of March 16, 2018.This information is current as Colitis

Models of Th1/Th17- versus Th2-Mediated Dichotomous Response to Nicotine in MurineReceptor in Colonic CD4 T Cells Mediate

7 NicotinicαCytokine-Induced Alterations of

Sergei A. GrandoSteve Marchenko, Vivian Gindi, Robert A. Edwards and Valentin Galitovskiy, Jing Qian, Alexander I. Chernyavsky,

ol.1002711http://www.jimmunol.org/content/early/2011/07/22/jimmun

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The Journal of Immunology

Cytokine-Induced Alterations of a7 Nicotinic Receptor inColonic CD4 T Cells Mediate Dichotomous Response toNicotine in Murine Models of Th1/Th17- versusTh2-Mediated Colitis

Valentin Galitovskiy,*,†,‡,1 Jing Qian,*,†,‡,1,2 Alexander I. Chernyavsky,*,†,‡

Steve Marchenko,*,†,‡ Vivian Gindi,*,†,‡ Robert A. Edwards,x and Sergei A. Grando*,†,‡

Ulcerative colitis (UC) and Crohn’s disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central

role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and

worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine

receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each

form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene

sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased

percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4+CD62L+ T cells in the

presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated

with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be

abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous

action of nicotine resulted from the up- and downregulation of anti-inflammatory a7 nAChR on colonic CD4 T cells induced by

cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help

explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in

regulating colonic inflammation. The Journal of Immunology, 2011, 187: 000–000.

Ulcerative colitis (UC) and Crohn’s disease (CD) arechronic inflammatory disorders of the gastrointestinaltract that result in significant morbidity. A search for

novel and more efficient approaches to treat patients with thesetwo forms of inflammatory bowel disease (IBD) is one of the mostimportant tasks of contemporary clinical medicine. UC is char-acterized by contiguous mucosal inflammation in the rectum andcolon causing epithelial cell destruction and sloughing leadingto ulceration. CD, in contrast, is manifested by transmural, gran-ulomatous inflammation occurring anywhere in the alimentarycanal but is usually centered in the terminal ileum and ascendingcolon.

Both UC and CD are epidemiologically related to smoking (1–4). Most patients with UC are non-smokers, and patients with ahistory of smoking usually acquire their disease after they havestopped smoking (5–7). Upon cessation of smoking, patients withUC experience more severe disease progression that can be ame-liorated by returning to smoking (8–10). In contrast, patients with CDexperience severe disease when smoking, requiring an immediateand complete cessation of any tobacco usage (3, 11).Nicotine appears to be the key mediator of the dichotomous

effect of smoking on UC and CD, because its administration intransdermal patches inhibits inflammation associated with UC butnot in patients with CD (8, 12–15). These observations suggestedthat the effects of smoking in patients with IBD are mediated, atleast in part, by the nicotinic acetylcholine receptors (nAChRs)activated by nicotine. The effects of nicotine are complex andinclude its specific actions on the CNS, gastrointestinal tract, andimmune system. It is thought that the therapeutic effects of nic-otine in UC are mediated by the nAChRs of gut immune cells(12). The nAChRs expressed by T cells play an important role inT cell development, differentiation, and function (reviewed in Ref.16). We have recently reported that, on the one hand, nicotinergicstimulation alters cytokine production, and, on the other hand, thatimmune cytokines themselves can alter the structure and functionof T cell nAChRs (17, 18).The pathogenesis of IBD is unknown but is thought to reflect an

interaction of genetic and environmental factors. Despite havinga common basis in overresponsiveness to mucosal Ags, the twodiseases have considerably different pathophysiologies. CD is as-sociated with a Th1/Th17 T cell-mediated response induced byIL-12, and possibly IL-23, with concomitant increased production

*Institute for Immunology, University of California, Irvine, Irvine, CA 92697;†Department of Dermatology, University of California, Irvine, Irvine, CA 92697;‡Department of Biological Chemistry, University of California, Irvine, Irvine, CA92697; and xDepartment of Pathology, University of California, Irvine, Irvine, CA92697

1V.G. and J.Q. contributed equally to this study.

2Current address: Department of Medical Microbiology and Parasitology, ZhejiangUniversity School of Medicine, Hangzhou, China.

Received for publication August 11, 2010. Accepted for publication June 18, 2011.

Address correspondence and reprint request to Prof. Sergei A. Grando, University ofCalifornia Irvine, 134 Sprague Hall, Irvine, CA 92697. E-mail address: sgrando@uci.edu

Abbreviations used in this article: CD, Crohn’s disease; DAI, disease activity index;FCM, flow cytometry; HAI, histology activity index; IBD, inflammatory bowel dis-ease; ICW, in-cell Western; i.r., intrarectal; LP, lamina propria; MC, mononuclearcell; nAChR, nicotinic acetylcholine receptor; qPCR, quantitative real-time PCR;TNBS, trinitrobenzene sulfonic acid; Treg, regulatory T cell; UC, ulcerative colitis.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002711

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of IL-2, IL-17, IL-18, and IFN-g, whereas UC is associated withan atypical Th2-mediated response characterized by NKT cellsecretion of IL-13 and increased production of IL-4 and IL-5(19–27).Many murine models of mucosal inflammation mimicking hu-

man IBD forms have been described, leading to a profound increasein our understanding of the immunologic basis of colonic in-flammation (28). Experimental animal models have convincinglydemonstrated that CD4 T cells play a central role in IBD (28).A hapten-induced colitis in mice caused by intrarectal (i.r.) in-stillation of trinitrobenzene sulfonic acid (TNBS) is a Th1 T cell-mediated colitis that captures many of the features of CD (29–32).In contrast, a hapten-induced colitis caused by i.r. instillationof oxazolone (4-ethoxymethylene-2-phenyl-2-oxazoline-5-one) isdriven by the production of Th2 cytokines and reproduces many ofthe features of UC (33, 34). The potential for nicotinergic drugsin regulating colonic inflammation has been recently documentedin vagotomized mice with experimental IBD, wherein the nAChRagonist nicotine alleviated and the antagonist hexamethoniumaggravated colitis (35, 36). However, recent studies on the clini-cal and immunologic outcomes of administration of nicotinergicagents to mice with TNBS-induced colitis gave conflicting results(37, 38), suggesting the need for an in-depth analysis of the nic-otinergic immunopharmacology in experimental IBDs.We postulated that the severity of gut inflammation in mice with

experimental IBD could be regulated through T cell nAChRs andthat the exact clinical outcome would depend on the particular typeof T cell-mediated experimental colitis. In the present study, wemeasured the clinical and the immunologic outcomes of treat-ing BALB/c mice with oxazolone- and TNBS-induced colitidesby nicotine. Nicotine attenuated oxazolone colitis and worsenedTNBS colitis, which was associated with the increased percentageof colonic regulatory T cells (Tregs) in mice with oxazolone- butnot TNBS-induced colitis. The dichotomous action of nicotinecould result from up- and downregulation of the expression ofanti-inflammatory a7 nAChR in colonic CD4 T cells under theinfluence of the cytokines characteristic of oxazolone and TNBScolitides, respectively.

