long-term ikk2/nf-kb signaling in pancreatic b-cells ... · heba h. salem,1,2 bernadette...

Heba H. Salem,1,2 Bernadette Trojanowski,1 Katja Fiedler,1 Harald J. Maier,1 Reinhold Schirmbeck,3 Martin Wagner,3

Bernhard O. Boehm,4,5 Thomas Wirth,1 and Bernd Baumann1

Long-Term IKK2/NF-kBSignaling in Pancreatic b-CellsInduces Immune-MediatedDiabetes

Type 1 diabetes is a multifactorial inflammatorydisease in genetically susceptible individualscharacterized by progressive autoimmunedestruction of pancreatic b-cells initiated by yetunknown factors. Although animal models of type 1diabetes have substantially increased ourunderstanding of disease pathogenesis,heterogeneity seen in human patients cannot bereflected by a single model and calls for additionalmodels covering different aspects of humanpathophysiology. Inhibitor of kB kinase(IKK)/nuclear factor-kB (NF-kB) signaling isa master regulator of inflammation; however, itsrole in diabetes pathogenesis is controversiallydiscussed by studies using different inhibitionapproaches. To investigate the potentialdiabetogenic effects of NF-kB in b-cells, wegenerated a gain-of-function model allowingconditional IKK2/NF-kB activation in b-cells. Atransgenic mouse model that expressesa constitutively active mutant of human IKK2dependent on Pdx-1 promoter activity (IKK2-CAPdx-1)spontaneously develops full-blown immune-mediated diabetes with insulitis, hyperglycemia,and hypoinsulinemia. Disease developmentinvolves a gene expression program mimicking

virus-induced diabetes and allergic inflammatoryresponses as well as increased majorhistocompatibility complex class I/II expressionby b-cells that could collectively promotediabetes development. Potential novel diabetescandidate genes were also identified. Interestingly,animals successfully recovered from diabetesupon transgene inactivation. Our data give thefirst direct evidence that b-cell–specificIKK2/NF-kB activation is a potential trigger ofimmune-mediated diabetes. Moreover, IKK2-CAPdx-1 mice provide a novel tool for studyingcritical checkpoints in diabetes pathogenesisand mechanisms governing b-cell degeneration/regeneration.Diabetes 2014;63:960–975 | DOI: 10.2337/db13-1037

Type 1 diabetes is an autoimmune disease in geneticallypredisposed individuals and presumably triggered and/oraccelerated by environmental factors (1). Analyses ofanimal models of type 1 diabetes have greatly improvedour knowledge about disease pathophysiology and ge-netic contribution. However, there is still an unmet needto understand islet cell pathology and ongoing in-flammatory processes within the islets of Langerhans.

1Institute of Physiological Chemistry, Ulm University, Ulm, Germany2Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt3Center for Internal Medicine, Ulm University Medical Center, Ulm University,Ulm, Germany4Division of Endocrinology, Diabetes and Metabolism, Ulm University MedicalCentre, Ulm University, Ulm, Germany5Lee Kong Chian School of Medicine, Nanyang Technological University, ImperialCollege London, Singapore

Corresponding author: Bernd Baumann, [email protected].

Received 2 July 2013 and accepted 27 November 2013.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1037/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

960 Diabetes Volume 63, March 2014

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In type 1 diabetes, inflammation contributes to theinduction and amplification of the immune insultagainst b-cells and, at later stages, to the stabilizationand maintenance of the insulitic process, thus pro-moting disease development and progression (2).Furthermore, b-cell response to stress and in-flammation is thought to be a critical determinant indisease outcome (2). Canonical inhibitor of kB (IkB)kinase 2 (IKK2)/nuclear factor-kB (NF-kB) signaling isthe master regulator of inflammatory responses andinnate immunity (3), and it is activated by viral andbacterial pathogens, various cytokines, and generalstress factors. Apart from its antiapoptotic function inmost cell types, the predominant role of NF-kB acti-vation in b-cells appears to be of a death-promotingnature (4,5).

The transcription factor NF-kB exists as homo- andheterodimers of five different subunits. In resting cells,NF-kB dimers are sequestered in the cytoplasm by IkBproteins. A key step in connecting extracellular stimulito NF-kB induction is the activation of the IKK complexcomposed of the catalytic subunits IKK1 and IKK2 andthe regulatory component NF-kB essential modulator.This complex phosphorylates IkB proteins, therebyinitiating their ubiquitination and subsequent protea-somal degradation, thus allowing nuclear translocationof NF-kB (4,5).

To mimic an inflammatory insult selectively in pancre-atic b-cells, we created a mouse model that allows theconditional expression of a constitutively active IKK2 allelein b-cells. Interestingly, prolonged IKK2/NF-kB activationin b-cells is sufficient to induce insulitis with marked hy-perglycemia, hypoinsulinemia, and reduced b-cell mass intransgenic animals. Intriguingly, upon switching off trans-gene expression, diabetes could be efficiently reversed.

RESEARCH DESIGN AND METHODS

Mice

Male mice were housed under specific pathogen-freeconditions at the animal facility of the University of Ulm.Pdx-1.tTA mice (C57BL/6) (6) and (tetO)7.IKK2-CA(constitutively active mutant of human IKK2) mice(NMRI) (7) were described previously. Pdx-1.tTA mice areknockin animals in which the coding sequence oftetracycline-dependent transactivator (tTA) has replacedthe endogenous Pdx-1 gene, thereby rendering this mouseline heterozygous for Pdx-1 (Pdx-1+/2). Control micegroup include wild-type and single-transgenic (tetO)7.IKK2-CA mice unless otherwise stated. Experiments wereperformed in accordance with institutional guidelines andGerman animal protection law.

Metabolic Studies

Blood glucose was measured using the One-Touch Ultraglucometer (LifeScan Inc., Mipitas, CA). Pancreaticinsulin was extracted by overnight agitation with coldacid ethanol (0.18 mol/L HCl in 70% ethanol) at 4°C.

