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Molecular Immunology 47 (2009) 79–86

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

eview

etinoids differentially regulate the progression of autoimmune diabetesn three preclinical models in mice

tanislava Stosic-Grujicic ∗, Tamara Cvjeticanin, Ivana Stojanovicepartment of Immunology, Institute for Biological Research “Sinisa Stankovic”, Belgrade University, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 2 October 2008eceived in revised form 9 November 2008ccepted 24 December 2008vailable online 10 February 2009

eywords:OD micetreptozotocin

a b s t r a c t

Retinoids have a variety of biological activities, including immunomodulatory action in a number ofinflammatory and autoimmune conditions. Considering the pathogenesis of type 1 diabetes mellitus(T1D), in this study we examined the potential role for retinoids, etretinate and all-trans-retinoic acid(ATRA) in preclinical models of human T1D. When administered prophylactically to CBA/H mice madediabetic with multiple low doses of streptozotocin (MLD-STZ), both drugs effectively prevented clinicalsigns of diabetes. Prevention of T1D was associated with reduced emergence of primed (autoreactive)CD4+ CD25+ T cells, but not with the emergence of Foxp3+ Treg in the peripheral compartments. More-over, the animals receiving ATRA exhibited reduced Th1/Th17 response and nitric oxide (NO) production

yclophosphamideetinoidsytokinesh1h17

in the peripheral lymphoid tissues, thus shifting the balance towards the anti-inflammatory cytokines.In NOD mice with spontaneous form of diabetes, ATRA prophylaxis, starting at a time point immediatelybefore T1D onset, markedly reduced hyperglycemia and incidence of the disease. However, administra-tion of ATRA to NOD mice in which the proportion and function of CD4+Foxp3+ Treg cells was abrogatedby cyclophosphamide (CY), failed to permit progression to T1D. These findings suggest that effectivenessof T1D suppression by retinoids depends on the presence of Tregs which down-modulate immunoinflam-

nd “c

matory events at the seco

. Introduction

Type 1 diabetes mellitus (T1D) is a T cell-mediated autoimmuneisorder resulting from the selective destruction of the insulin-roducing � cells of the pancreas (Atkinson and Eisenbarth, 2001).

n order to gain insight into T1D pathogenic mechanisms in humansnd to test novel therapeutic approaches for its treatment, vari-us preclinical models of the disease are now available such aspontaneous and accelerated diabetes in the non-obese diabeticNOD) mice, BioBreeding/Worcester rats, or diabetes induced inusceptible rodent strains by multiple low doses of streptozotocinMLD-STZ). Although pathogenic differences have been reportedo occur between these experimental models, both T cells and

acrophages are thought to exert their diabetogenic potential byeleasing proinflammatory cytokines of both innate and adaptivemmune systems (reviewed by Kolb, 1987). Several proinflamma-ory cytokines including MIF, IFN-�, TNF-�, IL-1, as well as IL-17

Hill and Sarvetnick, 2002; Miljkovic et al., 2005; Rabinovitch anduarez-Pinzon, 2007; Stosic-Grujicic et al., 2007a,b), have beenmplicated in the pathogenesis of T1D. These cytokines are thoughto contribute to diabetes development by recruiting effector cells

∗ Corresponding author. Tel.: +381 11 3643 236; fax: +381 11 2761 433.E-mail address: duta@eunet.rs (S. Stosic-Grujicic).

161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2008.12.028

heck-point” and allow diabetes progression.© 2009 Elsevier Ltd. All rights reserved.

to the endocrine pancreas and by enabling in situ release of othertoxic products such as free oxygen and nitrogen species. It isalso thought that the production of anti-inflammatory type 2/3cytokines, such as IL-4, IL-10 and TGF-� correlates with protectionfrom T1D (Rabinovitch and Suarez-Pinzon, 2007).

Vitamin A (retinol) and its metabolites (retinoids) are a groupof potent natural or synthetic molecules which exert a number ofbiological activities, including regulation of differentiation, prolif-eration, apoptosis and developmental changes. Dietary Vitamin Ais taken up in the intestine, delivered mainly to the liver, and insmaller portion to some extrahepatic tissues, such as adipose tis-sue, kidney, and bone marrow, where it is stored in its ester formfor meeting tissue needs. Retinyl ester is further secreted into thecirculation and transported as retinol bound to retinol-binding pro-tein to its target cells (Kurlandsky et al., 1995). Most of VitaminA actions depend on its active metabolites, principally all-trans-retinoic acid (ATRA) and 9-cis-RA (Kastner et al., 1995; Iwata et al.,2004), formed in the target tissues mainly through the intracel-lular oxidative metabolism (Duester, 2000). Importantly, dendriticcells from the gut-associated lymphoid organs produce ATRA from

retinol (Iwata et al., 2004). Similarly, following oral administration,synthetic RA derivative, etretinate, is absorbed in the small intes-tine and accumulates in fat, liver and gut, and to a lower level inthe kidneys, brain and testises (Vahlquist et al., 1986), where it isextensively metabolized to the pharmacologically active acid form.

