Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes

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1 r t i l e s Crosstlk between neutrophils, B-1 ells n plsmytoi enriti ells initites utoimmune ibetes Julien Din 1,2,6, Ynnik Simoni 1,2,6, Letiti Furio 2,3,6, Luie Beuoin 1,2, Birgitt Agerberth 4, Frnk Brrt & Agnès Lehuen 1,2 npg 213 Nture Ameri, In. All rights reserve. Type 1 ibetes evelops over mny yers n is hrterize ultimtely by the estrution of insulin-prouing pnreti bet ells by utoretive T ells. Nonetheless, the role of innte ells in the initition of this isese remins poorly unerstoo. Here, we show tht in young femle nonobese ibeti mie, physiologil bet ell eth inues the reruitment n tivtion of B-1 ells, neutrophils n plsmytoi enriti ells (pdcs) to the pnres. Ativte B-1 ells serete IgGs speifi for ouble-strne DNA. IgGs tivte neutrophils to relese DNA-bining theliiin-relte ntimirobil peptie (CRAMP), whih bins self DNA. Then, self DNA, DNA-speifi IgG n CRAMP peptie tivte pdcs through the Toll-like reeptor 9 myeloi ifferentition ftor 88 pthwy, leing to interferon- proution in pnreti islets. We further emonstrte through the use of epleting tretments tht B-1 ells, neutrophils n IFN- prouing pdcs re require for the initition of the ibetogeni T ell response n type 1 ibetes evelopment. These finings revel tht n innte immune ell rosstlk tkes ple in the pnres of young mie n les to the initition of T1D. T ells infiltrte the pnres n trget insulin-prouing bet ells uring type 1 ibetes (T1D) evelopment 1. However, T ells represent only one piee of the puzzle of the multiple immune ells implite in bet ell loss 2. Plsmytoi enriti ells (pdcs) re entrl meitors of ntivirl immunity through their bility to proue lrge mounts of interferon-α (IFN-α) n IFN-β 3. In ition to their ntivirl role, pdcs lso promote vrious utoimmune iseses suh s psorisis n systemi lupus erythemtosus (SLE) 4,. Regring utoimmune ibetes, pthogeni role for IFN-α n pdcs hs been propose, s IFN-α tretment of ptients with virl infetions or with leukemi hs been shown to be ssoite with inrese iniene of T1D 6,7. Aitionlly, IFN-α prouing pdcs hve been etete in the bloo of ptients with T1D t the time of ignosis 8. Furthermore, geneti nlysis supports ibetogeni role for IFN-α inue genes in preibeti hilren 9. In mie, trnsgeni non utoimmune-prone mie expressing IFN-α in bet ells evelop utoimmune ibetes 1. Subsequently, it ws shown tht IFN-β elertes the onset of the isese in nonobese ibeti () mie n breks self tolerne to bet ell ntigens in nonobese resistnt mie 11. This potentil role of IFN-α n IFN-β n pdcs in T1D n in other utoimmune iseses prompte us to investigte the funtions of pdcs in the initition of ibetes. It hs been foun tht uring the first postntl weeks, wves of physiologil bet ell eth our in roents 12 14, pigs 1 n humns 16. Given tht e ell lerne is efetive in mie 17, we speulte tht the umultion of bet ell ebris (for exmple, self DNA) n tivte pdcs in the pnres. RESULTS Innte ells infiltrte the pnres of young mie We initilly fouse on hrterizing immune ell infiltrtion in the pnres of femle mie strting t 2 weeks of ge (Fig. 1,b). As erly s 2 weeks of ge, vrious innte immune ells suh s neutrophils (Ly6G + CD11b + ) n pdcs (m927 + CD11 me ) n lso B ells (CD19 + ) infiltrte the islets. The presene of neutrophils n pdcs ws only trnsient, rehing mximum t 3 n 4 weeks of ge, respetively. Notbly, the reruitment of these innte ells ws speifi to the mie, s we i not observe suh ells in the islets of C7BL/6 or BALB/ mie (Supplementry Fig. 1,b). Histologil nlysis onfirme the presene of pdcs (B22 + CD11 + ), onventionl DCs (DCs) (B22 CD11 + ), B ells (B22 + CD11 ) n neutrophils (NIMP-R14 + ) surrouning or insie the islets t 3 but not t 6 weeks of ge (Fig. 1, n Supplementry Fig. 2). Together, these t revel tht innte ells infiltrte the pnreti islets of young mie, whih suggests they hve role in T1D initition. Pnreti IFN- sereting pdcs re ruil for T1D initition We nlyze IFN-α proution in the islets of mie beuse this ytokine is ssoite with tivte pdcs n utoimmunity 3. IFN-α ws etete in the islets only t 3 weeks of ge (Fig. 2). To etermine the ssoition of IFN-α with T1D evelopment, we nlyze the mrna expression of IFN-α, IFN-α inue gene prouts (ISG1, IRF7, IFIT1 n IFIT3) n proinflmmtory gene prouts 1 Institut Ntionl e l Snté et e l Reherhe Méile (INSERM), U986, Pris, Frne. 2 Université Pris Desrtes Sorbonne Pris Cité, Lbortoire Exellene Inflmex, Pris, Frne. 3 INSERM, U781, Neker Hospitl, Pris, Frne. 4 Deprtment of Meil Biohemistry n Biophysis, Krolinsk Institute, Stokholm, Sween. Dynvx Tehnologies Corportion, Berkeley, Cliforni, USA. 6 These uthors ontribute eqully to this work. Corresponene shoul be resse to A.L. or J.D. (gnes.lehuen@inserm.fr or julien.in@inserm.fr). Reeive 23 July; epte 19 November; publishe online 16 Deember 212; oi:1.138/nm.342 nture meiine VOLUME 19 NUMBER 1 JANUARY 213 6

2 A r t i l e s npg 213 Nture Ameri, In. All rights reserve. Figure 1 Innte immune ells infiltrte the pnres of mie in the first postntl weeks. (,b) FACS nlysis of CD4 + infiltrting ells from pnreti islets ollete from 2 to 6 weeks. Frequenies of pdcs (m927 + CD11 + ), DCs (m927 CD11 + ), neutrophils (Ly6G + CD11b + ), B ells (CD19 + ) n T ells (TCRβ + ) re shown in eh qurnt. Frequenies of positive ells mong CD4 + ells re represente in n bsolute number in b. Dt re men vlues ± s.e.m. n re representtive of four inepenent experiments with four poole mie for eh group. P <. for eh group ompre to 2-week-ol group. (,) Immunohistologil nlysis of DCs () n neutrophils () from the pnres of 3-week-ol mie. Zoome-in boxe re in shows DCs (CD11 + ) in re, B ells (B22 + ) in yellow n pdcs (CD11 + B22 + ) in ornge n in shows neutrophils (NIMPR14 + ) in re. Pnreti setions were prepre s esribe in the Online Methos. Representtive t from nine pnreses with ten setions for eh pnres from three inepenent experiments re shown. TCR-β, T ell reeptor-β. (interleukin-1β, IFN-γ n CXCL1) in the islets of mie t vrious ges. Expression of IFN-α n IFN-α inue gene prouts rehe mximum t 3 n 4 weeks 2 µm of ge, respetively (Supplementry Fig. 3). Proinflmmtory gene expression inrese ontinully between 4 n 8 weeks of ge in the islets of mie (Supplementry Fig. 3). mrna expression of IFN-α