citrullinated glucose- regulated protein 78 is an autoantigen ......citrullinated glucose-regulated...

Dieter Rondas,1 Inne Crèvecoeur,1 Wannes D’Hertog,1 Gabriela Bomfim Ferreira,1 An Staes,2,3

Abhishek D. Garg,4 Decio L. Eizirik,5 Patrizia Agostinis,4 Kris Gevaert,2,3 Lut Overbergh,1 andChantal Mathieu1

Citrullinated Glucose-Regulated Protein 78 Is anAutoantigen in Type 1 DiabetesDiabetes 2015;64:573–586 | DOI: 10.2337/db14-0621

Posttranslational modifications of self-proteins play a sub-stantial role in the initiation or propagation of the autoim-mune attack in several autoimmune diseases, but theircontribution to type 1 diabetes is only recently emerging. Inthe current study, we demonstrate that inflammatory stress,induced by the cytokines interleukin-1b and interferon-g,leads to citrullination of GRP78 in b-cells. This is coupledwith translocation of this endoplasmic reticulum chaperoneto the b-cell plasma membrane and subsequent secretion.Importantly, expression and activity of peptidylargininedeiminase 2, one of the five enzymes responsible for citrul-lination and a candidate gene for type 1 diabetes in mice, isincreased in islets from diabetes-prone nonobese diabetic(NOD) mice. Finally, (pre)diabetic NOD mice have autoanti-bodies and effector T cells that react against citrullinatedGRP78, indicating that inflammation-induced citrullinationof GRP78 in b-cells generates a novel autoantigen in type 1diabetes, opening new avenues for biomarker developmentand therapeutic intervention.

Type 1 diabetes is an autoimmune endocrine disease inwhich loss of central and peripheral tolerance toward b-cellantigens is proposed as the underlying mechanism. How-ever, b-cells themselves also contribute to trigger and/orpropagate the autoimmune attack, leading to a dialoguewith immune infiltrating cells that may amplify local in-flammation (insulitis) in genetically predisposed individuals(1). Insulin (or proinsulin) is probably the primary autoan-tigen in type 1 diabetes (2), but antigen spreading occurs as

the autoimmune assault progresses, with autoantibodiesappearing against several non–b-cell-specific autoantigens,such as GAD65 (3), islet antigen 2 (IA2) (4), heat shockprotein 60 (HSP60) (5), and chromogranin A (ChgA) (6).

During insulitis, local production of inflammatory media-tors, such as the cytokines interleukin (IL)-1b and interferon-g(IFNg), triggers b-cell oxidative and endoplasmic reticulum(ER) stress. These, and other signals, may lead to alternativesplicing and misfolding of b-cell proteins as well as posttrans-lational modifications (PTMs) (7–9). In other autoimmunediseases, like rheumatoid arthritis (RA), multiple sclerosis,and celiac disease, such posttranslationally modified proteinsbehave as autoantigens (10,11), but their relevance in type 1diabetes is only starting to be explored (12–15).

Building on our previous observation that the ERchaperone 78 kDa glucose-regulated protein (GRP78; alsonamed binding immunoglobulin protein [BiP]) is post-translationally modified in cytokine-exposed insulin-producing INS-1E cells (9), we now identify this modificationas citrullination and show that inflammation-inducedcitrullinated GRP78 is an autoantigen in type 1 diabetes.These findings suggest a novel role for GRP78 beyond itswell-known function in the ER, leading to the loss oftolerance to b-cells in type 1 diabetes.

RESEARCH DESIGN AND METHODS

Western blotting, mass spectrometry, GRP78 cloning,expression, purification, and in vitro citrullination areavailable in the Supplementary Data.

1Laboratory for Clinical and Experimental Endocrinology, KU Leuven, Leuven,Belgium2Department of Medical Protein Research, VIB, Ghent, Belgium3Department of Biochemistry, Ghent University, Ghent, Belgium4Laboratory for Cell Death Research and Therapy, KU Leuven, Leuven, Belgium5Laboratory of Experimental Medicine and Université Libre de Bruxelles Center forDiabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium

Corresponding author: Lut Overbergh, [email protected].

Received 18 April 2014 and accepted 28 August 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0621/-/DC1.

D.R. and I.C. contributed equally to this study.

L.O. and C.M. contributed equally to this study.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 64, February 2015 573

IMMUNOLOGYAND

TRANSPLANTATIO

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Reagents and AntibodiesPrimary antibodies were as follows: mouse anti-poly(ADP-ribose) monoclonal antibody (mAb) (Enzo Life Sciences,Antwerp, Belgium), rabbit anti-GRP78 and anti-CHOPpolyclonal antibody (pAb) (Santa Cruz Biotechnology,Santa Cruz, CA), rabbit anti-eIF2a pAb, anti–p-eIF2a(Ser51) pAb, anti-PERK mAb and anti–p-PERK(Thr980)mAb (Cell Signaling, Beverly, MA), and mouse anti-actinmAb (Sigma-Aldrich, Diegem, Belgium) for Western blot-ting; rabbit anti–b-catenin mAb (Cell Signaling Technol-ogy) and mouse anti-GRP78 pAb (Abcam, Cambridge,U.K.) for immunocytochemistry. Anti–cit(510)-GRP78was raised in rabbits against the following peptides: C-aminohexanoic acid-IDVNGIL[citrulline]VTAEDKG-amideand acetyl-IDVNGIL[citrulline]VTAEDKG-aminohexanoicacid-C-amide, through five subsequent injections (21stCentury Biochemicals). Specificity of the antibody wasconfirmed by dot blot against the citrullinated peptideand its native counterpart. Secondary antibodies were asfollows: donkey anti-rabbit horseradish peroxidase, don-key anti-mouse Alexa Fluor 488, and donkey anti-rabbitAlexa Fluor 555 (Invitrogen, Merelbeke, Belgium). Oval-bumin and rabbit peptidylarginine deiminase (PAD) en-zyme were from Sigma-Aldrich.