Materials and MethodsInduction of colitis

Oxazolone and TNBS colitides in BALB/c mice were induced usingstandard techniques (30, 34, 39). Specific pathogen-free 6- to 8-wk-oldfemale mice, weighting 20–25 g, were purchased from The Jackson Lab-oratory (Bar Harbor, ME) and maintained in the Animal Facility at theUniversity of California, Irvine. All experiments were approved by In-stitutional Animal Care and Use Committee. Animals were lightly anes-thetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and a 3.5-Frsilicon catheter (Harvard Apparatus, Holliston, MA) was inserted 4 cminto the lumen of the colon, through which 100 ml 3% oxazolone or 3%TNBS in 47.5% ethanol in saline was slowly infused. To ensure distri-bution of the instilled solutions within the entire colon and cecum, themice were held vertically for 30 s after removing the catheter. The micetreated i.r. with a vehicle, that is, 47.5% ethanol in saline, served as diseasecontrols. The a7 nAChR knockout mice used in the oxazolone colitisexperiments were from The Jackson Laboratory. The nicotine saline so-lution was freshly prepared from nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO) and s.c. injected into each mouse. For nicotinetreatment, BALB/c mice received nicotine twice a day at the total dose of7.5 mg/kg for 5 d starting 1 d prior to the administration of haptenatingagents. Mice were sacrificed on day 2 or day 5 after hapten infusion.Twenty mice per each treatment condition were used in experiments.

Evaluations of colitis severity

To grade the clinical severity of colitis, mice were assessed daily for bodyweight, stool consistency, rectal bleeding, and presence of blood in thestool by a guaiac paper test. The clinical disease activity index (DAI) was

computed based on scores for weight loss, stool consistency, hema-tochezia, and rectal bleeding using standard protocols (31, 40, 41), asdetailed in Table I. To grade the severity of intestinal histopathology, theentire colon was excised from each mouse, opened longitudinally, fixed in10% neutral buffered formalin, embedded in paraffin, cut into 5-mm sec-tions, and stained with H&E. The degree of inflammation and epithelialinjury was graded in a blinded fashion by the same pathologist (R.A.E.).The histology activity index (HAI) was assessed to quantify colitis severitybased on standard criteria for assessment of inflammation and injury inmice with experimental IBD, such as appearance and extent of inflam-matory cell infiltration, goblet cell mucin depletion, mucosal thickening,and the extent of damage (31, 34, 42, 43), as described in Table II.

Immunofluorescence staining

The 8-mm-thick cryostat sections of mouse colons were fixed with coldacetone, blocked with 5% FCS, and used in the indirect immunofluores-cence assays following a standard procedure (44). The V450 rat anti-mouse CD4 (BD Biosciences, San Jose, CA) and Alexa Fluor 488 anti-mouse CD25 (BioLegend, San Diego, CA) were used for double staining,and PE rat anti-mouse Foxp3 (BD Biosciences) was added for triplestaining. For the CD4/IL-17 double staining, we used V450 rat anti-mouseCD4 (BD Biosciences) and a combination of rat anti-mouse IL-17 andFITC-labeled rabbit anti-rat Ab (both from Abcam, Cambridge, MA). Toidentify the CD4 T cells expressing a4 or a7 nAChRs, we used rabbit anti-a4 or anti-a7 Abs (both from Abcam) visualized with Alexa Fluor 488goat anti-rabbit Ab (Invitrogen, Eugene, OR), and rat anti-mouse CD4 Ab(BioLegend) visualized with Alexa Fluor 555 goat anti-rat Ab (Invitrogen).The slides were washed three times, mounted using the ProLong Gold anti-fade reagent (Invitrogen), and analyzed with a Nikon Eclipse Ti fluores-cence microscope. Samples without primary Abs served as negative con-trols. In each slide, the positive cells were counted in 10 high-power 360microscopic fields.

Isolation of lamina propria and spleen mononuclear cells

Lamina propria (LP) mononuclear cells (MCs) were isolated from freshlyobtained colonic specimens using modification of previously describedprotocols (45). Briefly, the colons were opened longitudinally and cut intopieces. After incubation in DMEM with 2 mM DTT (Bio-Rad, Hercules,CA) for 30 min, vortexing, and passing through a cell strainer, the tissuewas digested in DMEM containing collagenase D/Dispase (5 mg/ml) andDNase I (5 mg/ml) (both from Roche, Indianapolis, IN). The LP cellsreleased from the colonic tissue were harvested and subjected to Percoll(Sigma-Aldrich) gradient separation. The LPMCs were collected at the40/80 interphase. Spleen MCs were extracted by Ficoll (Sigma-Aldrich)gradient centrifugation. The obtained MCs were washed and resuspendedin either the FACS buffer or culture medium.

