Targeted delivery of antigen to intestinal dendritic cells induces oral tolerance and prevents autoimmune diabetes in NOD mice

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1 Dietologi (218) 61: ARTICLE Trgeted delivery of ntigen to intestinl dendriti ells indues orl tolerne nd prevents utoimmune dietes in D mie Yulin Chen 1 & Jie Wu 2 & Jiji Wng 1 & Wenjing Zhng 1 & Bohui Xu 1 & Xiojun Xu 3 & Li Zong 1 Reeived: 12 ovemer 217 /Aepted: 19 Ferury 218 /Pulished online: 15 Mrh 218 # Springer-Verlg GmH Germny, prt of Springer ture 218 Astrt Aims/hypothesis The intestinl immune system is n idel trget to indue immune tolerne physiologilly. However, the effiieny of orl protein ntigen delivery is limited y degrdtion of the ntigen in the gstrointestinl trt nd poor uptke y ntigen-presenting ells. Gut dendriti ells (DCs) re professionl ntigen-presenting ells tht re prone to induing ntigen-speifi immune tolerne. In this study, we delivered the ntigen het shok protein 65-6 P277 (H6P) diretly to the gut DCs of D mie through orl vintion with H6P-loded trgeting nnoprtiles (Ps), nd investigted the ility of this ntigen to indue immune tolerne to prevent utoimmune dietes in D mie. Methods A trgeting P delivery system ws developed to enpsulte H6P, nd the ility of this system to protet nd filitte H6P delivery to gut DCs ws ssessed. D mie were immunised with H6P-loded trgeting Ps orlly one week for 7 weeks nd the onset of dietes ws ssessed y monitoring lood gluose levels. Results H6P-loded trgeting Ps proteted the enpsulted H6P from degrdtion in the gstrointestinl trt environment nd signifintly inresed the uptke of H6P y DCs in the gut Peyer s pthes (4.1 times higher uptke ompred with the ontrol H6P solution group). rl vintion with H6P-loded trgeting Ps indued ntigen-speifi T ell tolerne nd prevented dietes in 1% of D mie. Immune devition (T helper [Th]1 to Th2) nd CD4 + CD25 + FXP3 + regultory T ells were found to prtiipte in the indution of immune tolerne. Conlusions/interprettion In this study, we suessfully indued ntigen-speifi T ell tolerne nd prevented the onset of dietes in D mie. To our knowledge, this is the first ttempt t delivering ntigen to gut DCs using trgeting Ps to indue T ell tolerne. Keywords Autoimmune dietes. Dendriti ells. noprtiles. D mie. rl tolerne. rl vintion Eletroni supplementry mteril The online version of this rtile ( ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. Xiojun Xu xiojunxu2@163.om Li Zong zongcpu@126.om Deprtment of Phrmeutis, Chin Phrmeutil University, 24 TongJiXing, njing 219, People s Repuli of Chin Minigene Phrmy Lortory, Chin Phrmeutil University, njing, People s RepuliofChin Stte Key Lortory of turl Mediines, Chin Phrmeutil University, 24 TongJiXing, njing 219, People s Repuli of Chin Arevitions CFSE Croxyfluoresein suinimidyl ester CLSM Confol lser snning mirosopy ConA Connvlin A CS Chitosn DC Dendriti ell FITC-H6P FITC-lelled het shok protein 65-6 P277 FRET Fluoresene resonne energy trnsfer H6P Het shok protein 65-6 P277 H6P/RMCS Het shok protein 65-6 P277-loded RGD- nd mnnose-modified hitosn HSP Het shok protein i.g. Intr-gstri MCS Mnnose-modified hitosn

2 Dietologi (218) 61: P RCS RGD RMCS SGF SIF Th Treg Introdution noprtile RGD-modified hitosn Arginylglyylsprti id RGD- nd mnnose-modified hitosn Simulted gstri fluid Simulted intestinl fluid T helper Regultory T ell The ext use of type 1 dietes remins elusive. Usully, it is onsidered to e hroni utoimmune disese in whih pnreti et ells re destroyed or dmged y the ody s own immune system, using insulin defiieny [1]. In individuls with type 1 dietes, glyemi ontrol is hieved y dily injetions or ontinuous s.. infusion of insulin. However, there is urrently no ure for the ondition. Attempts to prevent or ure type 1 dietes using immunotherpy tht re eing investigted in linil trils inlude non-ntigen-speifi therpy (e.g. T/B ell depleting strtegies, nti-inflmmtory strtegies or ell therpy strtegies) nd ntigen-speifi therpy (e.g. orl/nsl insulin, lum-formulted GAD65, proinsulin peptides or DiPep277) [2, 3]. Antigen-speifi therpy hs een onsidered the Holy Gril of immunotherpy for type 1 dietes euse it trgets only et ell retive T ells without impiring