neurostimulation of the cholinergic antiinflammatory ... · rostimulation of the cholinergic...

3 4 | L E V I N E E T A L . | B I O E L E C T R O N M E D 1 : 3 4 - 4 3 , 2 0 1 4

INFLAMMATORY BOWEL DISEASE ANDRHEUMATOID ARTHRITIS: CLINICALCHARACTERISTICS AND CURRENTTREATMENT APPROACH

Inflammatory bowel disease (IBD),comprising Crohn’s disease and ulcera-tive colitis, and rheumatoid arthritis (RA)are debilitating chronic inflammatory dis-orders that together affect approximately1% to 1.5% of the population in devel-oped countries. These diseases have amajor negative impact on patients’ livesand an enormous social and economiccost as they often reach their greatest

severity during what should be the highlyproductive years of early adulthood andmiddle age (1,2). In the case of RA, it is in-creasingly appreciated that incompletelytreated systemic inflammation leads to ac-celeration of atherosclerosis with elevatedrisk of morbidity and mortality from car-diovascular disease (3). Both IBD and RAare typically treated in a step-care para-digm, beginning with older drugs such ascorticosteroids and immunosuppressiveagents (for example, azathioprine inCrohn’s disease and methotrexate in RA).Although many patients can be controlled

on these drugs, most will progress to tar-geted biological or small molecule thera-pies including first line agents such astumor necrosis factor (TNF) inhibitors,followed by a variety of second-lineagents. Although these targeted therapiesare quite efficacious and have resulted inmajor improvements in disease control,not all patients respond; and they are as-sociated with significant side effects andare costly, so there remains a significantunmet medical need for patients with RAand IBD.

A POTENTIAL ALTERNATIVE APPROACHTO RA AND IBD TREATMENT: THEINFLAMMATORY REFLEX AND THECHOLINERGIC ANTIINFLAMMATORYPATHWAY

The vagus nerve mediates the inflam-matory reflex, a mechanism the centralnervous system utilizes to regulate in-nate and adaptive immunity (4). The af-ferent arm of the reflex senses inflamma-

Neurostimulation of the Cholinergic AntiinflammatoryPathway in Rheumatoid Arthritis and Inflammatory BowelDisease

Yaakov A Levine,1 Frieda Koopman,2 Michael Faltys,1 Ralph Zitnik,1 and Paul-Peter Tak3,4,5

1Setpoint Medical Corporation, Valencia, California, United States of America; 2Academic Medical Centre/University of Amsterdam,Amsterdam, the Netherlands; 3Ghent University, Ghent, Belgium; 4Cambridge University, Cambridge, United Kingdom; and5GlaxoSmithKline, Stevenage, United Kingdom

Inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) cause significant morbidity and mortality. Despite significanttherapeutic advances, the medical need for patients with these disorders remains high. An important neural-immune regulatorymechanism termed the “inflammatory reflex,” and its efferent arm, the “cholinergic antiinflammatory pathway” regulate innateand adaptive immunity. An emerging body of evidence indicates that stimulation of this pathway with implantable medical de-vices is a feasible therapeutic approach in disorders of dysregulated inflammation. Herein we describe the underlying biologyand the preclinical experiments done in standard animal models that provided the rationale for testing in clinical trials. The pre-clinical development approach comprised elements of classic drug and medical device development, yet had unique featuresand challenges. “Bioelectronic medicines” having ideal characteristics of both drugs and medical devices hold great concep-tual promise for treatment of systemic diseases in the future. However studies being done today will help determine whether neu-rostimulation of the cholinergic antiinflammatory pathway (NCAP) has the potential in the nearer term to fulfill the needs of pa-tients, caregivers and payers for an additional potential treatment option for inflammatory disorders, and might thus become oneof the first feasible examples of a bioelectronic medicine.Online address: www.bioelecmed.orgdoi: 10.15424/bioelectronmed.2014.00008

Address correspondence to Paul P Tak, GlaxoSmithKline, Gunnels Wood Road, Stevenage

SG1 2NY, United Kingdom. Phone: +44-1438-763766; E-mail: [email protected].

Submitted October 7, 2014; Accepted for publication October 9, 2014; Published Online

(www.bioelecmed.org) December 2, 2014.

P R O C E E D I N G S

B I O E L E C T R O N M E D 1 : 3 4 - 4 3 , 2 0 1 4 | L E V I N E E T A L . | 3 5

tion both peripherally and in the centralnervous system, and downregulates theinflammation via efferent neural outflow.The efferent arm of this reflex has beentermed the “cholinergic antiinflamma-tory pathway” (CAP) (Figure 1). The re-flex serves as a physiological regulator ofinflammation by responding to environ-mental injury, pathogens and other exter-nal threats with an appropriate degree ofimmune system activation (4,5).

Our understanding of the CAP begantwo decades ago with the first studiesshowing communication between thebrain and the immune system (6,7). Traceyand colleagues demonstrated that sys-temic, hepatic, and splenic TNF produc-tion as well as the physiological manifesta-tions of endotoxemic shock in rodentswere worsened by vagotomy and amelio-rated by electrical stimulation of the cervi-cal vagus nerve (VNS). Further, based onin vitro experiments, they postulated thatthis effect was mediated directly by acetyl-choline acting through a specific α-7 nico-tinic acetylcholine receptor (α7nAChR) onmacrophages (8). It was later demon-strated that reducing the response to en-dotoxemia using NCAP required an intactspleen, and selective anatomical lesion ex-periments showed that an intact neuralpathway to the spleen from the cervicalvagus through the celiac plexus was alsonecessary for this effect (9). Within thespleen itself, nerve fiber synaptic vesiclesare found in close apposition to TNF- secreting macrophages (10), and to a re-cently described population of cluster ofdifferentiation (CD)4+CD44highCD62low

T cells (11). These T cells bear surface β adrenoceptors, and have the capacity tosynthesize and secrete acetylcholine. Thisunique T-cell population functions analo-gously to an intermediate neuron, sensingadrenergic neurotransmitters released bythe splenic nerve, and, in turn, transmit-ting the efferent signal to adjoining splenicmacrophages by local secretion of acetyl-choline. The α7nAChR expressed on thesurface of macrophages is essential for theNCAP effect, as demonstrated by antisenseoligonucleotide, targeted disruption (12)and adoptive transfer experiments (13).