Nicotine stimulation experiments

CD4+CD62L+ naive T cells from intact BALB/c mice were enriched byusing the CD4+CD62L+ T cell isolation kit II and MACS sorting (MiltenyiBiotec, Auburn, CA) following the protocol provided by the manufacturer.To stimulate T cells via the TCR/CD3 complex, 250 ml 2 3 106 cells/mlwere seeded in each well of the 24-well tissue culture plates (BD Falcon/BD Biosciences) coated for 2 h at 37˚C with 10 mg/ml anti-mouse CD3(clone 145-2C11) and 4.0 mg/ml anti-mouse CD28 (clone 37.51) Abs (bothfrom BD Biosciences) and cultured for 5 d in RPMI 1640 supplementedwith 10% FCS, 0.05 mM 2-ME, 10 mM HEPES buffer, 100 U/ml peni-cillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodiumpyruvate (all from Invitrogen) in a humid, 5% CO2 incubator at 37˚C in theabsence or presence of 100 mM nicotine added on a daily basis. SomeT cells were also exposed to 10 ng/ml IL-4 or IL-12 (both from PeproTech,Rocky Hill, NJ). Changes in Foxp3 expression were analyzed by flowcytometry (see below) and immunoblotting. Briefly, after incubation, 1 3106 cells were lysed in 1% Nonidet P-40 lysis buffer (Sigma-Aldrich), andthe proteins were separated on 4–12% SDS-PAGE and transferred to ni-trocellulose (Bio-Rad). After blocking with Odyssey blocking solution, themembranes were incubated overnight with rabbit anti-mouse Foxp3 Ab(Abcam) followed by a goat anti-rabbit secondary Ab conjugated with LI-COR IRDye 800CW (LI-COR Biosciences, Lincoln, NE). For proteinloading control, same membrane was reprobed with a b-actin Ab (Genway,San Diego, CA). The Odyssey infrared imaging system (LI-COR Bio-sciences) was used to scan the membranes and visualize protein bands. TheImageQuant 5.1 software (Molecular Dynamics, Sunnyvale, CA) was usedfor the semiqualitative analysis of protein bands.

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Cytokine stimulation experiments

After stimulation of CD4+CD62L+ naive T cells by CD3/CD28 without anyadditions (control) or in the presence of 10 ng/ml IL-4 or IL-12, as de-scribed above, the expression of a7 gene was quantitated at the mRNA andprotein levels by quantitative real-time PCR (qPCR) and in-cell Western(ICW) assays, respectively. Total RNA was extracted with the RNeasyMini Kit (Qiagen, Valencia, CA), and the a7 mRNA expression wasmeasured using the TaqMan gene expression assay (Mm01317884_m1) atthe Applied Biosystems 7500 system (Applied Biosystems, Carlsbad, CA)in accordance with the manufacturer’s protocol, as described by us in detailelsewhere (46). Ubiquitin C (Mm01198158_m1) was included as endog-enous reference gene, and the data were analyzed with a Sequence De-tection Software version 1.2.3 (Applied Biosystems). The a7 protein levelwas determined in situ by ICW, as described by us in detail elsewhere (17),using anti-a7 Ab (Abcam) and secondary IRDye 800CW goat anti-rabbitAb (LI-COR Biosciences). Sapphire 700 (LI-COR Biosciences) andDRAQ (Cell Signaling Technology, Danvers, MA) were used to normalizefor cell numbers per well. The receptor protein expression was quantitatedusing the LI-COR Odyssey imaging system. The results of both qPCR andICW assays were expressed as means 6 SD of a7 mRNA or protein rel-ative to that of control, that is, T cells stimulated by CD3/CD28 withoutIL-4 and IL-12, taken as 1.

Flow cytometry

Single-, double-, or triple-color flow cytometric analyses were performedusing a BD FACSCalibur bench top analyzer and WinMDI software (http://facs.scripps.edu/software.html). The CD3-PE/CY5, CD4-FITC, and CD8-PE (all from eBioscience, San Diego, CA) Abs were used as cell surfacemarkers of T cell subpopulations. FITC-IL-10, FITC-IL-4, PE-IL-17, PE-IFN-g, and Alexa Fluor 647 Foxp3 Abs (all from BD Biosciences) wereused for intracellular staining.

ELISA assays

IL-10, IL-17, and IFN-g were assayed by Ab sandwich ELISA in cellculture supernatants using the mouse ELISA Ready-SET-Go kit (eBio-science). ELISA assays of IL-10, IL-4, IL-12, IL-23, and IFN-g in colonlysates were performed using mouse ELISA immunoassay kits (R&DSystems, Minneapolis, MN). Total protein was extracted using a TotalProtein Extraction Kit (Millipore, Bedford, MA) in accordance with themanufacturer’s protocol.

Statistical analysis

All experiments were performed in duplicates or triplicates. Statisticalsignificance was determined using the Student t test. The differences weredeemed as significant when the calculated p value was ,0.05.

ResultsOxazolone and TNBS induce acute forms of UC- and CD-likecolitides, respectively, in BALB/c mice

Mice treated with either oxazolone or TNBS in both cases re-producibly developed a rapid-onset colitis marked by weight lossand diarrhea. Mice were evaluated daily to assess the DAI (Table I).Their colons were removed postmortem and examined macro- andmicroscopically. Macroscopic examination of colons from micetreated with either oxazolone or TNBS in both cases revealedstriking hyperemia and focal mucosal necrosis (Fig. 1A). Colonsfrom the TNBS-treated mice were hyperemic and containedpractically no feces due to profound diarrhea. The peak of clinicaldisease in both forms of colitis occurred on the fifth day after i.r.instillation of the haptenating agent (Fig. 1B). Histological exami-nation of the H&E sections of colons from mice with oxazolonecolitis showed extensive goblet cell depletion as well as densechronic inflammatory infiltrates in the LP, with focal granulomaformation (Fig. 1C). The inflammation observed in colons frommice with TNBS-induced colitis also featured goblet cell depletionand lymphocytic infiltrates (Fig. 1C). Thus, the macro- and mi-croscopic evaluations of colons from BALB/c mice treated withoxazolone and TNBS revealed clinical and pathologic correlationswith UC and CD, respectively, consistent with previous reports(29, 33, 47, 48).

Nicotine attenuates the oxazolone colitis and exacerbates theTNBS colitis

To evaluate the effects of nicotine on the development and severityof experimental colitis, the mice were daily treated with nicotinesolution administered s.c. starting on the day before instillation ofeach haptenating agent.Nicotine alleviated oxazolone-induced colitis, as nicotine-treated

mice showed significantly (p , 0.05) decreased DAI values ondays 4 and 5, and colons from nicotine-treated mice had only mildhyperemia restricted to the distal part (Fig. 1A, 1B). Histologically,nicotine treatment drastically diminished goblet cell mucin de-pletion and the intensity of lymphocytic infiltrates (Fig. 1C). Con-sequently, the calculated HAI value (Table II) in colons fromnicotine-treated mice was 2-fold less compared with that in colonsfrom untreated mice with oxazolone colitis (Fig. 1D).In marked contrast, the nicotine-treated mice with TNBS-

induced colitis showed significantly (p , 0.05) elevated DAIthroughout the treatment period, and their colons had more severemacroscopic and microscopic inflammation than did colons of theuntreated mice with TNBS colitis (Fig. 1A, 1B). Nicotine treat-ment was associated with the appearance of extensive mucosalnecrosis in the proximal colon, extensive crypt damage, completegoblet cell depletion, LP destruction, and massive lymphocyticinfiltration (Fig. 1C). HAI was significantly (p , 0.05) elevated(Fig. 1D).Thus, nicotine attenuated oxazolone-induced colitis but exac-

erbated TNBS-induced colitis, consistent with the notion thatsmoking/nicotine ameliorates UC and worsens CD (3, 12).

