The Effects of All-Trans Retinoic Acid on the Induction of Oral Tolerance in a Murine Model of Bronchial Asthma

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1 Originl Pper Int Arch Allergy Immunol 21;167: DOI: 1.119/ Received: December 19, 214 Accepted fter revision: July 1, 21 Published online: August 2, 21 The Effects of All-Trns Retinoic Acid on the Induction of Orl Tolernce in Murine Model of Bronchil Asthm Hirotk Skmoto Toshiyuki Koy Keisuke Tsukiok Kenjiro Shim Stoshi Wtnbe Hiroshi Kgmu Yosuke Kimur Tkuro Skgmi Tkshi Hsegw b Eiichi Suzuki b Ichiei Nrit Division of Respirtory Medicine, Deprtment of Homeosttic Regultion nd Development, Niigt University Grdute School of Medicl nd Dentl Sciences, nd b Deprtment of Generl Medicine, Niigt University Medicl nd Dentl Hospitl, Niigt, Jpn Key Words All-trns retinoic cid Bronchil sthm Orl tolernce Regultory T cell Abstrct Bckground: Active suppression induced by regultory T (Treg) cells is reported to be one of the mechnisms involved in orl tolernce. All-trns retinoic cid () hs been reported to ffect Treg cell differentition. The present study exmined the effects of on the induction of orl tolernce in murine model of bronchil sthm. Methods: BALB/c mice were sensitized to nd chllenged with ovlbumin (OVA) through feeding followed by OVA chllenges. In some study groups ws orlly dministered concomitntly with OVA feeding either in the presence or bsence of the retinoic cid receptor ntgonist LE13. Lung CD4 + T cells were isolted from mice exposed to nd/or OVA, nd trnsferred to control mice. Airwy hyperresponsiveness (AHR), cell counts nd cytokine levels in broncholveolr lvge (BAL) fluid, nd lung histology were ssessed. Results: Concomitnt dministrtion of with OVA meliorted AHR, irwy eosinophili, elevtion of cytokines in BAL fluid nd goblet cell metplsi. The proportion of Treg cells in the lungs ws incresed in mice treted with OVA nd, s compred to those treted with OVA only. Trnsfer of lung CD4 + T cells from mice treted with OVA nd induced suppression of AHR nd irwy inflmmtion. LE13 completely reversed the effects of on AHR, irwy llergic inflmmtion nd the number of Treg cells in the lungs. Conclusion: These dt suggested tht orl dministrtion of with OVA hd the potentil to enhnce orl tolernce in this murine model of bronchil sthm. These effects were medited, t lest in prt, by Treg cell expnsion. 21 S. Krger AG, Bsel Introduction Bronchil sthm is chrcterized by recurrent episodes of irwy obstruction, irwy hyperresponsiveness (AHR) to environmentl stimuli, cute-on-chronic irwy inflmmtion nd structurl chnges in the irwy wlls [1]. Therefore, sthm mngement is bsed on voiding exposure to llergens nd controlling irwy in- E-Mil krger@krger.com 21 S. Krger AG, Bsel /1/ $39./ Correspondence to: Dr. Toshiyuki Koy Division of Respirtory Medicine, Deprtment of Homeosttic Regultion nd Development, Niigt University Grdute School of Medicl nd Dentl Sciences 1-74 Ashimchi-Dori, Niigt City (Jpn) E-Mil med.niigt-u.c.jp

2 flmmtion. Currently, inhled corticosteroids (ICS) re the most potent gents used to suppress irwy inflmmtion. Indeed, there is substntil evidence for the efficcy of ICS in reducing irwy inflmmtion [2], meliorting AHR [3], irwy remodeling [4], lleviting clinicl symptoms [] nd improving prognosis [6]. However, severl reports hve indicted tht ICS hs little influence on disese history, even if disese severity is controlled [7, 8]. Moreover, the cost of sthm mngement is incresing rpidly. Allergen (ntigen)-specific immunotherpy is n immune-modifying therpy tht hs been recommended for the tretment of llergic rhinitis, venom hypersensitivity, some drug llergies, nd mild bronchil sthm [9]. Orl dministrtion of ntigen is clssiclly used to induce ntigen-specific systemic immunotherpy, nd is termed orl tolernce [1, 11]. It is now widely ccepted tht the mechnisms of orl tolernce include not only nergy nd poptosis of ntigen-specific T cells in the gut, but lso ctive suppression through the induction of ntigen-specific regultory T (Treg) cells [12, 13]. Most of the induced Treg cells re chrcterized by bundnt production of immunosuppressive cytokines, such s interleukin (IL)-1 nd trnsforming growth fctor-β (TGF-β), nd expression of the trnscription fctor forkhed box P3 (Foxp3) [14]. We previously reported tht both trnsfer of T helper (Th) 17 cells or dministrtion of IL-17 in the induction phse of orl tolernce bolished the therpeutic effects of orl tolernce by upregulting IL-6 production in Peyer s ptch (PP) in murine sthm model [1]. The vitmin A metbolite, retinoic cid (RA), hs lso been reported to enhnce the expression of α4β7 integrin nd C-C chemokine receptor type 9 on T cells upon ctivtion, imprinting them with gut tropism [16]. RA is key regultor of TGF-β-dependent immune responses nd ws shown to inhibit IL-6-driven induction of proinflmmtory Th17 cells nd to promote nti-inflmmtory Treg cell differentition; peripherl conversion of CD4 + T cells to Treg cells occurred primrily in gut-ssocited lymphoid tissue fter orl exposure to ntigen [17]. We therefore hypothesized tht orl dministrtion of lltrns RA () long with the ntigen would hve the potentil to enhnce the effects of orl tolernce. In the present study, we investigted the effect of on the induction of orl tolernce in murine irwy llergy model. Following chllenges with ovlbumin (OVA), we evluted AHR nd llergic inflmmtion. We lso investigted the modulting effects of trnsfer of CD4 + T cells from mice treted with orl nd OVA to nimls tht did not receive these compounds orlly, nd ssessed whether the effects of were medited through the RA receptor (RAR) by using the RAR ntgonist LE13. Mterils nd Methods Animls Eight-week-old femle BALB/c mice free of murine-specific pthogens were purchsed from CLEA Jpn Inc. (Tokyo, Jpn). The nimls were housed under specific pthogen-free conditions with 12: 12 h light:drk cycle. All experiments were conducted under protocol pproved by the Niigt University ethics committee for niml experiments. OVA-Induced Allergic Airwy Inflmmtion nd Orl Tolernce Mice were sensitized on dys nd 14 by intrperitonel injection of 2 μg OVA premixed with 2.2 mg of Al(OH) 3 in 1 μl of phosphte-buffered sline (PBS). After sensitiztion, the nimls were exposed to n OVA erosol (1 mg/ml in.9% sline) for 2 min on dys 28, 29 nd 3. From dys 37 to 41 the mice were dministered OVA (2 mg/dy or 1 mg/dy) by gvge once dy to induce orl tolernce, followed by OVA erosol exposure (s described bove) on dys 48, 49 nd. (2 μg/dy) ws dministered orlly long with OVA on dys mice received the sme volume of PBS or lone by gvge. In some experiments, n RAR ntgonist (LE13; 2 μg/dy) ws dministered intrperitonelly (i.p.) with orl + OVA on dys Twenty-four hours fter the finl OVA chllenge, AHR ws ssessed nd specimens of broncholveolr lvge (BAL) fluid, serum nd lungs were collected for further nlysis. Figure 1 shows summry of the experimentl protocols used in this study. In some mice, PP specimens were obtined 24 h fter the finl OVA feeding. Cell Preprtions from Lungs nd PPs nd Trnsfer of Lung Cells Lung cells were isolted s previously described [18] using collgense digestion. Lung CD4 + T cells were purified from lung cells (purity >98%) using mouse CD4 Dynbeds TM (Invitrogen, Crlsbd, Clif., USA). These lung CD4 + T cells were then dministered intrvenously ( 1 6 cells/mouse) to OVA-sensitized mice, followed by further OVA chllenges ( fig. 1 ). PPs were resected from the smll intestine, pssed through steel mesh to remove ny ggregtes, nd then wshed twice with PBS contining.2% BSA nd.2% NN 3 before use for flow cytometry. Airwy Responsiveness AHR ws ssessed by mesuring chnges in respirtory resistnce in response to incresing doses of inhled methcholine (MCh) using the Flexivent system (SCIREQ, Montrel, Que., Cnd), s previously reported [19]. BAL Fluid nd Lung Histology Immeditely fter the mesurement of AHR, BAL ws performed vi trchel tube, s previously described [2]. Lungs were fixed in 1% formlin nd processed for prffin embedding. 168 Int Arch Allergy Immunol 21;167: DOI: 1.119/ Skmoto et l.

