis (Figure 4A, B) and impaired the inflammatory state (Table 3), thus confirming previously reported findings ³³ . SPX did not alter the development of colitis; however the absence of the spleen abolished the deleterious effect of VXP (Figure 4A, B and Table 3). The beneficial effects of GAL on disease severity and inflammation were abolished in mice with VXP, SPX, or VXP and SPX (Figure 4A, B and Table 3) with the exception of the drug effect on IL-10 levels (Table 3). NRX resulted in comparable to VXP exacerbation of disease severity (Figure 4A, B) and inflammation (Table 3). These effects were abolished in mice simultaneously subjected to SPX (Figure 4A, B and Table 3). Furthermore, the beneficial effects of GAL were abrogated in mice with NRX, SPX or NRX and SPX, and no effect of these manipulations was observed on IL-10 levels (Figure 4 and Table 3).

In addition, beneficial effects of the MlmAChR agonist McN-A-343 i.c.v. infusion in the context of colitis were abolished in the absence of an intact vagus nerve or splenic nerve, or in mice with SPX (Figure 10). Previously, vagus nerve activation functionally associated with signaling along the splenic nerve has been demonstrated to result in an increase of ACh in the spleen ²¹ . Therefore, the inventors next examined the effects of GAL and McN-A-343 treatments on splenic ACh levels and the mediating role of the vagus nerve and the splenic nerve. GAL and McN-A-343 treatments were associated with a significant increase in ACh levels in the spleen of sham-operated control mice with DSS colitis (Figure 5). ACh levels in the VXP and the NRX group were significantly lower as compared to sham-operated controls in mice with DSS colitis and VXP and NRX significantly abolished the increase in splenic ACh release caused by GAL or McN-A-343 treatments (Figure 5). Together, these results demonstrate that neural signaling through the vagus nerve-to spleen axis is required for central cholinergic activation to alleviate colitis.

Central cholinergic activation decreases splenic DC cytokine production and regulates DC priming of CD4 ⁺ T cells. To gain further insight into the cellular mechanisms mediating cholinergic anti-inflammatory effects on colitis the inventors studied the role of DCs. IL-12p40 levels in the CD1 1c ⁺ DC- culture supernatant from colitic GAL-treated (Figure 6A) were significantly decreased as compared to colitic non-treated groups. Conversely, VXP or NRX increased IL-12p40 (Figure 6A) and no beneficial effect of GAL (Figure 6A) treatment was found in the absence of an intact vagus or splenic nerve. The inventors next performed ex vivo experiments to highlight the role of the a7nAChR in mediating cholinergic anti-inflammatory effects in spleen. Splenic CD1 1c ⁺ DCs were isolated from colitic mice subjected to sham-operation, VXP or NRX and a treatment with vehicle or GAL (i.p.). Then these cells were treated ex vivo with GTS-21 (a specific o7nAChR agonist). At all conditions the addition of GTS- 21 in the culture medium significantly decreased IL-12p40 production (Figure 6A). Apart from being an AChE inhibitor, GAL has properties of an allosteric positive modulator of nicotinic, including a7nAChR34. Therefore, the inventors evaluated the possibility that stimulation of the a7AChR on CD11 c ⁺ DC by GAL might contribute to anti-inflammatory effects. CD11c ⁺ DC isolated from non-colitic mice were activated with LPS and treated with GAL or vehicle ex vivo. The inventors did not observe any significant GAL effect on IL-12p40 (440±23 vs 435±41 pg/ml) levels in these settings. We next assessed whether GAL treatment could affect the ability of DCs to prime CD4 ⁺ T cells. CD4 ⁺ T cells isolated from naive mice were co-cultured with splenic DCs isolated from colitic mice treated with GAL in the presence or absence of the vagus or splenic nerve for 24h. When co-cultured with DCs isolated from colitic in vivo GAL- treated mice, CD4 ⁺ T cells released significantly lower amount of INF-γ and IL-17 as compared to CD4 ⁺ T cells co-cultured with DCs isolated from colitic in vivo saline- treated mice (Figure 6B, C). The inventors observed the same effects using colitic CD1 1c ⁺ DCs treated ex vivo with GTS-21 (Figure 6B, C). In the absence of the vagus nerve or splenic nerve, the effects of GAL were abolished (Figure 6B, C). However, the ex vivo effect of GTS-21 was manifested at any of these conditions (Figure 6B, C). Similar patterns of cholinergic efficacy were observed when instead of galantamine (i.p.) treatments we utilized i.c.v. infusions of the Ml mAChR agonist cN-A-343 at the above described experimental conditions (Figure 10A, B, C). Taken together, these findings indicate that a7nAChR-dependent decrease of IL-12p40 production from splenic DCs followed by sequential T cell down-regulation are important events mediating the anti-inflammatory efficacy of central cholinergic activation in the context of colitis.