Nicotine alters both peripheral and colonic T lymphocytepopulations in mice with experimental colitis

Because T cells play an important role in the pathogenesis of IBD(28), we analyzed major populations of T cells in the spleens andcolons of experimental mice. The expression of CD3, CD4, andCD8 was measured by flow cytometry.Untreated mice with oxazolone colitis showed an increased per-

centage of CD3+ T cells among splenic MCs, as well as of CD4T cells among splenic CD3+ T cells (Fig. 2A). In the nicotine-treated mice, the numbers of these cells returned to the normallevels characteristic of control BALB/c mice. Mice with oxazo-lone colitis also had increased CD4 T cells and decreased CD8+

T cells among LP CD3+ T cells. Treatment with nicotine led toa further increase of percentage of CD4 T cells among LP CD3+

T cells (Fig. 2B, 2C).Similar to oxazolone colitis, mice with TNBS colitis had an

increased percentage of splenic CD3+ and CD4 T cells (Fig. 2A).However, the percentage increase above the baseline in splenicCD3+ and CD4 T cells was greater than in oxazolone colitis.Alterations in LP T cell populations in TNBS colitis matchedthose in oxazolone colitis, that is, increased CD4 T cells, de-creased CD8+ T cells, and further elevation of CD4 T cells fol-lowing nicotine treatment (Fig. 2B, 2C).Thus, the differences between the effects of nicotine on oxa-

zolone- versus TNBS-induced colitis were as follows: 1) nor-

Table I. DAI scoring system used in this study

ScoreWeight Loss

(%)Stool

ConsistencyBlood in Stool(Hematochezia)

RectalBleeding

0 None Normal None Absent1 1–10 Loose stool Hemaoccult positive Present2 11–20 Diarrhea Gross blood n/a3 $20 n/a n/a n/a

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mailzation of the percentage of splenic CD3+ and CD4 T cells inthe former, and 2) additional elevation of the percentage of CD3+

T cells without changing the already elevated numbers of spleenCD4 T cells in TNBS colitis.

Distinct effects of nicotine on the numbers of Th17 T cells andFoxp3+ Tregs in the colons affected by oxazolone or TNBScolitis

The Treg/Th17 axis plays an important role in the control anddevelopment of IBD (49, 50). Therefore, to elucidate the role ofCD4 T cells in mediating dichotomous effects of nicotine on theseverity of oxazolone- and TNBS-induced colitides, we determinedrelative proportions of Tregs and Th17 T cells among LPMCs innicotine-treated versus untreated mice with each form of colitis.Whereas nicotine treatment was associated with a significant (p ,0.05) increase in colonic CD4+CD25+ T cells in both oxazolone-and TNBS-induced colitides (Fig. 3A), the percentage of CD25+

Foxp3+ Tregs among colonic CD4 increased .13-fold in oxazo-lone colitis but remained practically unchanged in TNBS colitis(Fig. 3B). Nicotine induced the opposite response regardingTh17-positive colonic CD4 T cells, with a significant decreasein oxazolone colitis and an increase in TNBS colitis (p , 0.05)(Fig. 3C).We also found that on day 2 both TNBS and TNBS plus nicotine

treatment groups featured similar T cell repertoire:∼80% of colonicT cells were CD4 positive, and ∼90% of CD4+ T cells were alsoCD17 positive (not shown). These findings are consistent with thelack of significant differences between DAI values in the TNBSand TNBS plus nicotine treatment groups on day 2 (Fig. 1B).Thus, the ability of nicotine to ameliorate oxazolone-induced

colitis was associated with an expansion of Tregs and reductionof CD4 Th17+ T cells among LPMCs. In contrast, the nicotine-dependent worsening of TNBS-induced colitis was associatedwith an elevation in LP CD4 Th17+ T cells. Therefore, we nextinvestigated the mechanism underlying the differential effects ofnicotine in these two forms of experimental IBD.

Nicotinergic stimulation of naive CD4+CD62L+ T cellsfacilitates Foxp3 expression

Because T cells express the nAChRs that meditate immuno-pharmacologic action of nicotine on these cells (17), we hypoth-esized that the immunomodulatory effects of nicotine in mice withexperimental colitis are mediated, in part, by nAChR-dependent

FIGURE 1. Clinical and histological correlations in mice with experi-

mental colitides treated with nicotine. A, Representative images of colons

dissected from control BALB/c mice treated i.r. with 47.5% ethanol in

saline (Control), mice with oxazolone-induced colitis either untreated (Ox)