3 OVA/lum i.p. Dys Scrifice N.S. (control) p.o. OVA 2 mg/dy p.o. OVA 1 mg/dy p.o. OVA 2 mg/dy + 2 μg/dy p.o. OVA/lum i.p. Fig. 1. Experimentl protocols for OVA sensitiztion nd chllenge, including orl OVA dministrtion, CD4 + T cell trnsfer, orl nd LE13 dministrtion, s described in Mterils nd Methods. Mice were treted with orl OVA (2 mg/ dy or 1 mg/dy) or orl OVA (2 mg/ dy) nd for dys fter the first OVA erosol chllenges, followed by secondry OVA chllenges, nd were scrificed 24 h fter the lst chllenge. b Lung CD4 + T cells were trnsferred from mice treted with orl OVA (2 mg/dy) nd to mice sensitized with OVA, followed by OVA chllenges. c The RAR ntgonist, LE13, ws dministered i.p. to mice treted with OVA nd. N.S. = Norml sline. b c Dys OVA/lum i.p OVA 2 mg/dy p.o. OVA 2 mg/dy + 2 μg/dy p.o OVA/lum i.p. CD4 + T cell purifiction trnsfer vi til vein Dys Scrifice 1 N.S. (control) p.o. 2 OVA 2 mg/dy + 2 μg/dy p.o. 3 OVA 2 mg/dy + 2 μg/dy p.o. + LE13 i.p. Mucus-contining goblet cells were detected by stining the prffin sections ( μm thick) with periodic cid-schiff (PAS). Histologicl nlyses were performed s previously described [21]. Flow Cytometry The surfce phenotypes of the lung CD4 + T cells were nlyzed by flow cytometry using three-color immunofluorescence test. This employed monoclonl ntibodies rised ginst CD4 (RM4 ), CD2 (PC61; both obtined from BD Biosciences, Sn Jose, Clif., USA) nd Foxp3 (FJK-16S; ebioscience, Sn Diego, Clif., USA). After wshing, the stining ws nlyzed on FACSClibur flow cytometer using CellQuest softwre (BD Biosciences). Mesurement of Cytokines in BAL Fluid Superntnts from BAL fluid were stored t 8 C prior to the mesurement of cytokines. Enzyme-linked immunosorbent ssy (ELISA) kits for the detection of IL-4, IL- nd IL-17 were obtined from ebioscience. IL-1 nd IL-13 ELISA kits were purchsed from R&D Systems (Minnepolis, Minn., USA). Sttisticl Anlysis The Mnn-Whitney U test ws used to determine the significnce of group differences. Dt were pooled from three independent experiments with 4 mice per group in ech experiment (n = 12). Comprisons for ll pirs were performed using the Kruskl- Wllis test. Significnce ws ssumed t p <. for ll tests. Vlues for ll mesurements were expressed s the men ± stndrd error of the men (SEM). Results Effect of on AHR, Airwy Inflmmtion nd Airwy Remodeling We initilly exmined the effects of orl OVA dministrtion in murine irwy llergy model. Mice tht were treted with 1 mg/dy OVA showed reduced AHR in response to MCh ( fig. 2 ), reduced irwy eosinophili ( fig. 2 b), lower levels of IL- nd IL-13 in BAL fluid ( fig. 2 c), nd reduced mucus production in the bronchi ( fig. 2 d, e) s compred with mice treted with PBS lone. On the other hnd, 2 mg/dy OVA did not induce significnt reduction of AHR, irwy eosinophili, Th2 cytokine production in BAL fluid nd goblet cell metplsi ( fig. 2 ). However, dministrtion of in ddition to 2 mg/dy OVA reduced AHR to similr level to tht Reinforces Orl Tolernce Int Arch Allergy Immunol 21;167: DOI: 1.119/