In contrast to the path through thespleen, vagal efferent fibers directly inner-vate the gut with the exception of the dis-tal colon, synapsing with the myentericplexus without an intermediate anatomicganglion (see Figure 1). Nerve fibers lie inclose apposition to macrophages and den-dritic cells in the gut wall. Intermediaryacetylcholine- producing T cells analogousto those observed in the spleen have notyet been identified (14). Indeed, in amodel of postoperative inflammatoryileus, VNS was able to reduce intestinalinflammation even in recombinase- activating gene (RAG)-1 knockout ani-mals lacking T cells, and in animals withsurgical denervation of the spleen. Inflam-mation in this model was not reduced by

VNS in animals with targeted disruptionof the α7nAChR, but this deficit could bereconstituted by bone marrow transplantfrom wild-type animals (15). In contrast,the spleen was required for resolution ofmucosal inflammation by central cholin-ergic agonists in a recent study in experi-mental colitis (16). Vagus nerve terminalshave been shown to interface with cholin-ergic myenteric fibers, yet the question ofprecisely which if any nonneuronal inter-mediate cells complete the pathway be-tween the vagus and macro phages in thegut is still being resolved (15).

Circulating monocytes produce TNFand other proinflammatory cytokines inresponse to in vitro exposure to bacteriallipopolysaccharide (LPS) in normal ro-

Figure 1. Schematic representation of the cholinergic antiinflammatory pathway. The reflexis activated in the circumventricular brainstem and vagal afferent fibers through signalingby inflammatory mediators. After integration, this sensory input will trigger the efferent armvia the celiac ganglion, modulating the immune response in the spleen. Adrenergic neu-rons in the spleen release norepinephrine that induces acetylcholine release by a subsetof splenic T cells. Acetylcholine ligands the α7nAChR expressed on cytokine-producingmacrophages thereby reducing the release of TNF, IL-1, IL-18, HMGB1, and other cytokines.During intestinal inflammation the vagal efferent fibers directly contact myenteric neuronsin the intestinal wall. Enteric neurons release acetylcholine, similarly reducing intestinalmacrophage cytokine release. Image from (14) (reproduced from Gut, The vagal innerva-tion of the gut and immune homeostasis, Gianluca Matteoli, Guy E Boeckxstaens,62:1214–22, © 2013, with permission from BMJ Publishing Group Ltd.).


N C A P I N R A A N D I B D

dents, normal human volunteers and RApatients. After VNS or in vitro exposure tocholinergic agonists, the ability of theseperipheral blood cells to release inflam-matory mediators in response to LPS isreduced markedly (17,18). In rodent mod-els, VNS reduced granulocytic infiltrationto sites of injury, including the muscularismucosa in inflammatory postoperativeileus (19), to the pancreas in pancreatitis(20), and to the lung following burn- induced acute lung injury (21). Experi-ments using carrageenan to induce in-flammation demonstrated that VNS orpharmacologic CAP reduced trafficking tosites of inflammation, driven by a reduc-tion in surface expression of the integrincomponent CD11b on neutrophils (22).

When taken together, these observationsdemonstrate that CAP activation reducesproduction of systemically active cyto -kines, chemokines and antibodies by resi-dent spleen cells, and causes circulatingcells which traverse the spleen to developan altered phenotype with reduced expres-sion of inflammatory mediators and adhe-sion molecules. As a result of this alteredphenotype, trafficking of immune cells toinflamed tissue is reduced. Further, uponentering inflamed tissue, these cells are lessable to release mediators that both directlydamage tissue, and induce other cells tomigrate and cause damage secondarily.

An increasing body of evidence indi-cates that the CAP also can be harnessedto reduce pathological inflammation.Neurostimulation of the cholinergic anti-inflammatory pathway (NCAP) usingVNS may be a feasible means of treatingdiseases including RA and IBD that arecharacterized by excessive and dysregu-lated inflammation (4,5,23). Implantablemedical devices are increasingly beingstudied and approved for use as therapiesfor systemic diseases that, in the past,were traditionally treated with medicines.One such application would utilize achronically implanted neurostimulator toreduce systemic and organ-specific in-flammation in diseases including IBD andRA, potentially offering a treatment alter-native to patients, physicians and health-care payers (24) (Figure 2).

PRECLINICAL DEVELOPMENT OFMEDICAL DEVICES VERSUS DRUGS,AND UNIQUE CHALLENGES WITH NCAPDEVICE DEVELOPMENT

Small molecule compounds or proteintherapeutic agents such as monoclonalantibodies developed as targeted thera-pies for RA and IBD are typically potentand highly selective molecules that inter-act with a single soluble mediator, recep-tor or signal transduction molecule. Char-acteristics of the interaction between thedrug molecule and its target are tradi-tionally first determined in in vitro assaysystems. The molecule is then studied in

a standard, accepted animal model, es-tablishing a relationship between deliv-ered dose and tissue exposure, relevantbiomarker effect and measures of efficacy.This concentration–activity–effect rela-tionship is then used to inform the choiceof doses selected for human studies.