enefiil immune responses. Peptide P277 is 24 mino id frgment of het shok protein (HSP)6/65 tht hs een found to e n utontigen for dietogeni T ell lones in D mie nd individuls with type 1 dietes [4, 5]. In previous work, we suessfully onstruted het shok protein 65-6 P277 (H6P) vine, sed on utontigen P277, ginst utoimmune dietes in D mie [6]. For vintion, utontigens n e delivered vi different routes, inluding intrperitonel, intrvenous, orl nd intrnsl dministrtion [7]. The dministrtion route of vine plys n importnt role in induing n ntigen-speifi immune response nd influenes linil effiy [8]. It is ommonly onsidered tht orl dministrtion of ntigens is more effiient for tolerne indution, euse the intestinl immune system hs predisposition to indue tolerne [9] nd its immunosuppressive environment n ffet the lymphoytes [1 12]. Dendriti ells (DCs) re professionl ntigen-presenting ells with the pity to instigte either inflmmtory or nti-inflmmtory dptive immunity. The diretion of the response is influened y DC phenotype: either n tivted phenotype or, onversely, tolerogeni phenotype [13]. It hs een reported tht DCs from gut Peyer s pthes tend to indue differentition of T helper (Th)2 ells nd seretion of nti-inflmmtory ytokines [14 16] nd re prone to induing regultory T ells (Tregs), whih migrte nd suppress dmging immune responses nd serete ntigen non-speifi ytokines suh s TGF-β, ontriuting to ystnder suppression [17]. In this study, we ttempted to indue immune tolerne in D mie through the orl delivery of H6P to the DCs in gut Peyer s pthes. However, it is diffiult for orl ntigens to

3 1386 Dietologi (218) 61: reh gut DCs euse of the poor stility of ntigens in the gstrointestinl trt nd the sorption rriers presented y the intestinl muus lyer nd the epithelil ell lyer [18], whih leds to low tretment effiieny. M ells re speilised epithelil ells tht re minly loted in the follile-ssoited epithelium of Peyer s pthes[19] nd hve high trnsytoti pilities to trnsport rod rnge of mterils from the intestinl lumen to the DCs in Peyer s pthes [2]. The speifi lotion nd funtion of M ells mke them n ttrtive trget for orl vine delivery to DCs. Thus, we developed delivery system sed on the M ell trgeting peptide rginylglyylsprti id (RGD) [21] nd DC-trgeting lignd mnnose [22]-modified hitosn (CS) nnoprtiles (Ps), to protet nd filitte H6P ntigen delivery to gut DCs, hoping to improve tretment effiieny. In this study, we imed to investigte whether orl H6P-loded trgeting Ps ould deliver H6P to DCs in gut Peyer s pthes nd prevent utoimmune dietes through the indution of T ell tolerne. Methods Mie Four-week-old femle D/LtJ mie were purhsed from Beijing HFK Biosiene (Beijing, Chin). Six- to 8-week-old femle BALB/ mie were otined from Qinglongshn Lortory Animl Center (njing, Chin). Mie were housed in pthogen-free fility with temperture (23 ± 1 C) nd light (12 h light/drk yle) ontrol, nd hd free ess to wter nd stndrd mouse how. Animl re nd ll experiments in this study were onduted in ordne with the Provision nd Generl Reommendtion of Chinese Experimentl Animls Administrtion Legisltion nd pproved y the Chin Phrmeutil University Institutionl Animl Cre nd Use Committee. The investigtors were not linded to the experimentl groups, unless otherwise noted. Regents Full detils of the regents used in this study re provided in the eletroni supplementry mteril (ESM) Methods. Synthesis of RGD- nd mnnose-modified hitosn Mnnosemodified hitosn (MCS) ws synthesised s previously desried [23]. RGD-modified hitosn (RCS) or RGD- nd mnnose-modified hitosn (RMCS) were synthesised y using rosslinking regent, -suinimidyl-3-(2- pyridyldithio)-propionte, to onjugte RGD to the mino groups of CS or MCS [24]. Detils re provided in the ESM Methods. Preprtion nd hrteristion of Ps Ps were prepred y eletrostti self-ssemly of oppositely hrged polyshrides t room temperture. Detils re provided in the ESM Methods. The prtile size, zet potentil vlue, ssoition effiieny nd drug loding of Ps were hrterised, nd detils re provided in the ESM Methods. In vitro relese of H6P from H6P-loded RMCS Ps The H6P relese profile of H6P-loded RMCS (H6P/RMCS) Ps ws seprtely evluted in.1 mol/l HCl (ph 1.) nd PBS (ph 