n IFN-α inue gene prouts ws not 1 µm observe in nonutoimmune C7BL/6 or BALB/ mie (Supplementry Figs. 3 n 4). Together, these t revel tht the IFN-α signture in young mie ws ssoite with B22 Hoehst the utoimmune-prone geneti bkgroun. We next fouse on pdcs n their puttive role in T1D initition. FACS nlysis showe tht pdcs isolte from the islets of mie proue IFN-α, whih peke t 3 n eline t 6 weeks of ge (Fig. 2b). Aoringly, the expression of IFN-α inue genes in 4-week-ol mie ws bolishe fter pdc epletion using m927 monolonl ntiboy (mab) ginst the BST2 ntigen between 2 n 3 weeks of ge (Fig. 2 n Supplementry Fig. ). To ssess the ibetogeni role of pdcs, we evlute the utoretive CD8 + T ell response ginst the bet ell ntigen islet-speifi gluose-6-phosphtse tlyti subunit-relte protein (IGRP) in 8-week-ol mie tht were eplete of pdcs or tht h been trete with Sigle-H speifi mab to prevent IFN-α proution by pdcs (Supplementry Fig. 6). Both tretments ministere between 2 n 3 weeks of ge le to rmti reution in the frequeny of NRP-V7-tetrmer + CD8 + T ells speifi for IGRP within the pnreti lymph noes (PLN) n to strong erese in IGRP retive IFN-γ + CD8 + T ells within the islets of 8-week-ol mie (Fig. 2 n Supplementry Fig. 7). Aoringly, pdc epletion in 2-week-ol mie prevente the evelopment of T1D up to 3 weeks of ge (Fig. 2e). Together, these t emonstrte tht pnreti pdcs proue IFN-α in young mie n subsequently inue utoimmune ibetes. To etermine whether Toll-like reeptor (TLR) 2 weeks 3 weeks 4 weeks 6 weeks m927.71% ±.2% 2.3% ± 4.% 3.8% ± 1.2% 28.3% ±.1% 4.41% ± 2.2% 27.6% ± 3.3%.86% ±.3% 14.1% ± 3.7% CD11 Ly6G Insulin CD11 B22 Hoehst Zoom CD11b CD11 B22 Hoehst 3.72% ± 1.1% 42.% ± 4.1% 12.9% ± 3.2% 39.9% ± 3.3% 1.16% ±.9% 42.3% ±.1%.17% ±.1% 26.9% ± 2.7% CD4 CD11 Hoehst 36.8% ± 4.1% 27.3% ± 3.6% 19.3% ± 2.8% CD19 2.7% ± 2.% CD11 B22 Hoehst pdc DC B ell TCR-β 2.83% ±.6% 2.84% ±.2% 8.24% ± 1.4% 34.1% ± 3.7% CD11b 2 µm Insulin NIMP-R14 Hoehst Neutrophil 1 µm Zoom pthwys were neessry for IFN-α expression in the islets of mie, we use My88 / mie, whih lk the bility to signl through TLRs (exluing TLR3). We file to etet IFN-α proution in the islets from 3-week-ol My88 / mie (Supplementry Fig. 8). Aoringly, the inrese in IFN-α inue gene expression observe in mie ws lso bsent in the islets of 4-week-ol My88 / mie (Supplementry Fig. 8b). To etermine the TLR involve, we trete 2-week-ol mie with IRS 94, n ntgonist of TLR7 n TLR9 pthwys. Suh tretment erese the expression of IFN-α inue genes in the islets of 4-week-ol mie (Supplementry Fig. 8b). Finlly, tretment of 2-week-ol mie with IRS 94 prevente the evelopment of T1D, wheres CpG 18, TLR9 gonist, inrese the iniene of T1D, onfirming the role of TLR7 n TLR9 n subsequent IFN-α proution in the initition of T1D in young mie (Fig. 2e). B-1 ells tivte pdcs in the pnres During psorisis n lupus evelopment, self DNA relese from ying kertinoytes forms immune omplexes with IgGs speifi for ouble-strne DNA (sdna), whih then tivte pdcs vi TLR9 (ref. 4). Thus, we investigte whether this mehnism of pdc tivtion tkes ple in T1D. First, we etete sdna-speifi IgGs b B ells ( 1 4 ) pdcs ( 1 3 ) Neutrophils ( 1 3 ) DCs ( 1 4 ) T ells ( 1 4 ) 66 VOLUME 19 NUMBER 1 JANUARY 213 nture meiine

3 r t i l e s npg 213 Nture Ameri, In. All rights reserve. IFN-α (pg ml 1 ) TT-NRP-V Spleen t low onentrtion in the ser of, BALB/ n C7BL/6 mie n t high onentrtion in serum from NZB/W F1 lupusprone mie use s positive ontrol (Fig. 3). Conversely, in the islets, we observe higher mount of sdna-speifi IgGs from mie s ompre to BALB/ or C7BL/6 mie (Fig. 3b). We lso observe higher mount of totl IgG in islets from mie ompre to BALB/ or C7BL/6 mie (Supplementry Fig. 9). Among B ell popultions, the CD + CD19 + CD1 me B-1 ells my proue high titers of sdna-speifi IgGs 18. Aoringly, we observe lrge popultion of B-1 ells in the islets of mie s erly s 2 weeks of ge (Fig. 3). Their frequeny mong the totl B ell popultion in the islets eline with the ge (Fig. 3 n Supplementry Fig. 1). Next, we performe peritonel lvges between 1 to 3 weeks of ge to eplete B-1 ells from pnreti islets of mie s previously esribe 19 (Supplementry Fig. 11). B-1 ell epletion lrgely reue the proution of sdna-speifi IgGs in the islets of 3-week-ol mie (Fig. 3b). Aitionlly, pnreti B-1 ells sorte from 3-week-ol mie proue sdna-speifi IgGs fter 7 of ulture, ontrry to pnreti B-2 ells (Fig. 3e). B-1 ell epletion lso strongly reue the frequeny of IFN-α sereting pdcs in the islets of 4-week-ol mie (Fig. 3f) n inhibite the ibetogeni T ell response within the PLN n islets of 8-week-ol b 2-week-ol 4-week-ol 4-week-ol, pdc eplete 2 ISG1 IRF Pnreti islets Events (% of mx) IFN-α.24% 11.3% pdc-eplete.83% CD8 TT-NRPV7 + CD8 + T ells (%) PLN IFN-γ pdc-eplete 1.1% TCR-β Isotype ontrol IFN-γ + CD8 + T ells (%) IFN-α + pdcs (%) IFN-α + pdcs (%) week-ol 3-week-ol pdc eplete + Sigle H mab Pnres Dibeti mie (%) IFIT IFIT3 mie (Fig. 3g). Aoringly, B-1 ell epletion in 2-week-ol mie prevente T1D evelopment (Fig. 3h). Colletively, these t unveil ruil role of pnreti B-1 ells in the tivtion of pdcs n T1D initition. Pnreti neutrophils tivte pdcs n initite T1D Our nlysis of the pnreti infiltrte of 3-week-ol mie revele the presene of neutrophils (Fig. 1). This group of innte ells is key plyer in the lerne of extrellulr pthogens 2. In iniviuls with SLE, neutrophils serete DNA-bining ntimirobil pepties (LL-37 in humns n CRAMP in mie) tht bin immune omplexes n potentite their stimultory effet on IFN-α sereting pdcs 21,22. We first observe tht neutrophil epletion between 2 n 3 weeks of ge using the NIMP-R14 mab ginst Ly6G ntigen (Supplementry Fig. 12) ttenute the expression of IFN-α inue genes in the pnres of 4-week-ol mie (Fig. 4). Injetion of CRAMP in 4-week-ol mie inrese the expression of IFN-α inue genes in the islets 24 h lter (Fig. 4). Moreover, oiniing with the presene of neutrophils, pek CRAMP mrna expression ws etete t 3 weeks of ge (Fig. 4b), n neutrophil epletion mrkely reue this expression (Fig. 4). FACS nlysis onfirme tht pnreti neutrophils were the soure of CRAMP in young mie (Fig. 4). No CRAMP expression ws Control pdc eplete CpG 18 IRS 94 Figure 2 Pnreti pdcs express IFN-α n re require for T1D evelopment in mie. () IFN-α proution in the islets of mie