The supernatant was collected, and the pellet was re-extracted. The pooled supernatant was used for insulinmeasurement. Insulin was determined in plasma samplescontaining protease inhibitor and pancreatic extractsusing the Ultra-Sensitive Mouse Insulin ELISA Kit(Chrystal Chem Inc.) following the manufacturer’sinstructions.

Protein Extraction, Western Immunoblotting,Luciferase Assay, and Electrophoretic Mobility ShiftAssay

Pancreata were snap-frozen in liquid nitrogen and pul-verized, and proteins were extracted for Western im-munoblotting and luciferase activity measurement (7).For immunoblotting, antibodies against IKK2 (Abcam)and extracellular signal–related kinase-2 (Santa CruzBiotechnology) were used. Electrophoretic mobility shiftassay was performed as described (7) using whole-cellextract from isolated islets.

Histology and Immunostaining

For paraffin sections, pancreata were excised, fixedovernight in 3.8% buffered formalin, dehydrated, paraf-fin embedded, cut in 3-mm sections, and further pro-cessed as previously described (8). For cryosections,10-mm slices from natively frozen pancreata were fixedwith 4% paraformaldehyde. Sections were incubated withthe primary antibodies: rabbit (Cell Signaling Technol-ogy) or guinea pig (Abcam) anti-insulin, goat anti-humanIKK2 (Santa Cruz Biotechnology), rabbit anti-RelA/p65(Laboratory Vision), anti–Pdx-1 and antichromogranin A(Abcam), anti-Ki67 (Thermo Scientific), rat anti-CD4 andanti-CD8 (Abcam), anti–CD11c-phycoerythrin and anti-B220 (BD Biosciences), and anti–CD25-phycoerythrinand anti–major histocompatibility complex (MHC) classII (MHC II)–FITC (eBioscience). Secondary antibodieswere coupled with Alexa Fluor (Invitrogen) for immu-nofluorescence and with horseradish peroxidase inimmunohistochemistry that was developed by 3-amino-9-ethylcarbazole (DakoCytomation). Immunofluorescentstainings were visualized as before (8), and other stain-ings were analyzed on a Leica DM IRB microscope (LeicaMicrosystems) equipped with ProgRes C14 digital camera(Jenoptik) and Openlab software (Improvision).

Detection of Apoptosis

In situ detection of DNA strand breaks was performedusing the TUNEL labeling method with the FragEL DNAFragmentation Detection Kit and colorimetric-TdT en-zyme (EMD Millipore) according to the manufacturer’sinstructions.

Islet Isolation

Pancreata were inflated in situ with 0.5 mg/mL ice-coldcollagenase XI solution (Sigma-Aldrich, St. Louis, MO)dissolved in PBS (with Ca/Mg). Pancreatic tissue wasdissected out and digested for 19–24 min at 37°C withsubsequent washing and centrifugation. Islets in the

diabetes.diabetesjournals.org Salem and Associates 961

Figure 1—Gain-of-function mouse model for conditional activation of IKK2 in pancreatic b-cells. A: Transgenic approach for doxycycline(Dox)-regulated expression of IKK2-CA in pancreatic b-cells. tTA protein is expressed under the control of Pdx-1 promoter and can bind toa bidirectional promoter [(tetO)7; 7xtetO] driving the transcription of luciferase and IKK2-CA transgene. This binding is inhibited by Dox,thereby shutting off transgene expression. The Pdx-1.tTA mice are knockin animals in which the coding sequence of tTA has replaced theendogenous Pdx-1 gene, thereby rendering this mouse line heterozygous for Pdx-1 (Pdx-1+/2). Animals were bred under Dox (0.1 g/L Doxin drinking water) to avoid developmental defects, which then had been withdrawn after weaning (3 to 4 weeks) to activate transgeneexpression. B: Luciferase activity was measured in pancreas (Pa), spleen (Sp), liver (Li), kidney (Ki), stomach (St), intestine (Int), lung (Lu),thymus (Thy), thalamus/hypothalamus (Tha), and brain (Br) of 26- to 32-week-old control (white bars) and IKK2-CAPdx-1 mice (black bars) (n = 2).

962 NF-kB Activation in b-Cells Induces Diabetes Diabetes Volume 63, March 2014

sediment were purified with Histopaque 10771 (Sigma-Aldrich), washed, and frozen in liquid N2.

RNA Extraction, Quantitative PCR, and Microarray

RNA was extracted with RNeasy kit (Quiagen) and cDNAsynthesis was done using the Transcriptor High FidelitycDNA Synthesis Kit (Roche). Quantitative real-time PCRwas performed with the Roche LightCycler 480 (Roche)using gene-specific primers and hydrolysis probesdesigned by the Roche Universal Probe Library system(Roche). Microarray analysis was performed with theMouse Gene 1.0 ST array (Affymetrix) and evaluatedwith the “Genesifter” sofware (Geospiza). The expressiondata are available at Gene Expression Omnibus superseries (accession number GSE47504).

Flow Cytometry

Pancreatic cells were isolated and purified as previouslydescribed (9) and subsequently stained with monoclonalantibodies using standard procedures. The followingantibodies were purchased from eBioscience: anti–MHCII (M5/114.15.2), anti–MHC class I (MHC I; AF6–88.5.5.3), anti-CD11b (M1/70), anti-CD3e (17A2), anti-CD25 (PC61.5), and anti-CD69 (H1.2F3); from BDBiosciences: anti-CD11b (M1/70), anti-CD8a (53–6.7),anti-CD4 (RM4–5), CD44 (IM7), anti-NK1.1 (PK136),and anti-CD11c (HL3). Anti-CD45 (30-F11), anti-CD19(6D5), anti-F4/80 (BM8), and anti-Ly6G (1A8) were fromBioLegend, while Ly6C (1G7.G10) was from MiltenyiBiotec. Fixable viability dye (eBioscience) was used toexclude dead cells. FACS was performed on an FACS-Canto II (BD Biosciences), and data were analyzed withFACSDiva 6.2 (BD Biosciences) and FlowJo softwares(Tree Star).