6.8). Detils re provided in the ESM Methods. Stility of H6P/RMCS Ps The stility of H6P/RMCS Ps in simulted gstri fluid (SGF; ontining pepsin, ph 1.2, USP 38) nd simulted intestinl fluid (SIF; ontining pnretin, ph 6.8, USP 38) ws tested y mesuring hnges in prtile size nd fluoresene resonne energy trnsfer (FRET) effiieny fter 6 h inution. Further detils re provided in the ESM Methods. Struturl integrity of the H6P ntigen The struturl integrity of H6P fter enpsultion in RMCS Ps ws ssessed y SDS-PAGE s previously desried [25], with some modifitions. The protetive effets of RMCS Ps on H6P ginst degrdtion in SGF (ontining pepsin, ph 1.2, USP 38) nd SIF (ontining pnretin, ph 6.8, USP 38) were lso ssessed in vitro using SDS-PAGE. Detils re provided in the ESM Methods. Evlution of FITC-lelled H6P delivery effiieny to Peyer s pthes The delivery effiienies of FITC-lelled H6P (FITC-H6P) loded in CS Ps or RCS Ps were evluted using onfol lser snning mirosopy (CLSM) s previously desried [26], with some modifitions. Detils re provided in the ESM Methods. Evlution of FITC-H6P uptke y DCs in Peyer s pthes The uptke of FITC-H6P loded in CS Ps, MCS Ps or RMCS Ps y DCs in Peyer s pthes ws evluted using flow ytometry. FITC-H6P solution ws used s ontrol. Detils re provided in the ESM Methods. Vintion Four-week-old femle D mie were rndomly divided into four groups (ten mie per group). Mie in three groups were given n orl gvge of H6P solution (H6P intr-gstri [i.g].), lnk RMCS Ps (RMCS Ps i.g.) or H6P/RMCS Ps (H6P/RMCS Ps i.g.), respetively. Mie in the fourth group were given n s.. injetion of H6P solution (H6P s..). Eh mouse ws immunised with dose of 1 μg H6P t 4, 5, 6, 7, 8, 9 nd 1 weeks of ge. Anlytil mesurements nd histologil nlysis The lood gluose levels of D mie were monitored from 7 to 23 weeks of ge. Mie were dignosed s hving dietes one the lood gluose level ws higher thn 11 mmol/l for two onseutive dys. Serum insulin levels were determined

4 Dietologi (218) 61: using ommeril ELISA kit. Insulitis nd insulin immunohistohemistry were nlysed s previously reported [27, 28]. Further detils re provided in the ESM Methods. Determintion of H6P-speifi ntiodies The presene of H6P-speifi ntiodies ws determined using ELISA. Detils re provided in the ESM Methods. Mehnisti studies Four-week-old femle D mie were rndomly divided into three groups (four mie per group) nd immunised with RMCS Ps i.g., H6P/RMCS Ps i.g. or H6P s.. The vintion proess ws s desried in the Vintion setion ove. Four weeks fter the lst dministrtion, T ell prolifertion responses to H6P were ssessed using roxyfluoresein suinimidyl ester (CFSE)-sed method; ytokine seretion fter H6P stimultion ws determined using ommeril ELISA kits; nd the presene of Tregs nd Th1 ells in pnreti drining lymph nodes ws nlysed using flow ytometry. Detils re provided in the ESM Methods. Sttistil nlyses Dt were evluted using SPSS Sttistis version 17 (SPSS, Chigo, IL, USA). Dt re expressed s mens ± SD. Sttistil signifine ws determined using the independent-smples t test. The log rnk test ws used to ompre dietes inidene urves. p <.5 indited sttistil signifine. o inlusion or exlusion riteri were pplied. Results Synthesis nd hrteristion of RMCS The syntheti sheme for RMCS is shown in ESM Fig. 1. The strutures of MCS, RCS nd RMCS were onfirmed using 1 H MR (Fig. 1). As shown in Fig. 1, the multiple peks within ppm (rrow) orrespond to the protons of mnnose overlpping with the protons of CS, whih onfirmed the onjugtion of mnnose with CS [23]. In Fig. 1, the onjugtion of RGD with CS ws onfirmed y the pperne of hrteristi peks t ppm (rrowhed), whih were ssigned to the protons of enzene ring in phenyllnine of the RGD peptide [24]. The hrteristi peks t ppm (rrow) nd ppm (rrowhed) in Fig. 1d demonstrted tht oth RGD nd mnnose were onjugted with CS. The degree of sustitution of mnnose in MCS ws 7.1%. The Fig. 1 1 HMRspetrof()CS, ()MCS,()RCSnd(d)RMCS. Representtive results re shown from three independent experiments. Arrows indite the onjugtion of mnnose nd rrowheds indite the onjugtion of RGD H H H H H CH3 m H H H HR H R=A or H m H H H2 n H n H H H H H H H H H H H H2 S S H CS d H H H H H H H H H H2 S S H MCS ppm