from 2 to 6 weeks of ge, mesure fter 36-h ulture. Dt represent seven inepenent mie for eh group from four inepenent experiments. P <. for eh group ompre to 2-week-ol group. (b) FACS nlysis of IFN-α by pdcs from islets of 3- or 6-week-ol mie. Frequeny (left) n bsolute number (right) of IFN-α + ells mong pdcs in pnreti islets (top) n the spleen (bottom). Dt re men vlues ± s.e.m. of four inepenent experiments with four poole mie for eh group. P <. for 3-week-ol group ompre to 6-week-ol group. () mrna expression of IFN-α inue genes s ssesse by quntittive PCR in the islets of mie trete with epleting m927 mab or isotype ontrol. Dt re men vlues ± s.e.m. of three inepenent experiments with four inepenent mie for eh group. P <.. () Anlysis of CD8 + T ells speifi for IGRP in 8-week-ol mie fter pdc epletion or bloke of IFN-α prouing-pdcs t 2 3 weeks of ge. Left, frequeny of NRP-V7 tetrmer-speifi ells mong the CD8 + T ell popultion from PLN. Right, frequeny of IFN-γ + ells mong the CD8 + T ell popultion fter re-stimultion with IGRP peptie. Representtive ot plots re shown, n vlues in the grph orrespon to four inepenent mie for eh group from two inepenent experiments. P <. for trete group ompre untrete group. (e) Iniene of ibetes in mie fter pdc epletion or TLR9 trgeting. Mie were trete with m927 mab, isotype ontrol, IRS 94 or CpG 18 t 2 weeks of ge over 2 weeks. P <. for eh trete group ompre to ontrol group (n = 12 mie per group). e 2 3 nture meiine VOLUME 19 NUMBER 1 JANUARY

4 A r t i l e s npg 213 Nture Ameri, In. All rights reserve. sdna-speifi lgg (A 4 ) f Events (% of mx) IFN-α Serum B-1 ell eplete Isotype ontrol NZB/W F1 (1/1) (1/1) BALB/ (1/1) C7BL/6 (1/1) IFN-α + pdcs ( 1 3 ) b sdna-speifi lgg (A 4 ) NZB/W F1 (serum 1/1) Pnreti islets BALB/ C7BL/6 B-1 ell eplete observe in other CD4 + or in CD4 ells in the pnreti islets (Supplementry Fig. 13). Histologil nlysis onfirme the presene of CRAMP-sereting neutrophils insie the islets of 3-weekol mie (Fig. 4e). Pnreti neutrophils ppere to relese neutrophil extrellulr trps (NETs), whih re typilly ssoite with CRAMP relese n tivtion of IFN-α sereting pdcs in ptients with SLE 21,22. We onfirme tht ex vivo isolte pnreti neutrophils proue NETs ontining DNA by stining with Ly6G-speifi ntiboy n SYTOX-Green, n these NETs were ssoite with CRAMP peptie (Fig. 4f n Supplementry Fig. 14). Neutrophil epletion t 2 weeks of ge reue the frequeny of IFN-α sereting pdcs in the islets of trete mie (Fig. ) n mpene the ibetogeni T ell response within the PLN n islets of 8-week-ol mie (Fig. b). Aoringly, neutrophil epletion in 2-week-ol mie inhibite T1D evelopment t lter ges (Fig. ). Together, these t support role for CRAMP-sereting neutrophils in the tivtion of pnreti pdcs n T1D initition. Neutrophils n B-1 ells ooperte to tivte pdcs We next investigte the moleulr interply between innte ells in the islets of young mie. Neutrophils re tivte vi vrious pthwys inluing Fγ reeptors (FγRs), TLRs or both. g TT-NRP-V7 CD CD21 CD19 CD B-1 ells 37%±4.8% CD4 ells Pnreti B-1 ells Spleni MZB ells CD1.227% 9.7% TT-NRPV7 + CD8 + T ells (%) B-1-eplete B-1-eplete.11%.2 4.4% CD8.3.1 PLN IFN-γ TCR-β IFN-γ + CD8 + T ells (%) Perentge of B-1 ells mong B ells First, we exlue the TLR pthwy beuse CRAMP mrna expression in the islets ws unltere in My88 / mie s ompre to WT mie (Fig. 4). As IgG-sereting B-1 ells hve been shown to tivte neutrophils vi FγRs 2, we performe in vitro ultures with neutrophils isolte from bone mrrow n tivte with phorbol-12-myristte-13-ette (PMA), immune omplexes (sdna plus ommeril sdna-speifi IgGs) or B-1 immune omplexes (sdna plus pnreti B-1 ell onitione meium), n mesure CRAMP expression on the surfe of neutrophils (Fig. ). As expete, PMA (use s positive ontrol) inue expression of CRAMP. Immune omplexes n B-1 immune omplexes lso inue CRAMP expression, whih ws bloke by the ition of mix of FγRII/III-speifi n FγRIV-speif mabs, reveling tht the neutrophils were tivte through FγRs (Fig. ). Notbly, our t obtine with severe ombine immunoefiieny mie, whih re evoi of T n B ells, emonstrte the requirement of B ells in the tivtion of pnreti neutrophils (Supplementry Fig. 1). These t strongly suggest tht in the pnreti islets of mie, CRAMP-prouing neutrophils re tivte by IgG-sereting B-1 ells vi FγRs. To etermine whether both neutrophils n B-1 ells re require for tivtion of pdcs, we ulture spleni pdcs with pnreti B-1 ell onitione meium, superntnt of bone mrrow isolte B-1 ell eplete Pnres h Dibeti mie (%) 8 e sdna-speifi lgg (A 4 ) Cell ulture NZB/W F1 (serum 1/1) B-1 ells B-2 ells Control B-1 ell eplete Figure 3 B-1 ells tivte pdcs in the pnres n prtiipte in the initition of T1D. (,b) sdna-speifi IgG proution in the serum () or in the islet superntnts (b) from 3-month-ol NZB/W F1 mie or, C7BL/6 n BALB/ mie t 3 weeks of ge. In some onitions, B-1 ells were eplete in mie s esribe in the Online Methos. Vlues re obtine by ELISA n re expresse s men bsorbne vlues t 4 nm with orrete utoff vlues. Dt re men vlues ± s.e.m. of two inepenent experiments with three inepenent mie for eh group. P <.. () FACS nlysis of B ell popultions in the islets of mie t 3 weeks of ge. Frequeny of B-1 ells (CD19 + CD + CD21 ) mong CD4 + ells re represente, n representtive expression of CD1 mong CD4 + CD19 + ells is shown. () FACS nlysis of B-1 ells in the islets of mie t vrious ges. Frequenies of B-1 ells mong CD4 + CD19 + ells re represente. Dt re men vlues ± s.e.m. of four inepenent experiments with four poole mie for eh group. P <.. (e) sdna-speifi IgG proution in B-1 n B-2 ell ultures from 3-week-ol. Dt re men vlues ± s.e.m. of two inepenent experiments with three inepenent mie for eh group. P <.. (f) FACS nlysis of IFN-α seretion by pdcs fter B-1 ell epletion in mie t 3 weeks of ge. Right, bsolute number of IFN-α + ells mong pdcs. Dt re men vlues ± s.e.m. of four inepenent experiments with four poole mie for eh group. P <., trete group ompre to untrete group. (g) Anlysis of CD8 + T ells speifi for IGRP in 8-week-ol mie fter B-1 ell epletion t 2 3 weeks of ge. Left, frequeny of NRP-V7 tetrmer-speifi ells mong CD8 + T ell popultion from PLN. Right, frequeny of IFN-γ + ells mong CD8 + T ell popultion fter re-stimultion with IGRP peptie. Representtive ot plots re shown, n vlues in the grphs orrespon to four inepenent mie for eh group from two inepenent experiments. P <. for trete group ompre untrete group. (h) Iniene of ibetes in mie fter B-1 ell epletion in mie. Mie were trete between 7 n 21 of ge. P <. for trete group ompre to ontrol group (n = 12 mie per group). 68 VOLUME 19 NUMBER 1 JANUARY 213 nture meiine