4 % chnge from bseline 1,2 1, Neb OT 2 OT 1 OT 2 + # ## % # Neb OT 2 OT 1 OT 2 + ## pg/ml # # # # Color version vilble online Methcholine (mg/ml) 1 b Mc Lym Neu Eos c IL-4 IL- IL-13 IL-4 IL-4 Neb OT 2 OT 1 OT 2 + Number (per mm BM) Neb OT 2 OT 1 OT 2 + ## ## d e Fig. 2. The effects of on AHR, irwy llergic inflmmtion nd goblet cell metplsi. Chnges in irwy resistnce in response to the indicted concentrtions of nebulized MCh. b Chnges in cell composition in BAL fluid. c Levels of the indicted cytokines in BAL fluid. d Representtive PAS stining of lung sections obtined 24 h fter the lst OVA chllenge. Originl mgnifiction 1. e Quntittive nlysis of PAS-positive cells in bronchil tissue. : OVA-sensitized mice with OVA chllenges; OT 2: OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/dy); OT 1: OVA-sensitized mice with OVA chllenges + orl OVA (1 mg/dy); OT 2 + : OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/dy) nd (2 μg/dy); : OVA-sensitized mice with OVA chllenges + (2 μg/dy). Dt represent the men ± SEM. p <., p <.1 compred to the control group; # p <., ## p <.1 compred to the OT 2 group. Mc = Mcrophges; Lym = lymphocytes; Neu = neutrophils; Eos = eosinophils; BM = bsement membrne; Neb = nonsensitized mice with OVA chllenges. observed in nimls dministered 1 mg/dy OVA ( fig. 2 ). Airwy eosinophili in the plus 2 mg/ dy OVA group ws significntly decresed in comprison to the group tht received OVA only ( fig. 2 b). The IL- nd IL-13 levels in BAL fluid were decresed in mice tht were treted with plus 2 mg/dy OVA, s compred to mice receiving only 2 mg/dy OVA only ( fig. 2 c). The number of PAS-positive cells ws lso significntly lower in the plus 2 mg/dy OVA group nd ws similr to the number observed in mice dministered 1 mg/dy OVA ( fig. 2 d, e). Interestingly, orl dministrtion of without OVA hd little effect on AHR, irwy eosinophili, cytokine levels in BAL fluid, or goblet cell metplsi ( fig. 2 ). Orl dministrtion of in combintion with 1 mg/dy OVA feeding hd no dditionl effects on AHR nd irwy inflmmtion compred to 1 mg/dy OVA feeding lone (dt not shown). A 2-μg dose of ws selected bsed on preliminry investigtion. We investigted the dose depen- 17 Int Arch Allergy Immunol 21;167: DOI: 1.119/ Skmoto et l.

5 OT OT Foxp OT Foxp3 OT CD4 c 2 CD4 CD2 + Foxp3 + /CD4 (%) 1 CD2 + Foxp3 + /CD4 (%) 1 1 b OT 2 OT 2 + d OT 2 OT 2 + Fig. 3. Anlysis of lung nd PP cells using flow cytometry. Representtive CD4 nd Foxp3 expression in lung cells. b CD2 + Foxp3 + cells, expressed s percentge of lung CD4 + cells. c Representtive CD4 nd Foxp3 expression in PP cells. d CD2 +Foxp3 + PP cells, expressed s percentge of CD4 + PP cells. : OVA-sensitized mice with OVA chllenges; OT 2: OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/dy); OT 2 + : OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/ dy) nd (2 μg/dy); : OVA-sensitized mice with OVA chllenges + (2 μg/dy). Dt represent the men ± SEM from three independent experiments (n = 6). p <. compred to the OT 2 group. dency of the effects of on AHR, irwy inflmmtion nd goblet metplsi nd found tht 2 μg of ws superior to μg nd 1, μg (online suppl. fig. 1; for ll online suppl. mteril, see doi/1.119/437326). Comprison of Lung nd PP Foxp3-Positive Treg Expnsion Previous reports