or treated with 7.5 mg/kg/day nicotine (Ox+N), and untreated (T) versus

nicotine-treated (T+N) mice with TNBS colitis. The treatment with nico-

tine started from the day before instillation of a haptenating agent (i.e., at

the day 0) and ended on the fourth day of experiment, that is, the day

before mice were sacrificed. Note that the colon from untreated mouse with

oxazolone colitis has several necrotic areas, whereas in the nicotine-treated

animal the inflammation is visible only in the distal segment. Whereas the

colon of mouse with TNBS colitis is diffusely hyperemic, it lacks overt

necrosis. In contrast, the mouse with TNBS colitis treated by nicotine has

developed necrotic areas, as seen in the proximal segment of colon. B,

Clinical evaluation of the effects of nicotine treatment on the severity of

colitis in mice with oxazolone- and TNBS-induced colitides. The DAI

values were computed as described in detail in Materials and Methods

(Table I). In oxazolone colitis, the differences are statistically significant

(p , 0.05) on days 4 and 5. In TNBS colitis, the statistical differences

between the DAI values of nicotine-treated versus untreated mice are

significant (p , 0.05) starting from the third day after instillation of the

haptenating agent. Each point represents the mean 6 SD of data obtained

in 20 mice. The DAI values in control mice instilled with the ethanol

solution without haptenating agents were equal or close to 0 (data not

shown). C, Representative images of the histologic findings in nicotine-

treated versus untreated mice with oxazolone- and TNBS-induced coli-

tides. The photomicrographs of H&E-stained 5-mm sections of matching

segments of colons were made at an original magnification of 310. Note

that the extensive goblet cell depletion with submucosal involvement and

lymphocytic infiltrations characteristic of oxazolone colitis were not

present in mice treated with nicotine. In contrast, nicotine treatment ag-

gravated colonic inflammation in TNBS colitis, leading to crypt de-

struction, goblet cells depletion, and extensive lymphocytic infiltration. D,

Histopathologic analysis of the effects of treatment with nicotine on the

colonic inflammation in mice with oxazolone- and TNBS-induced coli-

tides. The HAI values were computed as described in detail in Materials

and Methods (Table II). Nicotine treatment protected the colons from in-

flammation induced by oxazolone, as can be judged from an ∼2-fold de-

crease of HAI. Vice versa, the HAI value computed in mice with TNBS

colitis treated by nicotine significantly increased, indicating that nicotine

worsened TNBS-induced colonic inflammation. *p , 0.05 compared with

the HAI value computed in colons of the nicotne-untreated mice with the

respective form of colitis.

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alterations of T cell development and function. To test this hy-pothesis, we exposed naive CD4+CD62L+ T cells from intactBALB/c mice to 100 mM nicotine for 5 d to match the in vivonicotine treatment dose used in this study, and we then evaluatedchanges in the expression of the transcription factor Foxp3.By immunoblotting, prior to stimulation with anti-CD3/CD28

Abs, Foxp3 was undetectable in naive CD4+CD62L+ T cells (Fig.4A). CD3/CD28 stimulation upregulated Foxp3 expression, inkeeping with the previously reported acquisition of T regulatoryactivity by stimulated naive T cells (51, 52). In the presence ofnicotine, anti-CD3/CD28 stimulation resulted in a further increaseof Foxp3 (Fig. 4A). Flow cytometry (FCM) analysis gave similarresults. CD3/CD28 stimulation increased the number of Foxp3-positive T cells and nicotine intensified this effect (Fig. 4B).Thus, upregulation of Foxp3+ T cells is a normal outcome of

TCR stimulation of naive CD4+CD62L+ T cells in the presence ofnicotine. This effect of nicotine could provide a mechanism forelevation of colonic CD25+Foxp3+ Tregs in mice with oxazolonecolitis, but it remained unclear why such elevation did not occurin mice with TNBS colitis.

Contribution of the cytokine milieu to the dichotomous effect ofnicotine on the development of Foxp3 CD4 T cells inoxazolone- versus TNBS-induced colitides

It is well recognized that the cytokine milieu of oxazolone colitisdiffers from that of TNBS colitis, with IL-4 being the predominantcytokine of the former and IL-12 being the principal cytokine ofthe latter form of experimental IBD (53). It is also known thatnicotinergic signaling in non-neuronal cells can be altered by theenvironmental stimuli such as cytokines, hormones, and growthfactors (reviewed in Refs. 54, 55). Therefore, we hypothesizedthat the difference in the cytokine milieu could lead to the dicho-tomous effects of nicotine treatment on oxazolone- versus TNBS-induced colitis. To test this hypothesis, we reproduced in vitro themost distinctive features of oxazolone- and TNBS-specific gutinflammation using IL-4 and IL-12, respectively. IL-4 did not alterthe stimulatory effect of nicotine on Foxp3 expression in naiveCD4+CD62L+ T cells stimulated with anti-CD3/CD28 Abs (Fig.4C). In marked contrast, the presence of IL-12 eliminated theability of nicotine to upregulate Foxp3 (Fig. 4D). These resultswere consistent with an increased proportion of CD25+Foxp3+

Tregs among colonic CD4-positive cells in nicotine-treated micewith oxazolone- but not TNBS-induced colitis (Fig. 3B).

Contribution of the cytokine milieu to the dichotomous effect ofnicotine on pro- and anti-inflammatory cytokine production

Next, we asked whether the reciprocal effects of IL-4 and IL-12on nicotine-dependent changes in Foxp3 expression correlate withproduction of the pro- and anti-inflammatory cytokines IL-17/IFN-

g and IL-10, respectively. First, we sought to ascertain that thecytokine milieu of each form of experimental IBD correspondsto the expected profile. Indeed, we found significantly (p , 0.05)increased colonic levels of IL-12 and IL-23 in TNBS colitis, andIL-4 in oxazolone colitis (Fig. 5A). Treatment with nicotine fur-ther elevated IL-23 and IFN-g levels (p , 0.05) in TBS colitis.Although the differences in IL-10 levels did not significantlydiffer among tested groups (p . 0.05), we detected a tendency forelevation of IL-10 in the oxazolone plus nicotine group (data notshown).To identify pharmacologic effects of nicotine on cytokine pro-

duction by CD4+ T cells, we performed in vitro experiments. Asexpected, stimulation of naive CD4+CD62L+ T cells with anti-CD3/CD28 Abs upregulated secretion of IL-17 (Fig. 5B), an ef-fector cytokine of the Th17 lineage that plays a pathogenic role inIBD (56). This effect was blocked in the presence of nicotine (p,0.05). IL-4 did not change this effect of nicotine (p. 0.05), whichis consistent with the anti-inflammatory action of nicotine in micewith oxazolone colitis. On the contrary, IL-12 totally abolishednicotine-dependent inhibition of IL-17 production. Furthermore,in the presence of nicotine, IL-12 significantly (p , 0.05) raisedIL-17 above control levels (Fig. 5B). A similar pattern of IL-4– and IL-12–dependent modulation of nicotine effects was ob-served for IFN-g production (Fig. 5B), another proinflammatorycytokine characteristic of TNBS-induced gut inflammation (57).As was the case with IL-17 production, IFN-g production wasmaximal in the presence of both nicotine and IL-12 (p , 0.05).These observations help explain how nicotine treatment exacer-bates gut inflammation in mice with TNBS colitis.CD3/CD28 stimulation of naive T cells also induced very low

levels of IL-10 secretion (Fig. 5B). When the cells were stimulatedwith anti-CD3/CD28 Abs in the presence of nicotine, the con-centration of IL-10 in culture supernatant rose ∼5-fold (Fig. 5B).Simultaneous exposure to IL-4 did not alter the nicotine-dependentupregulation of IL-10 secretion (Fig. 5B). In marked contrast,stimulation with anti-CD3/CD28 Abs in the presence of bothnicotine and IL-12 not only abolished the stimulatory effect ofnicotine on IL-10 secretion, but also decreased it below controllevels (p , 0.05). The latter phenomenon suggested that wors-ening of TNBS colitis by nicotine could be explained by itsproinflammatory action in the presence of IL-12.We also performed FCM analyses of cytokine expression in

CD4+CD62L+ cells stimulated with anti-CD3/CD28 with andwithout nicotine. The results shown in Fig. 5C confirmed thatnicotine can enhance IFN-g and IL-10, but inhibit IL-17 expres-sion. About 85% of IL-10–producing cells expressed Foxp3 (datanot shown), which is consistent with a finding that nicotine en-hances Foxp3 expression (Fig. 4B). Production of IL-4 was notaffected.