Cell Lines and Culture ConditionsRat INS-1E cells, a gift from Prof. Wollheim (CMU,Geneva, Switzerland), were cultured as previously de-scribed (9). INS-1E cells were exposed to recombinantrat IFNg (500 units/mL; R&D Systems), recombinanthuman IL-1b (10 units/mL; R&D Systems), thapsigargin(Tg) (15 and 50 nmol/L; Sigma-Aldrich), tunicamycin(Tn) (2 and 5 mg/mL; Sigma-Aldrich), high glucose(HG) (25 mmol/L; Sigma-Aldrich), and palmitate (Pa)(0.5 mmol/L; Sigma-Aldrich). Mouse MIN6 cells, a giftfrom Dr. Miyazaki (Osaka University, Osaka, Japan),were cultured in DMEM (Invitrogen) containing 15%(volume for volume) FCS, 100 units/mL penicillin, 100mg/mL streptomycin, and 70 mmol/L b-mercaptoethanol.MIN6 cells were exposed to recombinant mouse IFNg (500units/mL; R&D Systems) and human recombinant IL-1b(10 units/mL).

Apoptosis MeasurementsThe percentage of living and apoptotic cells was assessedas previously described (9).

MiceC57Bl/6 mice were obtained from Harlan Laboratories(Horst, the Netherlands) and nonobese resistant (NOR)mice from The Jackson Laboratory (Bar Harbor, ME).Nonobese diabetic (NOD) mice have been inbred in ouranimal facility since 1989 and are kept under semi-barrier conditions. For all experiments, a mix of male andfemale mice was used. All animal manipulations werein compliance with the principles of laboratory care andapproved by the Institutional Animal Ethics Committee ofKU Leuven.

Islet Isolation and CulturePancreatic islets were isolated from 3- or 10-week-oldC57Bl/6, NOD, and NOR mice. Islet isolation and culturewere performed as previously described (16). C57Bl/6islets were exposed to recombinant mouse IFNg (1,000units/mL) and recombinant human IL-1b (50 units/mL).

ImmunofluorescenceImmunofluorescence on isolated islets was performed aspreviously described (17). Fixed INS-1E cells or sectionedislets were incubated with primary antibody in 1% BSAfor 1 h, followed by four washes in PBS before incubationwith the secondary antibody in 1% BSA for another hour.Nuclei were detected with DNA-binding dye DRAQ5TM(Biostatus Ltd., Leicestershire, U.K.). Specificity was con-firmed by including negative controls with secondary anti-bodies alone. INS-1E samples were observed under a ZeissLSM 510 microscope using a Plan-Neofluar 403/1.3 oilDIC lens. Images were acquired and processed using LSM510 software (Carl Zeiss AG, Jena, Germany). Mouse isletsections were observed under a Nikon Eclipse Ti micro-scope using a Plan-Fluor 403/0.75 DIC lens, and imageswere acquired and processed using Nis-Elements Viewer4.20 software (Nikon Instruments Inc.).

Cell Surface BiotinylationINS-1E or MIN6 cells were incubated with the indicatedstressors for 12–15 h and then treated as previously de-scribed (18).

GRP78 ELISATo determine GRP78 concentration in conditioned mediafrom INS-1E cells, the GRP78 ELISA kit (Enzo LifeSciences, Antwerp, Belgium) was used, according to themanufacturer’s protocol.

Two-Dimensional Gel Electrophoresis AnalysisTwo-dimensional gel electrophoresis (2D-GE) analysis wasperformed as previously described (9).

Quantitative RT-PCRQuantitative RT-PCR was performed as previously de-scribed (19).

Measurement of PAD ActivityTo determine PAD activity levels in islets and pancreatafrom C57Bl/6, NOR, and NOD mice, the antibody-basedassay for PAD activity (ABAP) (ModiQuest Research, Oss,the Netherlands) was used, according to the manufac-turer’s protocol.

Autoantibody ELISA Against Native or CitrullinatedGRP78Serum autoantibodies against two native and citrullinatedGRP78 peptides (amino acids 500–519 TFEIDVNGILRVTAEDKGTG and amino acids 295–314 AKRALSSQHQARIEIESFYE) were determined by ELISA as previously de-scribed (20). For the citrullinated forms, arg510 or arg306were replaced by citrulline (synthesized by PolyPeptide Lab-oratories, Strasbourg, France).