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In the immune system retinoids act as modulators for a varietyf inflammatory and immunological events (Ross and Hammerling,994). For example, in immunologically related rodent modelsetinoids have been shown to inhibit disease progress in adju-ant arthritis (Brinckerhoff et al., 1983), experimental autoimmunencephalomyelitis (Massacesi et al., 1991; Xiao et al., 2008), lupusPerez de Lema et al., 2004), autoimmune nephritis (Escribese etl., 2007), and inflammatory bowel disease (Osanai et al., 2007).ecently, by utilizing dietary approach, it has been shown thatitamin A inhibits the progression of T1D in NOD mice (Zuninot al., 2007). The beneficial effect of this molecule in T1D wasscribed solely to its effects on suppressing inflammatory immuneells and reducing the oxidative damage in the islets that con-ribute to loss of � cells. In line with this, we and others havehown in vitro that Vitamin A and its metabolites modulate theunction of lymphocytes, macrophages and neutrophils, and theroduction of several cytokines (Stosic-Grujicic and Simic, 1992;tosic-Grujicic and Ejdus, 1994; Semba, 1998; Kang et al., 2000).urthermore, possible therapeutic value of retinoids was suggestedy showing the direct protective effects of retinoic acid on IL-1-nd IFN-�-induced cytotoxicity of pancreatic � cells (Kang et al.,004). However, it is now apparent that retinoids may modulatehe autoimmune disease outcome by various mechanisms, includ-ng generation of immunoregulatory T (Treg) cells and inhibitionf Th17 cell differentiation (Kang et al., 2007; Xiao et al., 2008).e therefore examined possible mechanisms by which retinoidsodulate autoimmune reactions in vivo by using three preclin-

cal models of T1D which differ in some respect to each other:pontaneous and CY-accelerated diabetes in the non-obese dia-etic (NOD) mice, and MLD-STZ-induced diabetes in susceptibletrain of mice. The NOD mouse provides a spontaneous model,n which aberrant immunoregulation of CD4+CD25+Foxp3+ regu-atory T cell (Treg) is associated with the shift from “benign” toaggressive” insulitis (around 12 weeks of age), resulting in 80% ofemales developing hyperglycemia by 30 weeks of age (Pop et al.,005). CY-induced NOD model is characterized by the synchronousnset of T1D shortly after CY treatment (1–2 weeks), accompaniedy a rapid reduction of naturally occurring CD4+CD25+Foxp3+ TregBrode et al., 2006). Finally, while STZ at high dose induces non-mmune “toxic” form of diabetes, multiple low doses of STZ retrievehe expression of � cell autoantigen, thus synchronously triggeringcell specific immunoinflammatory form of diabetes in susceptible

odent strains (Elias et al., 1994).In the present study we examined the effects of syn-

hetic Vitamin A derivative, etretinate and an active Vitaminmetabolite, all-trans-retinoic acid and demonstrated powerful

ntidiabetogenic effects in both chemically induced and sponta-eous models of disease. Simultaneously, ex vivo studies carriedut in diabetes-induced mice indicated that ATRA dampenedhe immunoinflammatory diabetogenic processes by suppres-ion of both Th1 and Th17 response. Moreover, T1D suppressiony ATRA depends on the presence of Treg within target tissuehich down-modulate immunoinflammatory events at the second

check-point” of diabetes progression.

. Materials and methods

.1. Mice

Inbred CBA/H mice and NOD-ShiltJ mice, originally obtainedrom the Jackson Laboratory (Bar Harbor, ME, USA), were kept

nder non-specific pathogen free conditions at the animal housef the Institute for Biological Research “Sinisa Stankovic”, Univer-ity of Belgrade, Serbia. Studied mice were age-matched (8–12eeks of age) males or females (depending upon certain exper-

ment) in a body weight ranging from 20 to 25 g. This study

munology 47 (2009) 79–86

was approved by the local Institutional Animal Care and UseCommittee.

2.2. Induction of diabetes and in vivo treatments

To induce immunoinflammatory (MLD-STZ) diabetes in geneti-cally susceptible CBA/H male mice, streptozotocin (Sigma–Aldrich,St. Louis, MO, USA) was injected i.p. at a dose of 40 mg/kg bodyweight (bw), daily for 5 consecutive days. For induction of accel-erated diabetes with cyclophosphamide (CY, Sigma–Aldrich), thedrug was dissolved in water and injected i.p. at a dose of 250 mg/kgbw into 8-week-old euglycemic female NOD mice. To evaluate theimpact of retinoids on both MLD-STZ-induced and CY-induced dia-betes, the mice were treated either i.p. or p.o. with etretinate (a kindgift from Hofman La-Roche, Basel, Switzerland) (30–90 mg/kg bw),or all-trans-retinoic acid (ATRA, Sigma–Aldrich) (2–20 mg/kg bw).Treatment with both retinoids started on day 0 of either STZ- orCY-induced diabetogenic challenge. Control animals were treatedwith diluent DMSO/H2O under similar experimental conditions, orleft untreated. Blood glucose levels sampled from tail vein bloodwere monitored on a weekly basis using a handheld glucometer(GlucoSure Plus, ApexBio, Taiwan), and mice were defined as dia-betic when nonfasting blood glucose levels in CBA/H and NOD miceexceeded 10 mmol/l and 11.8 mmol/l, respectively. To evaluate theimpact of retinoids on the development of spontaneous T1D inthe NOD mouse, hyperglycemic NOD mice (with glycemia above11.8 mmol/l for 2 consecutive days) were treated i.p. with 10 mg/kgbw ATRA or its vehicle from the 12th week of age until the end ofexperiment. Mice defined as diabetic were used immediately afterbecoming overtly diabetic. An additional control group of NOD micewas euglycemic at the beginning of ATRA treatment.