Table II. HAI scoring system used in this study

Score InfiltrateExtent of Inflammatory

Infiltrate

Goblet CellMucin

DepletionMucosalThickness

Length of BowelDamage (%)

0 Normal/physiologic None None Normal None1 Minimal elevation Single or rare, scattered

fociMinimal Minimal, focal

thickening,20

2 Expanded within or beyondlamina propria

Patchy, moderatelyabundant

Moderate Moderate, multifocalthickening

21–40

3 Crypt abscesses or submucosalinvolvement

Extensive Extensive Extensive 41–60

4 Inflammatory crypt destructionwith erosion of the mucosa

n/a n/a n/a .61

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Differential expression of a7 nAChR by colonic CD4 T cells intwo forms of experimental IBD

Inflammatory cytokines can modify expression levels of distinctnAChR subtypes in immune cells (18, 58), and specific nAChRsubtypes exhibit diverse effects on inflammatory cytokines, inhi-biting production of proinflammatory cytokines via both a4 anda7 nAChRs (17, 59). Therefore, the dichotomous effects of nic-otine in oxazolone- and TNBS-induced colitis might reflect dif-ferences in the repertoire of nAChRs expressed by colonic T cellsin each form of colitis. To test this hypothesis, we quantified theproportion of CD4 T cells expressing a4 versus a7 in in-flammatory infiltrates of oxazolone and TNBS colitis. The a4expression in colonic CD4 T cells in both forms of experimentalIBD was similarly low, but the level of a7 expression differeddramatically (Fig. 6A). Fewer than 3% of colonic CD4 T cells inTNBS-induced colitis expressed a7, whereas approximately two-thirds of CD4 T cells expressed this receptor in oxazolone coli-tis, that is, the frequency of a7 expression in oxazolone colitis

FIGURE 2. The effects of nicotine treatment of mice with oxazolone-

and TNBS-induced colitides on the peripheral and colonic populations of

T lymphocytes. The FCM assays with T cell marker Abs were performed

using splenocytes and LPMCs freshly isolated from intact (control) and

nicotine-treated and untreated mice with experimental colitides at the end

of nicotine treatments, as detailed in Materials and Methods. The cells

were triple stained for CD3, CD4, and CD8 and examined by flow cy-

tometry. The contour plots were generated after gating on lymphocytes

(by forward and side scatter) for CD3 staining or gating on CD3 T cells

during the analyses of CD4/CD8 staining. Shown on the graphs are rep-

resentative dot blots for the CD3 T cell population of spleen MCs (A) and

the CD4 and CD8 T cell subpopulations of CD3 T cells from spleen (B)

and colon (C). Note that the treatment with nicotine decreased the per-

centage of spleen CD3 and CD4 T cells to the normal levels in mice with

oxazolone colitis, and it further elevated the percentage of CD3 T cells

without altering the already elevated numbers of spleen CD4 T cells in

mice with TNBS colitis.

FIGURE 3. The effects of nicotine treatment of mice with experimental

IBD on the percentage of Tregs and Th17 cells among colonic CD4

T cells. The cryosections of colons from mice with oxazolone- and TNBS-

induced colitides treated with nicotine were subjected to a double or tri-

ple immunofluorescence staining for CD4/CD25, CD4/CD25/Foxp3, and

CD4/Th17 on day 5 after hapten treatment, as described in Materials and

Methods. The results are expressed as means 6 SD of the percentage of

CD25- (A), CD25/Foxp3- (B), and IL-17-positive cells (C) among colonic

CD4 T cells, taken as 100%. The data were computed in at least 10 dif-

ferent microscopic fields at magnification of360 in the triplicates of tissue

sections from three mice in each group. *p , 0.05 compared with un-

treated mice with the respective form of colitis.

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exceeded that in TNBS colitis by .30-fold (Fig. 6A). Hence, thetherapeutic action of nicotine on oxazolone colitis could beexplained by its ability to inhibit the proinflammatory function ofmost CD4 T cells by activating their a7 nAChR. The pivotal roleof a7 nAChR in mediating the therapeutic activity of nicotine inoxazolone-induced colitis was confirmed by the absence of suchactivity in knockout mice lacking a7 (Fig. 6B).

Alterations of a7 nAChR expression in normal murine CD4T cells by IL-4 and IL-12

To elucidate possible causes for differential expression of a7nAChR by colonic CD4 T cells in the two forms of experimentalIBD, we measured changes in a7 expression under influence ofIL-4 and IL-12, the principal inflammatory cytokines characteristicof oxazolone- and TNBS-induced gut inflammation, respectively.The a7 gene expression at the mRNA level was analyzed by qPCRand at the protein level by ICW. Following 5 d anti-CD3/CD28stimulation of naive CD4+CD62L+ T cells from intact BALB/cmice in the presence of IL-4 or IL-12, qPCR revealed that IL-12reduced the a7 mRNA expression to undetectable levels. In markedcontrast, IL-4 increased a7 mRNA by ∼1.5-fold (Fig. 6C). By ICW,IL-12 reduced the protein level of a7 by 70% (p , 0.05), whereasIL-4 increased it by ∼.3-fold (Fig. 6C). Neither cytokine altered theexpression level of a9 nAChR (data not shown). Thus, IL-4 and IL-12 could enhance the anti- and proinflammatory effects of nicotinein oxazolone- and TNBS-induced colitides, respectively, by selec-tively up- and downregulating expression of the anti-inflammatorya7 nAChR in colonic CD4 T cells.