Central muscarinic cholinergic activation decreases serum SAP level and colonic Th1/Th17 cytokine release via the vagus nerve and the splenic nerve.

Infusion with McN-A-343 (5ng/kg/day) started one day before induction of DSS-colitis decreased all colonic inflammatory markers studied. Decreased severity of colitis in McN-A-343-treated mice as compared to vehicle-treated mice was evident in the 2.1- fold decrease in the SAP levels (Figure 1 1A) and the colonic increase in IFN-γ was 2.7-fold lower (Figure 11 B), respective values for IL- 17 (Figure 11 C), IL~12p70 (Figure 11 D), and IL-23 (Figure 1 1 E) were 2.4, 3.1 and 2.2 fold-lower. No significant changes were detected in IL-4 levels. Together these findings confirm that centrally- acting MlmAChR agonist treatments result in disease-alleviating and counter- inflammatory effects in the DSS experimental models of colitis as assessed by alterations in the Th1/Th17 cytokine profile.

To study whether the beneficial effects of central cholinergic activation in the context of colitis depend on the vagus nerve-to spleen axis the inventors performed a series of experiments with mice subjected to vagotomy (VXP), splenic neurectomy (NRX) and/or splenectomy (SPX). VXP significantly exacerbated the severity of colitis (Figure 1 ), thus confirming our previously reported findings ³³ . SPX did not alter the development of colitis; however the absence of the spleen abolished the deleterious effect of VXP (Figure 1 1 ). The beneficial effects of McN- A-343 on disease severity and inflammation were abolished in mice with VXP, SPX, or VXP and SPX (Figure 11). NRX resulted in comparable to VXP exacerbation of disease severity (Figure 11). These effects were abolished in mice simultaneously subjected to SPX (Figure 1 1 ). Furthermore, the beneficial effects of McN-A-343 were abrogated in mice with NRX, SPX or NRX and SPX (Figure ). No effect of these manipulations was observed on colonic IL-4 levels. These results demonstrate that neural signaling through the vagus nerve and the splenic nerve is required for central cholinergic activation to alleviate colitis, by altering specifically the Th1/Th 7 cytokines and with no effect on IL-4.

Central cholinergic activation decreases splenic CD11c+ DCs IL-12p70 and IL-23 release via the vagus and the splenic nerves. Some studies have reported a major role of the spleen in the context of colitis pathogenesis ^{51 ,52} while others have failed to demonstrate that ⁵³ . DC and macrophage regulation in the spleen during the development of experimental colitis has been previously shown ⁵⁴ ' ²¹ and here we indicated the implication of specific splenic DC-derived cytokines. To gain further insight into the cellular mechanisms mediating cholinergic anti-inflammatory effects on colitis the inventors studied the role of splenic CD11 c ⁺ DCs. A significant decrease of IL-12p70 and IL-23 was found in the supernatant of splenic CD 1c+ DCs isolated from colitic McN-A-343-infused mice (Figure 12) as compared to colitic vehicle-treated groups. Conversely, VXP or NRX treatments increased the two cytokines and no beneficial effect of McN-A- 343 infusion was found in the absence of an intact vagus nerve or splenic nerve. To confirm the role of the a7nAChR we isolated splenic CD1 1c ⁺ DCs from sham- operated, VXP or NRX-colitic mice. In both last conditions, addition of GTS-21 (a specific a7nAChR agonists, 100μΜ) in the culture medium significantly decreased the production of IL~12p70 and IL-23 (Figure 12). No synergistic affect was observed in CD1 1c ⁺ DCs isolated from colitic cN-A- 343-infused mice and treated in vitro with GTS-21 . No effect on splenic CD1 1 c ⁺ DCs proliferation was observed (data not shown) in line with previously published findings ⁵⁴ .