Statistical Analysis

Values are given as mean 6 SEM. Statistical analysis wasperformed with the Prism software (GraphPad) usingtwo-tailed Student t test. Data with P values of #0.05were considered statistically significant.

RESULTS

Generation of Conditional, Gain-of-Function MouseModel for Canonical NF-kB Signaling in Pancreaticb-Cells

To directly explore the biological consequences of ca-nonical NF-kB activation in pancreatic b-cells, we gen-erated IKK2-CAPdx-1. This was achieved by crossing

transgenic mice expressing the tTA under the control ofPdx-1 promoter (Pdx-1+/2) (6) with mice carrying theluciferase-(tetO)7-IKK2-CA minigene (7) (Fig. 1A). Thetransgenic expression system was not activated untilweaning in order to avoid any effects of IKK2-CA activityon pancreas development. Measurement of luciferaseactivity, the coexpressed reporter gene, revealed strongtransgene activity in the pancreas of IKK2-CAPdx-1 miceafter doxycycline withdrawal and only minor activity inthe intestine (Fig. 1B). Pancreatic IKK2-CA expressionwas confirmed by immunoblot and could be switched offby doxycycline (Fig. 1C), thus allowing conditional regu-lation of IKK2 activity. Immunofluorescence stainingdemonstrated a mosaic expression of IKK2-CA exclu-sively in the islets of IKK2-CAPdx-1 mice (Fig. 1D), cor-relating with obvious reduction in insulinimmunoreactivity (Fig. 1D).

Electrophoretic mobility shift assays showed strongbasal NF-kB activity in IKK2-CAPdx-1 mice relative totheir Pdx-1+/2 littermates, demonstrating transgenefunctionality (Fig. 1E). NF-kB activity was also elevatedin Pdx-1+/2 mice compared with controls. Furthermore,IKK2 activation led to nuclear translocation of the NF-kBsubunit RelA in b-cells of IKK2-CAPdx-1 mice, while al-most no nuclear RelA was detected in islets of eitherPdx-1+/2 or control mice (Fig. 1F).

IKK2/NF-kB Activation in b-Cells Induces Full-BlownDiabetes

We then assessed the physiological and histologicalconsequences of IKK2/NF-kB signaling in pancreaticb-cells. Interestingly, at the age of 11 weeks, some ani-mals started to develop hyperglycemia, and at;24 weeksof age, all IKK2-CAPdx-1 mice showed substantial eleva-tion in fed (538.5 6 16 vs. 154.9 6 7 mg/dL in Pdx-1+/2

mice) and fasting (423.3 6 34 vs. 96.9 6 4 mg/dL inPdx-1+/2 mice) blood glucose levels, with several animals.600 mg/dL (Fig. 2A and B). Hyperglycemia was ac-companied by a 57% reduction of plasma insulin levelscompared with Pdx-1+/2 mice (Fig. 2C), indicating thatthese mice were overtly diabetic. Animals displayedclinical signs of diabetes, including polyuria, polydipsia,weight loss, and general sickness, and in severe cases,mortality was recorded. Furthermore, immunohisto-logical analyses indicated an extensive loss of insulin-positive b-cells in IKK2-CAPdx-1 mice, with the remainingb-cells appearing degranulated with only faint residual

High luciferase activity was detected in pancreas of IKK2-CAPdx-1 mice and only minor activity in their intestine. C: Strong IKK2 expressionwas detected by Western blot in pancreatic extracts of IKK2-CAPdx-1 mice after doxycycline (Dox) withdrawal and disappeared after Doxreadministration; extracellular signal–related kinase-2 (ERK2) was used as loading control. D: Immunofluorescence staining of paraffin-embedded pancreatic sections showing mosaic expression of transgenic human IKK2 in islets of 12-week-old IKK2-CAPdx-1 mice. Thestaining shows that IKK2-positive cells have reduced or even no insulin immunoreactivity. Sections were costained with DAPI (blue) fornuclei. Scale bar, 50 mm. E: An electrophoretic mobility shift assay of whole-cell islet extracts with NF-kB and SP1-specific probes showingstrong activation of NF-kB in 12-week-old IKK2-CAPdx-1 mice. SP1 serves as a quality control. F: Immunohistological staining of paraffin-embedded pancreatic sections showing IKK2-CA–induced nuclear localization of RelA in b-cells of 12-week-old IKK2-CAPdx-1 mice. Scalebar, 50 mm. Controls denoted in this figure were single transgenic for IKK2-CA transgene. RLU, relative light unit.


insulin immunoreactivity (Fig. 2D) and reduced Pdx-1expression (Supplementary Fig. 1A). Costaining withIKK2 revealed that most of the IKK2-CA–expressing cellswere no longer positive for insulin. Interestingly, fewinsulin-negative cells still expressed nuclear Pdx-1, whilemore such cells retained the expression of the endocrinemarker chromogranin A (Supplementary Fig. 1A and B).

This implicates that IKK2/NF-kB activation in b-cellsleads to suppression of insulin expression and thus lossof their functionality in addition (or prior) to theirdemise. In accordance, pancreatic insulin content andinsulin transcription had dropped to ;3 and 26% ofthe values of the Pdx-1+/2 littermates, respectively(Fig. 2E and F).