2.3. Cytokine production and detection of cytokines levels

Spleen mononuclear cells (SMNC) and pancreatic lymph nodecells (PLNC) were prepared from vehicle- or ATRA-treated MLD-STZ-induced mice on day 15 and 22 diabetes post-induction(p.i.). The cells were cultured for 48 h in RPMI-1640 mediumsupplemented with 5% FCS, 1 mM sodium pyruvate, 0.1 mg/mlpenicillin/streptomycin and 5 × 10−5 M 2-mercaptoethanol (allfrom Sigma–Aldrich). For collecting supernatants for ELISA, SMNC(5 × 105/ml) and PLNC (3 × 106/ml) were incubated in culturemedium in the presence or absence of polyclonal mitogenic stimu-lator concanavalin A (ConA, 1 �g/ml, Pharmacia, Uppsala, Sweden)at 37 ◦C in a humidified atmosphere with 5% CO2. Cell-free super-natants were collected after 24 h and stored at −20 ◦C untildetermination of cytokine concentration. The secretion of IL-17,IFN-�, TNF-�, IL-1�, IL-6, IL-10 and IL-4 from SMNC was deter-mined by sandwich ELISA according to manufacturer’s instructions.Mouse IL-17, IL-1�, TNF-� and matching antibody pairs were fromBD Pharmingen (San Diego, CA, USA), IL-6, IL-10 and matching anti-body pairs were from eBioscience (San Diego, CA, USA), while IFN-�,IL-4 and matching antibody pairs were from R&D (Minneapolis, MN,USA). The results were calculated using standard curve which wasmade on the basis of known concentrations of applied recombinantcytokines.

2.4. RNA isolation and RT-PCR

Total RNA from PLNC was isolated with RNA Isolator (Fermentas,Vilnius, Lithuania) according to manufacturer’s instructions. RNA

(1 �g) was reverse transcribed using Moloney leukemia virusreverse transcriptase and random primers (both from Fermentas).PCR amplification of cDNA (1 �l per 20 �l of PCR reaction) wascarried out in Real-time PCR machine (Applied Biosystems, UK)using SYBRGreen PCR master mix (Applied Biosystems) as follows:

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3.2. In vivo treatment with ATRA prevents cellular changesinduced by MLD-STZ and modulates cytokine and NO production

To elucidate the mechanisms by which retinoids prevent diseaseonset, ex vivo studies were performed on peripheral compartments

Fig. 1. The effect of etretinate and ATRA on MLD-STZ-induced hyperglicemia. (A)Blood glucose levels in mice receiving, respectively, MLD-STZ (40 mg/kg/day for 5days) in conjunction with 7 daily i.p. injections of vehicle only (♦), or two different

S. Stosic-Grujicic et al. / Molec

0 min at 50 ◦C for dUTP activation, 10 min at 95 ◦C for initialenaturation of cDNA followed by 40 cycles (15 s of denaturationt 95 ◦C and 60 s for primer annealing and chain extension step).rimer pairs for IL-17 were 5′-GGGAGAGCTTCATCTGT-3′ and 5′-ACCCTGAAAGTGAAGGG-3′ (GenBank accession no. NM 010552.3),oxp3 5′-AGGAGCCGCAAGCTAAAAGC-3′ and 5′-TGCCTTCGTGCC-ACTGT-3′ (GenBank accession no. NM 054039.1), IFN-� 5′-ATCAGCAACAACATAAGCGTCA-3′ and 5′-CTCCTTTTCCGCTTCC-GA-3′ (GenBank accession no. NM 008337.2), TNF-� 5′-CCACGT-GCAAACCAC-3′ and 5′-TGGGTGAGGAGCACGTAGT-3′ (GenBankccession no. NM 013693.2), IL-4 5′-ATCCTGCTCTTCTTTCTCG-′ and 5′-GATGCTCTTTAGGCTTTCC-3′ (GenBank accession no.M 021283.1), IL-10 5′-TGTGAAAATAAGAGCAAGGCAGTG-3′

nd 5′-CATTCATGGCCTTGTAGACACC-3′ (GenBank accession no.M 010548.1) and �-actin 5′-GGACCTGACAGACTACC-3′ and 5′-GCATAGAGGTCTTTACGG-3′ (NM 007393.2). The expression of

hese genes was calculated according to the formula 2−(Cti−Cta)

here Cti is cycle threshold of the gene of interest and Cta is cyclehreshold value of �-actin.

.5. Isolation of pancreatic-infiltrating leukocytes

Whole pancreas samples were torn into small pieces in coldanks’ balanced salts solution without calcium and magnesium

BDSL, Kilmarnock, UK). The tissues were digested by shaking withml of 1 mg/ml collagenase V (Sigma–Aldrich) on 37 ◦C for 10 min.fter washing, digested tissue was forced through a cell strainer,entrifuged on 500 × g for 5 min, resuspended in 3 ml of cultureedium and laid upon the same volume of Lymphocyte separationedium (ICN Biomedicals, Costa Mesa, CA, USA). After centrifuga-

ion on 400 × g for 18 min, a ring consisted of mononuclear cells wasollected and washed two times before staining for cytofluorimetricnalysis.