5 1388 Dietologi (218) 61: Tle 1 Chrteristis of RMCS Ps nd H6P/RMCS Ps P Size (nm) Zet potentil (mv) Assoition effiieny (%) Drug loding (%) RMCS ± ±.4 H6P/RMCS ± ± ± ± 2.6 Dt re mens ± SD n=3 degree of sustitution of RGD in RCS nd RMCS ws 6.% nd 5.6%, respetively. Preprtion nd hrteristion of H6P/RMCS Ps H6P/ RMCS Ps were formulted y intermoleulr rosslinking etween positively hrged RMCS nd negtively hrged dextrn sulphte. H6P ws positively hrged t ph 3. nd inorported into Ps through eletrostti intertion. As neither n orgni solvent nor high-energy input is required in the eletrostti self-ssemly of n H6P/RMCS P, this P is prtiulrly suitle for the delivery of iologil mromoleulr drugs. The hrteristis of RMCS Ps nd H6P/RMCS Ps re listed in Tle 1. A trnsmission eletron mirosope imge of H6P/RMCS Ps is shown in Fig. 2. The in vitro relese profiles of H6P from H6P/RMCS Ps t ph 1. nd ph 6.8 re shown in Fig. 2. The results indited tht less thn 4.1% H6P ws relesed from H6P/ RMCS Ps t ph 1. (simulting the ph of the stomh). At ph 6.8, however, urst relese of H6P from H6P/ RMCS Ps ws oserved within.5 h, followed y sustined relese. The stility of H6P/RMCS Ps in SGF or SIF ws evluted y prtile size nd FRET nlysis, respetively. The prtile size of H6P/RMCS Ps remined lmost unhnged in SGF, ut inresed to ± 1.9 nm within.5 h in SIF (Fig. 2). Although the swelling or ggregtion of Ps n e monitored y hnges in prtile size, ny lekge of the enpsulted H6P my not e preisely refleted. FRET nlysis n ope with this issue without ny pretretment. Sine FRET signl ours only when the two proes re in lose proximity (<1 nm), ny lekge of FITC-H6P redues FRET effiieny [29]. The fluoresene spetrum of FRET d Fluoresene intensity Cumultive relese (%) Wvelength(nm) Fig. 2 () Trnsmission eletron mirosope imge of H6P/RMCS Ps. Sle r, 5 nm. () The in vitro relese profiles of H6P from H6P/ RMCS Ps t ph 1. (tringles) or ph 6.8 (squres). () Prtile size of H6P/RMCS Ps in SGF (tringles) or SIF (squres) t different time points. (d) Fluoresene emission spetr of lnk RMCS Ps (green line), FITC-RMCS Ps (purple line), rhodmine B isothioynte- RMCS Ps (lue line) nd FRET RMCS Ps (red line) with exittion Time (h) Time(h) e FRET effiieny Prtile size (nm) Time(h) t 493 nm. Down rrow indites the emission intensity of FITC deresed t 518 nm, up rrow indites the emission intensity of rhodmine B isothioynte inresed t 58 nm. (e) The FRET effiieny of FRET RMCS Ps in SGF (tringles) or SIF (squres) t different time points. Representtive results re shown from three independent experiments. Error rs indite SDs

6 Dietologi (218) 61: RMCS Ps is shown in Fig. 2d. The emission intensity of FITC deresed t 518 nm nd the emission intensity of rhodmine B isothioynte inresed t 58 nm, implying energy trnsfer from the donor to the eptor. As shown in Fig. 2e, the FRET effiieny of Ps inresed in SGF, suggesting the formtion of more tightly pked Ps tht proteted the loded H6P from degrdtion. In SIF, the FRET effiieny of Ps deresed oviously within.5 h, nd then deresed grdully. The results suggest tht FITC-H6P strted to e relesed from Ps in SIF. Struturl integrity of H6P The struturl integrity of H6P is very importnt for retining its tivity. Therefore, the impt of the preprtion proess on the struture of H6P nd the protetive effets of RMCS Ps on H6P ginst degrdtion in SGF or SIF were ssessed using SDS-PAGE. As shown in Fig. 3, the nds of H6P relesed from H6P/RMCS Ps (lne 3) were similr to those of ntive H6P (lne 2), inditing tht H6P ws not degrded during the preprtion proess. The first prerequisite for effetive orl protein delivery is to protet the protein from the hrsh onditions in the gstrointestinl environment. As shown in Fig. 3, ntive H6P ws degrded in SGF (Fig. 3, lne 3) or SIF (Fig. 3, lne 3). However, H6P relesed from H6P/RMCS Ps fter pretretment with SGF (Fig. 3, lne 4) or SIF (Fig. 3, lne 4) showed trget H6P nd nd no degrdtion nds. The results suggest tht H6P/RMCS Ps effetively protet H6P from degrdtion in SGF or SIF. RCS Ps inresed FITC-H6P delivery effiieny to Peyer s pthes The properties of FITC-H6P/CS Ps nd FITC-H6P/RCS Ps re listed in ESM Tle 1. A CLSM kd Fig. 3 () Evlution of the struturl integrity of H6P relesed from RMCS