described the potency of Treg cell induction by RA in vitro nd in vivo [17, 22, 23]. To investigte the expnsion of Treg cells in the lungs, cells were hrvested nd stined for CD4, CD2 nd Foxp3. As shown in figure 3 nd b, the rtio of Foxp3 + Treg cells to the totl count of CD4 + T cells ws incresed in the plus 2 mg/dy OVA group s compred to the 2 mg/ dy OVA group or lone. To determine the effects of combined OVA nd tretment on the gut, cells were hrvested from PPs of treted mice nd stined for CD4, CD2 nd Foxp3. The rtio of Foxp3 + Treg cells to the totl count of CD4 + T cells ws not significntly incresed by the plus 2 mg/dy OVA tretment s compred to the 2 mg/dy OVA group or the group tht received lone (fig. 3c, d). Reinforces Orl Tolernce Int Arch Allergy Immunol 21;167: DOI: 1.119/

6 % chnge from bseline 1, PBS trnsfer Lung CD4 trnsfer (OT) Lung CD4 trnsfer (OT + ) % PBS trnsfer Lung CD4 trnsfer (OT) Lung CD4 trnsfer (OT + ) pg/ml PBS trnsfer Lung CD4 trnsfer (OT) Lung CD4 trnsfer (OT + ) Color version vilble online 1 Methcholine (mg/ml) 1 b Mc Lym Neu Eos c IL-4 IL- IL-13 IL-1 IL-17 PBS Trnsfer (OT) Trnsfer (OT + ) Number (per mm BM) PBS Trnsfer (OT) Trnsfer (OT + ) d e Fig. 4. The effects of lung CD4 + T cells from OVA-sensitized mice treted with orl nd/or OVA (nd OVA chllenges) on AHR, irwy llergic inflmmtion nd goblet cell metplsi. Chnges in irwy resistnce in response to the indicted concentrtions of nebulized MCh. b Chnges in the cell composition of BAL fluid. c Levels of the indicted cytokines in BAL fluid. d Representtive PAS stining of lung sections obtined 24 h fter the lst OVA chllenge. Originl mgnifiction 1. e Quntittive nlysis of PAS-positive cells in bronchil tissue. Trnsfer (OT): trnsfer of CD4 + lung T cells from mice treted with OVA (2 mg/dy); trnsfer (OT + ): trnsfer of CD4 + lung T cells from mice treted with OVA (2 mg/dy) nd (2 μg/ dy). Dt represent the men ± SEM. p <., p <.1 compred to the trnsfer (OT) group. Mc = Mcrophges; Lym = lymphocytes; Neu = neutrophils; Eos = eosinophils; BM = bsement membrne. Effects of Lung CD4 + T Cell Trnsfer on AHR, Airwy Inflmmtion nd Airwy Remodeling To investigte whether CD4 + T cells hd the potentil to modify AHR nd llergic irwy inflmmtion, lung CD4 + T cells were trnsferred from mice tht received either nd 2 mg/dy OVA or only 2 mg/dy OVA to control mice sensitized with OVA; these nimls then underwent dditionl OVA exposures. Trnsfer of these cells from mice exposed to nd OVA suppressed AHR to MCh, wheres trnsfer from mice tht received OVA lone did not suppress AHR s compred to PBS-treted mice ( fig. 4 ). The percentge of eosinophils in BAL fluid ws decresed in recipients of lung CD4 + T cells from mice tht received nd OVA s compred to mice tht only received OVA ( fig. 4 b). Trnsfer of lung CD4 + T cells from mice treted with nd OVA suppressed IL- nd IL-13 levels in BAL fluid ( fig. 4 c). Goblet cell metplsi ws drmticlly reduced in recipients of lung CD4 + T cells from mice tht received nd OVA s compred to recipients of cells from mice tht only received OVA (fig. 4d, e). 172 Int Arch Allergy Immunol 21;167: DOI: 1.119/ Skmoto et l.