.6. Immunofluorescence analysis

Single-cell suspensions were prepared from various organs asescribed above. Cells (3 × 105 per sample) were stained for surfacearkers with 0.5 �g of FITC labelled anti-CD4 antibody (eBio-

cience). Treg cells were detected by Mouse Regulatory T celltaining Kit (PE-Cy5 Foxp3 FJK-16 s, FITC CD4, PE CD25) accord-ng to manufacturer’s instructions (eBioscience). Stained cells wereetected on FACSCalibur and analyzed by CellQuestPro softwareBD Biosciences, San Diego, CA).

.7. Assay for nitric oxide release

Peritoneal cells (PC) were collected by rinsing the peritoneal cav-ty of vehicle- or ATRA-treated MLD-STZ-induced mice on day 15nd 22 diabetes p.i. with 3 ml cold PBS. The cells (1.5 × 106/ml) werencubated for 48 h in culture medium at 37 ◦C in a humidified atmo-phere with 5% CO2. Cell-free supernatants were collected and theoncentration of the stabile NO-metabolite, nitrite was determineds previously described (Maksimovic-Ivanic et al., 2002). Briefly,liquots of cell-free culture supernatants were mixed with an equalolume of Griess reagent (a 1:1 mixture of 0.1% naphthylethylene-iamine dihydrochloride and 1% sulfanilamide in 5% H3PO4). After0 min, the absorbance at 570 nm was determined in a microplateeader and nitrite concentration was calculated from a standardurve of NaNO2.

.8. Statistical analysis

Data are expressed as mean values ± S.D. Statistical anal-sis of differences was evaluated using ANOVA, followed by

munology 47 (2009) 79–86 81

Student–Newman–Keuls test for multiple comparisons, or the Stu-dent’s t-test, as appropriate. Differences in diabetes incidence wereanalyzed by �-square test. A p < 0.05 was considered to be signifi-cant.

3. Results

3.1. Treatment with etretinate or ATRA prevents development ofMLD-STZ-induced diabetes

To determine whether retinoids modulate T1D, we first studiedtheir in vivo effects in a model of immunoinflammatory diabetesin MLD-STZ-exposed CBA/H mice. Control MLD-STZ-injected micethat received the vehicle developed persistent hyperglycemia thatstarted from ∼2 weeks after the first injection of the STZ (Fig. 1). Pro-phylactic i.p. treatment with either 30 or 90 mg/kg bw of syntheticretinoid etretinate from day 0 to 7 p.i. significantly inhibited MLD-STZ-induced hyperglycemia in a dose-dependent way (Fig. 1A).Similarly, mice treated with the main Vitamin A metabolite, ATRAunder the same experimental conditions (i.p. 2 mg/kg bw/day)exhibited a reduction in blood glucose levels (Fig. 1B). This pro-tection did not depend on continuous application of the drug,because none of the mice developed hyperglycemia throughoutthe entire follow-up period after treatment withdrawal. Moreover,ATRA (10 mg/kg bw/day) showed a similar protective effect whengiven p.o. (Fig. 1B).

doses of etretinate: 30 mg/kg (�) and 90 mg/kg (�). (B) Blood glucose levels in micereceiving MLD-STZ only (♦), or mice treated with MLD-STZ in conjunction with 7daily i.p. injections of ATRA (20 mg/kg/day, �) or 7 daily p.o. applications of ATRA(10 mg/kg/day, �). Results from a representative of two independent experimentswith similar results, are presented as the means ± S.D. for 5–7 mice per group. *,p < 0.05 and **, p < 0.01 refers to treatment with MLD-STZ.

82 S. Stosic-Grujicic et al. / Molecular Immunology 47 (2009) 79–86

Table 1Effect of ATRA on cellular changes in the draining lymph nodes induced by MLD-STZ treatment.

Treatment group Day 15 p.i. Day 22 p.i.

PLNC (×106) CD4+CD25+ (%) Foxp3+/CD4+CD25+ (%) PLNC (×106) CD4+CD25+ (%) Foxp3+/CD4+CD25+ (%)

MLD-STZa

Exp. 1 9.2 ± 2.8 13.2 ± 1.1 83.1 ± 1.3 3.8 ± 1.7 10.7 ± 1.2 66.0 ± 3.5Exp. 2 11.0 ± 1.6 79.9 ± 4.1

ATRAb + MLD-STZExp. 1 3.4 ± 1.9* 10.3 ± 1.2* 78.0 ± 4.5 3.8 ± 0.9 9.4 ± 0.7 58.8 ± 2.4

*

days.STZ diabetes postinduction.ntrol MLD-STZ-induced diabetic animals.

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Fig. 3. The effect of ATRA on proinflammatory cytokine production obtained fromsplenocytes. The levels of indicated cytokines were determined by ELISA in the 48 hculture supernatants of ConA (1 �g/ml)-stimulated SMNC. The cells were obtainedfrom ATRA-treated (20 mg/kg/day, black columns) and/or MLD-STZ-induced (white

Exp. 2 6.9 ± 0.3 80.1 ± 2.7

a MLD-STZ was administered i.p. at daily dose of 40 mg/kg/day for 5 consecutiveb ATRA was administered i.p. at daily dose of 20 mg/kg/day from the day of MLD-* P < 0.005 (Student’s t-test) refers to ATRA-treated MLD-STZ-induced mice vs. co

n day 15 and 22 during progression of the MLD-STZ-inducediabetes. Concordant with the clinical status, in control diabeticice the absolute number of draining PLNC was high on day 15

ostinduction, but decreased 2.5 fold on day 22 postinductionTable 1). However, in ATRA-treated MLD-STZ-induced mice theransient increase in the PLNC number was not observed. In addi-ion, the early selective increase in the proportion of CD4+CD25+

ells induced by MLD-STZ administration was prevented by in vivoTRA treatment (Table 1). This implied that ATRA is capable to

imit lymphocyte activation and proliferation (and/or recruitment)uring the induction of disease by MLD-STZ. Surprisingly, ATRAreatment did not increase the frequency of Foxp3+ cells amongD4+CD25+ cells when compared with vehicle-treated animalsTable 1). Consistent with this, by using a real-time PCR assay, no sig-ificant differences in Foxp3 mRNA expression was found betweenLNC isolated from ATRA-treated versus diabetic mice (Fig. 2).