In contrast, electrically active im-plantable medical devices (for example,cardiac pacemakers, spinal cord stimula-tors for pain reduction) typically alterphysiologic activity of an entire organ ororgan system by inducing depolarizationof excitable neural or muscle tissues in aselective manner. Depolarization is elic-

Figure 2. Neurostimulation of the cholinergic antiinflammatory pathway with an implantablemedical device: A potential alterative therapeutic approach for RA and IBD. Signals fromthe device pass through the efferent vagus nerve and are relayed directly to the intestine,and across the celiac ganglion, traversing the splenic nerve to terminate in the spleen(panel 1). Neurotransmitter release in the end organs results in reduced activation of resi-dent immune cells (panel 1), as well as reduced production of inflammatory mediatorsand activation of circulating immune cells (panel 2). These effects should result in attenua-tion of inflammation with improvement in signs and symptoms, and reduction in joint andintestinal mucosal damage (panels 3 and 4).



ited by applying electrical charge, whichcan be altered by modulating the stimula-tion parameters of the device includingthe amplitude (measured as output cur-rent), waveform, frequency and durationof the delivered electrical pulse. The timeduration of each stimulation and the dutycycle (ratio between the “on” and “off”time of the device) can also be altered.Together these parameters determine theoverall therapeutic effect of the device ontissue function and disease pathophysiol-ogy in appropriate animal models, whichthen in turn informs selection of stimula-tion parameters used in clinical studies.

Activating the CAP using VNS to pro-duce a therapeutic effect in an animalmodel is more complex than either in-ducing neural depolarization alone ordelivering a molecule to alter a singlecellular target alone. Successful interven-tion using NCAP requires intermittentinduction of vagus nerve depolarization,releasing neurotransmitters that modu-late immune cell function and ultimatelyreduce pro inflammatory mediator re-lease (see Figure 1). Because the mecha-nism of NCAP action is unconventional,preclinical development required a non-traditional, hybrid approach between thetechniques typically used in inflamma-tion drug development and those used inneuromodulation device development.In both RA and IBD, standard well- characterized animal models were used,

but a progression from more typicalpharmacologic, genetic and anatomicmanipulations to direct electrical nervestimulation was required.

NCAP EFFECT IN MODELS OF RAThe collagen-induced arthritis (CIA)

model is used in drug development to as-sess preclinical efficacy and help guidedecisions on which candidate moleculesshould be advanced into clinical develop-ment for RA (25). In the classical CIAmodel, autoimmunity against native jointstructure proteins is induced in rodentsby repeated injections of bovine collagen,often in the presence of an immune-acti-vating compound such as Freund’s adju-vant. Animals rapidly develop synovitisthat can be assessed either by direct visu-alization or caliper measurement of jointswelling. Although the correlation is im-perfect as with any animal model, thepathological findings in the rodent jointduring CIA exhibit a reasonable degree ofsimilarity to those seen in RA patients, in-cluding infiltration of the synovium withimmune effector cells, formation of an in-flammatory synovial outgrowth termed a“pannus,” damage to articular cartilageand peri-articular erosions of the bone.Improvement in these parameters in-duced by exposure to a test compoundestablishes preclinical efficacy (26).

The CIA model was utilized to provideevidence supporting the advancement of

NCAP toward clinical development(Table 1). Two studies (27,28) demon-strated that surgical vagotomy worsensand the α7nAChR agonist amelioratesCIA disease severity. However, four keystudies definitively demonstrated the im-portance of the pathway in affecting thecourse and severity of CIA and togetherprovided the rationale to study the thera-peutic potential of NCAP in RA patients.

Owing to the critical role of theα7nAChR in mediating the CAP effect,we studied the course of CIA in micewith targeted disruption of the α7nAChRreceptor gene (29). When compared withwild-type littermates, α7nAChR knock-out animals had a greater cumulative in-cidence of disease onset, accompanied byworsened clinical disease severity and ra-diographic evidence of bone destruction,increased histological joint inflammation,systemic monocyte chemotactic peptide(MCP)-1 and TNF levels, and increased invitro release of Th1 cytokines from cul-tured splenocytes (Figures 3A, B). Consis-tent with the antiinflammatory effect ofthe CAP, clinical arthritis was exacer-bated by vagotomy in CIA (30). Con-versely, in another set of experiments, wedemonstrated that the course and sever-ity of CIA were ameliorated by systemictreatment with nicotine (a nonselectiveligand of the α7nAChR) or with the selec-tive α7nAChR agonist AR-R17779 (Fig-ures 3C–E) (30).

Table 1. CAP activation and/or vagotomy in preclinical rheumatoid arthritis models.

Intervention Model Species Result Reference

Cervical vagotomy; and CIA Mouse Vagotomy worsens and nAChR agonists improve arthritis score. (48)nAChR agonists nicotine nAChR agonists improve bone degradation, synovial inflammation, TNF.or AR-R17779

α7nAChR Knockout CIA Mouse Knockout worsens incidence and severity of arthritis, inflammation, joint (29)destruction and proinflammatory cytokines. Decrease in the collagen-specific Th1-associated IgG2a, but not IgG1.

Surgical vagus suspension CIA Rat Improves paw volume, arthritis score, bone erosions, histology, serum TNF. (31)

Vagus nerve stimulation CIA Rat Improves paw volume, arthritis score, histology, bone metabolic markers. (32)

Cervical vagotomy and CIA Mouse Vagotomy worsens and nicotine improves arthritis score, inflammation and (27)nicotine bone destruction, TNF, IL-6 expression and HMGB1 translocation.

Cervical vagotomy and CIA Mouse Vagotomy worsens and nicotine improves arthritis score; nicotine reduces (28)nicotine Th17cells, IL-17A and RORγτ expression; and nicotine increases IL-4 and

GATA3 expression.

CIA, collagen-induced arthritis; HMGB1, high-mobility group protein B1; IgG, immunoglobulin G; IL, interleukin; RORγ, RAR-related orphanreceptor γ; TNF, tumor necrosis factor; WT, wild type.



Evidence that directly activating signal-ing through the vagus nerve itself couldimprove CIA outcome was first providedin a study using surgical suspension of thecervical portion of the nerve against thesternocleidomastoid muscle in rats (31).The apposition of muscle and nerve in-duced chronic mechanical vagus activa-tion, which, when measured electrophysio-

logically, appeared similar to the pattern ofnerve activation achieved with direct stim-ulation using standard electrodes. CIA wasinduced in sham-operated animals and an-imals that underwent full surgical suspen-sion of the nerve and both groups were fol-lowed for three months. When comparedwith the sham-operated group, animalswith surgical suspension had statistically

significant improvements in paw volume,clinical arthritis score, semiquantitative ra-diographic assessment of bone erosions,histological evidence of erosions and in-flammation and reduced serum TNF levels.