7 % chnge from bseline 1, OT OT + OT + + LE13 % OT OT + OT + + LE13 pg/ml Color version vilble online 1 Methcholine (mg/ml) 1 b Mc Lym Neu Eos c IL-4 IL- IL-13 IL-1 IL-17 OT OT + 16 OT + + LE13 Number (per mm BM) CD2 + Foxp3 + /CD4 1 1 d e f Fig.. Effects of LE13 on AHR, irwy llergic inflmmtion nd goblet cell metplsi. Chnges in irwy resistnce in response to the indicted concentrtions of nebulized MCh. b Cell composition in BAL fluid. c Cytokine levels in BAL fluid. d Representtive dt of PAS stining of lung sections obtined 24 h fter the lst OVA chllenge. Originl mgnifiction 1. e Quntittive nlysis of PAS-positive cells in bronchil tissue. f CD2 +Foxp3 + lung cells, expressed s percentge of lung CD4 + cells. OT: OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/dy); OT + : OVA-sensitized mice with OVA chllenges + orl OVA (2 mg/dy) nd (2 μg/dy); OT + + LE13: OVAsensitized mice with OVA chllenges + orl OVA (2 mg/dy) nd (2 μg/dy) + LE13 (2 μg/dy). Dt represent the men ± SEM. p <., p <.1 compred to the OVA + group. Mc = Mcrophges; Lym = lymphocytes; Neu = neutrophils; Eos = eosinophils; BM = bsement membrne. Effects of LE13 on AHR, Airwy Inflmmtion nd Airwy Remodeling To determine whether the effects of were medited by the RAR, n ntgonist of this receptor (LE13) ws dministered i.p. concomitntly with OVA nd. As shown in figure, LE13 reversed the inhibitory effects of. LE13 lso incresed the percentge of eosinophils nd the levels of IL- nd IL-13 in BAL fluid ( fig. b, c). Goblet cell metplsi ws greter in mice tht received LE13 with nd OVA, s compred to those receiving nd OVA only ( fig. d, e). Moreover, the rtio of Foxp3 + Treg cells to the totl count of CD4 + T cells ws significntly decresed in the group tht received LE13, OVA nd, s compred to the group tht only received OVA nd ( fig. f). In mice tht received LE13 without nd OVA, there ws no significnt chnge in AHR or irwy eosinophili (dt not shown). Discussion The present study found tht ugmented the orl tolernce induced by OVA. LE13-medited RAR blockde reversed the inhibitory effects of, indi- Reinforces Orl Tolernce Int Arch Allergy Immunol 21;167: DOI: 1.119/

8 cting the involvement of this receptor. We lso demonstrted tht the increse of lung Treg cells plyed role, t lest in prt, in the suppression of AHR, irwy eosinophili nd goblet cell metplsi. This represented novel pproch to the investigtion of the function of in model of sthm. In this study, orl dministrtion of, in ddition to OVA, enhnced the induction of orl tolernce. Orlly dministered my be bsorbed in the gut mucos nd could subsequently modulte the immune system. Interestingly, our dt suggested tht the therpeutic effects of in this orl tolernce model required concomitnt dministrtion of ntigen, becuse dministrtion of without OVA hd no effects on the therpeutic outcomes. It is presumbly importnt for locl ntigen-presenting cells to recognize ntigens under RArich milieu for upregultion of orl tolernce. Indeed, dendritic cells produce RA from dietry vitmin A using retinldehyde dehydrogense; they lso express RAR nd hve been shown to respond to RA [24]. Moreover, intrperitonel injection of in ddition to orl OVA ws reported to be ineffective for the meliortion of AHR nd irwy llergic inflmmtion becuse dministered by this route ppered to be rpidly metbolized in the liver [2]. RA plys n indispensble role in the gut by modulting CD13 + dendritic cells [26, 27] nd CD4 + T cells [16]. In prticulr, RA is known to be strong inducer of Tregs, n effect medited by enhnced TGF-β-driven phosphoryltion of SMAD3 [28, 29], inhibition of CD4 + CD44high memory T cells [3], or induction of histone H4 cetyltion t the Foxp3 locus [31]. In this study, lung Tregs expressing CD4, CD2 nd Foxp3 were incresed by exposure to orl OVA, nd subsequent erosol OVA chllenges; lung CD4 + T cells from mice treted with OVA nd lso sustined the suppression of AHR, irwy inflmmtion nd goblet cell metplsi in mice sensitized nd chllenged with OVA. According to previous studies employing doptive trnsfer of CD4 + CD2 + T cells or dministrtion of