Figure 2—Expression of IKK2-CA in b-cells results in diabetes development. Animals were kept under doxycycline during breeding,doxycycline was removed after weaning, and mice were then analyzed at the indicated ages (A) or at 24–28 weeks (B–F ). A: Fed bloodglucose levels of 8, 11–13, 14–17, and 24- to 28-week-old control (n = 9, 13, 7, and 28), Pdx-1+/2 (n = 6, 12, 3, and 17), and IKK2-CAPdx-1

mice (n = 5, 12, 7, and 28). The upper detection limit of the glucometer is 600 mg/dL. B: Overnight-fasted blood glucose levels of control(n = 21), Pdx-1+/2 (n = 16), and IKK2-CAPdx-1 mice (n = 22). C: Fed plasma insulin level of control (n = 12), Pdx-1+/2 (n = 6), andIKK2-CAPdx-1 mice (n = 12). D: Representative immunofluorescent images of paraffin sections of IKK2-CAPdx-1 and Pdx-1+/2 pancreatastained for insulin (red) and IKK2 (green) and costained with DAPI for nuclei in blue. Scale bar, 50 mm. E: Pancreatic insulin content ofcontrol, Pdx-1+/2, and IKK2-CAPdx-1 mice (n = 6–9/group). F: Quantitative RT-PCR of Ins2 mRNA of control, Pdx-1+/2, and IKK2-CAPdx-1

mice (fold upregulation vs. control; n = 8–12/group). Hprt was used as a reference gene. Results were analyzed by Student t test and-presented as the mean 6 SEM. **P < 0.01; ***P < 0.001.




Figure 3—IKK2/NF-kB activation in b-cells induces inflammation, insulitis, and increased islet antigen presentation. A: Hematoxylin-eosinstaining of paraffin sections from pancreas of Pdx-1+/2 and IKK2-CAPdx-1 mice showing peri-insulitis (I) and invasive insulitis (II) inIKK2-CAPdx-1 mice. B: Flow cytometric analysis of pancreatic cells from control (white bars) and IKK2-CAPdx-1 (black bars). The data arerepresented as relative to the number of CD45+ cells in the control samples, which is set to one (n = 5/group from two independentexperiments). Immune cells were analyzed for the expression of surface markers CD3+ (T cells), CD19+ (B cells), and CD11b+/CD11c+

(dendritic cells [DCs]), CD11b+/F4/80+ (macrophages [Mf]), and NK1.1+ (natural killer cells [NK]). MHC II expression on pancreatic cells ofthe myeloid (C ) and lymphoid lineage (D), analyzed by flow cytometry and gated as indicated. Elevated MHC II levels show that infiltrated


Pdx-1 is an important transcription factor for b-cellfunction, and its heterozygosity, present in the Pdx-1+/2

knockin mouse, was shown to be associated with someb-cell defects (6). Indeed, Pdx-1+/2 mice showed slightelevation in fed blood glucose level (Fig. 2A) and re-duction in insulin transcription and pancreatic contentcompared with control littermates (Fig. 2E and F); how-ever, in contrast to IKK2-CAPdx-1 mice, they were notdiabetic.

IKK2-CA–Induced Diabetes Is Associated WithInflammation and Leukocytic Infiltration

Invasion of pancreatic islets by leukocytes is the hallmarkof immune-mediated diabetes. Unlike Pdx-1+/2 mice,islets of IKK2-CAPdx-1 showed marked peri-insulitis(perivascular, periductal, and peri-islet infiltrates) andinsulitis (Fig. 3A), reminiscent of the insulitic process inhumans. Of note, similar to human histopathology, notall islets showed mononuclear infiltration (Supplemen-tary Fig. 2).

Flow cytometry revealed that hematopoietic (CD45+)cells in IKK2-CAPdx-1 pancreata increased fourfold (Fig.3B) and that infiltrating leukocytes were activated CD4+

and CD8+ T, B, and dendritic cells as well as macrophagesand few natural killer cells (Fig. 3B–D and SupplementaryFig. 3A). This was further confirmed on the histologicallevel (Fig. 3E and F and Supplementary Figs. 3B and Cand 4). The majority of infiltrating T cells was of theCD4+ type (Fig. 3B and F). Furthermore, we detectedhyperexpression of MHC I and II by b-cells of IKK2-CAPdx-1 mice (Fig. 3G, Supplementary Fig. 5A–C), aspreviously reported in diabetic patients (10). This sug-gests increased antigen presentation capacity of b-cells inthese mice, which might be critical for phenotype de-velopment. In accordance, there was transcriptionalupregulation of the MHC I and II components, H2-Q4and H2-Aa (Fig. 3H), respectively.

The insulitic process in IKK2-CAPdx-1 mice was asso-ciated with elevated expression of inflammatory cyto-kines and chemokines like tumor necrosis factor (Tnf),Ccl5, Ccl2, and Cxcl10 and the adhesion molecule in-tracellular adhesion molecule-1 (Fig. 3I). This in-flammatory profile is reminiscent of those found in isletsfrom patients, in animals with type 1 diabetes (11,12),and in normal human islets subjected to cytokines orenteroviruses (13–15).

IKK2-CAPdx-1 Mice Show Signs of Apoptosis andEndoplasmic Reticulum Stress

As apoptosis is thought to be the major cause of b-celldeath in diabetes (16), we performed TUNEL assays thatshowed the presence of apoptotic cells only in IKK2-CAPdx-1 islets (Fig. 4A and B). This indicates that apo-ptosis may account at least in part for the reduced b-cellmass in IKK2-CAPdx-1 mice. b-Cells are susceptible toendoplasmic reticulum (ER) stress, and ER stress-mediated apoptosis in b-cells has been implicated in thepathogenesis of diabetes (17). Moreover, islets fromprediabetic NOD mice (18) and patients with type 1 di-abetes (19) exhibit signs of ER stress. Consistent withthat, the ER stress-related factors Ddit3/Chop and Atf3were upregulated in IKK2-CAPdx-1 mice (Fig. 4C). In ad-dition, other stress-associated apoptosis-inducing factorslike Nos2 (iNOS) and Myc were also elevated in IKK2-CAPdx-1 pancreata (Fig. 4D and E).