574 GRP78 Is an Autoantigen in Type 1 Diabetes Diabetes Volume 64, February 2015

IFNg MeasurementSplenocytes from 10-week-old and new-onset diabeticNOD mice and age-matched C57Bl/6 and NOR mice werecultured in flat-bottom 96-well plates (1 3 106 cells/well)in the absence or presence of the indicated stimuli. IFNglevels were measured in cell culture supernatant after48 h of culture, using Meso Scale Discovery technology(Rockville, MD), according to the manufacturer’s protocol.

StatisticsStatistical analyses of data were performed using GraphPadPrism 6 (GraphPad Software, San Diego, CA). Data areexpressed as means6 SEM and were analyzed by a Kruskal-Wallis test followed by a Dunn multiple comparisons test,unless stated otherwise in the figure legend. P values ,0.05were considered significant.

RESULTS

GRP78 Is Translocated to the b-Cell Surface UponCytokine ExposureWe investigated cytokine-mediated regulation of GRP78at both transcriptional and translational levels after 12–15 hexposure and at cytokine concentrations that inducedonly minor apoptosis (Fig. 1A). No significant increasein GRP78 mRNA (Fig. 1B) and total protein expression(Fig. 1C, center panel, and Fig. 1D) were observed ascompared with control INS-1E cells. However, a detailedanalysis of the plasma membrane fraction revealed a lim-ited presence of GRP78 at the cell surface under basalconditions, which was increased upon IL-1b+IFNg expo-sure, but not upon single cytokine exposure (Fig. 1C, bot-tom panel, and Fig. 1E). This was confirmed in MIN6 cellsafter 12 h treatment (Fig. 1F), a preapoptotic time point(Fig. 1G). In parallel with the increased plasma membranetranslocation of GRP78, we also observed an increase inGRP78 secretion upon cytokine exposure of INS-1E cells(Fig. 1H).

These observations were confirmed by immunocyto-chemistry showing increased GRP78 staining on theplasma membrane of nonpermeabilized cytokine-exposedINS-1E cells (Fig. 2A), and a clear increase in colocaliza-tion between GRP78 and the plasma membrane markerb-catenin in cytokine-exposed INS-1E cells (Fig. 2B). Im-portantly, a similar membrane translocation was observedin cytokine-exposed C57Bl/6 mouse islets (Fig. 2C), againat an early and preapoptotic time point (Fig. 1I). Thus,surface expression of GRP78 upon cytokine exposure isnot solely taking place in clonal b-cell models but also inprimary b-cells, and is not a consequence of nonspecificchanges associated with cell death, increasing the rele-vance of these findings.

GRP78 Is Translocated to the Plasma Membrane UponChemical ER Stress, but Not Upon Metabolic StressCytokine exposure of INS-1E cells and primary rat, mouse,and human b-cells is known to induce ER stress–dependentapoptosis (21–25). We further evaluated the contribution

of ER stress to the observed cytokine-induced GRP78 mem-brane translocation by investigating the effect of the chem-ical ER stressors Tg and Tn. Cytokines (12–15 h) induceda clear activation of the PERK-eIF2a-CHOP pathway, at thedose tested (Supplementary Fig. 1). For Tg, there wasa dose-response effect both in terms of apoptosis (Fig.3A) and ER stress induction (Fig. 3B and SupplementaryFig. 1), with minor ER stress at 15 nmol/L but a clear in-duction of the PERK-eIF2a-CHOP branch at 50 nmol/L Tg,toward levels similar to those observed upon cytokine ex-posure. Of note, Xbp1 splicing was induced by Tg at 50nmol/L, which was not the case upon cytokine exposure(Supplementary Fig. 1B). INS-1E cells were more resistantto Tn, with less apoptosis (Fig. 3A) and an intermediateactivation of PERK-eIF2a-CHOP and Xbp1 splicing, both at2 and 5 mg/mL (Supplementary Fig. 1), as compared withthe higher doses of Tg and cytokines. As illustrated in Fig.3C–E, 15 nmol/L Tg and 2 mg/mL Tn did not induce mem-brane translocation of GRP78. Importantly, the higherdose of 50 nmol/L Tg led to a clear membrane transloca-tion, which was paralleled by more marked expression ofER stress markers (see above). A similar effect was ob-served with 5 mg/mL Tn, although less pronounced.

Finally, upon metabolic stress (Pa [0.5 mmol/L] or thecombination of HG [25 mmol/L] + Pa), most of the ERstress markers were increased, although the increase inCHOP mRNA and protein was less marked than thatobserved with chemical ER stressors or cytokines (Sup-plementary Fig. 1). On the other hand, the total andmembrane-associated GRP78 protein levels remained un-altered (Fig. 3F–H). Taken together, these findings indi-cate that GRP78 membrane translocation occurs uponinflammation- and severe chemical–induced ER stress,but not upon metabolic stress.