Central cholinergic activation decreases splenic CD11c+ DCs IL-12p70 and IL-23 release via the NF- Β pathway. The inventors have previously demonstrated an increase of splenic ACh levels after central activation of the CAIP, accompanied by a decrease of splenic CD 1c+ DCs IL-12p40 release ⁵⁴ . In parallel, nicotine inhibits NF-κΒ production from lipopolysaccharide (LPS)-stimulated splenocytes ⁵⁵ ' ⁵⁶ .. To provide insight into the transduction pathway by which central cholinergic activation decreases IL-12p70 and IL-23 in splenic CD1 c ⁺ DCs, we pretreated isolated splenic CD 1 c+ DCs with an NF-κΒ activator (betulinic acid) or an inhibitor (BAY 1 1-7082) for 24 hours. The beneficial effect of McN-A-343 infusion or treatment with GTS-21 was reversed in the presence of betulinic acid (Figure 13A, B). Conversely, the deleterious effect of VXP and NRX was abolished in the presence of BAY 11-7082 (Figure 13C, D). These results highlight the importance of the NF-κΒ pathway in splenic CD11 c ⁺ DCs as a mediating event of IL-12p70 and IL-23 release during colitis and its suppression by central muscarinic cholinergic activation of the vagus nerve-to spleen axes.

Central cholinergic activation regulates splenic CD11c ⁺ DCs priming of CD4 ⁺ CD25 ^" T cells via the vagus and splenic nerve. Recently the inventors demonstrated that the central cholinergic activation decreases splenic CD11 c ⁺ DCs cytokine production, MHC class II marker and that this effect was dependent on neural signals along the vagus and the splenic nerve ⁵⁴ . Moreover, T cell regulation in the spleen during the development of experimental colitis has been previously shown ⁵⁷ . To gain further insight into the cellular mechanisms the inventors studied the T-cell priming function of splenic CD1 1 c ⁺ DCs in this context. To determine the contribution of CD4 ⁺ CD25 ^" T ceils to the decreased cytokines production observed in the spleen following activation of the CAIP during colitis, we isolated CD4 ⁺ CD25 ^_ T cells from the spleen of naive animals and determine the splenic CD1 1 c ⁺ DCs priming of CD4 ⁺ CD25 ^" T cells and their cytokine release. CD4 ⁺ CD25 ^" T cells, isolated from naive mice were co-cultured for 24h with splenic CD11 c+ DCs isolated from colitic mice infused with McN-A-343. CD4 ⁺ CD25 ^" T cells released significantly lower amount of IFN-Y (Figure 14A), IL-17 (Figure 14B) as compared to CD4 ⁺ CD25 ^~ T cells co- cultured with splenic CD11c ⁺ DCs isolated from in vivo saline-treated colitic mice. Conversely, naive CD4 ⁺ CD25 ^" T cells released significantly more IFN-γ and IL-17 when they were co-culture with splenic CD11c ⁺ DCs isolated from vagotomized or neurectomized colitic mice. No beneficial effect of McN-A-343 infusion was found in the absence of an intact vagus and splenic nerve. None of the treatment had an effect on the IL-4 levels (Figure 14C). To evaluate the role of the a7nAChR we used splenic CD1 1c ⁺ DCs isolated from sham-operated, VXP, and NRX-colitic mice. At all conditions, addition of GTS-21 in the culture medium significantly decreased the production of IFN-γ and IL-17. No synergistic affect was observed in DCs isolated from colitic McN-A-343 treated mice and treated in vivo with GTS21. No drug effect on T-cell proliferation was observed within a 24h period of time (Figure 14D).

Central cholinergic activation regulates splenic CD11c ⁺ DCs priming of CD4 ⁺ CD25 T cells via IL-12 and IL-23. The p40 subunit is shared by the IL-12 and IL-23 cytokine. To elucidate the mechanisms by which central cholinergic activation decrease T-cell priming, we pretreated isolated splenic CD1 c ⁺ DCs with anti p19- mAb or p35-mAb. In the presence of anti p19-mAb, the deleterious effect of VXP and NRX was abolished in terms of the IL-17 cytokine profile (Figure 5A) whereas in the presence of anti p35-mAb only lFN-γ levels were affected (Figure 15B). Conversely, addition of IL-12p70 or IL-23 recombinant proteins restore the level of IFN-γ and IL-17 in CD4 ⁺ CD25- T cells conditioned with splenic CD11 c ⁺ DCs isolated from colitic mice treated with McN-A-343 or GTS-21 (Figure 15C, D).