IKK2-CA–Induced Diabetes Is Reversible

To examine the possibility of reverse remodeling thediabetic phenotype, doxycycline was readministered for30 days to diabetic IKK2-CAPdx-1 mice (Fig. 5A). Diabetesin IKK2-CAPdx-1 mice was confirmed by elevated fed andfasted blood glucose values and reduced plasma insulinlevels (Fig. 5B and C). We observed a clear reduction infed blood glucose values already within the first 10 days,which virtually completely normalized in all animals by30 days (Fig. 5B). Consistently, fasting blood glucose andfed plasma insulin levels were also restored (Fig. 5C).Doxycycline-dependent transgene inactivation was con-firmed by immunofluorescence staining (Fig. 5D) andWestern blot (Fig. 1C). Diabetic IKK2-CAPdx-1 mice alsoregained normal structured islets showing virtual ab-sence of infiltrating cells upon doxycycline treatment(Fig. 5D and E). Insulin immunostaining demonstratedthe reappearance of b-cell–rich islets and prominentb-cell regranulation, which strongly stained for insulin(Fig. 5D). Importantly, the reversion of the diabetesstatus was accompanied by increased levels of Ki67 im-munoreactivity in islets of IKK2-CAPdx-1 mice (Fig. 5Fand Supplementary Fig. 6), indicating that proliferationis involved in b-cell regeneration in this model. Fur-thermore, transcription of insulin, MHC I/II molecules,and various inflammatory markers was effectively nor-malized (Fig. 5G–I).

cells in IKK2-CAPdx-1 were evidently activated. Data are representative of two experiments with five mice per group (C ) or one experimentwith two mice per group (D). Pdx-1+/2 mice were included in the control group in B–D, as they show the same pattern. E: Immunohis-tochemical staining of paraffin sections from Pdx-1+/2 and IKK2-CAPdx-1 mice (n = 3/group) for the B-cell marker B220. F: Immunofluo-rescence staining of cryosections from pancreas of Pdx-1+/2 and IKK2-CAPdx-1 mice (n = 3/group) for CD4, CD8, or CD11c (DC marker)together with insulin and DAPI. G: Immunofluorescence staining of cryosections from pancreas of Pdx-1+/2 and IKK2-CAPdx-1 mice forinsulin (red) and MHC II (green) (n = 3/group) showing the expression of MHC II by some b-cells (arrows, inset). Quantitative RT-PCR formRNA transcripts involved in antigen presentation (H) and inflammation (I) from control (white bars), Pdx-1+/2 (gray bars), and IKK2-CAPdx-1

(black bars) mice (fold upregulation vs. control; n = 8–12). Hprt was used as a reference gene. Results were analyzed by Student t test andpresented as the mean6 SEM. Animals were 24–28 weeks old. Scale bars, 50 mm. *P < 0.05; **P < 0.01; ***P < 0.001. Icam1, intracellularadhesion molecule-1.










IKK2-CAPdx-1 Mice Exhibit Gene Expression ProfileMimicking Antiviral and Allergic InflammatoryResponses

To gain insight into molecular events induced by IKK2/NF-kB activation and possibly involved in diabetes de-velopment, we performed gene expression profiling ofpancreatic islets isolated from 11-week-old IKK2-CAPdx-1

animals. This age represents a critical checkpoint inphenotype development at which some animals alreadyshowed hyperglycemia, while others were still normo-glycemic (Fig. 6A). In this analysis, including samplesfrom both types, 288 transcripts were found to beupregulated, 46 of which are known NF-kB targets(Supplementary Table 2) and 28 to be downregulated

(threshold, 1.5-fold; P , 0.05 in t test with Benjamini-Hochberg correction) (Supplementary Table 1, selectedgenes, and Table 1). Several of those genes were verifiedby quantitative PCR (qPCR) as depicted in Fig. 6B. Theincreased expression of various cytokines, chemokines,adhesion molecules, and antigen presentation moleculesclearly demonstrated an activation of innate and adap-tive immune responses in islets of IKK2-CAPdx-1 mice(Table 1, Supplementary Table 1, and Fig. 6B). However,aside from these factors typically detected in other type 1diabetes models, other less-characterized genes werehighly upregulated (Table 1, Supplementary Table 1, andFig. 6B). The T-cell–directed chemokine Ccl17 (alsoknown as thymus and activation-regulated chemokine)

Figure 4—Islets of IKK2-CAPdx-1 mice show signs of apoptosis and ER stress. A: TUNEL assay on paraffin sections of pancreas from24- to 26-week-old control, Pdx-1+/2, and IKK2-CAPdx-1 mice (n = 3/group). Sections were counterstained with methyl green. Arrowheadsindicate the presence of apoptotic nuclei (TUNEL-positive nuclei) in the islets of IKK2-CAPdx-1 mice. Dashed line marks the islet area. Scalebar, 50 mm. B: Quantification of TUNEL staining in A. A total of >400 islets from three animals per group were analyzed. C–E: QuantitativeRT-PCR for mRNA transcripts encoding stress and apoptosis-inducing genes in pancreata of 24- to 26-week-old control, Pdx-1+/2, andIKK2-CAPdx-1 (fold upregulation vs. control; n = 6–10/group). Hprt was used as a reference gene. *P < 0.05; **P < 0.01; ***P < 0.001.Results were analyzed by Student t test and presented as the mean 6 SEM. ND, not detectable.