GRP78 Is Posttranslationally Modified in Cytokine-Exposed b-CellsBesides the above described membrane translocation ofGRP78, we observed extensive PTM of GRP78 uponcytokine exposure of INS-1E cells (Fig. 4A and previouslydescribed [9]) and C57Bl/6 mouse islets (Fig. 4B). Thiswas also the case, although to a lesser degree, for IL-1bor IFNg exposure alone (Fig. 4A). Of particular interest,2D-GE analysis of the plasma membrane fraction of con-trol and cytokine-exposed INS-1E (Fig. 4C) and MIN6cells (Fig. 4D) not only demonstrated the presence ofthe three different cytokine-responsive GRP78 isoforms,but also showed a cytokine-mediated upregulation of nu-merous acidic GRP78 isoforms as compared with controlcells. On the other hand, metabolic- and chemical-induced(both at low and high concentrations) ER stress did notinduce detectable PTMs of GRP78 (Fig. 4E), confirmingthe specific effects of proinflammatory cytokines.

GRP78 Is Citrullinated in Cytokine-Exposed INS-1ECellsIn order to identify the nature and site of cytokine-induced PTMs in GRP78, we subjected the three different

diabetes.diabetesjournals.org Rondas and Associates 575

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GRP78 isoforms observed in INS-1E cells to massspectrometry (MS) analysis. Sequence coverage rangedfrom 62.54 to 74.46% (n = 2). When comparing theresulting peptide profiles of isoform 1 (I1) versus isoform2 (I2) and I1 versus isoform 3 (I3), one specific peptide,VTAEDKGTGNK (AA511–521), was identified exclusivelyin I1. This was confirmed by a quantitative differentialanalysis using trypsin digestion in combination with differ-ential N-butyrylation and endoproteinase Lys-C (endo Lys-C)

digestion combined with differential N-propionylation(Fig. 5A and B). As the peptide upstream of VTAEDKGTGNKcould not be identified using both methods, we hy-pothesized the presence of a PTM in this region, possi-bly on Arg510, which would prevent tryptic digestion inI2 and I3, thereby rendering the resulting peptide(AA493–521) too long for retrieval and detection byMS. Of note, also a second peptide of interest wasfound, which was identified almost exclusively in I2 and

Figure 1—Cytokine exposure induces membrane translocation of GRP78 in insulin-secreting INS-1E and MIN6 cells. A: Apoptosis levelsin INS-1E cells exposed for 12–15 h (white bars) or 24 h (black bars) to IL-1b (10 units/mL) and/or IFNg (500 units/mL) (n = 3–8independent experiments, each biological replicate is the mean of two technical duplicates). B: GRP78 mRNA expression in INS-1Ecells treated for 12–15 h as described above (n = 7, each biological replicate is the mean of two technical duplicates). C: Total andplasma membrane-associated (PM) GRP78 protein expression in INS-1E cells exposed to the indicated stressors. A representativeWestern blot from four independent experiments is shown. D and E: The relative intensities of the different protein bands were quantifiedby densitometry and expressed as a ratio (n = 4). F: Total and PM GRP78 protein levels in control and cytokine-exposed MIN6 cells. Arepresentative Western blot from two independent experiments is shown. G: Apoptosis levels in MIN6 cells exposed for 12 h (white bars)and 24 h (black bars) to IL-1b and IFNg (n = 5–8 independent experiments, each biological replicate is the mean of two technicalduplicates). H: GRP78 protein concentration in the culture medium of control and cytokine-exposed INS-1E cells. Data are expressed asmeans 6 SEM and were analyzed by a two-tailed paired Student t test (n = 10 independent experiments). I: Apoptosis levels in C57Bl/6mouse islets exposed for 24 h (white bars) and 72 h (black bars) to IL-1b (50 units/mL) and IFNg (1,000 units/mL) (n = 3–5 independentexperiments, each biological replicate is the mean of two technical duplicates). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001vs. respective control (Ctrl).


Figure 2—Microscopic imaging of cytokine-induced GRP78 membrane translocation in INS-1E cells and mouse islets of Langerhans. A:Unpermeabilized control and cytokine-exposed INS-1E cells (15 h) were stained for GRP78 (green), and the nuclei (blue) were stained withHoechst 33342. Images shown are representative for two independent experiments. Bar, 10 mm. Control and cytokine-exposed INS-1Ecells (15 h) (B) and intact mouse islets (24 h) (C ) were stained for GRP78 (green) and b-catenin (red). Nuclei (blue) were stained with Hoechst33342. Images shown are representative for three independent experiments. Scale bars, 20 mm.


I3, whereas hardly in I1 (AA307–324) (Fig. 5A and B).Since this would point to the presence of a PTM in thebasic but not acidic forms, we did not further analyze therelevance of this peptide.

Based on these findings, we then investigated thenature of PTM present in the identified region. We didnot succeed in retrieving by MS the longer, Arg510-containing peptide (AA493–521) or a spiked tryptic pep-tide with heavy label (AA493–521) used as internal control.This is probably caused by insolubility and inability toanalyze on a C18 column and forced us to use insteadspecific enzymatic and antibody-based assays. We focusedon three potential PTMs consistent with an acidic shift in

isoelectric point without change in molecular weight anddescribed to occur in GRP78 in other cell types, namelyADP ribosylation, phosphorylation, and citrullination(26–29). Both ADP ribosylation and citrullination occuron Arg residues, whereas phosphorylation may occur onThr 518.