5 r t i l e s npg 213 Nture Ameri, In. All rights reserve week-ol ISG1 IFIT1 4-week-ol neutrophils pretivte with B-1 immune omplexes, or both. As positive ontrol of pdc tivtion, CpG 18 inue IFN-α proution in pdcs, whih ws inhibite by IRS 94 (Fig. e). Soluble IC inue moerte proution of IFN-α, whih ws potentite by the ition of CRAMP s previously esribe 21. B-1 ell onitione meium lso inue moerte IFN-α proution tht ws potentite by the ition of neutrophilonitione meium (Fig. e). This strong IFN-α proution inue by the ition of both superntnts ws inhibite by IRS94 (Fig. e). These t emonstrte tht B-1 ells sereting sdna-speifi IgG n CRAMP-sereting neutrophils ooperte to stimulte IFN-α proution by pdcs through TLR9. Bet ell eth initites the innte ell tivtion se Our t revele tht n innte ell rosstlk tkes ple in the pnres of young mie n les to the initition of T1D. However, the initil event tht triggers this rosstlk remine unler. We investigte the puttive role of physiologil bet ell eth tht ours spontneously in young mie uring orgnogenesis n fter wening 13. To mimi this event, we inue bet ell eth by injeting streptozotoin (STZ). On y 1 fter STZ injetion in 6-week-ol mie, we observe n inrese in the expression of IFN-α inue genes in the islets (Fig. 6). This inrese ws epenent on pdcs, s revele by m927 epleting mab tretment (Fig. 6b). STZ tretment file to inue expression of IFN-α inue genes n reruitment of pdcs, neutrophils or B-1 ells in the islets of non utoimmune-prone C7BL/6 n BALB/ mie (Supplementry Figs. 16 n 17) week-ol without neutrophils IRF7 IFIT3 4-week-ol + CRAMP Figure 4 Neutrophils from the pnres of young mie proue NET-ssoite CRAMP t 3 weeks of ge. () mrna expression for IFN-α inue genes in the islets of mie fter neutrophil epletion or CRAMP peptie injetion. Dt re men vlues ± s.e.m. b CRAMP reltive expression CRAMP reltive expression week-ol WT 3-week-ol WT 3-week-ol WT neutrophil eplete 3-week-ol My88 / 8 Events (% of mx) f 3.1% ± 1.% (MFI = 127) BM 2.6% ± 4.8% (MFI = 24) Spleen 92.1% ± 3.3% (MFI = 1,746) Pnres CRAMP Isotype ontrol CRAMP-speifi pab µm Sytox Ly6G e Insulin CRAMP NIMP-R14 Hoehst 2 µm 1 µm Zoom of two inepenent experiments with four inepenent mie for eh group. P <.. (b) mrna expression for CRAMP ws nlyze in the islets of mie t vrious ges. () CRAMP mrna expression in the islets of WT mie eplete or not eplete of neutrophils or in the islets of My88 / mie. Dt re men vlues ± s.e.m. of two inepenent experiments with four inepenent mie for eh group. P <.. () FACS nlysis of CRAMP surfe expression by neutrophils of mie t 3 weeks of ge. Dt re the frequeny of CRAMP + ells n men fluoresene intensity (MFI) of CRAMP expression CD4 + CD11b + Ly6G + ells. Dt re men vlues 1 µm Sytox CRAMP Merge ± s.e.m. of four inepenent experiments with four poole mie for eh group. P <. for eh group ompre to bone mrrow (BM) group. (e) Immunohistologil nlysis of pnreti neutrophils in 3-week-ol mie. Zoome-in boxe re shows NETs relese by the neutrophil. Representtive t from six pnreses with ten setions for eh pnres from three inepenent experiments re shown. (f) The bility of neutrophils to form CRAMP-ssoite NETs ex vivo when isolte from islets of t 3 weeks of ge ws etermine by immunostining. Representtive t from five fiels of ells from the islets of eight poole mie from three inepenent experiments re shown. Merge We next emonstrte tht STZ tretment t 6 weeks of ge inue the proution of sdna-speifi IgGs n inrese the frequeny of CRAMP-expressing neutrophils in the islets of mie 24 h lter (Fig. 6, n Supplementry Fig. 17b). This inrese proution of sdna-speifi IgGs n the reruitment of CRAMP + neutrophils in the islets inue by STZ were similr to tht whih we observe in 3-week-ol mie, suggesting tht bet ell eth oul be require for the tivtion of pnreti pdcs in young mie. To test this hypothesis, we trete 7--ol mie with single injetion of Z-VAD, pn-spse inhibitor. This tretment brogte sdna-speifi IgG seretion, CRAMP-proution by neutrophils n IFN-α inue gene expression in islets of 4-week-ol mie (Fig. 6e g) n prevente T1D evelopment up to 3 weeks of ge (Fig. 6h). Altogether, our stuy emonstrte tht in young mie, physiologil bet ell eth initites the tivtion of innte immune ells, whih les to the lol proution of IFN-α, the evelopment of ibetogeni T ell responses n utoimmune ibetes. DISCUSSION Despite inrese knowlege of the pthogenesis of T1D, the erly stges of isese pthogenesis remin poorly efine. Here, we emonstrte tht in young mie, when physiologil bet ell eth ours, innte immune ell rosstlk tkes ple in the pnres tht is ruil for T1D evelopment (Supplementry Fig. 18). We propose tht bet ell ebris (tht is, self DNA) form immune omplexes with sdna-speifi IgGs serete by B-1 ells. Neutrophils proue DNA-bining peptie tht potentites these immune omplexes, inuing IFN-α seretion by pnreti pdcs through TLR9. nture meiine VOLUME 19 NUMBER 1 JANUARY

6 A r t i l e s npg 213 Nture Ameri, In. All rights reserve. b m927 TT-NRP-V7 Neutrophil Neutrophil eplete eplete IFN-α + pdcs ( 1 3 ) 4.% ± 1.7% 3.8% ± 1.7% 17.6% ± 4.7% 3.