Finally, we extended these observations,establishing the practicality and effective-ness of traditional electrical VNS in the CIAmodel using a chronic implantable rodent

Figure 3. Both the α7nAChR agonist AR-R17779 and nicotine improve, while targeted α7nAChR gene disruption exacerbates CIA. CIA wasinduced in male and female α7nAChR deficient C57BL/6 mice and wild-type littermates. Clinical arthritis scores are shown over time asmean + SE (A). Significance between groups was determined by comparing the area under the curve (AUC) from d 20 to d 26 (B), **p <0.005 for t test versus saline. Beginning on d 20 after immunization with type II collagen, male DBA/1 mice were treated with nicotine (400g/kg; n = 8) or saline, or AR-R17779 (1, 2.5, or 5 mg/kg; n = 15 per dose group) or saline for 7 d. Clinical arthritis scores are shown over timeas mean + SE (C, D). Significance between groups was determined by comparing the AUC from d 20 to d 26 (not shown). Arthritis scoreswere significantly lower in mice treated with nicotine or AR-R17779 at 2.5 mg/kg or 5 mg/kg compared with scores in control animals. In-cidence of arthritis in mice treated with AR-R17779 (5 mg/kg) or saline are plotted cumulatively over time (E), *p ≤ 0.05 for Kaplan-Meiersurvival test versus saline. Panels A and B from a figure in (29) (adapted by permission from BMJ Publishing Group Limited. Annals of theRheumatic Diseases. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine re-ceptor α7 subunit gene knockout mice. Marjolein A van Maanen, Susanne P Stoof, Gregory J LaRosa, Margriet J Vervoordeldonk, Paul PTak. 69:1717–23, © 2010). Panels C–E adapted by permission from a figure in (30) (Stimulation of Nicotinic Acetylcholine Receptors Attenu-ates Collagen-Induced Arthritis in Mice. Marjolein A van Maanen, Maria C Lebre, Tom van der Poll, Gregory J LaRosa, Daniel Elbaum, Mar-griet J Vervoordeldonk, Paul P Tak. Arthritis & Rheumatism. Vol. 60, issue 1. Copyright © 2009 by the American College of Rheumatology).



system we developed with a cuff lead anal-ogous to those used in humans treatedwith implantable VNS devices (32). In thesestudies, animals were immunized on d 0and d 6, and treatment was initiated afterthe disease had become semiestablished ond 9. When compared with implanted butunstimulated animals, VNS stimulation de-livered at 3.0-mA output current, 10-Hzpulse frequency, 200-microsecond pulsewidth for 60 s once daily over d 9 throughd 16 significantly reduced clinical manifes-tations as assessed by ankle diameter, andreduced the histological severity of inflam-mation, pannus formation, cartilage dam-age and bone resorption (Figure 4). Theseimprovements were accompanied by a re-duction in circulating proinflammatory me-diators, and changes in important media-tors of bone metabolism including anincrease in osteoprotegerin (OPG), and amarked decrease in serum receptor activa-tor of nuclear factor (NF)-κB ligand(RANKL), the major regulator of osteoclastsurvival, maturation, and function (33).

These observations in animal models ofRA were supported by experiments in

human biology models. Pretreatment ofsynovial tissue–derived fibroblast-likesynoviocytes with acetylcholine, nicotineor AR-R17779 (all resulting in α7nAChRactivation) reduced production of theproinflammatory cytokines interleukin(IL)-6 and IL-8 significantly (34-36).Moreover, it has been suggested that useof “snuff” (smokeless, nicotine- containingtobacco) is associated with lower diseaseactivity in RA patients [37].

NCAP EFFECT IN INTESTINALINFLAMMATION MODELS

Similar to the CIA model in RA drugdevelopment, rodent models of intestinalmucosal inflammation can be used to testpreclinical efficacy and guide decisionson development of candidate moleculesfor use in clinical studies in inflammatorybowel disease (38). Mechanistically, themodels fall broadly into four categories:(a) direct mucosal irritant-mediated in-flammation (for example, dextran sulfatesodium [DSS]); (b) exposure to mucosalhaptenating agents which cause delayedtype hypersensitivity (DTH)-mediated in-

flammation (for example, oxalazone,dinitrobenzene or trinitrobenzene sul-fonic acid [DNBS/TNBS]); (c) sponta-neous colon and small intestinal inflam-mation occurring in IL-10 knockout mice;and (d) induction of mucosal inflamma-tion by T cell transfer into recombinase-activating gene (RAG) knockout micelacking native T cells) (39–42). Notably,none of these models by themselves fullyemulate the pathology of either Crohn’sdisease or ulcerative colitis, and theyhave been generally less predictive ofeventual outcomes in IBD clinical studiesthan the CIA model has been for RA (38).The effect of therapeutic intervention inall of these models is assessed by changesin levels of systemic inflammatory medi-ators, clinical assessment of weight loss,stool frequency and stool characteristics,morphometric or semiquantitative scor-ing of ulceration and inflammation inwhole-intestine preparations, endoscopicscoring of disease, gut histology and levelof inflammatory mediators and immunecell products (for example, leukocytemyeloperoxidase [MPO]) in gut tissue.