Because proinflammatory cytokines contribute to the patho-enesis of T1D development, we next examined whether ATRAreatment may influence MLD-STZ-induced cytokine production.ompared with vehicle-treated animals, a marked reduction in

FN-� and IL-17 mRNA expression was detected in PLNC isolatedrom ATRA-treated versus diabetic mice (Fig. 2). Moreover, in ATRA-reated PLNC a significant up-regulation of anti-inflammatoryytokine IL-4 mRNA was observed. Consistent with these findings,MNC obtained from ATRA-treated mice showed decreased secre-ion of proinflammatory cytokines into culture supernatants upononA-restimulation, including IFN-�, as well as IL-17 (Fig. 3). Theseata indicated that ATRA strongly inhibited the priming/expansionf pathogenic Th1 and Th17 responses in vivo, thus shifting a bal-nce towards protective Th2 response.

We have previously shown that NO mediates islet cell destruc-

ion in MLD-STZ-induced diabetes in mice (Lukic et al., 1991). Onhe other hand, it has been shown in vitro that RA affects cytokine-

ediated destruction of � cells by inhibition of NO generationKang et al., 2004). We therefore assessed the NO production by

ig. 2. The effect of ATRA on proinflammatory cytokine and Foxp3 expression inLNC. The expression of genes in PLNC obtained from ATRA-treated (20 mg/kg/day,lack columns) and/or MLD-STZ-induced (white columns) mice by day 22 postin-uction was determined by real-time PCR. Expression levels are shown as arbitrarynits normalized to expression of the housekeeping gene �-actin. *, p < 0.05 referso treatment with MLD-STZ.

columns) mice by day 15 postinduction (A) and day 22 postinduction (B). The resultsare expressed as the means ± S.D. for 4–5 mice per group. *, p < 0.05 refers to treat-ment with MLD-STZ.

peritoneal macrophages as the main NO producers and the firstcells to accumulate in islets during MLD-STZ-induced disease. Theresults clearly show (Table 2) that peritoneal macrophages isolated

from MLD-STZ-induced control mice produced high levels of NO,as revealed by nitrite formation by these cells, while macrophagesfrom ATRA-treated mice exhibited impaired NO production.

Table 2Effect of ATRA on NO production by peritoneal cells induced by MLD-STZ treatment.

Treatment group Nitrite accumulation (�M)a

Day 15 p.i. Day 22 p.i.

MLD-STZ 9.9 ± 5.4 8.9 ± 0.9ATRA + MLD-STZ 0.9 ± 1.0* 3.8 ± 2.8*

a Nitrite accumulation was measured in the 48 h culture supernatants of peri-toneal cells of ATRA-treated (i.p. 2 mg/kg/day, for 7 consecutive days) and/orMLD-STZ-treated mice.

* P < 0.005 (Student’s t-test) refers to ATRA-treated MLD-STZ-induced mice vs.control MLD-STZ-induced diabetic animals.

S. Stosic-Grujicic et al. / Molecular Immunology 47 (2009) 79–86 83

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ig. 4. The effect of ATRA on spontaneous and CY-induced T1D development in Nreated with ATRA (i.p., 20 mg/kg/day) for 3 weeks. (B) Accelerated form of T1D in Nas given for 3 weeks from the day of CY administration. Blood glucose levels were mice (glycemia over 11.8 mmol/l) compared to the number of mice in experimental

.3. ATRA prevents development of spontaneous T1D in NODice, but does not counteract accelerated diabetogenesis induced

y CY challenge

We next examined whether ATRA may show a comparable ben-ficial effects in spontaneous model of autoimmune diabetogenesiss in immunoinflammatory MLD-STZ-induced diabetes. Indeed,oncurring with the observed antidiabetogenic effect of Vitamin-rich diet in NOD mice (Zunino et al., 2007), ATRA also turnedut to abolish the development of the disease in this model of dia-etes. When long-term i.p. ATRA treatment was applied accordingo the experimental design described in Section 2 to euglicemicOD mice, or to mildly hyperglycemic NOD mice immediatelyfter becoming overtly diabetic (blood glucose levels 7.0 ± 0.7 and2.3 ± 1.9 mmol/l, respectively), none of the mice (0/12) becameiabetics by the end of the study (13–19-week old) (Fig. 4A). Impor-antly, prolonged treatment with the drug appeared to be wellolerated by the mice as judged from their behaviour and generalppearance. However, in contrast to MLD-STZ-induced diabetes,he beneficial effect of ATRA in NOD mice depended upon contin-ous application of the drug, because in a group of ATRA-treatedildly hyperglycemic 16-week-old mice the severe hyperglicemia

ppeared soon (1 week) after treatment disconnection (Fig. 4A).Selective effect of CY on CD4+Foxp3+ Treg has been recently