We initially investigated ADP ribosylation and phos-phorylation of GRP78, but the absence of a positive signalin 2D Western blots of cytokine-exposed INS-1E cells withanti–poly(ADP-ribose) (Fig. 6A) and the absence of GRP78staining by Pro-Q Diamond in control- and cytokine-exposed INS-1E cells (Fig. 6B), as well as persistence of themodified isoforms upon treatment with calf intestinal

Figure 3—Chemical ER stress, but not metabolic stress, induces membrane translocation of GRP78 in insulin-secreting INS-1E cells. A:Apoptosis levels in INS-1E cells exposed for 12–15 h (white bars) or 24 h (black bars) to Tg (15 or 50 nmol/L) or Tn (2 or 5 mg/mL) or HG (25mmol/L), Pa (0.5 mmol/L), or the combination (HG+Pa) (n = 5–9 independent experiments, each biological replicate is the mean of twotechnical duplicates). B: GRP78 mRNA expression in INS-1E cells treated for 12–15 h as described above (n = 4–10, each biologicalreplicate is the mean of two technical duplicates). C and F: Total and plasma membrane-associated (PM) GRP78 protein expression in INS-1E cells exposed to the indicated stressors. A representative Western blot from four independent experiments is shown. D, E, G, and H: Therelative intensities of the different protein bands were quantified by densitometry and expressed as a ratio (n = 4–7). *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001 vs. respective control (Ctrl).


Figure 4—Cytokine exposure induces PTM of GRP78 in INS-1E cells and in intact mouse islets. Representative images from 2D differencegel electrophoresis analysis (selected region of 24 cm, pH 4–7, 12.5% SDS-PAGE) of GRP78 in control and cytokine-exposed INS-1E cells(A) and intact mouse islets (B) with corresponding three-dimensional view and Graph view of the DeCyder analysis. For each of the threeisoforms (I1, I2, and I3), the fold increase or decrease is shown, and statistical analysis was performed using a two-tailed, unpaired Studentt test (n = 4 independent experiments; *P < 0.05 and **P < 0.01 vs. control). Representative images of 2D-GE analysis (selected region of24 cm, pH 4–7, 12.5% SDS-PAGE) of intracellular and membrane-associated GRP78 in control and cytokine-exposed INS-1E cells (onerepresentative experiment out of five independent experiments is shown) (C ) and MIN6 cells (one representative experiment out of twoindependent experiments is shown) (D). E: Representative 2D-GE analysis (selected region of 24 cm, pH 4–7, 12.5% SDS-PAGE) withcorresponding three-dimensional view of GRP78 in total lysates from control and differentially exposed (as indicated) INS-1E cells (onerepresentative experiment out of three independent experiments is shown).


alkaline phosphatase (Fig. 6C) or l-phosphatase (data notshown), argued against both modifications.

To verify the implication of citrullination, an antibodythat specifically recognizes citrullinated GRP78 at Arg510(selection based on the obtained MS data, see Fig. 5) wasraised in rabbits. 2D Western blots from control andcytokine-exposed INS-1E cells with anti–cit510-GRP78clearly indicated that the cytokine-induced acidic isoformsof GRP78 correspond to citrullinated GRP78 at residueArg510, whereas no reactivity was observed against themost basic, nonmodified GRP78 isoform 1 (Fig. 6D).

Padi2 Expression and Activity Are Upregulated inNOD MiceNext, we investigated the potential role of citrullinationin the diabetes-prone NOD mouse. Elevated Padi2 mRNAexpression was observed in islets of 3- and 10-week-oldprediabetic NOD mice as compared with islets fromage-matched C57Bl/6 and NOR control mice (Fig. 7Aand B). No differences between the strains were ob-served for Padi1, Padi3, Padi4, and Padi6 expression,

which were either low or undetectable. Further analysisin other tissues revealed an overall very low expressionof Padi2 in the immune-related tissues thymus, lymphnodes, and spleen. Except for kidney, no elevated levelsof Padi2 were observed in the other tissues analyzed inNOD as compared with C57Bl/6 mice. In addition, NORmice showed even lower/undetectable Padi2 expressionin most of the tissues analyzed (Fig. 7C). Furthermore,elevated Padi2 mRNA expression in NOD islets corre-sponded to higher PAD activity in total pancreases(Fig. 7D and E) and islets (Fig. 7F and G) of 3- and 10-week-old NOD mice, compared with both C57Bl/6 andNOR mice, indicating a marked increase of PAD activityin islets of NOD mice immediately before and duringinsulitis. Of note, 3-week-old NOD mice did not showany sign of inflammation in the islets, as measured by IL-1b (Fig. 7H) and IFNg (Fig. 7I) mRNA expression. In 10-week-old NOD mice, on the other hand, clear signs ofimmune infiltration were observed, as evidenced by highexpression of IFNg and IL-1b mRNA, confirming previ-ous findings from our group (30).