Figure 5—Recovery from diabetes, alleviation of the inflammatory status, and regeneration of pancreatic islets upon transgene in-activation. A: Experimental design. Mice were kept under doxycycline (Dox; 0.1 g/L Dox in drinking water) until weaning and discontinueduntil 24–28 weeks to activate transgene expression. The diabetic phenotype was confirmed by fed and fasted blood glucose as well asplasma insulin measurements (before Dox). Dox (1 g/L) was then readministered in drinking water for 30 days (except for F ) to inactivatetransgene expression. Fed blood glucose was monitored during that time after which animals were analyzed (after Dox). B: Fed bloodglucose monitoring before and during Dox readministration of control (white circles), Pdx-1+/2 (white squares), and IKK2-CAPdx-1 (whitetriangles) mice (n = 7–9/group from two independent experiments). Significance is relative to the Pdx-1+/2 group at the specified time


was the most prominent one. Ccl17 together with Ccl22(also known as macrophage-derived chemokine), anotherelevated chemokine, are known ligands for the Ccr4receptor, which has been linked to the developmentof autoimmune diabetes (20). Also, a cluster ofinflammation-related serine proteases called serine pro-tease inhibitor clade A member 1 (Serpina1) and differentmembers of the cationic protein products of eosinophils,the eosinophil-associated ribonucleases (Ears), wereupregulated. Kyoto Encyclopedia of Genes and Genomesanalysis clearly revealed a signature reflecting antiviralresponses and reactions to infections. This was repre-sented by the upregulation of different interferon (IFN)-regulated genes, especially involved in the type 1 IFNresponse including signal transducer and activator oftranscription 1 (Stat1), IFN regulatory factor (Irf) 7, Irf8,and Irf5 as well as IFN-inducible genes like Ifih1 and Ifit1.Furthermore, genes involved in oxidative stress (Nox1)and tissue remodeling (Mmp12) were also elevated (Table1 and Supplementary Table 1). Taken together, the ob-served gene expression profile in the IKK2-CAPdx-1 modelmimics to a great extent that detected in virus-inducedimmune-mediated diabetes and, importantly, points toother potential novel candidate genes for diabetes.

Ccl17 Expression Precedes Phenotype Development

In an attempt to identify candidate genes involved inphenotype initiation, we followed up the temporal ex-pression pattern of distinct genes. Interestingly, Ccl17was massively upregulated in islets of 8-week-old IKK2-CAPdx-1, when animals were asymptomatic, while therewas only mild upregulation of Ear2, Serpina1a, Irf7, andMadcam1 and no elevation of MHC I/II molecules(Fig. 7A). These data support the hypothesis thatCcl17 might be a critical initial effector of IKK2/NF-kB–mediated diabetes development. In addition, qPCRanalysis of highly diseased IKK2-CAPdx-1 mice (;24weeks old) revealed sustained upregulation of Ccl17,Ear2, and Serpina1a that was completely normalized inthe reverse-remodeling experiment (Fig. 7B).

DISCUSSION

In this study, we describe a novel mouse model ofimmune-mediated diabetes, the IKK2-CAPdx-1 model thatis based on genetic activation of proinflammatory

IKK2/NF-kB signaling, specifically in b-cells. IKK2-CAPdx-1 animals develop severe hyperglycemia andhypoinsulinemia mirroring substantial b-cell loss that isassociated with marked islet inflammation. Similar to thehuman immunopathology, infiltrates are not seen in allpancreatic islets and include different kinds of immunecells (21). This model is the first animal model to activateIKK2/NF-kB in b-cells and to prove directly its capabilityto trigger diabetes development on its own. IKK2-CAPdx-1

islets show a gene expression signature reflecting theactivation of innate immunity and type I IFN response,which could generate a proinflammatory microenviron-ment sufficient to recruit different immune cells. Theseinfiltrating cells are able to contribute to and/or amplifythe inflammatory insult, thereby promoting b-cell de-struction as observed in diabetic subjects and NOD mice(1,2). Furthermore, increased expression of MHC I and IIin transgenic b-cells indicates that IKK2/NF-kB signalingis capable of increasing islet antigen presentation to in-filtrating T cells, which could participate in the autoim-mune insult. In addition, MHC II expression by b-cellscan promote their death, as seen by MHC II ligation inantigen-presenting cells (22). This may allow for an in-tensive dialogue between b-cells and immune cells thatfinally promotes b-cell destruction in a T-cell–dependentmanner. The main T-cell subtype in our model is theCD4+ type, in contrast to the concept that CD8+ T cellspredominate in humans (21), However, there is a con-siderable heterogeneity within diabetic patients (e.g.,even no autoreactive T cells were found in a subset ofrecent-onset patients) (23). In addition, CD4+ T cells canmediate b-cell death in transgenic NOD mice (24), anddiabetes development was shown to require the presenceof both CD4+ and CD8+ T cells (25).

The IKK2-CAPdx-1 phenotype is in line with thereported role of NF-kB as a mediator of cytokine-inducedb-cell destruction (4,5) and its activation in islets ofprediabetic NOD mice (18). Consistent with this notion,resistance to streptozotocin (STZ)-induced diabetes wasachieved by b-cell–specific inhibition of NF-kB (26,27),and various natural products were found to inhibit STZ-induced diabetes and protect against b-cell damage throughNF-kB inhibition (4). However, b-cell–specific repression ofNF-kB in normal mice elicits hyperglycemia and defectiveglucose-stimulated insulin secretion and accelerates

point. At 20 and 30 days after Dox administration, Pdx-1+/2 mice had significantly higher blood glucose values relative to the controllittermates. C: Overnight-fasting blood glucose (top) and fed plasma insulin levels (bottom) before and after doxycycline readministration.Immunofluorescence (D) and hematoxylin-eosin stainings (E) of paraffin sections from Pdx-1+/2 and IKK2-CAPdx-1 mice after Dox read-ministration showing regenerated b-cells with normal insulin content and near absence of infiltrating cells. F: Immunofluorescence stainingof insulin (red), Ki67 (green), and DAPI (blue) of paraffin sections from Pdx-1+/2 and IKK2-CAPdx-1 mice 10 days after Dox readministration.The staining shows the presence of increased Ki67-positive b-cells (arrows) in the islets of IKK2-CAPdx-1 mice as compared with Pdx-1+/2

ones. Scale bars in D–F, 50 mm. Quantitative RT-PCR for insulin mRNA (G), MHC I (H2-Q4) and MHC II (H2-Aa) molecules (H), and in-flammatory genes (I) in pancreata of control (white bars), Pdx-1+/2 (gray bars), and IKK2-CAPdx-1 (black bars) mice after Dox readminis-tration (fold upregulation vs. control; n = 6–10/group from 2 independent experiments). Hprt was used as a reference gene. *P< 0.05; **P<0.01; ***P < 0.001. Results were analyzed by Student t test and presented as the mean 6 SEM. Icam1, intracellular adhesion molecule-1.