Pharmacologic activation usingα7nAChR-selective and nonselective CAPactivators, and CAP inhibition by vago-tomy have been studied extensively in themucosal irritant, hapten-mediated, and IL-10 knockout models (Table 2). In general,vagotomy causes worsening in disease pa-rameters, and pharmacologic CAP activa-tion ameliorates disease activity. However,there are notable exceptions: Eliakim et al.(43) found that nicotine improved colitis,but worsened jejunal inflammation in theIL-10 knockout model. The authors con-sidered this finding to be consistent withthe well- accepted clinical observation thatsmoking tends to lessen disease activity inulcerative colitis, and exacerbate its coursein Crohn’s patients. Moreover, in contrastto the CIA model findings noted abovewhere α7nAChR-selective agonists havetherapeutic efficacy, Snoek, et al. foundthat this class of agents were either ineffec-tive, or actually exacerbated disease in ahapten model (44). Despite these conflict-ing observations, the preponderance of ev-idence from vagotomy and pharmacologic

Figure 4. NCAP decreases joint swelling and histological indices of joint damage in CIA.Rats implanted with vagus nerve cuff electrodes had CIA induced or were injectedwith saline (Control) and were followed for 15 d. Animals underwent active (NCAP) orsham electrical stimulation (Sham NCAP) once daily from study d 9. Ankle diameterover time is shown as mean + SE (A). *p ≤ 0.05 for ANOVA versus CIA/Sham NCAP. Anklejoints were harvested on study d 16 and scored on a scale of 0-5 for inflammation,pannus formation, cartilage damage and bone resorption (B). Data are shown asmean + SE score. *p ≤ 0.05 for t test versus CIA/Sham NCAP. Image adapted from fig-ures in (32) (Neurostimulation of the Cholinergic Anti-Inflammatory Pathway Amelio-rates Disease in Rat Collagen- Induced Arthritis. Yaakov A Levine, Frieda A Koopman,Michael Faltys, April Caravaca, Alison Bendele, Ralph Zitnik, Margriet J Vervoordeldonk,Paul Peter Tak. PLOS ONE. 2014;9:e104530. Permission not required for adaptation. Availablefrom: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0104530).



intervention studies predicts that CAP ac-tivation may have beneficial effects on in-testinal mucosal inflammation, and there-fore might also be expected improveclinical outcomes in IBD.

More recent studies have used electricalVNS delivered by chronically implantedcuff leads similar to those we used in therodent CIA models, supporting the feasi-bility of this approach, and providing a ra-tionale to study NCAP delivery using im-plantable medical devices in IBD.Meregnani, et al. used a smaller diameterversion of an approved and commerciallymarketed VNS helical coil lead that wasimplanted on the vagus nerve in rats, andconnected to a subcutaneously implantedconnection block. Stimulation was deliv-ered by external leads connected percuta-neously to the implanted block in awakeand mobile animals using an externalpulse generator set to deliver an outputcurrent of 1-mA, at 5-Hz pulse frequency,500- microsecond pulse width, and a duty

cycle of 10 s on/90 s off for 3 h per day(45). In these experiments, VNS wasbegun on the same day as initial TNBS ex-posure. Compared to implanted animalswithout VNS treatment, VNS reducedTNBS-induced reductions in body weight,colonic lesional edge MPO and histologicindices of inflammation. Lesional levels ofmRNA for TNF, IL-6 and IL-1β were notreduced by VNS treatment, nor were in-tralesional histologic inflammation scoresor MPO levels.

Another investigative group reportedon the effects of VNS delivered using ahelical cuff electrode similarly implantedand linked to a subcutaneous connectionblock, which was then connected to an ex-ternal pulse generator (46). TNBS colitiswas induced concomitantly with VNS ini-tiation. VNS was given for 3 h per day at0.25-mA output current, 20-Hz pulse fre-quency, 500-microsecond pulse width, andduty cycle 30 s on/5 min off. VNS signifi-cantly improved TNBS-induced body

weight reductions, clinical disease activityand colonic tissue histology, MPO, in-ducible nitric oxide synthetase, IL-6 andTNF. VNS increased acetylcholine levels inmucosal tissue, and reduced phosphory-lated forms of NF-κB p65, ERK, JNK andp38 MAPK, all of which are important sub-cellular intermediate signaling moleculesthat modulate cytokine secretion (Figure 5).

TAKING NCAP FROM PRECLINICALMODELS TO HUMAN TRIALS

As summarized above, an understand-ing of the neuroanatomy and immunol-ogy of the CAP and identification of keycomponents of the signaling path (for ex-ample, the α7nAChR) enabled testing ofthe hypothesis that modulating the activa-tion state of the CAP could affect diseaseseverity in classical inflammation modelsof RA and IBD. In both cases, evidence ofeffect first came from experiments inwhich pharmacologic agonists or antago-nists ameliorated or exacerbated disease

Table 2. CAP activation and/or vagotomy in preclinical colitis models.

Intervention Model Species Result Reference

Nicotine TNBS Rat Improves macroscopic colon score, MPO activity, histology. (43)

Nicotine TNBS Rat Improves clinical score, macroscopic colon score, MPO activity, histology, (49)colonic tissue LTB4 and TNF levels.

Nicotine IL-10 knockout Mouse Improves clinical score and histology in colon; worsens jejunal histology. (50)

Neostigmine DNBS Rat Improves clinical score, macroscopic colon score, MPO activity, histology. (51)

Vagotomy or nicotine DSS Mouse Vagotomy worsens and nicotine improves clinical score, macroscopic (52)colon score, MPO activity, histology, colonic tissue IL-6, SAP.

Vagotomy DNBS Mouse Vagotomy worsens clinical score, macroscopic colon score, MPO activity, (52)histology, colonic tissue IL-6 and TNF, SAP.

nAChR agonist (AN) TNBS Mouse nAChR antagonist worsens and nAChR agonist improves histological (53)or antagonist (CHD) scores, MPO, TNF and NF-κB in lamina propria mononuclear cells.

Choline or α7nAchR DSS or DSS with Mouse Choline decreases macroscopic scores, MPO, CRP, IL-6 and IL-1β while (54)antagonist (DMI) reactivation α7nAchR antagonist worsens macroscopic scores and blocks

therapeutic benefit of antidepressant.

α7nAChR knockout DSS Mouse Knockout and vagotomy worsen MPO, CRP, IL-6 and IL-1β; and knockout (54)± vagotomy prevents therapeutic benefit of choline.