Ps. Lne 1: mrker; lne 2: ntive H6P; lne 3: H6P relesed from RMCS Ps. () Evlution of the protetive effet of RMCS Ps on H6P in SGF. Lne 1: mrker; lne 2: ntive H6P; lne 3: ntive H6P pretreted with SGF; lne 4: H6P relesed from RMCS Ps fter tretment with SGF. () Evlution of the protetive effet of RMCS Ps on H6P in SIF. Lne 1: mrker; lne 2: ntive H6P; lne 3: ntive H6P pretreted with SIF; lne 4: H6P relesed from RMCS Ps fter tretment with SIF. Representtive results re shown from three independent experiments imge of Peyer s pth is shown in Fig. 4, nd the omprtments of the Peyer s pth re outlined nd lelled. Fig. 4 shows representtive CLSM imges of Peyer s pth treted with FITC-H6P/CS Ps (Fig. 4) or FITC-H6P/RCS Ps (Fig. 4) nd villus region treted with FITC-H6P/RCS Ps (Fig. 4d). As shown in Fig. 4e, the fluoresene intensity of FITC in the Peyer s pth treted with FITC-H6P/RCS Ps ws signifintly higher thn tht following tretment with FITC-H6P/CS Ps (p <.1). In ddition, the distriution of FITC-H6P in the Peyer s pthwsoviouslyhigher thn tht in the villus region fter tretment with FITC-H6P/RCS Ps (Fig. 4e). These results suggest tht RGD-modified Ps my inrese FITC-H6P delivery effiieny to Peyer s pthes through M ell-medited trnsytosis. on-speifi sorption in the villus region ws present, ut the intensity ws quite low. Evlution of FITC-H6P uptke y DCs in Peyer s pthes The properties of FITC-H6P/CS Ps, FITC-H6P/MCS Ps nd FITC-H6P/RMCS Ps re shown in ESM Tle 1. FITC-H6P uptke y DCs in Peyer s pthes (Fig. 5,) ws nlysed sed on FITC fluoresene in CD11 + ells s desried previously [3]. In Fig. 5, 14.1 ± 1.% of CD11 + ells in Peyer s pth treted with FITC-H6P/MCS Ps were positive for FITC, whih ws signifintly higher thn tht of FITC-H6P/CS Ps (p <.1). The enhned uptke of FITC-H6P y CD11 + ells might e ttriuted to the DC-trgeting effet of mnnose-modified Ps. Moreover, for FITC-H6P/RMCS Ps, 18.5 ± 1.2% of CD11 + ells in the Peyer s pth were positive for FITC (4.1 times higher uptke ompred with the ontrol H6P solution group). These results indite tht FITC-H6P enpsulted in RMCS Ps ws most effetively delivered into CD11 + ells in the Peyer s pth. rl vintion with H6P/RMCS Ps prevented utoimmune dietes in D mie Priortotheonsetofdietes, 4-week-old D mie were treted with H6P i.g., RMCS Ps i.g., H6P/RMCS Ps i.g. or H6P s.. Mie treted with H6P i.g. or RMCS Ps i.g. strted to develop dietes t 13 weeks of ge, nd 9% were dieti t 23 weeks of ge (Fig. 6). In ontrst, the dietes inidene t 23 weeks in the H6P/RMCS Ps i.g. group nd the H6P s.. group ws % nd 4%, respetively; differenes etween groups were sttistilly signifint (Fig. 6). At 23 weeks of ge, nine mie in the RMCS Ps i.g. group exhiited hyperglyemi (Fig. 6), severe insulitis (Fig. 6,e), loss of et ells nd loss of et ell funtion (Fig. 6d,e). By ontrst, in the H6P/ RMCS Ps i.g. group, no mie showed hyperglyemi (Fig. 6) nd mie hd predominntly norml islet struture (Fig. 6,e) nd et ell funtion (Fig. 6d,e). The results indite tht orl vintion with H6P/RMCS Ps prevented the onset of utoimmune dietes in D mie.

7 139 Fig. 4 () A CLSM imge of Peyer s pth, with the omprtments outlined nd lelled. FAE, follile-ssoited epithelium; F, follile; GC, germinl entre; IFR, intrfolliulr region; SED, suepithelil dome. (,) Lolistion of FITC-H6P on Peyer s pth t 1 h fter injetion of FITC-H6P/CS Ps () or FITC-H6P/RCS Ps () into the losed ilel loop. (d) Lolistion of FITC-H6P on the villus region t 1 h fter injetion of FITCH6P/RCS Ps into the losed ilel loop. FITC-H6P (green), DAPI stining (lue). Sle rs, 2 μm. (e) The fluoresene intensity of FITC in CLSM imges ws quntified using Imge J2x softwre version (Rwk Softwre, Stuttgrt, Bden-Württemerg, Germny). The results of three independent experiments re displyed. Mens nd SDs re shown s lines. p<.1, p<.1 Dietologi (218) 61: FAE SED F GC IFR IFR FITC DAPI Merge d Pe -H6 ye P/ r s CS p FI TC t Ps h Pe H6P ye /R r s C p S FI t Ps TC h -H vi 6P llu /R s re CS gi on P s FI TC Fluoresene intensity e However, it should e noted tht leukoyti infiltrtion ws oserved in some pnreti islets in the H6P/RMCS Ps i. g. group (Fig. 6,e), suggesting tht the protetive effets indued y H6P/RMCS Ps might weken over time.