Figure 5—Quantitative mass spectrometric analysis of the three GRP78 isoforms I1, I2, and I3 using trypsin digestion combined withdifferential N-butyrylation and endo Lys-C digestion combined with differential N-propionylation. For each dataset, the ratios (light [I1]/heavy [I2 or I3]) were converted to their log2 value, in order to render a normal distribution. In the volcano plots, the size of the fold change iscompared with the statistical significance level. Comparison between the most basic isoform of GRP78 (I1) and the first more acidic isoform(I2) (A) and between I1 and the second more acidic GRP78 isoform (I3) (B) with a sequence coverage of 77.67 and 71.70%, respectively(494 and 456 out of 654 amino acids of the GRP78_RAT sequence, respectively, with omission of the 18–amino acid signal peptide). Theidentified tryptic peptides are marked in red, the identified endo Lys-C peptides in bold, and the signal peptides in blue. The two peptideswith the highest z score and fold change are depicted on the volcano plot and underlined in the sequence.


NOD Mice Have Circulating Autoantibodies andAutoreactive T Cells Against Citrullinated GRP78To evaluate whether citrullinated GRP78 contributes to theautoimmune response in NOD mice, serum samples fromprediabetic and new-onset diabetic NOD mice and age-matched C57Bl/6 and NOR mice were analyzed for the pres-ence of autoantibodies against the native and citrullinatedpeptide containing the epitope of interest (p500–519).Serum levels of anti-GRP78 antibodies recognizing thiscitrullinated epitope were significantly higher in new-onsetdiabetic NOD mice as compared with age-matched C57Bl/6or NOR mice (Fig. 8A, right panel). Furthermore, in diabeticNOD mice, significant higher serum antibody levels to thecitrullinated peptide were found compared with those ofthe native peptide. No such differences were observed inthe case of another irrelevant (citrullinated) GRP78 peptide(p295–314) tested (Supplementary Fig. 2). These findingsprovide evidence that this specific citrullinated epitope isimportant for autoantibody generation during type 1 diabetesdevelopment in NOD mice.

Next, to determine if NOD mice have autoreactiveT cells against native or citrullinated GRP78, freshly isolatedsplenocytes from prediabetic and new-onset diabetic NODmice and age-matched C57Bl/6 and NOR mice werestimulated with various concentrations of native and in

vitro citrullinated recombinant mouse GRP78 protein.Secretion of IFNg was used as a measure of effector T-cellactivation. Whereas little to no effector T-cell activation wasobserved in the three different strains when culturingsplenocytes with different concentrations of nativeGRP78 (Fig. 8B, red line and top right graph), a clear,dose-responsive increase in IFNg secretion was observedwhen splenocytes from prediabetic and diabetic NOD micewere cultured in the presence of citrullinated GRP78 (Fig.8B, blue line and bottom right graph). C57Bl/6 splenocyteswere unresponsive to citrullinated GRP78, whereas a minorIFNg response was detected in NOR splenocytes. Absenceof an IFNg response against both the PAD enzyme aloneand the control protein ovalbumin, either native or in vitrocitrullinated (at 0.1, 1, and 5 mg/mL) (data not shown),suggests that the observed autoreactive T-cell response isspecifically generated against citrullinated GRP78.

DISCUSSION

The role of posttranslationally modified proteins is wellestablished in several human autoimmune diseases, andevidence for similar phenomena in the development oftype 1 diabetes is accumulating (12–14). Most impor-tantly for this study, McGinty et al. (15) recently demon-strated the relevance of citrullination in patients with

Figure 6—GRP78 is citrullinated upon cytokine exposure of INS-1E cells. A: Representative 2D Western blot (11 cm, pH 4–7, 4–12.5%SDS-PAGE) of cytokine-exposed INS-1E lysate detected with anti–poly(ADP-ribose) antibody (top panel) followed by anti-GRP78 (bottompanel) (one representative experiment out of three independent experiments is shown). B: 2D-GE gel of cytokine-exposed INS-1E cells (24cm, pH 4–7, 12.5% SDS-PAGE) stained using Sypro Ruby to visualize all proteins (top panel) and Pro-Q Diamond dye to detect phos-phoproteins (bottom panel) (one representative experiment out of two independent experiments is shown). C: 2D-GE analysis of GRP78 incontrol and cytokine-exposed INS-1E cells (selected region of pH 4–7, 12.5% SDS-PAGE is shown), treated or not with calf intestinalalkaline phosphatase (CIAP) (one representative experiment out of two independent experiments is shown). D: 2D-GE analysis of citrullinatedGRP78 in control and cytokine-exposed INS-1E cells with anti–cit510-GRP78 (top panels) and total GRP78 Ab (bottom panels) (onerepresentative experiment out of seven independent experiments is shown).


http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0621/-/DC1

type 1 diabetes, by showing an increased response tocitrullinated GAD65 peptides. We show that the ER chap-erone GRP78 is citrullinated specifically upon exposure ofb-cells to inflammatory stress. This is paralleled by trans-location of GRP78 to the b-cell plasma membrane andeventually its secretion. Under these circumstances, a spe-cific cross-talk between the b-cell and the immune systemis initiated, resulting in the generation of autoantibodiesand induction of T-cell autoreactivity against citrullinatedGRP78 (Fig. 8C). Importantly, we also observed a markedupregulation of Padi2 in islets of NOD mice, providinga strong argument for PADI2 being the diabetes suscep-tibility gene in the recently identified Idd25 locus onmouse chromosome 4 (31) and adding to a potentialrole for citrullination in type 1 diabetes (15).