% ± 3.1% CD11 CD8.191% 12.4% Neutrophil eplete.8% TT-NRPV7 + CD8 + T ells (%) IFN-α m927 PLN IFN-γ TCR-β Neutrophil eplete 1.6% IFN-γ + CD8 + T ells (%) Pnres Figure Neutrophils n B-1 ells ooperte to promote IFN-α proution by pdcs. () FACS nlysis of IFN-α seretion by pdcs fter neutrophil epletion in mie t 3 weeks of ge. Frequeny of pdcs (left), frequeny (mile) n bsolute number of IFN-α + ells mong pdcs (right) re represente. Dt re men vlues ± s.e.m. of four inepenent experiments with four poole mie for eh group. P <., trete group ompre to untrete group. (b) Anlysis of CD8 + T ells speifi for IGRP in 8-week-ol mie fter neutrophil epletion t 2 3 weeks of ge. Left, frequeny of NRP-V7 tetrmer-speifi ells mong CD8 + T ell popultion from PLN. Right, frequeny of IFN-γ + ells mong CD8 + T ell popultion fter restimultion with IGRP peptie. Representtive ot plots re shown, n vlues in the grphs orrespon to four inepenent mie for eh group from two inepenent experiments. P <. for trete group ompre 1 1 Neutrophil eplete Neutrophil eplete e IFN-α (pg ml 1 ) Dibeti mie (%) 1 Events (% of mx) CRAMP Control Neutrophil eplete FγR bloking 2, 2, 4 1, 3 1, CpG B-1 ell sup lc Neutrophil sup CRAMP + IRS IRS 94 IFN-α (pg ml 1 ) CRAMP + ells (%) Control +PMA FγR bloking untrete group. () Iniene of ibetes in mie fter neutrophil epletion in mie. Mie were trete with NIMP-R14 mab or isotype ontrol between 7 n 21 of ge. P <. for trete group ompre to ontrol group (n = 12 mie per group). () FACS nlysis of surfe expression of CRAMP by neutrophils fter in vitro ulture. Bone mrrow neutrophils were e to PMA, immobilize immune omplexes (IC) or B-1 IC n ulture for 2 min. In some onitions, neutrophils were preinubte with FγR-bloking mabs. Dt re men vlues ± s.e.m. of four inepenent experiments. P <., eh group ompre to ontrol group. (e) IFN-α proution evlute by ELISA in in vitro pdc ulture in the presene of neutrophil or B-1 ell onitione meium. pdcs were isolte from the spleen of 3-week-ol mie n were inubte with CpG 18, soluble IC or CRAMP (left) or ulture with neutrophil-onitione meium n/or B-1 ell onitione meium (right), ll in the presene or bsene of TLR7 n TLR9 inhibitor (IRS 94 ). Dt re men vlues ± s.e.m. of three inepenent experiments with four poole mie for eh group. P <.. B-1 IC IC This ytokine retes n inflmmtory milieu fvorble for the ibetogeni ptive response n utoimmune ibetes. Asie from the key role of IFN-α sereting pdcs in ntivirl efense, growing boy of eviene rgues for pthogeni role for them in severl utoimmune iseses suh s psorisis n lupus 23. In T1D, iffering results re foun in the literture onerning the role of IFN-α n pdcs in isese pthogenesis. The expnsion of IFN-α prouing pdcs hs been oumente in ptients with T1D roun the time of ignosis 8, n IFN-α tretment of ptients with heptitis infetion or with leukemi hs been shown to inue ibetes evelopment 6,7,24. Reent stuies revele tht mie hrbor high IFN-α levels in PLN before the onset of ibetes 2 n tht pdc epletion prevents T1D evelopment in these mie 26. mie hrbor substntilly more spleni pdcs in omprison to C7BL/6 mie, n pdcs proue more IFN-α fter in vitro stimultion thn C7BL/6 pdcs 27. Proxilly, oler stuies showe tht IFN-α tretment reues ibetes iniene in mie or Biobreeing (BB) rts, strin of rts tht spontneously evelop utoimmune ibetes However, tretment with n inuer of IFN-α n IFN-β proution suh s poly(i:c) n prevent 32,33 or elerte the isese epening on the ose, time, urtion n route of ministrtion (orl, intrperitonel or subutneous). The proinflmmtory role of type I IFNs hs been extensively oumente; IFN-α n IFN-β inue DC mturtion, tivte immunoglobulin-sereting B ells, enhne CRAMP expression by neutrophils n boost effetor T ell responses 37,38. Type I IFNs lso iretly ffet pnreti bet ells by inuing ytokine n hemokine seretion n mjor histoomptibility omplex lss I expression, enhning their suseptibility to ttk from ibetogeni T ells 39. The protetive role of IFN-α tretment remins unler; one hypothesis is tht type I IFN n boost the tivity of regultory T ells It hs been lso reporte in some ibetes moels tht pdcs n exert isese-protetive effets ginst the evelopment of T1D 44,4. However, the preise mehnism leing to T1D prevention n the iret role of pdcs were not preisely resse. In our previous stuies 46,47, we emonstrte tht uring virl infetion of 6-week-ol mie, pdcs n inhibit T1D evelopment by two omplementry pthwys but t ifferent times n lotions. Inee, 1 2 fter infetion, pdcs trnsiently proue IFN-α in the pnres to mpen virl replition, voiing tissue mge, n then migrte to the PLN 7 VOLUME 19 NUMBER 1 JANUARY 213 nture meiine

7 r t i l e s npg 213 Nture Ameri, In. All rights reserve. e ISG Time fter tretment () IFIT week-ol 4-week-ol 4-week-ol + ZVAD ISG1 IRF Time fter tretment () IFIT1 IRF Time fter tretment () IFIT Time fter tretment () IFIT3 f sdna-speifi lgg (A 4 ) b ISG week-ol +SZT (y 1) +SZT (y 1) without pdc IFIT1 + Z-VAD IRF g 3-week-ol + Z-VAD CRAMP + neutrophils ( 1 4 ) IFIT3 + Z-VAD sdna-speifi lgg (A 4 ) h Dibeti mie (%) week-ol + SZT 6-week-ol + SZT CRAMP + neutrophils ( 1 4 ) Figure 6 Initil bet ell eth is require to inue innte ell tivtion n T1D evelopment. (,b) mrna expression for IFN-α inue genes in islets from 6-week-ol mie fter injetion of STZ. In some onitions (b), pdcs were eplete 1 before the injetion of STZ. Dt re men vlues ± s.e.m. of two inepenent experiments with four inepenent mie for eh group. P <.. () DNA-speifi IgG proution in the islet superntnts from 6-week-ol mie 12 h fter STZ injetion. Dt re men vlues ± s.e.m. of two inepenent experiments with two inepenent mie for eh group. P <.. () FACS nlysis of CRAMP expression by pnreti neutrophils from 6-week-ol mie 12 h fter STZ injetion. Absolute number of CRAMP + ells mong CD4 + CD11b + Ly6G + ells is represente. Dt re men vlues ± s.e.m. of four inepenent experiments with two poole mie for eh group. P <.. (e) mrna expression for IFN-α inue genes in islets of 2- or 4-week-ol mie fter Z-VAD tretment. Dt re men vlues ± s.e.m. of two inepenent experiments with four inepenent mie for eh group. P <.. (f) DNA-speifi IgG proution in the islet superntnts from 3-week-ol mie trete or not with Z-VAD. Dt re men vlues ± s.e.m. of two inepenent experiments with two inepenent mie for eh group. P <.. (g) FACS nlysis of CRAMP expression by pnreti neutrophils from 3-week-ol mie trete or not with Z-VAD. Absolute number of CRAMP + ells mong CD4 + CD11b + Ly6G + ells is represente. Dt re men vlues ± s.e.m. of four inepenent experiments with two poole mie for eh group. P <.. (h) Iniene of ibetes in mie fter bloke of poptosis by Z-VAD tretment. Mie were trete with Z-VAD t 7 of ge. P <. for trete group ompre to ontrol group (n = 12 mie per group). Control Z-VAD + SZT 3 n proue trnsforming growth ftor-β, whih inues regultory T ells t lter time points. However, to be effiient, these mehnisms of protetion require the stimultion of invrint nturl killer T ells t the time of infetion. Altogether, these stuies support the notion tht pdcs oul hrbor both pthogeni n protetive funtions uring utoimmune ibetes evelopment, epening on the ourse of the isese, the infetious ontext n the loliztion