Vagotomy DSS Mouse Vagotomy worsens clinical and macroscopic scores, MPO activity; (55)increases colonic NF-κB signaling; increases splenic tissue levels of TNF, IL-6, MCP-1; and decreases CD4+FoxP+ Treg cells. Effect of vagotomy can be adoptively transferred via CD4+CD25– T cells.

α7-nAchR agonist DSS or TNBS Mouse AR-R17779 and GSK 1345038A either are ineffective or worsen disease (44)

Nicotine Oxalazone or Mouse Oxalazone colitis improves; there is no change in TNBS colitis. (56)TNBS

DSS, dextran sulfate sodium; MPO ,myeloperoxidase; DNBS/TNBS, dinitrobenzene/trinitrobenzene sulfonic acid; LT, leukotriene; IL, interleukin;CD, Cluster of Differentiation; Fox, forkhead box; TNF, tumor necrosis factor; MCP, monocyte chemotactic peptide; NF, nuclear factor; AN,anabaseine; CHD, chlorisondamine diiodide; DMI, desmethylimipramine; SAP, serum amyloid P.



manifestations, and from demonstratingthe exacerbating effect of blocking thepathway by anatomic destruction of theinvolved nerves or targeted disruption ofthe α7nAChR gene. Finally, progressionfrom pharmacologic, anatomic, or geneticexperiments to studies of the effect of di-rect stimulation of the vagus nerve pro-vided further confirmation. The summa-tion of the evidence for the importance ofthe pathway in improving or exacerbatingdisease burden these models is shownschematically in Figure 6. Indeed on thebasis of this body of evidence there was a

Figure 5. NCAP reduces weight loss, disease activity level, and systemic and intestinal inflammatory mediator levels in TNBS colitis. Rats im-planted with vagus nerve cuff electrodes had colitis induced by intrarectal administration of TNBS or received saline control, and were fol-lowed for 6 d. Rats then received VNS or sham VNS for 3 h per day from experimental d 1 to d 6 (0.25 mA, 20 Hz, 500-ms pulse width, 30 s on,5 min off continuously). Body weight (A) and disease activity index (DAI) (B) are shown as mean ± SE. MPO, inducible nitric oxide synthase(iNOS), TNF and IL-6 were measured from d-6 colonic homogenates (C). *P < 0.05 versus the control group and VNS group; ΔP < 0.05 versusthe TNBS group (B). MPO (U/mL), iNOS activity (U/mL), TNF-α (pg/mg tissue) and IL-6 levels after 6 d of VNS in TNBS rats. Data presented asmean ± SE. *P < 0.05 versus control group; #P < 0.05 versus TNBS group (C). Image adapted from a figure and a table in (46) (Involvement ofMAPK/NF-κB Signaling in the Activation of the Cholinergic Anti-Inflammatory Pathway in Experimental Colitis by Chronic Vagus Nerve Stimu-lation. Peng Sun, Kewen Zhou, Sheng Wang, Ping Li, Sijuan Chen, Guiping Lin, Yan Zhao, Tinghuai Wang. PLOS ONE. 2013:8:e69424. Permissionnot required for adaptation. Available from: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0069424).

Figure 6. Experimental manipulation of the cholinergic inflammatory pathway influencesdisease burden in preclinical models of RA and IBD. Surgical, genetic, or pharmacologicdisruption of the CAP increases, while electrical and pharmacologic stimulation of theCAP decreases disease burden in preclinical models of RA and IBD.



clear rationale to study electrically activeimplantable vagus nerve stimulation de-vices in RA and IBD. Early studies by ourgroup and others are underway (NCT01569503, NCT 01552941), and further de-finitive studies are being planned.

TRANSLATION OF BIOELECTRONICMEDICINES—OPPORTUNITIES ANDCHALLENGES

An effort to increase the therapeuticarmamentarium in systemic diseasestypically treated with systemically ad-ministered drugs has resulted in the con-cept of bioelectronic medicines (47). Suchtherapies would have structural andfunctional characteristics of both classicalmedicines and classical medical devices.They would be embedded within thebody, and would deliver electrical orother kinds of physical stimulation in aprecise and targeted manner that woulddirectly affect the disease pathophysiol-ogy. Ideally the therapeutic entity wouldbe able to monitor the function of the tis-sue or organ system continuously, adjust-ing its therapeutic effect in a precise waythat would maximize safety and efficacy.

To produce a therapy that fulfills all ofthese requirements, significant furtheradvances in basic science and engineer-ing will be necessary. However, the useof NCAP for RA and IBD represents anearly step in the search for an ideal bio-electronic medicine, and challenges facedduring development of NCAP can be ofhelp in guiding future efforts. For exam-ple, our experience indicates that greatattention must be paid to creating im-planted hardware that correctly fits theanatomy of and interacts with the modelspecies in a reliable and robust way, andto ensuring the effects of the surgery andthe chronic implant do not interfere withbiomarkers and disease measurements.Further, in contrast to drug developmentwhere the relationship between pharma-cokinetic and pharmacodynamic param-eters and disease activity in the model isoften well understood, with implanteddevices this is usually not the case. Simi-larly, the optimal way to extrapolate de-vice stimulation parameters from animal

to human studies in complex systemicdiseases is not yet well studied.

Despite these challenges, the prospectof creating an entirely new way to treatserious systemic illnesses with high med-ical need such as RA and IBD remains ex-citing. Along the way to development ofan idealized bioelectronic medicine,NCAP therapy is now being studied todetermine if it can fulfill its promise ofproviding an alternative to drug therapy,with a potential for lower cost and bettersafety, thus better fulfilling the needs ofpatients, providers and health-care payers.

DISCLOSUREYA Levine, M Faltys and R Zitnik

are employees of SetPoint Medical Cor-poration. P-P Tak is a consultant to andhas received research grants from SetPoint Medical and is an employee ofGlaxoSmithKline.