DiscussionTo our knowledge, for the first time we demonstrated in this studythat the dichotomous effect of smoking that improves UC andworsens CD can be reproduced by nicotine treatment in mice withoxazolone and TNBS colitides. We also showed that the differen-ces in the repertoire of nAChRs expressed by colonic CD4 T cellsplay an important role in modifying the outcome of nicotinergictreatment of gut inflammation. The inflammatory milieu charac-teristic of UC/oxazolone colitis facilitated expression of the anti-inflammatory a7 nAChR by colonic CD4 T cells, thus improvingcolitis, whereas the milieu in CD/TNBS colitis abolished a7 ex-pression, which could exacerbate gut inflammation. These findingsunderscore the potential for nicotinergic drugs in regulating co-lonic inflammation.It is well known that smoking affects both UC and CD by

ameliorating UC and worsening CD. Because both smoking andpure nicotine can alter production of Th1- and Th2-type cytokines

(60–62), we hypothesized that the nicotinergic agonist nicotinewould mimic the effects of smoking in the mouse models of UC-and CD-like colitides. As expected, oxazolone and TNBS inducedacute forms of UC- or CD-like colonic inflammation, respectively,and mice with each form of experimental IBD demonstrated di-rectly opposing responses to nicotine treatment. The analysis ofperipheral and colonic T cell subpopulations revealed that clinicaland histological features of experimental IBD types correlatedwell with the immunopathologic characteristics of each form ofcolitis (19–27). Clinical and histological improvement of micewith oxazolone colitis was associated with a relative increase ofCD25+Foxp3+ Tregs and a reciprocal decrease of CD4 IL-17+

cells among LPMCs, indicating that in this form of IBD thetherapeutic effect of nicotine stems from skewing the Treg/Th17balance toward the immunosuppressive Tregs. The opposite wasobserved in mice with nicotine-treated TNBS colitis, indicatingthat the Treg/Th17 balance was skewed toward proinflammatoryTh17 cells.To elucidate the mechanisms responsible for the dichotomous

outcomes following nicotine treatment of mice with oxazolone-versus TNBS-induced colitides, we investigated the direct effect ofnicotine on freshly isolated murine naive CD4+CD62L+ T cells.In vitro nicotine stimulation of naive T cells activated via TCR/CD3 cross-linking resulted in upregulated expression of Foxp3+,indicating that facilitated development of Tregs from naive T cellsis a normal outcome of nicotinergic stimulation. Furthermore,nicotine upregulated production of the immunosuppressive cyto-kine IL-10 and inhibited that of IL-17, an effector cytokine ofTh17 cells. Upregulated production of IL-10 provided indirectevidence that the Foxp3+ Tregs induced by nicotine were func-tional cells that could produce and respond to IL-10, long knownto suppress intestinal inflammation in mice with experimentalcolitis (63). These observations are in keeping with earlier reportsthat treatment with nicotine can induce “suppressor” T cells (64,65), inhibit IL-18-induced T cell proliferation (66), and influencecytokine profiles and subsequent cell cycling/apoptotic responsesof PBMCs from IBD patients (37). These results, taken together,suggest that the anti-inflammatory action of nicotine in mice withoxazolone-induced colitis resulted from stimulating production ofTregs. This observation, however, did not explain why nicotineworsened TNBS colitis and prompted additional mechanisticstudies.The induction and function of Foxp3+ T cells can both affect

and be affected by inflammatory cytokines (67, 68). Therefore,differences between nicotine effects on mice with oxazoloneversus TNBS colitis might reflect differences in the gut in-

FIGURE 4. The nicotinergic effects on the develo-

pment of Tregs from the naive CD4+CD62L+ T cells

stimulated with anti-CD3/CD28 Abs. The enriched

naive CD4+CD62L+ T cells isolated from the spleens of

intact BALB/c mice were cultured in growth medium

alone (condition 1) or stimulated with anti-CD3 and

anti-CD28 Abs in the absence (condition 2) or presence

of nicotine (Nic; condition 3), 10 ng/ml IL-4, 100 mM

nicotine plus 10 ng/ml IL-4 or IL-12, or 100 mM nic-

otine plus 10 ng/ml IL-12 as detailed in Materials and

Methods. After 5 d in culture, the expression of Foxp3

protein was analyzed by immunoblotting (A) and FCM

(B–D). The graph in A demonstrates changes of Foxp3

concentration after its normalization for the level of the

housekeeping protein b-actin. In B–D, the changes in

Foxp3 expression are shown as an overlay histogram

representing different treatment conditions.

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flammatory environment produced by each haptenating agent.Because it is well known that IL-4 is an effector cytokine inoxazolone colitis (53), and that IL-12 plays a crucial role in TNBScolitis, these cytokines could modulate the nicotinergic effects onthe T cells mediating colonic inflammation in mice with each formof colitis. To test this hypothesis, we determined whether IL-4and IL-12 alter the ability of nicotine to upregulate Foxp3 ex-pression by naive CD4+CD62L+ T cells stimulated with anti-CD3/CD28 Abs. Although IL-4 did not alter the nicotine effect, IL-12 completely abolished the nicotine-dependent upregulation ofTregs. Decreased numbers/function of Tregs skew the dynamic

equilibrium within the Treg/Th17 axis toward Th17 cells (69).Hence, worsening of colonic inflammation by nicotine/smokingmay be explained by a loss of control of Th17 cells playing thepathogenic role in both TNBS-induced colitis in mice (70) andpatients with CD (71).To elucidate the molecular mechanisms allowing cytokines to

diversify the immunopharmacologic action of nicotine, we investi-gated the role of T cell nAChRs. Immunologic stimulation has beenshown to alter the expression and function of nAChRs in T cells,and different nAChR subtypes are known to exhibit diverse im-munoregulatory effects due to differential regulation of pro- and

FIGURE 5. The influence of IL-4 and IL-12 on nicotinergic regulation of IL-10, IL-17, and IFN-g secretion. A, Colons from mice with oxazolone- or

TNBS-induced colitides were collected on day 5 after hapten treatment. Total protein was extracted, and tissue levels of IL-4, IL-12, IL-23, and IFN-g were

detected using mouse ELISA immunoassay kits, as described in Materials and Methods. *p , 0.05 compared with control mice; #p , 0.05 compared with

untreated mice with the respective form of experimental colitis. B, The naive CD4+CD62L+ T cells from intact BALB/c mice were stimulated with anti-

CD3 and anti-CD28 in the presence or absence of 100 mM nicotine with or without 10 ng/ml IL-4 or IL-12 for 5 d, after which the cell culture supernatants

were collected and analyzed by ELISA for the presence of IL-10, IL-17, and IFN-g, as described inMaterials and Methods. *p, 0.05 compared with naive

CD4+CD62L+ T cells stimulated by TCR/CD3 cross-linking without any additions; #p , 0.05 compared with cells stimulated by anti-CD3/anti-C28 in the

presence of nicotine given alone; +p, 0.05 compared with cells stimulated with anti-CD3/anti-C28 in the presence of relevant cytokine without nicotine. C,

The naive CD4+CD62L+ T cells from intact BALB/c mice were stimulated with anti-CD3 and anti-CD28 in the presence or absence of 100 mM nicotine

with and without 10 ng/ml IL-4 or IL-12 for 5 d, after which the expression of IL-4, IL-10, IL-17, and IFN-g was analyzed by FCM, as described in

Materials and Methods.