In previous studies, we have shown that GRP78 isposttranslationally modified in INS-1E cells exposed tocytokines (9), a PTM that we now identified as citrullina-tion. Cytokines contribute to b-cell dysfunction and deathat least in part through inducing ER stress (23–25). How-ever, citrullination of GRP78 is not induced by chemicalER stressors (Tg or Tn) or by metabolic stress via expo-sure to HG and/or Pa, suggesting that cytokine-inducedGRP78 citrullination occurs through a mechanism inde-pendent of its ER stress–inducing capacities. Preliminarydata suggest that this citrullination is not mediated by

direct upregulation of the PADI2 enzyme by cytokines(data not shown) but needs to be the consequence ofincreased PAD activity in b-cells exposed to cytokines.Since PAD activity is highly Ca2+ dependent, changes in Ca2+

fluxes induced in cytokine-exposed b-cells might play a role.The present findings, together with the recent report onposttranslationally modified GAD65 (15), add type 1 diabetesto the list of autoimmune diseases involving citrullination,i.e., RA, multiple sclerosis, and systemic erhythematosus (32).This suggests that citrullination is not a specific disease-related event but rather an inflammation-dependent pro-cess occurring preferentially in autoimmune target tissues.The role of citrullination in the induction of auto-antigenicity has been best described in RA, with severalcitrullinated autoantigens, including GRP78, already iden-tified (20,33). These citrullinated peptide epitopes are bet-ter accommodated in the HLA pocket of HLA-DR4–typeindividuals, determining the strength of the immune re-sponse to citrullinated peptides and providing a molecularbasis for the genetic predisposition of HLA-DR4 individualsto RA (34). This mechanism has recently also been de-scribed in type 1 diabetes (15), where HLA-DR4 is an im-portant risk allele for the disease (35).

In addition to citrullination, cytokine-induced trans-location of GRP78 to the b-cell plasma membrane may bea crucial step for GRP78 to become an autoantigen. This

Figure 7—NOD islets have high Padi2 mRNA expression and PAD activity. A and B: Padi1, 2, 3, 4, and 6 mRNA expression in islets ofLangerhans of respectively 3- and 10-week-old C57Bl/6 (black bars), NOR (gray bars), and NOD (white bars) mice (n = 5–8). C: Padi2mRNAlevels in different tissues of 3-week-old C57Bl/6 (black bars), NOR (gray bars), and NOD (white bars) mice (n = 5–10, each sample consistsof tissue isolated from a single mouse). D and E: Pancreatic PAD activity in respectively 3- and 10-week-old C57Bl/6 (black bars), NOR(gray bars), and NOD (white bars) mice. Data are expressed as means 6 SEM and were analyzed by a one-way ANOVA followed bya Bonferroni multiple comparisons test (n = 4–9). F and G: Islet PAD activity in respectively 3- and 10-week-old C57Bl/6 (black bars), NOR(gray bars), and NOD (white bars) mice (n = 4). All replicates refer to biological replicates with samples (islets or pancreas) obtained fromindividual mice. The PAD activity experiments were performed at least three times. ND, not detectable. IL-1b (H) and IFNg (I) mRNA levels in3- and 10-week-old C57Bl/6 (black bars), NOR (gray bars), and NOD (white bars) mice (n = 5–8, each sample consists of islets isolated froma single mouse). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Figure 8—NOD mice have circulating autoantibodies and autoreactive T cells against citrullinated GRP78. A: Comparison between theserum levels of antinative and anticitrullinated peptide (AA500–519) antibodies in prediabetic and diabetic NOD (NOD DM) mice and age-matched C57Bl/6 and NOR mice grouped according to age. Each dot indicates the value of a single mouse. Four independent experimentswere performed, each containing samples of all experimental groups. B: IFNg response of splenocytes from prediabetic (n = 9, light graybars) and diabetic NOD (n = 11, white bars) mice and age-matched C57Bl/6 (n = 8, black bars) and NOR (n = 11, dark gray bars) micestimulated with various concentrations of native (red) and citrullinated (blue) recombinant mouse GRP78. Four independent experimentswere performed, with two to three animals per group per experiment. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. C: Proposedmodel for the role of b-cell citrullinated GRP78 in autoantibody generation and T-cell activation. Exposure of b-cells to cytokines inducescitrullination, membrane translocation, and secretion of GRP78. Citrullinated membrane–associated or secreted GRP78 is taken up andprocessed by B cells and antigen-presenting cells (APCs), resulting in the generation of specific anticitrullinated GRP78 autoantibodies andrelease of IFNg by activated effector T cells, respectively.


translocation is shown to be an early event in response toinflammation, suggesting that it is an active process atleast in part independent from “protein leakage” by apo-ptotic b-cells. GRP78 membrane translocation, but notcitrullination, was found to be ER stress dependent andparalleled to the increased CHOP expression. Membrane-associated GRP78 has been described in different tumor celltypes (36–38) as well as in exocrine pancreatic cells (39) andproliferating endothelial and monocytic cells (40,41). Inthese models, membrane-associated GRP78 acts, amongother functions, as a cell surface signaling receptor for dif-ferent ligands such as activated a2-macroglobulin (42,43)and coxsackie A9 virus (44,45) and is found associatedwith the major histocompatibility complex class I (MHC-I)(44). Of interest, changes in the topography of membrane-associated GRP78, caused by PTMs, may convert GRP78into a receptor with autoantigenic properties. This hasbeen observed in cancer cells where GRP78 is modified byO-linked glycosylation (38), leading to the generation ofGRP78 autoantibodies. The present observations that in-flammation induces extensive GRP78 citrullination, trans-location to the plasma membrane, and secretion underscoreits putative function as an autoantigen in type 1 diabetes.Furthermore, based on the proposed transmembranemodel for GRP78 (46), the reactive p500–519 epitope,against which GRP78 autoantibodies are generated inNOD mice, is located in the extracellular domain, thusbeing exposed to infiltrating immune cells.