Effect on DNBS-induced colitis. In the context of DNBS colitis, results were similar (Table 5). Methods

Animals. Male C57BL/6 (7-9 weeks old) were purchased from Charles Rivers (Canada) and maintained in the animal care facility at the University of Manitoba under specific pathogen-free conditions. No differences in food intake or body weight were observed between the groups. All experiments were approved by the University of Manitoba animal ethics committee (10-073) and conducted under the Canadian guidelines for animal research.

Induction of DSS and DNBS colitis. DSS (molecular weight [MW], 40 kilodaltons: ICN Biomedicals Inc) was added to the drinking water in a final concentration of 5% (wt/vol) for 5 days ^22,23 . Controls were all time-matched and consisted of mice that received normal drinking water only. Mean DSS consumption was noted per cage each day. For the DNBS study, mice were anaesthetized with Isoflurane (Abbott, Abbott Park, IL). A 10cm long PE-90 tubing (ClayAdam, Parsippany, NJ), attached to a tuberculin syringe, was inserted 3.5cm into the colon. Colitis was induced by administration of 100μΙ of 5mg of DNBS solution (ICN) in 50% ethanol and left for 3 days ²⁴ . Control mice (without colitis) received saline administration. Mice with colitis were supplied with 6% sucrose in drinking water to prevent dehydration. Alternatively, 2, 4-dinitrobenzen-sulfonic-acid (DNBS) colitis was induced by administration of 100μΙ of 4mg of DNBS solution (Sigma, Oakville, Canada) in 30% ethanol for 3 days.

Surgical procedures and drug treatments. Mice were anaesthetized using ketamine (150 mg/kg, i.p) and xylazine (10 mg/kg, i.p). I.c.v. implantation of the cannula, splenectomy (SPX), splenic neurectomy (NRX) or subdiaphragmatic bilateral vagotomy (VXP) was performed on the same day ⁵ . In sham-operated group: mice implanted with the cannula received vehicle; mice were anaesthetized and laparotomy performed but the spleen was not removed ; splenic nerve was exposed but not cut; vagal trunks were exposed but not cut, however, a pyloroplasty was performed. The completeness of vagotomy was verified during post-mortem inspection of vagal nerve endings using microscopic inspection associated with a Bielschowsky silver staining ²⁵ . The completeness of neurectomy was verified post-mortem by noradrenaline enzyme- linked immunosorbent assay in sham-operated and NRX animals. Mice were allowed to recover for 10 days. One day before initiation of colitis pharmacological treatments started: (GAL, 1 -4 mg/kg/day, intraperitoneal (i.p.)); Huperzine-A: (H-A, 0.4 mg/kg/day, i.p., Sigma, Oakville, ON); Atropine Methyl Nitrate (AMN, injected 20 min prior to GAL, 4 mg/kg/day, i.p., Sigma); Atropine sulfate (AS, injected 20 min prior GAL, 4 mg/kg/day, i.p. Sigma). Micro-osmotic pumps (Alzet, Cupertino, California, USA) were filled with vehicle (saline 0.2%), M1 mAChR agonist McN-A-343 (Sigma) or M2 mAChR antagonist methoctramine (MTT, 5 ng/day, Sigma) solution and placed as previously described ²⁴ .

Characterization of inflammation. Disease activity index (DAI) and macroscopic scores and colonic damage were determined using a previously described scoring system for DSS colitis ⁵ ' ²⁶ and for DNBS ⁵ ' ²⁷ . Formalin-fixed colon segments coming from the splenic flexure were stained with hematoxylin-eosin ⁵ . Myeloperoxidase (MPO) activity was determined following an established protocol ²⁸ . Serum C-reactive protein (CRP) and cytokine levels were determined using ELISA commercial kit (R&D Systems, Minneapolis, Minnesota, USA). The amount of acetylcholine in the spleen was measured using the acetylcholine assay kit following manufacturer's recommendations (Molecular Probes Invitrogen).

Alternatively, samples were collected 5 days post-DSS activation and blood was collected by intracardiac puncture under isoflurane anesthesia. Serum amyloid protein (SAP) and colonic cytokine levels were determined using ELISA commercial kit (R&D Systems, Minneapolis, MN).