8 Dietologi (218) 61: rl vintion with H6P/RMCS Ps tivted n H6Pspeifi Th2-type humorl immune response Serum nti-h6p IgG ntiody ws mesured to evlute the ntigen delivery effiieny of H6P/RMCS Ps. As shown in Fig. 7, orlly dministered H6P/RMCS Ps produed high levels of nti-h6p IgG ntiody, whih were mintined for more thn 12 weeks fter the lst immunistion. At 8 weeks of ge, the serum nti-h6p IgG ntiody level of mie treted with H6P/ RMCS Ps ws lmost equivlent to tht of mie treted with n s.. injetion of H6P solution, nd ws 4.8-fold higher thn those treted with H6P i.g. The results suggest tht H6P/ RMCS Ps effetively deliver nd relese H6P in lymphoid tissues, induing high nd ontinuous ntigen-speifi humorl immune response. To identify the type of immune response, the serum isotype levels of IgG1 nd IgG2 were determined. The IgG2 isotype is ssoited with Th1-type response, while the IgG1 isotype is ssoited with Th2-type response [31]. As shown in Fig. 7, the nti-h6p ntiodies in the H6P/RMCS Ps i.g. group nd the H6P s.. group were lmost exlusively of the IgG1 sulss. The results suggest tht vintion with H6P tivted n H6P-speifi Th2-type response. Spontneous T ell prolifertion in response to H6P ws downregulted nd the seretion of Th1-type ytokines ws inhiited fter H6P/RMCS Ps vintion Splenoytes isolted from D mie were stimulted with H6P nd onnvlin A (ConA), respetively. The prolifertive responses of T ells to H6P nd ConAwere mesured y nlysing the CFSE dilution of gted CD3 + ells. As shown in Fig. 8, for the H6P/ RMCS Ps i.g. group, the perentge of proliferted T ells ws signifintly lower thn tht of the RMCS Ps i.g. group (p <.1) fter H6P stimultion. However, oth groups showed similr prolifertive responses to ConA (Fig. 8), inditing tht there ws no generl inhiition of T ell retivity indued y H6P vintion. The results indite tht the spontneous T ell prolifertive response to H6P in D mie ws downregulted fter H6P vintion. Splenoytes isolted from D mie were stimulted in vitro with H6P nd the sereted ytokines were mesured. As shown in Fig. 8 f, splenoytes from mie treted with H6P/RMCS Ps i.g. produed higher levels of Th2-type ytokines (IL-4, IL-1) nd lower levels of Th1-type ytokines (IF-γ, IL-2) thn those from mie treted with lnk RMCS Ps i.g. (ll p <.1). TGF-β plys pivotl role in the Fig. 5 FITC-H6P uptke y DCs in Peyer s pthes fter 1 h inution with FITC-H6P solution, FITC-H6P-loded CS Ps, MCS Ps nd RMCS Ps. () CD11 + ells (R2) were gted sed on llophyoynin (APC) fluoresene. () FITC-H6P uptke in gted ells ws nlysed sed on FITC fluoresene. SSC, side stter. () The perentge FITC-H6P uptke y DCs in three independent experiments is presented. Mens nd SDs re shown s lines. p<.1 SSC SSC R2 1.2% APC-CD11 FITC-H6P solution FITC-H6P/CS Ps FITC-H6P/MCS Ps FITC-H6P/RMCS Ps R3 R3 R3 R3 3.6% 1.% 13.9% 18.2% Doule-lelled DCs (%) FITC-H6P solution FITC-H6P/CS Ps FITC-H6P/MCS Ps FITC-H6P/RMCS Ps FITC

9 1392 Dietologi (218) 61: Fig. 6 rl vintion with H6P/RMCS Ps prevents utoimmune dietes in D mie. () The frequeny of dietes-free mie over time. Purple line, H6P i.g.; red line, RMCS Ps i.g.; green line, H6P s..; lue line, H6P/RMCS Ps i. g. (n=1 mie per group). () Blood gluose of D mie t 23 weeks of ge. Results from ten mie re displyed for eh group; mens nd SDs re shown s lines. () Frequeny of islets with vrious grdes of insulitis. White, (no infiltrte); light grey, 1( 25%); mid grey, 2 (25 5%); drk grey, 3 (5 75%); lk, 4 (>75%). At lest 5 islets were sored for eh group. (d) Serum insulin levels of D mie t 23 weeks of ge. Results from ten mie re displyed for eh group; mens nd SDs re shown s lines. (e) Representtive H&E stining nd insulin immunohistohemil results from D mouse pnres setions otined t 23 weeks of ge. Sle rs, 6 μm. p<.5, p<.1, p<.1 Dietes free (%) Frequeny of islet sore (%) e Age (weeks) H6P i.g. RMCS Ps i.g. H6P i.g. H6P s.. H6P/RMCS Ps i.g. RMCS Ps i.g. d Serum insulin (pmol/l) H6P i.g. Blood gluose (mmol/l) RMCS Ps i.g. H6P s.. H6P s.. H6P/RMCS Ps i.g. H6P i.g. RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. H6P/RMCS Ps i.g. Insulin H&E indution of Tregs from nive T ells to tret utoimmune diseses [32, 33]. As shown in Fig. 8g, splenoytes from mie treted with H6P/RMCS Ps i.g. mie produed higher levels of TGF-β thn those from mie treted with lnk RMCS Ps Fig. 7 () Serum nti-h6p IgG levels in D mie efore (t 4 weeks of ge) nd fter vintion with H6P i.g. (white rs), RMCS Ps i.g. (grey rs), H6P s.. (lk rs) nd H6P/ RMCS Ps i.g. (striped rs). () Serum nti-h6p IgG1 (lk rs) nd IgG2 (white rs) levels in D mie t 12 weeks of ge. The results re expressed s mens ± SD of ten mie Asorne t 45 nm Age (weeks) Asorne t 45 nm H6P i.g. RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g.