escribed in NOD mice showing that Treg control CY-induced dia-etes (Brode et al., 2006). In order to examine whether ATRA may

ounteract diabetogenesis in the conditions of Treg depletion, therug was tested in the model of CY-induced NOD diabetes. Asxpected, most of the mice treated with vehicle (32/41, 78.1%)eveloped acute form of T1D from 7–14 days after CY injectionFig. 4B). Somewhat surprisingly, however, the incidence of the dis-

able 3ffect of ATRA on lymphoid population in the spleen and PLN of NOD and CY-treated NOD

reatment group Day 7 Day 14

PLNC (×106)

ontrol 1.9 ± 0.8TRA 1.4 ± 0.1 7.3 ± 2.0Y 0.07 ± 0.02 6.9 ± 0.4TRA + CY 0.01 ± 0.01 3.4 ± 0.5

reatment group Day 7 Day 14

Splenocytes (×106)

ontrol 71.3 ± 14.1TRA 64.7 ± 1.3 97.5 ± 56.5Y 18.5 ± 6.1 123.5 ± 33.2TRA + CY 2.9 ± 0.9 141.3 ± 58.3

ice. (A) Euglycemic (♦) or mildly hyperglycemic (�) NOD mice were continuouslyice was induced with CY (♦) as described in Section 2. ATRA (�) (i.p., 20 mg/kg/day)red on a weekly basis. Incidence of T1D (%) represents the number of hyperglycemic

(10–15).

ease was similar in the CY-treated NOD mice that continuouslyreceived ATRA from day 0 of CY-induced diabetogenic challenge,with 10 out of 12 (83.3%) of them becoming diabetic 14 days afterCY (p > 0.05 versus the group of mice treated with CY + vehicle by�-square) (Fig. 4B).

3.4. Differential effects of ATRA on lymphoid populations in thespontaneous versus CY-induced NOD mouse model

To further examine the effect of ATRA on lymphoid populationsin the spontaneous versus CY-induced NOD mouse model, mononu-clear cell populations were extracted from spleen and PLN as wellas the pancreas and analyzed by flow cytometry. In keeping withprevious studies (Brode et al., 2006), CY treatment caused a signif-icant drop in the PLN and spleen cellularity, which remained lowon day 7 after CY administration, but then returned or exceededpre-treatment levels on day 14 (Table 3). Interestingly, while inNOD mice ATRA induced an increase of cellularity of both organsafter 14 days treatment, similar changes were not observed in theATRA + CY-treated mice (Table 3). Further analysis of the CD4+ T cellpopulation performed on day 14 following CY treatment indicatedthat the profile of CD4+Foxp3+ Treg differed markedly betweenspleen and PLN versus islets infiltrates. In accordance with tem-poral profile observed by others (Brode et al., 2006), mice treatedwith CY alone showed recovered levels of CD4+Foxp3+ Treg in thespleen and PLN equivalent to those seen in untreated control mice

(Table 3). In both spontaneous and CY-accelerated NOD mice, ATRAtreatment did not significantly influence the relative proportion ofany of the CD4+ T cell population examined. However, since in spon-taneous NOD mice ATRA induced an expanding absolute number ofboth splenocytes and PLNC, it is tempting to speculate that in this

mice.

Day 14

CD4+ CD4+CD25+ CD4+Foxp3+

49.6 ± 0.9 7.6 ± 0.6 8.6 ± 0.653.2 ± 0.4 7.0 ± 1.0 7.5 ± 1.054.4 ± 1.4 8.7 ± 0.7 9.9 ± 1.551.6 ± 1.4 9.7 ± 0.6 8.8 ± 0.7

Day 14

CD4+ CD4+CD25+ CD4+Foxp3+

21.5 ± 0.2 3.2 ± 0.2 4.3 ± 0.116.6 ± 2.5 3.2 ± 0.6 3.6 ± 0.743.6 ± 3.2 7.0 ± 3.0 11.1 ± 1.56.31 ± 1.86 1.8 ± 0.7 3.0 ± 0.1

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4 S. Stosic-Grujicic et al. / Molec

odel of diabetes the drug up-regulates actual number of Treg,t least in the peripheral compartments. In contrast to peripheralymphoid tissue, although by day 14 after CY administration theelative proportion of CD4+ and CD4+CD25+ in the pancreas was.6 ± 1.8, and 9.2 ± 1.3%, respectively, the percentage of CD4+Foxp3+

emained low (0.4 ± 0.04%). Since CD25 is not a unique marker forreg, but also for activated CD4+ cells, this indicates the loss of Tregnd simultaneous emergence of primed autoreactive CD4+CD25+ Tells within the pancreas.

. Discussion

In this study, we used prophylactic retinoid approach to alterhe immune response in mice and inhibit the progression of T1D.lthough one previous study did examine the effect of high-levelitamin A on the development of diabetes in NOD mice (Zuninot al., 2007), no other studies have, to our knowledge, examinedhe effect of retinoids on the development of MLD-STZ-inducediabetes, nor of CY-accelerated NOD mice. We found that ATRA

nhibited the disease development in both immunoinflammatoryLD-STZ-induced model and spontaneous NOD model, but not

n Foxp3+ Treg-depleted CY-induced NOD mouse model. Interest-ngly, in a model of spontaneous diabetes in BioBreeding/Worcesterats (Driscoll et al., 1996) Vitamin A deficiency produced a high

ortality rate before diabetes developed, but a retinoic acid-fedontrol rats had a reduced incidence of diabetes, thus suggestinghat retinoid deficiency and excess can influence the T1D via differ-nt pathways.