REFERENCES1. Scott DL, Wolfe F, Huizinga TW. (2010) Rheuma-

toid arthritis. Lancet. 376:1094–108.2. Cheifetz AS. (2013) Management of active Crohn

disease. JAMA. 309:2150–8.3. Gkaliagkousi E, et al. (2012) Cardiovascular risk

in rheumatoid arthritis: pathogenesis, diagnosis,and management. J. Clin. Rheumatol. 18:422–30.

4. Andersson U, Tracey KJ. (2012) Reflex principlesof immunological homeostasis. Annu. Rev. Im-munol. 30:313–35.

5. Tracey KJ. (2009) Reflex control of immunity. Nat.Rev. Immunol. 9:418–428.

6. Watkins LR, et al. (1995) Blockade of interleukin-1induced hyperthermia by subdiaphragmatic vago-tomy: evidence for vagal mediation of immune-brain communication. Neurosci. Lett. 183:27–31.

7. Bianchi M, et al. (1995) An inhibitor of macrophagearginine transport and nitric oxide production(CNI-1493) prevents acute inflammation and endo-toxin lethality. Mol. Med. 1:254–266.

8. Borovikova L, et al. (2000) Vagus nerve stimula-tion attenuates the systemic inflammatory re-sponse to endotoxin. Nature. 405:458–62.

9. Huston JM, et al. (2006) Splenectomy inactivatesthe cholinergic antiinflammatory pathway dur-ing lethal endotoxemia and polymicrobial sepsis.J. Exp. Med. 203:1623–8.

10 Rosas-Ballina M, et al. (2008) Splenic nerve is re-quired for cholinergic antiinflammatory pathwaycontrol of TNF in endotoxemia. Proc. Natl. Acad.Sci. U. S. A. 105:11008–13.

11. Rosas-Ballina M, et al. (2011) Acetylcholine- synthesizing T cells relay neural signals in avagus nerve circuit. Science. 334:98–101.

12. Wang H, et al. (2003) Nicotinic acetylcholine re-ceptor [alpha]7 subunit is an essential regulatorof inflammation. Nature. 421:384–8.

13. Olofsson PS, et al. (2012) alpha7 nicotinic acetyl-choline receptor (alpha7nAChR) expression inbone marrow-derived non-T cells is required forthe inflammatory reflex. Mol. Med. 18:539–43.

14. Matteoli G, Boeckxstaens GE. (2013) The vagalinnervation of the gut and immune homeostasis.Gut. 62:1214–22.

15. Matteoli G, et al. (2014) A distinct vagal antiinflammatory pathway modulates intestinalmuscularis resident macrophages independent of the spleen. Gut. 63:938-4.

16. Ji H, et al. (2014) Central cholinergic activation ofa vagus nerve-to-spleen circuit alleviates experi-mental colitis. Mucosal Immunol. 7:335–47

17. Bruchfeld A, et al. (2010) Whole blood cytokineattenuation by cholinergic agonists ex vivo andrelationship to vagus nerve activity in rheuma-toid arthritis. J. Intern. Med. 268:94–101.

18. Parrish WR, et al. (2008) Modulation of TNF re-lease by choline requires alpha7 subunit nicotinicacetylcholine receptor-mediated signaling. Mol.Med. 14:567–74.

19. de Jonge WJ, et al. (2005) Stimulation of thevagus nerve attenuates macrophage activationby activating the Jak2-STAT3 signaling pathway.Nat. Immunol. 6:844–51.

20. van Westerloo DJ, et al. (2006) The vagus nerveand nicotinic receptors modulate experimentalpancreatitis severity in mice. Gastroenterology.130:1822–30.

21. Krzyzaniak MJ, et al. (2011) Efferent vagal nervestimulation attenuates acute lung injury follow-ing burn: The importance of the gut-lung axis.Surgery. 150:379–89

22. Huston JM, et al. (2009) Cholinergic neural sig-nals to the spleen down-regulate leukocyte traf-ficking via CD11b. J. Immunol. 183:552–9.

23. Van Maanen M, Vervoordeldonk M, Tak P. (2009)The cholinergic anti-inflammatory pathway: to-wards innovative treatment of rheumatoidarthritis. Nat. Rev. Rheumatol. 5:229–32.

24. Koopman FA, et al. (2011) Restoring the balanceof the autonomic nervous system as an innova-tive approach to the treatment of rheumatoidarthritis. Mol. Med. 17:937–48.

25. Bevaart L, Vervoordeldonk MJ, Tak PP. (2010)Evaluation of therapeutic targets in animal mod-els of arthritis: how does it relate to rheumatoidarthritis? Arthritis Rheum. 62:2192–205.

26. Hegen M, Keith JC Jr, Collins M, Nickerson- Nutter CL. (2008) Utility of animal models foridentification of potential therapeutics for rheuma-toid arthritis. Ann. Rheum. Dis. 67:1505–15.

27. Li T, et al. (2010) The vagus nerve and nicotinicreceptors involve inhibition of HMGB1 releaseand early pro-inflammatory cytokines functionin collagen-induced arthritis. J. Clin. Immunol.30:213–20.

28. Wu S, et al. (2014) Attenuation of collagen in-duced arthritis via suppression on Th17 response



by activating cholinergic anti-inflammatory path-way with nicotine. Eur. J. Pharmacol. 735:97-104.

29. van Maanen M, Stoof S, Larosa G, VervoordeldonkM, Tak P. (2010) Role of the cholinergic nervoussystem in rheumatoid arthritis: aggravation ofarthritis in nicotinic acetylcholine receptor alpha7subunit gene knockout mice. Ann. Rheum. Dis.69:1717–23.

30. van Maanen MA. (2009) Stimulation of nicotinicacetylcholine receptors attenuates collagen- inducedarthritis in mice. Arthritis Rheum. 60:114–122.

31. Zhang P, Han D, Tang T, Zhang X, Dai K. (2008)Inhibition of the development of collagen-inducedarthritis in Wistar rats through vagus nerve sus-pension: a 3-month observation. Inflamm. Res.57:322–328.