of the ells. B ells re propose to be involve in T1D pthogenesis s self ntigen presenting ells, but there is little eviene for ibetogeni role for utontiboies 48. Our t revele tht the innte-like CD + B-1 ells hve n s yet unesribe role in T1D initition through the proution of sdna-speifi IgGs n the tivtion of neutrophils n pdcs in the pnres. A previous publition showe tht peritonel B-1 ells prtiipte in T1D evelopment in mie, but their mehnism of tion remine unknown 19. Notbly, the number of irulting CD + B ells is higher in hilren with very reent onset of T1D, s ompre with ptients with long-term isese or ontrols 49. An ltere B ell reeptor signling threshol hs lso been observe in ptients with T1D s ompre to helthy ontrols. High frequenies of CD + B ells hve been reporte in ptients with other utoimmune iseses, suh s Sjögren s isese 1 n rheumtoi rthritis 2. Similrly, mie hrbor n elevte frequeny of self-retive B ells ompre to C7BL/6 n BALB/ mie 3. The role of neutrophils in utoimmunity hs been esribe in smll-vessel vsulitis, SLE, psorisis n mouse moel of skin inflmmtion 21,22,4,. In these stuies, neutrophils t by prouing the DNA-bining pepties LL-37 n CRAMP, whih form omplexes with self DNA n sdna-speifi IgGs, tivting IFN-α proution by pdcs. DNA-bining pepties re relese by neutrophils uring NETosis 2. Our stuy emonstrtes tht neutrophils relese NET-ssoite CRAMP in pnreti islets, whih promotes ibetes initition in mie. We show tht spontneous or STZ-inue bet ell eth is require for the tivtion of IFN-α sereting pdcs probbly by relesing self DNA essentil to the formtion of immune omplexes, s shown in other iseses 6. Previous stuies in mie provie eviene tht bet ell eth nturlly ourring uring the first postntl weeks initites the tivtion of DCs n the priming of utoretive T ells 14. Notbly, these wves of physiologil bet ell eth lso our in pigs 1 n humns 16. Bet ell ebris oul umulte nture meiine VOLUME 19 NUMBER 1 JANUARY

8 A r t i l e s npg 213 Nture Ameri, In. All rights reserve. in the pnres of mie beuse of efet in phgoyti lerne in this geneti bkgroun 17. Previous t inite tht proinflmmtory responses n utoimmune isorers n be eliite by efetive lerne of poptoti ells 7. During infny, the immune system my be provoke with physiologil mssive rebuiling of the pnreti islets vi progrmme ell eth n my initite the ibetogeni proess in n utoimmune geneti bkgroun 8. Finlly, the interply between B-1 ells, neutrophils n pdcs seems to be ommon feture of utoimmune iseses. This new spet of the pthogenesis of utoimmune iseses is likely to open new therpeuti venues irete towr the trgeting of innte immune ells. One of the mjor nees is to set up more effiient tools tht llow the etetion of these primry ibetogeni events. Approhes fouse on pdcs woul be more seletive n tolerble thn trgeting of the IFN-α response, whih n isturb ntivirl responses. Methos Methos n ny ssoite referenes re vilble in the online version of the pper. Note: Supplementry informtion is vilble in the online version of the pper. Aknowlegments We thnk N. Chrles (INSERM UMR-S 699, Pris Dierot University) for sdnaspeifi immunoglobulin ELISA, M. Colonn (Deprtment of Pthology n Immunology, Wshington University Shool of Meiine) for m927 mab, S. Meheri (Biology of Host Prsite Intertions Unit, Psteur Institute) for NIMP- 14 mab, S. Muller (CNRS, Institut e Biologie Moléulire et Cellulire, UPR921) for serum from NZB/W F1 mie, J. Rveth (Lbortory of Moleulr Genetis n Immunology, Rokefeller University) for FγRIV-speifi mab, N. Thieblemont (CNRS UMR 8147, Pris Desrtes University) for My88 / mie n ll for their expertise. We thnk F. Boutillon for tehnil ssistne n the stff of the INSERM U986 mouse fility for help in niml re. Y.S. is supporte by otorl fellowship from the Region Ile e Frne. This work ws supporte by funs from INSERM, Centre Ntionl e l Reherhe Sientifique, ANR-- PCOD9-1, ANR-9-GENO-23 n Lbex INFLAMEX to A.L. A.L. is reipient of n APHP-CNRS Contrt Hospitlier e Reherhe Trnsltionelle. AUTHOR CONTRIBUTIONS J.D. initite n le the whole projet, oorinte with ifferent investigtors, esigne n performe experiments, nlyze the t n wrote the mnusript; Y.S. esigne n performe experiments, nlyze the t n wrote the mnusript; L.F. i onfol mirosopy experiments, nlyze the t n wrote the mnusript; L.B. provie tehnil ssistne; B.A. n F.B. provie intelletul input n key regents for neutrophil n pdc nlysis; n A.L. ws responsible for projet plnning, t nlysis, isussion n writing. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Publishe online t Reprints n permissions informtion is vilble online t reprints/inex.html. 1. Coppieters, K.T. et l. Demonstrtion of islet-utoretive CD8 T ells in insuliti lesions from reent onset n long-term type 1 ibetes ptients. J. Exp. Me. 29, 1 6 (212). 2. Lehuen, A., Din, J., Zone, P. & Cooke, A. Immune ell rosstlk in type 1 ibetes. Nt. Rev. 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Bet-ell prolifertion n poptosis in the eveloping norml humn pnres n in hyperinsulinism of infny. Dibetes 49, (2). 17. O Brien, B.A. et l. A efiieny in the in vivo lerne of poptoti ells is feture of the mouse. J. Autoimmun. 26, (26). 18. Bumgrth, N. The ouble life of B-1 ell: self-retivity selets for protetive effetor funtions. Nt. Rev. Immunol. 11, (211). 19. Kenll, P.L., Woowr, E.J., Hulbert, C. & Thoms, J.W. Peritonel B ells govern the outome of ibetes in non-obese ibeti mie. Eur. J. Immunol. 34, (24). 2. Mntovni, A., Csstell, M.A., Costntini, C. & Jillon, S. Neutrophils in the tivtion n regultion of innte n ptive immunity. Nt. Rev. Immunol. 11, (211). 21. Lne, R. et l. Neutrophils tivte plsmytoi enriti ells by relesing self- DNA peptie omplexes in systemi lupus erythemtosus. Si. Trnsl. Me. 3, 73r19 (211). 22. Gri-Romo, G.S. et l. Netting neutrophils re mjor inuers of type I IFN proution in peitri systemi lupus erythemtosus. Si. Trnsl. Me. 3, 73r2 (211). 23. Lne, R. & Gilliet, M. Plsmytoi enriti ells: key plyers in the initition n regultion of immune responses. Ann. NY A. Si. 1183, (21). 24. Fbris, P. et l. Type 1 ibetes mellitus in ptients with hroni heptitis C before n fter interferon therpy. Aliment. Phrmol. Ther. 18, 49 8 (23). 2. Li, Q. et l. Interferon-α initites type 1 ibetes in nonobese ibeti mie. Pro. Ntl. A. Si. USA 1, (28). 