Isolation of dendritic cells and culture. Briefly, the spleens were digested in 2mg/ml" ¹ collagenase D (Roche Diagnostics, Meylan, France) in RPMI 1640 for 30min at 37°C. EDTA at 5mM was added during the last 5min to disrupt DC-T cell complexes, and the cell suspension was filtered. Total splenocytes after RBC lysis with ACK lysis buffer (150mM NhUCI, 10mM KHCOs, 0.1 m EDTA) were incubated with CD 1 c microbeads (Miltenyi Biotec, Auburn, CA) for 15min at 48°C. The cells were then washed, resuspended in cell separation buffer (Dulbecco's Phosphate- Buffered Saline [D- PBS] without Ca21 and Mg21 containing 2% FBS and 2mM EDTA, Life Technology) and passed through magnetic columns for positive selection of DC. After passing consecutively through two columns, the collected DCs preparations showed greater than 95% purity. DC isolated from different groups of mice were cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS, 25mg/m|- ¹ gentamicin, 2mM L-glutamine in 12-well plates at 1.10 ⁺⁶ cells/well for 24hrs, and the supernatants were measured for IL- 2p40 by ELISA (R&D Systems).

In some experiments lipopolysaccharide (LPS, Sigma) was added to the cultures at a final concentration of 100 ng/ml ^" . In a separated set GAL or the a7nAChR agonist GST-21 were added to medium at a final concentration of 10 ^_5 M.

T Cell co-culturing with DCs. CD1 c ⁺ DCs isolated from colitic mice receiving the different treatments were cultured for 24 hrs before being co-cultured with CD4 ⁺ T cell isolated from naive mice at ratio of 1 :3 (DC:T)29 in plate coated with 10 pg/ml ^" of anti-CD3 and 2 g/ml ^"1 of anti-CD28. Cell culture supernatants were collected at 24 hrs, interferon (INF)-y and IL-17 levels were analyzed by ELISA (R&D Systems).

Isolation of splenic CD11c+ cells and culture. 5 days post-DSS activation, splenic CD1 1c ⁺ DC were isolated from different groups of mice and were cultured for 24hrs, the supernatant was measured for IL-12p70 and IL-23 by ELISA (R&D Systems). In a separate set of experiment the o;7nAChR agonist GTS-21 (100pM) or betulinic acid (a specific NF- Β activator, 10μΜ) or BAY 11-7082 (a specific NF- B inhibitor, 10μΜ) were added to medium.

T Cell co-culturing with DCs. CD1 1c+ DCs isolated from colitic mice receiving the different treatments were cultured for 24hrs before being co-cultured with CD4+/CD25- T cell isolated from naive mice at ratio of 1 :3 (DCs:T cells) ²⁷ in plates coated with ^g/ml ^'1 of anti-CD3 and 2μg/m|- ¹ of anti-CD28. Cell culture supernatants were collected at 24hrs, and interferon-gamma (IFN-v), IL-4 and IL- 17 levels were analyzed by ELISA (R&D Systems). In neutralization experiments, these cultures were treated with 0pg/ml anti-IL-12p35 or anti-IL-23p19 (R&D Systems) neutralizing mAb to block the potential activities of endogenous sources of these cytokines. In a separated set, recombinant (r) IL~12p70 or rfL- 23protein (25ng/m , R&D Systems) were added to the cell culture medium.

Induction of acute DNBS colitis. Mice were anaesthetized with lsoflurane→(Abbott, Toronto, Canada). A 10cm long PE-90 tubing (ClayAdam), attached to a tuberculin syringe, was inserted (intrarectaliy) 3.5 cm into the colon. Colitis was induced by intra-rectal administration of 10ΟμΙ of 4mg of DNBS solution (ICN) in 30% ethanol and the mice left for 3 days. Mice were killed on day 3 and samples were taken. Control mice (without colitis) received saline administration. Mice with colitis were supplied with 6% sucrose in drinking water to prevent dehydration.