32. Levine YA, et al. (2014) Neurostimulation of thecholinergic anti-inflammatory pathway amelio-rates disease in rat collagen-induced arthritis.PLoS One. 9:e104530.

33. Lacey DL, et al. (2012) Bench to bedside: elucida-tion of the OPG-RANK-RANKL pathway andthe development of denosumab. Nat. Rev. Drug.Discov. 11:401–19.

34. Zhou Y, et al. (2012) Nicotine inhibits tumor necro-sis factor-alpha induced IL-6 and IL-8 secretion infibroblast-like synoviocytes from patients withrheumatoid arthritis. Rheumatol. Int. 32:97–104.

35. van Maanen MA, et al. (2009) The alpha7 nico-tinic acetylcholine receptor on fibroblast-like syn-oviocytes and in synovial tissue from rheuma-toid arthritis patients: a possible role for a keyneurotransmitter in synovial inflammation.Arthritis Rheum. 60:1272–81.

36. Waldburger JM, Boyle DL, Pavlov VA, Tracey KJ,Firestein GS. (2008) Acetylcholine regulation ofsynoviocyte cytokine expression by the alpha7nicotinic receptor. Arthritis Rheum. 58:3439–49.

37. Andersson ML, Bergman S, Soderlin MK, BARFOTstudy group (2013) The effect of snuff (smokelesstobacco) on disease activity and function inrheumatoid arthritis: experiences from the BetterAnti-Rheumatic FarmacOTherapy, a longitudinalmulticenter study on early rheumatoid arthritis. J. Clin. Rheumatol. 19:14–18.

38. Valatas V, Vakas M, Kolios G. (2013) The value ofexperimental models of colitis in predicting effi-cacy of biological therapies for inflammatorybowel diseases. Am. J. Physiol. Gastrointest. LiverPhysiol. 305:G763–85.

39. Ostanin DV, et al. (2009) T cell transfer model ofchronic colitis: concepts, considerations, andtricks of the trade. Am. J. Physiol. Gastrointest.Liver Physiol. 296:G135–46.

40. Perse M, Cerar A. (2012) Dextran sodium sulphatecolitis mouse model: traps and tricks. J. Biomed.Biotechnol. 2012:718617.

41. te Velde AA, Verstege MI, Hommes DW. (2006)Critical appraisal of the current practice in murineTNBS-induced colitis. Inflamm Bowel Dis 12:995–9.

42. Rennick DM, Fort MM. (2000) Lessons from ge-netically engineered animal models. XII. IL-10-deficient (IL-10(-/-) mice and intestinal inflam-

mation. Am. J. Physiol. Gastrointest. Liver Physiol.278:G829–33.

43. Eliakim R, Karmeli F, Rachmilewitz D, Cohen P,Fich A. (1998) Effect of chronic nicotine adminis-tration on trinitrobenzene sulphonic acid-inducedcolitis. Eur. J. Gastroenterol. Hepatol. 10:1013–9.

44. Snoek SA, et al. (2010) Selective alpha7 nicotinicacetylcholine receptor agonists worsen disease inexperimental colitis. Br. J. Pharmacol. 160:322–33.

45. Meregnani J, et al. (2011) Anti-inflammatory ef-fect of vagus nerve stimulation in a rat model ofinflammatory bowel disease. Auton. Neurosci.160:82–9.

46. Sun P, et al. (2013) Involvement of MAPK/NF-κBsignaling in the activation of the cholinergic antiinflammatory pathway in experimental colitis bychronic vagus nerve stimulation. PLoS One. 8:e69424.

47. Famm K, Litt B, Tracey KJ, Boyden ES, Slaoui M.(2013) Drug discovery: a jump-start for electro-ceuticals. Nature. 496:159–61.

48. van Maanen M, et al. (2009) Stimulation of nicotinicacetylcholine receptors attenuates collagen-inducedarthritis in mice. Arthritis Rheum. 60:114–22.

49. Sykes AP, et al. (2000) An investigation into the ef-fect and mechanisms of action of nicotine in in-flammatory bowel disease. Inflamm. Res. 49:311–9.

50. Eliakim R, Fan QX, Babyatsky MW. (2002) Chronicnicotine administration differentially alters jejunaland colonic inflammation in interleukin-10 defi-cient mice. Eur. J. Gastroenterol. Hepatol. 14:607–14.

51. Miceli PC, Jacobson K. (2003) Cholinergic path-ways modulate experimental dinitrobenzene sul-fonic acid colitis in rats. Auton. Neurosci. 105:16–24.

52. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H,Verdu EF, Collins SM. (2006) The vagus nerve: atonic inhibitory influence associated with inflam-matory bowel disease in a murine model. Gas-troenterology. 131:1122–30.

53. Bai A, Guo Y, Lu N. (2007) The effect of thecholinergic anti-inflammatory pathway on exper-imental colitis. Scand. J. Immunol. 66:538–45.

54. Ghia JE, et al. (2009) Reactivation of inflamma-tory bowel disease in a mouse model of depres-sion. Gastroenterology. 136:2280–8.

55. O’Mahony C, van der Kleij H, Bienenstock J,Shanahan F, O’Mahony L. (2009) Loss of vagalanti-inflammatory effect: in vivo visualizationand adoptive transfer. Am. J. Physiol. Regul. In-tegr. Comp. Physiol. 297:R1118–26.

56. Galitovskiy V, et al. (2011) Cytokine-induced al-terations of alpha7 nicotinic receptor in colonicCD4 T cells mediate dichotomous response tonicotine in murine models of Th1/Th17- versusTh2-mediated colitis. J. Immunol. 187:2677–87.

Cite this article as: Levine YA, Koopman F, Faltys M,Zitnik R, Tak P-P. (2014) Neurostimulation of thecholinergic antiinflammatory pathway in rheuma-toid arthritis and inflammatory bowel disease. Bio-electron. Med. 1:34–43.

neurostimulation of the cholinergic antiinflammatory ... · rostimulation of the cholinergic...

Documents