Another key observation that was made in the present studys that the impact of ATRA on immunopathological events duringiabetes development is mainly due to down-regulation of bothh1 and Th17 responses that may have escaped immune regulationy Treg and/or counteracting Th2 responses. The different disease-uppressing effects of ATRA noted between NOD and CY-NOD maye associated with the role of Treg that otherwise antagonize func-ion of pathogenic T cells, especially Th17 cells. These findings areompatible with the recent report demonstrating that retinoidsroduced by mucosal dendritic cells promote TGF-�1-dependenteneration of Foxp3+ Treg population, but inhibit Th17 differen-iation (Kang et al., 2007). Moreover, the authors show a directunction of in vivo ATRA treatment in differentiation of naive Foxp3−

cells into a Foxp3+ Treg cells which preferentially migrate tohe small intestine and other nonlymphoid tissue sites. Therefore,ur apparently unexpected results that ATRA treatment does not

ncrease the frequency of Foxp3+ T cells within the lymphoid tissuesested, raised the possibility that ATRA directly induces generationf T regulatory cells that suppress diabetogenic T cells, but due torafficking receptor shift migrate to nonlymphoid tissues. In sup-ort to this, it was recently reported that upon Ag priming Foxp3+

egulatory T cells undergo the second switch in homing receptorsor migration to nonlymphoid tissues, such as small intestine, liver,kin or bone marrow (Lee et al., 2007). The other possibility is thatTRA acts on the (auto)Ag reactive primed T cells, but needs col-

aboration of the immune suppressive function of Foxp3+ Treg cellso switch the balance towards diabetes resistance. These variousossibilities of ATRA-mediated disease suppression warrant further

nvestigation in the future.Although pathogenic Th1 and Th17 responses were strongly

nhibited by short-term ATRA treatment under MLD-STZ-inducednflammatory conditions, the treatment had insignificant influencen the Foxp3+ Treg population. Consistent with these findings, Xiao

t al. (2008) also observed upon RA treatment of EAE in mice, aemarkable reduction of proinflammatory Th17 and Th1 responsesut not measurable enhancement of the differentiation of Foxp3+

reg. They suggested that this insignificant in vivo expansion ofreg by RA might be due to strong induction of proinflammatory

munology 47 (2009) 79–86

cytokines, IL-6, IL-1 and TNF-�, during the inflammatory processinduced by Ag/CFA immunization, as in vitro addition of thesecytokines into Th17 differentiation conditions strongly inhibitedthe development of Foxp3+ Treg by RA, but could not abrogate theinhibitory effects of this retinoid on Th17 cells. Similarly, whereasRA greatly reduced Th17 cell development in vivo in mice infectedwith Listeria monocytogenes, no effect on CD4+Foxp3+ T cells wasobserved (Mucida et al., 2007). The authors suggested that TGF-� might be a limiting factor of Treg cell differentiation, since RAcombined with TGF-� can drive differentiation of Foxp3+ T cells invitro. Herein we show suppression of MLD-STZ-induced diabetesby ATRA in the face of ongoing inflammation. Alternatively, periph-eral Treg numbers were transiently elevated for a very short period,but the frequency were normalized thereafter due to a concomitantincrease in CD4+ Foxp3− T cells.

Our results show that spontaneous T1D onset can be reversedwhen ATRA treatment is applied to NOD mice immediately afterthe first signs of hyperglycemia. On the other hand, the presenceof mild insulitis in the islets of NOD mice receiving ATRA (notshown) suggests that this intervention did not completely inhibitthe pathogenesis of T1D in these mice. However, the absence ofhyperglycemia observed upon continuous ATRA treatment stronglyimplies that ATRA can play an important role in the regulation andprogression of the disease. Moreover, in line with the storage andmetabolism of ATRA within the body (Kurlandsky et al., 1995; Iwataet al., 2004), both i.p. and oral administration of the drug results ingood compliance. Unlike MLD-STZ-induced diabetes, in NOD micethe immunosuppressive effects of such daily systemic ATRA admin-istration ceased when retinoid treatment was abrogated. This maybe due to sustained autoantigenic stimulus and ongoing inflam-mation in NOD mice, accompanied with simultaneous defect inthe maintenance and/or expansion of counteracting Treg cells (Popet al., 2005). It is well known that the onset of hyperglicemia inNOD mice is preceded by a protracted preclinical period of isletinflammation associated with the activation, recruitment, and localaccumulation of diabetogenic T cells. These processes are driven byislet-antigen loaded antigen-presenting cells in pancreatic lymphnodes (Hoglund et al., 1999). The insulitis begins in female NODmice at around 3 weeks of age. However, during this period miceremain diabetes-free, despite this extensive, but “benign” insulitis.The progression to overt diabetes begins at around 8–10 weeks ofage, when � cells are efficiently destroyed by “aggressive” insuli-tis. Present findings thus suggest that retinoids down-modulateimmunoinflammatory events at the second “check-point” whichallow diabetes progression. Progression of benign to aggressiveinsulitis corresponds with a temporal decrease in Treg cells resid-ing in the pancreatic lymph nodes (PLN) and islets of NOD femalemice (Pop et al., 2005). Immunoregulation of autoreactive T cellsin general is highly complex and likely involves a heterogeneousgroup of immunoregulatory T (Treg) cells (reviewed by Colemanet al., 2007). CY-induced NOD diabetes is associated with a selec-tive reduction of CD4+CD25+Foxp3+ Treg cells (Brode et al., 2006).By using this model, we showed that the deficiency of this pool ofimmunoregulatory effectors is associated with the failure of ATRAto successfully counteract the progression of T1D. Furthermore, wefound that selective decline of Treg cells from peripheral lymphoidtissues recovered by day 14, whereas in pancreatic infiltrates thispopulation failed to recover. It is becoming more apparent thatFoxp3 is not only a phenotypic marker of CD4+CD25+ Treg, but itis also essential to their development and function (Fontenot et al.,2003). Together, these data strengthen the hypothesis that the loss