Isolation of splenic CD11 c ⁺ cells and culture. 5 post activation associated with DSS, the spleens were digested in 2mg/ml-1 collagenase D (Roche Diagnostics, Meylan, France) in RPMI 1640 for 30min at 37°C. EDTA at 5mM was added during the last 5min to disrupt DC-T cell complexes, and the cell suspension was filtered. Total splenocytes after RBC lysis with ACK lysis buffer (150mM NH4CI, 10mM KHC03, 0.1 mM EDTA) were incubated with CD11c ⁺ microbeads (Milteny Biotech, Auburn, CA) for 15min at 48°C. The cells were then washed, re- suspended in cell separation buffer (Dulbecco's Phosphate-Buffered Saline [D-PBS] without Ca21 and Mg21 containing 2% FBS and 2mM EDTA, Life Technology, Grand Island, NY) and passed through magnetic columns for positive selection. After passing consecutively through two columns, the collected splenic CD11c ⁺ cell preparations showed greater than 95% purity. Splenic CD1 1c ⁺ cell isolated from different groups of mice were cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS, 25mg/ml-1 gentamicin, 2mM L-glutamine in 12-well plates at 1.10 ^+δ cells/well for 24hrs, and the supernatants were measured for IL- 12p70 and IL-23 by ELISA (R&D Systems). In a separate set the a7nAChR agonist GTS-21 or betu!inic acid (a specific NF- B activator, 10DM) or Bay 1 1 -7085 (a specific NF-κΒ inhibitor, 10μΜ) were added to medium at a final concentration of 10 ^~6 M.

T Cell isolation and proliferation. 5 post-activation associated with DSS, CD4 ⁺ cells were isolated from freshly isolated spleen by magnetic microbeads selection according to manufacturer's protocol (MACS, Milteny Biotech). Briefly, splenocytes were incubated for 15 min in 4°C with microbeads and washed twice in washing buffer (10 min) and depleted using MACS LD column. The obtained CD4 ⁺ T cells were washed twice in washing buffer (10 min) resuspended and labeled with anti-CD25 microbeads. Subsequently, the cells were incubated for 5 min in 4°C. The cells were then incubated for the next 15 min at 4°C and washed twice in washing buffer. Then CD4 ⁺ /CD25 ^" T cells were isolated using MACS MS column. After the last centrifugation, the selected subpopulations of CD4 CD25 ^" T cells were resuspended in culture medium (RPMI 1640 supplemented with 10% FCS, l-Glutamine 200 mM, HEPES 1 M, antibiotic-antimycotic) and were immediately used in assay.

For T-cell proliferation, cells were labeled with CFSE 5x10 ⁺⁴ of labeled cells were cultured for 24h, and then proliferation assay was assessed by CFSE dilution, using FACS and FlowJo program (TreeStar, Ashland, OR)

Statistical analysis. Results are presented as means ±SEM. Statistical analysis was performed using one or two way ANOVA followed by the Tukey-Kramer multiple comparisons post hoc analysis and a p value of <0.05 considered significant with n=8 to 12 depending on the groups tested (Prism 4, GraphPad).

The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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H20 DSS 5%

Vehicle Galantamine

Vehicle AMN AS Vehicle AMN AS Vehicle AMN AS

CRP

(pg/ml 2.9±2.5 2.4±1.8 3.7±2.6 35.9±1.8 ^a 37.2±1.9 ^a 36.2±1.3 ^a 30.1-tl.8 ^b 29.7±l.l ^b 36.8±1.3 ^C serum)

IL-l-p

(Pg/mg 5.5 ±2.3 5.4±2.1 5.2±2.7 40±2.7 ^a 38.4±2.8 ^a 39.2±3. 10.1-bl.8 ^b 11.4±1.9 ^b 38.9- 4.3 ^c protein)

IL-6

(Pg/mg 3.9±2.4 4.5±1.9 3.7±2.6 67.4±5.7 ^a 64.7±6.1 ^a 69.2±7.4 ^a 33.4±4.5 ^b 36.6±3.9 ^b 59.9±7.8 ^C protein)

TNF-a

(Pg/mg 7.5±2.9 8.2±1.3 9.1±1.4 40.3±3.8 ^a 37.3±3.1 ^a 39.1±7.1 ^a 18.3±2.7 15.5±2.4 ^b 32.7±4.3 ^C protein)

IL-10

(Pg/mg BLS* BLS* BLS* 8.4±4.5 ^a 9.7±4.1 ^a 8.9±3.1 ^a 8.8±4.5 ^b 7.7±3.7 ^b 9.4±4.4 ^C protein)

Table 1. Galantamine anti-inflammatory effects in DSS-induced colitis are mediated through central mAChRs. AMN (atropine methyl nitrate); AS (atropine sulfate); *BLS (below the lowest standard); ^a <0.05, as compared to non DSS-treated group (H20), ^b <0.05 as compared to vehicle DSS-treated group, ^C P<0.05 as compared to GAL DSS-treated group. See Material and Methods for details. H20 DSS 5%