Figure 6—Validation of selected microarray data by real-time PCR. A: Fed and fasted blood glucose values from 11- to 13-week-old control(white circles), Pdx-1+/2 (white squares), and IKK2-CAPdx-1 (white and black triangles) mice (n = 12–13/group). Black triangles representsamples used in the microarray analysis. B: Quantitative RT-PCR analysis of isolated islets from 11- to 13-week-old control, Pdx-1+/2, andIKK2-CAPdx-1. Samples used for the qPCR assays include those used in the array analysis and additional samples. Shown is the foldupregulation vs. control animals (n = 5–8/group). Actb was used as a reference gene. Results were analyzed by Student t test and presentedas the mean 6 SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Il12b, interleukin-12b; Madcam1, mucosal addressin cell adhesion molecule-1.


diabetes development in NOD mice (4,5), which mayimplicate that the extent/level, context, and timing ofNF-kB activation dictate the overall outcome of diabetespathogenesis.

Our gene expression data suggest that activation ofIKK2/NF-kB in b-cells is sufficient to initiate differentcellular mechanisms formerly shown to affect b-cellfunction/survival and induce diabetes in humans andanimals (16,17,28–32). These diverse factors (TNF-a,Atf3, C/EBP homologous protein, Nos2, c-myc, Stat1,and Nox-1) are known to induce inflammation, oxidativestress, ER stress, and nitric oxide production (Fig. 7C)that together may finally funnel in b-cell dysfunctionand/or apoptosis in our model. However, we cannot ex-clude other unknown effects of IKK2/NF-kB signalingthat might interfere with b-cell function and promotedisease development (Fig. 7C).

The expression profile in our model is further pointingto the involvement of innate immunity and type I IFNresponse (Fig. 7C), characteristic for viral infection, andis similarly detected in pancreata and islets from patientsat clinical onset and long-standing diabetes (12). Fur-thermore, the gene-expression program induced by en-teroviral infection of human islets (14,15) is alsoprominently mimicked by the IKK2-CAPdx-1 model, sug-gesting that IKK/NF-kB is a critical downstream effectorin this context. Viral infections have been proposed asa triggering factor of type 1 diabetes in humans andanimals (33), and importantly, they activate Toll-like andnucleotide-binding oligomerization domain–like recep-tors, which are well-known inducers of NF-kB. Indeed,NF-kB signaling is activated in b-cells by enterovirusinfection (15) and double-stranded RNA treatment (34)and mediates at least partially the deleterious effects ofthese insults. Recently, Irf7, the master regulator of typeI IFN–dependent immune responses and upregulated inour model, has been implicated in type 1 diabetes path-ogenesis (35). Similarly, Ifih, another candidate gene fortype 1 diabetes that is expressed in human islets andcan be induced by inflammatory cytokines (13) and bydouble-stranded RNA in rat b-cells (36), is elevated inIKK2-CAPdx-1 mice. Therefore, it is well conceivable thatNF-kB activation in b-cells on its own could create aninnate immune response similar to an antiviral response,which finally culminates in diabetes development. How-ever, we cannot exclude a possible role of Pdx-1 haplo-insufficiency on promoting b-cell susceptibility toIKK2/NF-kB–induced inflammation and phenotypedevelopment.

We also identified other novel diabetes-associatedcandidate genes. Ccl17, the most upregulated one, isa chemokine involved in immune and allergic in-flammatory responses (20,37–42). The early markedupregulation of Ccl17 at normoglycemia supports the ideaof being a foremost effector of IKK2/NF-kB signaling thatcontributes to diabetes development. Consistently, viralinfection of human B cells induces NF-kB–dependent

Table 1—List of selected genes differentially regulated inislets of 11-week-old IKK2-CAPdx-1 mice as compared withPdx-1+/2 littermates

Class Name Fold

Chemokines and their receptors Ccl17 24.59Ccl22 4.68Ccl5 3.10Ccl19 2.60Cxcl10 2.63Cxcl16 2.22Ccr2 3.43Ccr7 3.08Ccr5 2.25

Interleukins and their receptors Il1a 2.61Il12b 2.25Il1b 1.90Il6 1.72Il1r2 2.13Il2ra 2.14Il2rg 2.77Il12rb2 1.52

Adhesion molecules Itgax 4.64Itgae 3.64Itga2 2.38Madcam1 4.19Vcam1 2.51Icam1 2.30

Antigen presentation and processing Ctss 2.11

MHC I H2-M2 4.74H2-Q8 3.17

MHC II H2-Ab1 2.39H2-Aa 2.14

Antiviral response (IFN responsive) Irf7 2.60Irf8 2.34Irf5 1.84Ifit1 2.28Ifih1 1.74

Innate immunity Clec7a 3.10Tlr3 1.53

STAT-mediated signal transduction Stat1 2.51Jak2 1.53

TNF signaling Tnf 1.55Tnip3 1.95Traf1 1.79Tnfssf13b 1.67Tnip1 1.65

Miscellaneous Ear2 10.06Ear10 7.63Ear1 6.20Serpina1e 9.63Serpina1a 9.22Serpina1b 6.52Mmp12 4.92Nox1 4.06

Icam1, intracellular adhesion molecule-1; Il, interleukin; Il1r,interleukin-1 receptor; Il2r, interleukin-2 receptor; Jak2, Januskinase 2; Madcam1, mucosal addressin cell adhesionmolecule-1; Tlr, Toll-like receptor; Traf1, TNF receptor–associated factor 1; Vcam1, vascular cell adhesion molecule-1.