The possible role for citrullinated GRP78 as an auto-antigen in type 1 diabetes is supported by the presentobservations showing the generation of autoantibodies aswell as CD4+ T-cell autoreactivity against citrullinatedGRP78 in NOD mice. No T-cell reactivity is observed inC57Bl/6 mice, whereas NOR mice show a minor T-cell re-sponse. This supports the idea that inflammation is neces-sary to initiate this process, as low levels of IFNg and IL-1bare detected in islets of 10-week-old NOR mice, a phenom-enon referred to as protracted, noninvasive insulitis (47). Amarked upregulation of PAD2, a key enzyme for proteincitrullination, is observed exclusively in islets from NODmice, as compared with NOR and C57Bl/6 islets. PAD ac-tivity is further increased in NOD islets with increasing ageand insulitis, suggesting that inflammation plays a role forthis phenomenon, perhaps by increasing cytosolic Ca2+

concentrations due to cytokine-mediated calcium depletionfrom the ER (24). Interestingly, expression of Padi2 wasvery low in NOD thymus and not different from C57Bl/6 andNOR thymus. This may explain the escape of citrullinatedGRP78 from thymic tolerization in developing thymo-cytes, thereby clarifying why citrullinated GRP78 can berecognized as a b-cell–specific autoantigen whereas nativeGRP78 is ubiquitously expressed. A similar mechanismhas been proposed for chromogranin A, where exposureof a naturally occurring cleavage product (peptide WE14)to transglutaminase, expressed in b-cells, forms high- andlow-molecular weight aggregates, thus rendering the pep-tide highly antigenic in NOD mice (13).

Whether these observed processes are also applicableto other ER chaperones or heat shock proteins, such asHSP60, requires further investigation. It will be of utmostimportance to determine whether similar processesare involved in human type 1 diabetes. The knowledgethat inflammation-mediated b-cell stress is taking placein human type 1 diabetes (48) and the high overlapbetween autoantigens identified to date in NOD miceand type 1 diabetes patients (49) are in support of thishypothesis.

In conclusion, local inflammation in the islets inducescitrullination of GRP78 in the stressed b-cells, turningcitrullinated GRP78 into an autoantigen. This modifiedGRP78 is recognized by both B and T cells, thus propa-gating and amplifying the ongoing autoimmune attackagainst the b-cells. Our findings support and providemechanistic evidence for the concept that inflammation-induced b-cell stress initiates a specific communicationbetween the b-cell and the immune system, which willaggravate and accelerate the development of type 1 dia-betes. A genetic predisposition for increased citrullinationin the islets, as shown here for NOD mice, is expected tofurther exacerbate this process. This proposed mechanism(Fig. 8C), implicating tissue-specific and inflammation-induced protein modification, cell surface translocation,and secretion, would also explain why different tissue-specific autoimmune diseases can have similar autoantigens.

Acknowledgments. The technical assistance of Frea Coun, MartineGilis, Jos Laureys, Willem Van Den Berghe, Wim Werckx, and Farah-DeborahLok (Laboratory of Clinical and Experimental Endocrinology, KU Leuven) is greatlyappreciated. The authors thank Katleen Lemaire (Gene Expression Group, KULeuven) and Monique Beullens (Laboratory for Biosignaling and Therapeutics, KULeuven) for advice on GRP78 cloning in pET vector. The authors thank the CellImaging Core (KU Leuven) for providing technical assistance with confocalmicroscopy.Funding. This work was supported by Juvenile Diabetes Research FoundationInternational (17-2012-129 and 17-2013-515), the European Community’s Sev-enth Framework Programme NAIMIT (Natural Immunomodulators as NovelImmunotherapies for Type 1 Diabetes) under grant agreement 241447, theKU Leuven (Geconcerteerde Onderzoeksactie GOA 12/24 and an F+ fellowshipfor D.R.), and the Flemish Research Foundation (G.0619.12, a postdoctoral fel-lowship for G.B.F. and W.D. and a clinical research fellowship for C.M.).Duality of Interest. No potential conflicts of interest relevant to this articleare to be reported.Author Contributions. D.R. and I.C. designed and performed research,analyzed data, and wrote the manuscript. W.D., G.B.F., A.S., and A.D.G. per-formed research. D.L.E. analyzed data and wrote and edited the manuscript. P.A.and K.G. edited the manuscript. L.O. and C.M. designed research and wrote andedited the manuscript. L.O. and C.M. are the guarantors of this work and, assuch, had full access to all the data in the study and take responsibility for theintegrity of the data and the accuracy of the data analysis.

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