10 Dietologi (218) 61: Fig. 8 Four weeks fter vintion with RMCS Ps i.g., H6P s.. or H6P/RMCS Ps i.g., splenoytes were isolted from D mie for T ell prolifertion nd ytokine ssy. (,) T ell prolifertion following o-ulture with H6P () orcona() ws nlysed using CFSE-sed method. ( g) Cytokine levels in the superntnt of spleen ells fter H6P stimultion. Results from four mie re displyed for eh group; mens nd SDs re shown s lines. p<.5, p<.1 Counts Counts M1 2.9% CFSE M1 69.6% CFSE RMCS Ps i.g. RMCS Ps i.g. M1 14.1% M1 72.9% H6P s.. H6P s.. H6P/RMCS Ps i.g. M1 13.7% H6P/RMCS Ps i.g. M1 71.1% Prolifertion (%CD3 + ells) Prolifertion (%CD3 + ells) RMCS Ps i.g RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. IL-4 (pg/ml) f RMCS Ps i.g. 8 H6P s.. H6P/RMCS Ps i.g. d IL-1 (pg/ml) g RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. e IF-γ (pg/ml) RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. IL-2 (pg/ml) TGF-β (pg/ml) 1 5 RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. i.g. (p <.1). These results suggest tht the prevention of utoimmune dietes is ssoited with the inhiition of Th1-type ytokine seretion nd upregultion of Th2-type ytokines. rl vintion with H6P/RMCS Ps inresed the frequeny of Tregs nd deresed the frequeny of Th1 ells Tregs ply n importnt role in ntigen-indued tolerne [34]. After vintion of D mie with H6P/RMCS Ps i.g., the per ent of CD4 + CD25 + FXP3 + T ells ws signifintly inresed (p <.5) in pnreti drining lymph nodes (Fig. 9), wheres the per ent of CD4 + T-et + T ells ws mrkedly deresed (p <.1) (Fig. 9) ompred with the lnk RMCS Ps i.g. ontrol. Disussion For type 1 dietes immunotherpy, orl immunistion is n ttrtive route of vintion. A numer of prelinil studies hve suessfully prevented or treted utoimmune dietes through the orl delivery of utontigen [35 37]. However, the experimentl nimls in these studiesneededtotkelrgedoseofntigenforlongtime

11 1394 Dietologi (218) 61: Fig. 9 (,) Four weeks fter vintion with RMCS Ps i.g., H6P s.. or H6P/RMCS Ps i.g., CD4 + CD25 + FXP3 + Tells() nd CD4 + T-et + T ells () inthe pnreti drining lymph nodes of D mie were nlysed y flow ytometry. Results from four mie re displyed for eh group; mens nd SDs re shown s lines. p<.5, p<.1 FXP3 Q2-UL 5.6% Q2-LL 85.% CD25 RMCS Ps Q2-UR 4.3% Q2-LR 5.1% Q2-UL 5.1% Q2-LL 83.9% H6P s.. Q2-UR 4.1% Q2-LR 6.9% H6P/RMCS Ps i.g. Q2-UR 7.9% Q2-UL 6.3% Q2-LL 82.% Q2-LR 3.8% CD25 + FXP3 + (%CD4 + ) RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. Q1-UL Q1-UR Q1-UL Q1-UR Q1-UL Q1-UR.% 5.4%.% 3.2%.%.4% T-et Q1-LL.% CD4 Q1-LR 94.6% Q1-LL.% Q1-LR 96.8% Q1-LL.% Q1-LR 99.6% CD4 + T-et + (%CD4 + ) RMCS Ps i.g. H6P s.. H6P/RMCS Ps i.g. nd the therpeuti effet ws limited. In D mie, orl dministrtion of 1 mg insulin twie week for 5 weeks nd then weekly until 1 yer of ge hs een reported to redue the inidene of dietes, ut ould not ompletely prevent the onset of dietes [35]; in hildren genetilly suseptile to type 1 dietes, only high orl dose of ntigen (67.5 mg of insulin dily for 3 12 months) induednimmuneresponse[38]. Therefore, we elieve tht low delivery effiieny of the ntigen my e the min reson for the low effiieny of orl ntigen therpy. In previous study, genetilly modified Ltoous ltis tht expressed H6P ws used s vetor for intestinl delivery of H6P, ut the reominnt L. ltis ws dministered for 36 weeks nd % of D mie still developed dietes [39]. In the urrent study, we oserved tht the onset of dietes in D mie ws 1% prevented through the orl dministrtion of H6P/RMCS Ps one week for 7 weeks. This idel outome my e ttriuted to the high ntigen delivery effiieny of trgeting H6P/RMCS Ps. The vintion dose of H6P ws hosen sed on our previous study [6]. We hose to immunise the D mie from 4 to 1 weeks of ge (i.e. one weekly for 7 weeks), efore the onset of dietes, euse this ws prevention study nd we thought tht tht multiple drug deliveries would e ssoited with etter outome. β1 integrins re overexpressed t the pil surfe of humn nd mouse M ells. In ddition, it hs een demonstrted tht grfting RGD peptide tht trgets β1 integrins onto Ps signifintly inreses their trnsport y M ells [21]. Mnnose reeptor is 175 kd trnsmemrne protein tht is highly expressed on ntigenpresenting ells suh s DCs. It medites the endoytosis, proessing nd presenttion of ntigens tht expose mnnose residues. Mnnose-modified Ps hve een