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inflammatory cytokines through the signaling pathway couples bydistinct nAChR subtypes (16, 17, 72–78). In keeping with the well-established immunosuppressive/anti-inflammatory function of a7nAChR (reviewed in Ref. 79), we observed a dramatic difference inthe percentages of colonic CD4 T cells expressing a7 in oxazoloneversus TNBS colitis, 67 versus 3%, respectively, and confirmed theabilities of IL-4 and IL-12 to exhibit reciprocal effects on a7 geneexpression in murine CD4 T cells at the mRNA and protein levels.The pivotal role of a7 in mediating therapeutic effects of nicotine inoxazolone colitis was confirmed in experiments with a7 nAChRknockout mice that showed no improvement due to nicotine treat-

ment. The proinflammatory action of nicotine that aggravatedTNBS colitis could be mediated via the alternative nicotinergicsignaling pathways activated in colonic CD4 T cells that lackeda7 nAChR. The reported proinflammatory/immunostimulatoryeffects of nicotine include increased secretion of IL-12 by Th1T cells (80), enhanced Con A-induced production of IFN-g (62),and protection of lymphocytes from cortisol-induced apoptosis(81). It has been proposed that the nicotinergic proinflammatorycascade is mediated by a9 nAChR (82), whose expression wasnot affected by either IL-4 or IL-12. Thus, upregulated expres-sion of a7 by colonic T cells, facilitated by IL-4, could mediatean anti-inflammatory effect of nicotine on oxazolone colitis and,vice versa, downregulation of this receptor by IL-12 couldworsen TNBS colitis. These chains of events can be summarizedas follows:

IL-4→↑a7 signaling→↑Tregs↔↑IL-10; ↓IL-17→improvement of oxazolone colitisIL-12→↓a7 signaling→↓Tregs↔↓IL-10; ↑IL-17→aggravation of TNBS colitis

A recent discovery that the efferent vagus nerve modulates theimmune response and controls inflammation through nAChRsexpressed by inflammatory cells gave rise to a concept of a“cholinergic (nicotinic) anti-inflammatory pathway” (83), andopened a novel avenue for treatment of inflammatory diseaseswith nicotinic agonists (79, 84, 85). The goal is to avoid the un-desired side effects of the therapeutic doses of nicotine (86),which, unfortunately, is not effective in UC at low doses (12).The following nicotinic agonists that avoid the toxicity of nicotineare currently under investigation: AR-R17779 ((2)-spiro[1-aza-bicyclo[2.2.2]octane-3,59-oxazolidin-29-one]), GTS21 (3-[(2,4-dime-thoxy)benzylidene]-anabaseine), CAP55, Exo2 (exo-2-(2-pyridyl)-7-azabicyclo[2.2.1] heptane), PNU-282987 ([N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride]), and 4OHGTS(3-(4-hydroxy,2-methoxybenzylidene)anabaseine), some of whichhave already been successfully used in various in vitro and in vivomodels of inflammation (reviewed in Refs. 84, 87). In IBD asso-ciated with elevated levels of IL-4, such as UC, the therapeuticeffect may be achieved from a7 agonists, whereas a7 antagonistsmay be therapeutic in the IBD type associated with elevated IL-12,such as CD. By now, both nicotinic agonists and antagonists (35,36) have been demonstrated to be therapeutic in experimental IBD,and it also has been shown that administration of a7 agonists to theIBD type caused by IL-12, such as dextran sulfate sodium-inducedcolitis (88), worsen gut inflammation (38).In conclusion, our data indicate that the dichotomous clinical and

immunopharmacologic effects of smoking/nicotine in UC and CDcan be explained, in part, by the ability of the cytokine milieucharacteristic of each type of IBD to modify in a unique fashion thenicotinergic signaling by altering the repertoire of T cell nAChRs.Although there is significant potential for developing selectiveimmunomodulation using nicotinergic agents that target nAChRsin T cells, an equally broad and specific range of nAChR-relatedoutcomes can be expected, which may be both beneficial andharmful to the host. Identification of the nAChR subtypes coupledto specific functions of a particular T cell subset involved in eachtype of IBD should allow selective immunomodulation. Thus,future in-depth analysis of cholinergic immunopharmacology ofdistinct forms of gut inflammation should be rewarded with noveltherapeutic strategies.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 6. Variations in the expression of a4 and a7 nAChRs by CD4

T cells. A, The expression of a4 and a7 nAChRs by colonic CD4 T cells in

different forms of experimental IBD. The percentages of CD4 T cells

expressing a4 or a7 in the colonic inflammatory infiltrates of BALB/c

mice with oxazolone (Ox)- and TNBS (T)-induced colitides were com-

puted in 10 microscopic fields of double-stained cryostat sections of colons

from three mice in each group (n = 3). *p, 0.05 compared with mice with

oxazolone-induced colitis. B, Clinical evaluation of nicotine (5 mg/kg/d)

treatment on the severity of oxazolone colitis in a7 knockout (KO) mice

(B6.129S7-Chrna7tm1Bay/J; stock no. 003232). The DAI values were

computed as described in detail in Materials and Methods. Each point

represents the mean 6 SD of data obtained in five mice. The differences

between the nicotine-treated and untreated groups are not significant (p .0.05). C, The effects of IL-4 and IL-12 on a7 nAChR expression. The

enriched naive CD4+CD62L+ T cells from intact BALB/c mice were

stimulated with anti-CD3/anti-CD28 Abs alone (control) or in the presence

of 10 ng/ml either IL-4 or IL-12 for 5 d in culture, and the expression of a7

mRNA and protein was detected by qPCR and ICW, respectively, as de-

tailed in Materials and Methods. The results are expressed as means 6 SD

of relative receptor mRNA or protein levels in cells exposed to cytokines

compared with those in controls, taken as 1. *p , 0.05 compared with

control group; #p , 0.05 compared with cells exposed to IL-12.

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