nti-cd2 ntibody for the depletion of Tregs, nturlly occurring nd induced Treg cells both hve the potentil to modulte AHR nd irwy llergic inflmmtion [32 34]. In the present study, it ws not techniclly possible to isolte dequte numbers of CD4 + CD2 + cells nd we therefore trnsferred the entire CD4 + cell popultion. Our dt indicted tht these CD4 + T cells were ble to sustin the inhibitory effects on AHR nd irwy inflmmtion, suggesting tht the CD4 + T cells trnsferred in the present study contined sufficient number of regultory fetures. In our previous study, AHR nd llergic inflmmtion were meliorted through orl ntigen-medited induction of gut Treg cells, inhibited by endogenous nd exogenous IL-17 [1]. Orl dministrtion of with OVA ws thought to induce gut Treg cells, which presumbly moved to the lung fter the ntigen chllenges. However, in the current study, the number of Treg cells in PPs ws not significntly incresed fter orl dministrtion of in ddition to OVA feeding compred to OVA feeding lone. Although our results did not conclusively demonstrte tht the Treg cells found in the lung originted in the gut, they showed tht orl dministrtion of in ddition to OVA induced Treg cells in the lung fter OVA chllenges. Future work should be conducted to investigte the trnsfer of Treg cells using cells isolted from the gut. Severl reports hve described the inhibitory effects of orl ntigen dministrtion on irwy llergic inflmmtion [3, 36]. These studies involved the dministrtion of ntigen prior to the sensitiztion or chllenge phses. Orl tolernce is difficult to induce fter the estblishment of robust immune system; indeed, our dt showed tht lower dose (2 mg/dy) of orl OVA hd much less effect on AHR, irwy inflmmtion or goblet cell metplsi thn the higher dose (1 mg/dy). However, s bronchil sthm ptients never receive immunotherpy before the occurrence of their disese in the clinicl setting, it is worth noting tht the ddition of could ugment the effects of orl tolernce in this therpeutic model. Although RA is involved in modulting the function nd mturtion of eosinophils [37], bsophils [38] nd mst cells [39], s well s dendritic cells nd T cells, there re few reports describing its effects on irwy llergic inflmmtion. Mret et l. [4] reported tht systemic dministrtion of liposome-encpsulted during the sensitiztion phse induced more sustined levels of RA (s compred with conventionl formultion); this ugmented irwy eosinophili, the levels of Th2 cytokines nd chemokines, nd serum IgE levels. On the other hnd, systemic dministrtion of from immuniztion to the chllenge phses meliorted irwy llergic inflmmtion by modulting Th2 differentition [41]. In the present study, orl dministrtion of concomitnt to OVA ugmented the orl tolernce. From previous reports, hs multiple effects for llergic models, nd this study represents unique pproch to investigting the novel functions of. In summry, we investigted the role of in modulting the induction of orl tolernce in murine irwy llergy model. After chllenges with OVA, we 174 Int Arch Allergy Immunol 21;167: DOI: 1.119/ Skmoto et l.

9 evluted AHR nd llergic inflmmtion. We lso investigted the modulting effects of CD4 + T cell trnsfer from mice tht were treted with orl nd/or OVA, nd ssessed whether the effects of were medited through the RAR using LE13. These dt suggested tht orl dministrtion of concomitnt with ntigen ws useful for ugmenting the effects of orl tolernce vi the modultion of Treg cell induction in this murine sthm model. Acknowledgements The uthors re grteful for the expert help of Dr. Nofumi Imi, Ms. Keiko Ymgiw nd Ms. Kori Tkhshi for performing the histologicl studies nd cring for the nimls. Conflict of Interest The uthors declre tht they hve no competing interests. References 1 Busse WW, Lemnske RF Jr: Asthm. New Engl J Med 21; 344: Jeffery PK, Godfrey RW, Adelroth E, Nelson F, Rogers A, Johnsson SA: Effects of tretment on irwy inflmmtion nd thickening of bsement membrne reticulr collgen in sthm: quntittive light nd electron microscopic study. 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