widely used for DC-trgeted delivery nd show enhned effiieny over other systems [22]. In this study, RGD nd mnnose were used to modify CS, nd we oserved tht theinvivouptkeofh6pydcsinpeyer s pthes ws enhned signifintly fter its enpsultion in RMCS Ps. The hrteristis of the DCs tht tke up the ntigen (e.g. whether they re immture, tivted or tolerogeni DCs) remin to e nlysed in future studies. A further re of future study is identifying the distriution of the remining ntigen, whether in gut-ssoited lymphoid tissue or within the irultion. Mehnisms of ntigen-indued tolerne inlude deletion nd/or nergy of effetor T ells, immune devition (Th1 to Th2) nd indution of Tregs [4]. How these mehnisms re interrelted is not ompletely understood. Tregs nd immune devition ould e outomes of nergy; lterntively, Tregs ould indue nergy in effetor T ells. In this study, orl H6P/RMCS Ps inresed the seretion of Th2-type ytokines nd ttenuted the seretion of Th1-type ytokines. These results re in line with those of previous report, in whih vintion of D mie with H6P ginst utoimmune dietes ws ssoited with Th1 to Th2 ytokine shift [6]. In ddition, in the urrent study, orl H6P/RMCS Ps were ssoited with high levels of nti-h6p IgG1 ntiody. This ould e indued y HSP65 in the H6P ntigen, whih hs out 5% homology with the humn homologue HSP6 [41]. HSP6 n indue the prolifertion of B ells [42]. The tivted B ells hve een reported to not only produe ntiody, ut lso to inhiit spontneous Th1 utoimmunity [43].

12 Dietologi (218) 61: Whether the H6P/RMCS Ps vintion promotes the genertion of H6P-speifi IgA in the gut lumen ws not nlysed in the urrent study. Beuse the H6P ntigen is inorported into Ps, we think tht nti-h6p IgA is unlikely to interfere with the uptke of Ps upon repeted dministrtion. In reent yers, the entrl role of CD4 + CD25 + FXP3 + Tregs in mintining peripherl immune tolerne hs een onfirmed [44]. f linil importne is the ility of Tregs to exert ntigennon-speifi ystnder suppression. In ddition, ystnder suppression does not depend on the tolerising utontigen neessrily eing the primry driver of pthology. Severl studies hve suessfully prevented or treted dietes in niml models y induing ntigen-speifi Tregs [36, 45]. In this study, we oserved tht orl H6P/RMCS Ps inresed the frequeny of CD4 + CD25 + FXP3 + Tregs in pnreti drining lymph nodes t 4 weeks fter immunistion. However,whether these Tregs endure for long time remins to e further studied. In ddition, the hrteristis of these Tregs (e.g. whether they re de novo indued or n expnsion of pre-existing Tregs) is unler. f note, s.. injetion of H6P did not inrese the frequeny of Tregs. These results re in line with the theory tht either systemi or muosl dministrtion of ntigen n indue tolerne, ut tht the muosl route ppers more likely to indue Treg responses [4]. verll, the mehnisms of tion of orl H6P/RMCS Ps ginst utoimmune dietes in D mie my involve immune devition (Th1 to Th2) nd indution of Tregs. More detiled studies need to e performed to further eluidte the mehnism of tion of H6P/RMCS Ps, inluding T ell responses in the gut-ssoited lymphoid tissues, pnreti drining lymph nodes nd pnres t different time points. In this study, we demonstrted tht H6P/RMCS Ps effiiently deliver H6P to DCs in Peyer s pthes, indue ntigen-speifi T ell tolerne nd prevent the onset of utoimmune dietes in D mie. The results suggest tht RMCS Ps re suitle for enpsulting iologil mromoleules nd my serve s promising pltform for orl delivery of utontigens to indue tolerne. Aknowledgements We thnk Y. Xing (mnger of flow ytometry t the Chin Phrmeutil University) for her sientifi dvie nd tehnil ssistne with flow ytometry. Dt vilility The dtsets generted during nd/or nlysed during the urrent study re ville from the orresponding uthor on resonle request. Funding This study ws supported y tionl turl Siene Foundtion of Chin (o /H38) nd the Postgrdute Reserh & Prtie Innovtion Progrm of Jingsu Provine (KYCX17_676). Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Contriution sttement YLC, JW, XJX nd LZ ontriuted to the oneption nd design of the study. YLC, JJW, WJZ nd BHX performed the experiments nd nlysed the results. YLC, JW, XJX nd LZ drfted the mnusript. All uthors revised the mnusript ritilly nd gve finl pprovl of the sumitted version. 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