Vehicle MCN-A-343 MTT Vehicle McN-A-343 MTT

CRP

(pg/ral 2.5±2.1 1.25±1.9 2Λ±1.9 35.3±2.9 ^a 25.4±4.3 ^a ' ^b 21.4±3.1 ^a ' ^b serum)

IL-l-p

(pg/mg 5.9±1.6 4.5±1.6 5.4±1.5 35.2±3.1 ^a 12.3±2.1 ^a ' ^b 14.3±2.5 ^{a, b} protein)

IL-6

(Pg/mg 4.4±2.7 4.0±3.7 2.9±2.1 54.2±3.1 ^a 36.4±3.7 ^{a, b} 29.8±4.3 ^{¾ b} protein)

TNF-a

(Pg/mg 3.5±3.9 3.9±3.0 4.7±3.7 47.4±4.1 ^a 19.7±4.1 ^¾ " 20.2±3. ^b protein)

EL-10

(Pg/mg BLS* BLS* BLS* 8.3±3.3 ^a 7.9±4.1 ^a 8.9±3.9 ^a protein)

Table 2. Central administration of a MlmAChR agonist or a M2mAChR antagonist reduces inflammation in mice with DSS-induced colitis. MTT (methoctramine); *BLS (below the lowest standard; ^a <0.05 as compared to non DSS-treated group (H20), Vo.05 as compared to vehicle DSS- treated group. See Material and Methods for details.

Table 3: Galantamine ant i- inflammatory effects in mice with DSS-induced colitis are dependent on vagus nerve and splenic nerve signaling and the spleen. VXP (vagotomy); NRX (splenic neurectomy); SPX (splenectomy); *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; ^a <0.05 as compared to sham- vehicle-DSS-treated group, ^b <0.05 as compared to VXP-DSS-treated group , ^C <0.05 as compared to NRX-DSS-treated group ^d P<0.05 as compared to sham-GAL-DSS-treated group. ^C P<0.05 as compared to VXP-GAL-DSS-treated group , ^f <0.05 as compared to NRX-GAL- DSS-treated group. See Material and Methods for details.

Table 4: The centrally-acting AChE inhibitor huperzine-A reduces the severity of DSS-induced colitis and inflammation.

disease induction. Macroscopic score, myeloperoxidase (MPO) activity, serum C- reactive protein (CRP), and cytokine profile in colonic tissue were determined 5 days after DSS induction. H-A significantly decreased all the markers, but IL-10. aP<0.05 vs saline-treated non DSS (control) group, ^b P<0.05vs saline-treated DSS group. Values are shown as means ± SEM, n>8. *BLS (below the lowest standard) ^c

pg/ml (DC:T cell _{36 8±6} 44.2+6.5 45±3.8 44.1±7.6 48.4±7.9 41.5±8.9 co-culture

medium]

Supplementary Table 1. Central administration of a MlmAchR agonist alleviates the severity of 2, 4 dinitrobenzene sulfonic acid (DNBS) -induced colitis. Vagotomy (VXP) or splenic neurectomy (NRX) were performed 10 days prior to initiating McN-A-343 (5 ng kg/day, i.e. v.) treatment and/or colitis induction as described in Material and Methods. Serum amyloid protein (SAP); colonic interferon-gamma (IFN-γ), interleukin (IL)-17, IL-12p70, IL-23 and IL-4. IL-12p70 and IL-23 production from splenic CDl lc ⁺ dendritic cells (DC). Splenic CDl lc ⁺ DCs were isolated from McN- A-343 (5 ng/kg/day, i.c.v. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 5 post-colitis induction. Effect of McN-A343 (in vivo) treatment on splenic CDl lc ⁺ DCs function and sequential CD4 ⁺ CD25T cell activation. Splenic CDl lc ^"1" DCs isolated from different colitic group were cultured in for 24h before being co- cultured with CD4 ⁺ CD25 cells isolated from naive mice. The levels of IFN-γ and IL-17 and IL-4 were measured in media at 24hrs. ^a <0.05 as compared to sham- saline-DNBS -treated group, ^b P<0.05 as compared to VXP-DSS-treated group or NRX-DSS-treated group. P<0.05 as compared to NRX- DSS-treated group.