Figure 7—Gene-expression kinetics during diabetes development in IKK2-CAPdx-1 mice. A: Quantitative RT-PCR from islets isolated from8-week-old control, Pdx-1+/2, and IKK2-CAPdx-1 mice. Shown is the fold upregulation vs. control animals (n = 4–6/group). Actb was usedas a reference gene. B: Quantitative RT-PCR from pancreata of 24- to 26-week-old animals before (diseased) and after doxycyclinereadministration (reversed). Shown is the fold upregulation vs. control animals (in diseased state, n = 6–10/group; in reversed state, n = 4–5/group). Hprt was used as a reference gene. Results were analyzed by Student t test and presented as the mean 6 SEM. C: Proposedmodel for IKK2/NF-kB–induced diabetes. Activation of IKK2/NF-kB in b-cells is sufficient to induce gene expression programs involved ininnate immunity, type I IFN response, and antigen presentation. This may subsequently result in oxidative stress, ER stress, and nitric oxide(NO) production that finally funnels in b-cell dysfunction and death that is probably mediated by apoptosis. In addition, there was a massiveincrease in the chemokine Ccl17, formerly shown to be involved in autoimmunity and allergic inflammatory processes. Moreover, a directeffect of IKK2/NF-kB activation on b-cell function via unknownmechanisms may also contribute to diabetes development in this model. *P<0.05; **P < 0.01. CHOP, C/EBP homologous protein; Madcam1, mucosal addressin cell adhesion molecule-1.


expression of Ccl17 and Ccl22 (also elevated in our model)(37), and IKK/NF-kB inhibition prevents cytokine-inducedCcl17 production in keratinocytes (38). Ccl17 is mainlyproduced by dendritic cells (39) and plays an importantrole in T cell development, trafficking, and activation.Furthermore, it was highlighted as a novel biomarker forallergic inflammatory diseases like asthma (40) and atopicdermatitis (41), in which it is strongly coexpressed withCcl22 (42).

Both Ccl17 and Ccl22 preferentially attract CD4+

T cells via the Ccr4 receptor. Cells expressing Ccl17were detected within infiltrated islets from prediabeticNOD mice, and Ccr4-positive T cells were shown to becritically involved in autoimmune diabetes de-velopment (20). Additionally, neutralizing Ccl22 anti-bodies inhibited insulitis and diabetes, whereastransgenic Ccl22 expression accelerated disease de-velopment (20). However, autoimmune diabetes wasprevented by Ccl22-mediated regulatory T-cell re-cruitment in another study (43). Furthermore, nosignificant difference in plasma Ccl17 was detected intype 1 diabetes patients (44). Yet, this study wasconducted in a very small cohort of diabetic Japanesesubjects, which certainly does not exclude a differentpattern in other patient subsets.

Ears, also prominently induced in our model, re-present a subgroup of the RNase A family secreted byrodent eosinophils (45) and also expressed by macro-phages. Ears participate in host defense by their anti-bacterial and antiviral activity together with chemotaxisto dendritic cells (45). The Serpina1 family of genesencodes for inhibitors of serine proteases. In humans, itis represented by a single gene, called a1-antitrypsin(AAT) for which expression was enhanced by proin-flammatory cytokines in islet cells (46). AAT is an acute-phase protein with anti-inflammatory, tissue-protective,and antiapoptotic properties that is able to prolongislet allograft survival and to inhibit cytokine and STZ-induced b-cell apoptosis (47,48) as well as diabetes de-velopment in NOD mice (49). Although decreasedfunctional activity of serum AAT was shown in diabetes,serum levels are variable, probably reflecting changesin the inflammatory status of the disease (50). Towhat extent these newly identified genes contributeto human disease pathology, however, needs furtherinvestigation.

One striking aspect of the IKK2-CAPdx-1 model is theenormous recovery potential from the diabetes statusincluding the resolution of insulitis and the efficient re-generation of insulin-positive b-cells after transgene in-activation. A possible rationale is that a portion of b-cellsis not destroyed but, rather, has lost its functional anddifferentiated state and therefore can regain its func-tionality upon IKK2-CA inactivation with the consequentresolution of proinflammatory mediators and disap-pearance of immune cells. Furthermore, the increase ofKi67-positive b-cells during the regeneration phase

suggests that proliferation is also involved in the resto-ration of b-cell mass in this model as found in othermodels; however, other mechanisms cannot be excluded.This finding implies that the IKK/NF-kB system might bea potential target for clinical intervention. Furthermore,the IKK2-CAPdx-1 mouse model could be valuable forassessing mechanisms and identifying genes involved inb-cell regeneration as well. Since diabetes passes throughrelapsing-remitting onsets, our model is further poten-tially useful for evaluating clinical therapeutics at dif-ferent stages of the human disease presentation throughswitching the system off and on.

In summary, the IKK2-CAPdx-1 model representsa novel conditional mouse model of immune-mediateddiabetes with very distinct checkpoints of b-cell de-struction and regeneration. This model phenocopiesmajor aspects of the human disease and may representa valuable tool for improving preclinical drug assessmentfor diabetes treatment.

Acknowledgments. The authors thank Ute Leschik, Melanie Gerstenlauer,and Bianca Ries (University of Ulm) for excellent technical assistance andKarlheinz Holzmann (Genomics-Core Facility, University Hospital Ulm) forperforming the microarray analysis.

Funding. This work was supported by grants GRK-1041-P3 (DeutscheForschungsgemeinschaft) and BIU-C6 (Boehringer Ingelheim Ulm UniversityBioCenter) to B.B.

Duality of Interest. No potential conflicts of interest relevant to thisarticle were reported.

Author Contributions. H.H.S. and B.B. designed and performed theexperiments, analyzed data, and wrote the manuscript. B.T. and K.F. per-formed research and analyzed data. H.J.M. contributed to discussion andreviewed and edited the manuscript. R.S. and M.W. contributed to discussion,performed the experiments, and reviewed and edited the manuscript. B.O.B.and T.W. designed research and reviewed and edited the manuscript. H.H.S.and B.B. are the guarantors of this work and, as such, had full access to all thedata in the study and take responsibility for the integrity of the data and theaccuracy of the data analysis.

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