of CD4+CD25+Foxp3+ T cells in the target tissue of CY-treated NODmice directly contributes to the development of aggressive insulitisand overt diabetes.

It is well established that Th1-type cytokines correlate withT1D, whereas Th2/3-type cytokines correlate with protection from

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he disease (Rabinovitch and Suarez-Pinzon, 2007). In agreementith this, MLD-STZ-induced disease resulted in a higher IFN-�

esponse over an IL-4 response, which is indicative of Th1 cellspresent results and Stosic-Grujicic et al., 2007b). Also, IL-1 andNF-� have a prominent proinflammatory functions within theancreas and act downstream of Th1 cell-mediated autoimmunity.oncordantly, we have previously demonstrated that blocking theiological activities of these cytokines is a successful approach tomeliorate autoimmune-mediated inflammation in several mod-ls of T1D, including MLD-STZ-immunoinflammatory diabetes, andpontaneous and CY-accelerated NOD models (Stosic-Grujicic et al.,007a). Recently, it was shown that Th1 cells, and also Th2 cells,ay block differentiation of CD4+ T cell producing IL-17 (Th17 cells),hich is a newly identified phenotype that is crucial for the induc-

ion of autoimmune disorders like T1D (Wynn, 2005; Bettelli et al.,007). It is a matter of debate how retinoids suppress T1D. Pos-ible mechanisms by which administration of retinoids can affecthe disease development may be either direct or indirect. Previouseports indicate that both APC and naive T cells are the target sites ofA (Kang et al., 2000; Iwata et al., 2003). The authors demonstratedhat treatment with retinoids, beside modulation of Ag-presentingunction, led to the inhibition of Th1 signature cytokine, IFN-�, andn increase of IL-4 production (Kang et al., 2000). Moreover, theuppressive effects of Vitamin A on TNF-� and IL-1 production wereescribed (Aukrust et al., 2000). More recently, several independentroups reported that Vitamin A or its metabolite RA, inhibit the dif-erentiation of Th17, but enforce the generation of Foxp3+ regulatorycells (Kang et al., 2007; Mucida et al., 2007; Xiao et al., 2008). Simi-

arly to this, we here presented that ATRA suppressed Th1, as well ash17 response, but enhanced Th2 cytokine IL-4 in vivo, thus shiftinghe balance towards the anti-inflammatory cytokines. In addition,ur results obtained in a model of long-term depletion of Treg by CYuggest that this indirect effect may be mediated through Foxp3+

egulatory T cells that antagonize Th1/Th17-mediated proinflam-atory responses which otherwise lead to diabetes progression.

ince islets provide a key site for immunoregulation of � cell spe-ific T cell reactivity (Herman et al., 2004), it can be speculatedhat retinoids may also down-modulate local priming of autoreac-ive T cells and progression to T1D. It appears that RA suppressesL-17 cell differentiation by dendritic cells, but also may operateirectly on Th17 cells via the reduction of ROR�t (Mucida et al.,007). In addition, results presented herein clearly show that inivo treatment with ATRA markedly suppressed NO generation inLD-STZ-induced diabetic mice. In agreement with this, the direct

rotective effect of RA from cytokine-mediated toxicity has beenemonstrated on RINm5F rat insulinoma cells in vitro (Kang et al.,004). The molecular mechanism of RA protection was shown to beediated by the inhibition of NF-�B activation and reduced NO pro-

uction, which correlated well with direct effects of RA in the reg-lation of cytokine-induced iNOS expression in these cells. Since inddition to iNOS, NF-�B has been implicated in the transcriptionalegulation of various other genes, including stress-response pro-eins, cytokines, superoxide dismutase, heme oxyganase, it is possi-le that retinoids influence induction of these genes either directly,r indirectly by interaction with some other transcription factors.

In conclusion, this study demonstrated that retinoids exhibiteneficial effects in immunoinflammatory MLD-STZ-induced dia-etes as well as in spontaneous form of autoimmune diabetes

n NOD mice by inhibiting pathogenic Th1/Th17 T cell responses.owever, ATRA did not suppress the disease of NOD mice in the

onditions of depleted CD4+CD25+Foxp3+ Treg cell population, thus

ndicating that retinoids down-modulate the disease progression athe second checkpoint in which Treg cells are believed to contribute.evertheless, having in mind a favourable effects of retinoids, our

tudy indicate that their potential use for treatment of human T1Ds worthy of further investigation.

munology 47 (2009) 79–86 85

Acknowledgments

This work was supported by the Ministry of Science and Technol-ogy of the Republic of Serbia (Grant No. 143029B). We acknowledgethe kind assistance of Dr. Ferdinando Nicoletti (University of Cata-nia, Italy) in providing the NOD mice.

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