26. Li, Q. & MDevitt, H.O. The role of interferon α in initition of type I ibetes in the mouse. Clin. Immunol. 14, 3 7 (211). 27. Peng, R.H., Pek, E., Xi, C.Q., Tennyson, N. & Clre-Slzler, M.J. Heightene interferon-α/β response uses myeloi ell ysfuntion n promotes T1D pthogenesis in mie. Ann. NY A. Si. 179, (26). 28. Sobel, D.O. & Ahvzi, B. α-interferon inhibits the evelopment of ibetes in mie. Dibetes 47, (1998). 29. Sobel, D.O. et l. Low ose poly I:C prevents ibetes in the ibetes prone BB rt. J. Autoimmun. 11, (1998). 3. Bro, S.A., Mlone, M., Drn, S., Ppoll, M. & Nelson, L. Ingeste interferon α suppresses type I ibetes in non-obese ibeti mie. Dibetologi 41, (1998). 31. Tnk-Ktok, M. et l. Orl use of interferon-α elys the onset of insulin-epenent ibetes mellitus in nonobese ibetes mie. J. Interferon Cytokine Res. 19, (1999). 32. Serreze, D.V., Hmguhi, K. & Leiter, E.H. Immunostimultion irumvents ibetes in /Lt mie. J. Autoimmun. 2, (1989). 33. Zhou, R., Wei, H. & Tin, Z. NK3-like NK ells re involve in protetive effet of polyinosini-polyytiyli i on type 1 ibetes in nonobese ibeti mie. J. Immunol. 178, (27). 34. Ewel, C.H., Sobel, D.O., Zeligs, B.J. & Bellnti, J.A. Poly I:C elertes evelopment of ibetes mellitus in ibetes-prone BB rt. Dibetes 41, (1992). 3. Sobel, D.O. et l. Poly I:C inues evelopment of ibetes mellitus in BB rt. Dibetes 41, 1 2 (1992). 36. Hung, X., Hultgren, B., Dybl, N. & Stewrt, T.A. Islet expression of interferon-α preees ibetes in both the BB rt n streptozotoin-trete mie. Immunity 1, (1994). 37. Theofilopoulos, A.N., Kono, D.H., Beutler, B. & Bl, R. Intrellulr nulei i sensors n utoimmunity. J. Interferon Cytokine Res. 31, (211). 38. Desmet, C.J. & Ishii, K.J. Nulei i sensing t the interfe between innte n ptive immunity in vintion. Nt. Rev. Immunol. 12, (212). 39. Lng, K.S. et l. Toll-like reeptor enggement onverts T-ell utoretivity into overt utoimmune isese. Nt. Me. 11, (2). 4. Aune, T.M. & Piere, C.W. Ativtion of suppressor T-ell pthwy by interferon. Pro. Ntl. A. Si. USA 79, (1982). 72 VOLUME 19 NUMBER 1 JANUARY 213 nture meiine

9 r t i l e s 41. Mujtb, M.G., Soos, J.M. & Johnson, H.M. CD4 T suppressor ells meite interferon τ protetion ginst experimentl llergi enephlomyelitis. J. Neuroimmunol. 7, 3 42 (1997). 42. Teige, I., Liu, Y. & Isszeh-Nviks, S. IFN-β inhibits T ell tivtion pity of entrl nervous system APCs. J. Immunol. 177, (26). 43. González-Nvjs, J.M., Lee, J., Dvi, M. & Rz, E. Immunomoultory funtions of type I interferons. Nt. Rev. Immunol. 12, (212). 44. Kre, H. et l. Tretment with grnuloyte olony-stimulting ftor prevents ibetes in mie by reruiting plsmytoi enriti ells n funtionl CD4 + CD2 + regultory T-ells. Dibetes 4, (2). 4. Sxen, V., Onr, J.K., Mgnusen, A.F., Munn, D.H. & Ktz, J.D. The ounterviling tions of myeloi n plsmytoi enriti ells ontrol utoimmune ibetes in the nonobese ibeti mouse. J. Immunol. 179, 41 3 (27). 46. Din, J. et l. Virl infetion prevents ibetes by inuing regultory T ells through NKT ell plsmytoi enriti ell interply. J. Exp. Me. 28, (211). 47. Din, J. et l. NKT ell-plsmytoi enriti ell oopertion vi OX4 ontrols virl infetion in tissue-speifi mnner. Immunity 3, (29). 48. Mllone, R. & Brezr, V. To B or not to B: (nti)boies of eviene on the rime sene of type 1 ibetes? Dibetes 6, (211). 49. De Filippo, G. et l. Inrese CD + CD19 + B lymphoytes t the onset of type 1 ibetes in hilren. At Dibetol. 34, (1997).. Hbib, T. et l. Altere B ell homeostsis is ssoite with type I ibetes n rriers of the PTPN22 lleli vrint. J. Immunol. 188, (212). 1. Shiri, T., Ok, T. & Hirose, S. Geneti regultion of CD + B ells in utoimmune isese n in hroni lymphoyti leukemi. Ann. NY A. Si. 61, 9 26 (1992). 2. Burstero, S.E., Csli, P., Wiler, R.L. & Notkins, A.L. Monoretive high ffinity n polyretive low ffinity rheumtoi ftors re proue by CD + B ells from ptients with rheumtoi rthritis. J. Exp. Me. 168, (1988). 3. Thoms, J.W., Kenll, P.L. & Mithell, H.G. The nturl utontiboy repertoire of nonobese ibeti mie is highly tive. J. Immunol. 169, (22). 4. Kessenbrok, K. et l. Netting neutrophils in utoimmune smll-vessel vsulitis. Nt. Me. 1, (29).. Guiui, C. et l. Autoimmune skin inflmmtion is epenent on plsmytoi enriti ell tivtion by nulei is vi TLR7 n TLR9. J. Exp. Me. 27, (21). 6. Kono, H. & Rok, K.L. How ying ells lert the immune system to nger. Nt. Rev. Immunol. 8, (28). 7. Hnym, R. et l. Autoimmune isese n impire uptke of poptoti ells in MFG-E8 efiient mie. Siene 34, (24). 8. To, J.A. Etiology of type 1 ibetes. Immunity 32, (21). npg 213 Nture Ameri, In. All rights reserve. nture meiine VOLUME 19 NUMBER 1 JANUARY

10 npg 213 Nture Ameri, In. All rights reserve. ONLINE METHODS Mie n tretments. Femle BALB/, C7BL/6, n My88 / 9 mie were bre n house in speifi pthogen free onitions. For pdc n neutrophil epletion, WT mie were injete i.p. on ys 14 n 21 fter birth, respetively, with µg of epleting m927 mab per mouse (from M. Colonn) or 2 µg of epleting NIMP-R14 mab per mouse (from S. Meheri). For inhibition of IFN-α proution by pdcs, WT mie were injete i.p. on ys 14 n 21 fter birth, with 2 µg of nti Sigle-H mab in 2 µl of PBS (ebio44, ebiosiene). The sme tretment ws performe with respetive isotype ontrol. Inhibition of TLR7 n TLR9 ws performe using 1 µg per mouse of IRS 94 injete i.p. on ys 14 n 21 fter birth. TLR9 tivtion ws performe by intrvenous injetion with 1 µg per mouse of CpG 18 (Invivogen) on y 14 fter birth. For B-1 ell epletion we use protool previously esribe with some moifitions 19,6. Briefly, 1-week-ol mie were trete with i.p. injetion of µl wter every 2 over 14 ; PBS ws injete s negtive ontrol. Apoptosis inhibition ws performe using Z-VAD (Sigm) s previously esribe 14. Mie were injete i.p. with 1 µl per mouse of Z-VAD t 1 µm on ys 14 n 21 fter birth. Bet ell eth ws inue by one i.p. injetion with 1.6 mg of STZ (Sigm) per mouse. The syntheti mouse theliiin peptie CRAMP (Innovgen) ws injete i.p. in 4-week-ol mie, 2 µg per mouse. This stuy ws pprove by the lol ethis ommittee on niml experimenttion of Pris Desrtes University (CEEA34.JD.46.12). Preprtion of B-1 ell onitione meium. B-1 ells ( 1 3 in 1 µl) sorte s CD + CD19 + ells from pnreti islets of ten poole 3-week-ol mie were inubte in RPMI omplete meium, n ell-free superntnt ws reovere fter 7 n store t 8 C before ition to neutrophil or pdc in vitro ultures. Preprtion of plte-boun immune omplexes. Immobilize immune omplexes were prepre s follows. Briefly, immune omplex overe surfes were prepre by inubting 96-well Mxisorp F96 (Nun Interntionl) ELISA pltes with 2 µg ml 1 genomi sdna (Promeg) in PBS for 12 h t 37 C, followe by bloking with 1% BSA in PBS for 1 h n further 1-h inubtion with nti-sdna IgG2b mab (DNA11-M, Gentur) t 1 µg/ml. Alterntively, to form B-1 immune omplexes, nti-sdna IgG2b mabs were reple by pnreti B-1 ell onitione meium. Prllel wells prepre without the ition of nti-sdna IgGs serve s ontrols. Neutrophil stimultion. Neutrophil tivtion by immobilize immune omplexes (with nti-sdna IgG2b mab) or B-1 immune omplexes ws hieve by plting the ells on the immune omplex ote surfes without ny itionl stimulus. Neutrophils (2 1 in 1 µl) sorte s Ly6G + CD11b + ells from bone mrrow of 3-week-ol mie were inubte in omplete RPMI. In some onitions, neutrophils were preinubte uring 1 h t 37 C with mix of bloking nti-fγrii/iii (lone 24G2, BD) n nti-fγriv mabs (lone 9E9, from J. Rveth) t 1 µg ml 1. Neutrophils were inubte for 2 min for evlution of CRAMP expression by FACS or for 6 h to generte neutrophilonitione meium to stimulte pdcs in vitro. In vitro pdc tivtion by neutrophils n B-1 ells. Purifie spleni pdcs from 3-week-ol mie (1 1, Miltenyi kit) were stimulte in 1 µl omplete RPMI with neutrophil-onitione meium (1:3 vol/vol) n/or B-1 ell-onitione meium (1:3 vol/vol) in the presene or bsene of TLR7/9 inhibitor (IRS 94 1 µg ml 1 ). As positive ontrol, pdcs were inubte with CpG 18 ( µg ml 1 ), soluble immune omplexes (3 µg/ml sdna + 1 µg ml 1 nti-sdna IgGs, inubte 3 min t room temperture) or CRAMP ( µg ml 1 ). After 36 h of ulture, superntnts were reovere n IFN-α proution ws mesure by ELISA (PBL). Dibetes ignosis. Mie were teste every y for ibetes. Overt ibetes ws efine s two positive urine gluose tests, onfirme by bloo sugr level of >2 mg L 1. The Glukotest kit ws purhse from Rohe. Preprtion of pnreti islets. Mie were kille n pnret were perfuse with 3 ml of solution of ollgense P (1. mg ml 1, Rohe), then issete free from surrouning tissues. Pnret were then igeste t 37 C for 1 min. Digestion ws stoppe by ing HBSS % FCS followe by extensive wshes. Islets were then purifie on isontinuous Fioll grient n isrupte ing 1 ml of ell issoition buffer (GIBCO) for 1 min t 37 C. After nother wsh, ells were resuspene, ounte n use. Flow ytometry. Cell suspensions were prepre from vrious tissues n were stine t 4 C in PBS ontining 2% FCS n.% EDTA fter FγRII/III bloking. Surfe stining ws performe with ntiboies ll from BD Phrmingen or ebiosiene (nti-cd11 (lone N418), nti-cd11b (lone M1/7), nti-ly6g (lone 1A8), nti-cd4 (lone 3-F11), nti-cd19 (lone 1D3), nti-tcrβ (lone H7-97), nti-cd8 (lone 3-6.7), nti IFN-γ (lone XMG1.2), nti-cd (lone 3-7.3), nti-cd21 (lone 8D9), nti-cd1 (lone 1B1)) exept m927 mab n NIMP-R14 mab, whih were onjugte in our lbortory n were use t the onentrtion of 1 μg ml 1. For CRAMP surfe stining, ells were stine sequentilly with rbbit nti-cramp pab (from B. Agerberth, 1 µg ml 1 ) n nti-rbbit-pe pab ( , ebiosienes,. µg ml 1 ). For intrellulr IFN-α stining, the ell suspension ws inubte 4 h t 37 C with Brefelin A. After fixtion n permebiliztion (BD Fix&Perm), ells were first stine with nti IFN-α (lone RMMA-1, 1 µg ml 1 PBL); then the surfe stining ws performe. For NRP-V7 tetrmer (from the Ntionl Institutes of Helth tetrmer ore fility) stining, ells were stine with tetrmers for 4 min t room temperture followe by surfe stining for 1 min t 4 C. De ells were exlue using Fixble Vibility Dye stining (ebiosiene). Stine ells were nlyze n/or sorte on FACS Ari flow ytometer (BD Biosienes). Histology. Pnreses from mie were ollete, embee in tissuefreezing meium (Jung) n store t 2 C. Tissues were ut into -µm setions in ryostt (Lei). Frozen setions were fixe in ol etone. Stining with primry ntiboies ws performe for 1 h with the following ntiboies: nti-ly6g (lone NIMP-R14, from S. Meheri, 1 µg ml 1 ), nti-b22 (lone RA3-6B2, from BD, 1 µg ml 1 ) n nti-cd11-pe (lone N418, from ebiosienes, 1 µg ml 1 ) mabs n insulin (A64, Dko, 1 µg ml 1 ) or CRAMP (from B. Agerberth, 1 µg ml 1 ) pabs. After wshing, seon-step regents were pplie: nti rt biotin (A117, Invitrogen, 1 µg ml 1 ) or nti guine pig A488 (A1173, Invitrogen, 1 µg ml 1 ) pabs. If neessry, thir-step regent ws pplie: streptviin-pe or streptviin-apc (ebiosiene, 1 µg ml 1 ). Nulei were stine with Hoehst (H1399, Invitrogen, µg ml 1 ). Controls with isotype ontrol stining were negtive (t not shown). Mirosopy. NET proution by neutrophils ws ssesse s follows. Briefly, pnreti or bone mrrow neutrophils were seee on poly-l-lysine ote glss (Polysine, Kinler GmhH) t onentrtion of 1 6 ells per ml for 1 h t 37 C in RPMI with 2% FCS. Then, ells were stine with nti Ly-6G onjugte ntiboy (lone NIMP-R14) for 1 min on ie n immeitely fixe in 2% prformlehye n ounterstine for DNA with SYTOX Green (Invitrogen, 1 nm). In some experiments, fter fixtion, neutrophils were further stine with nti-cramp pab (from B. Agerberth, 1 µg ml 1 ) n then ounterstine for DNA with SYTOX Green. Slies were nlyze using Lei TCS SP AOBS onfol mirosope. Quntittive PCR. Cells were ollete in lysis buffer (RLT, Qigen) buffer with 1% of β-merptoethnol. mrna ws isolte using RNesy Mini Kit (Qigen) n reverse trnsribe with Supersript III (Invitrogen). Quntittive PCR ws performe with SYBR Green (Rohe) n nlyze with LightCyler 48 (Rohe). Dt were normlize to Gph housekeeping gene. Detetion of IFN- or nti-sdna IgGs in the pnreti islets. Pnreti islets were reovere by hnpiking uner polrize mirosope without ny prior ensity seprtion. Fifty islets from the sme mouse were then ulture for 48 h in 1 µl of RPMI omplete meium. Superntnts were use for IFN-α-quntifition by ELISA (PBL) or for nti-sdna IgG quntifition by ELISA (Clbiohem). Sttistil nlyses. Dibetes iniene ws plotte oring to the Kpln- Meier metho. Inienes between groups were ompre with the log-rnk test. For other experiments, omprison between mens ws performe using the non-prmetri Mnn-Whitney U-test. Reporte vlues re mens ± s.. s inite. P vlues <. were onsiere sttistilly signifint. All t were nlyze using GrphP Prism v softwre. nture meiine oi:1.138/nm.342