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Ort, Datum: Bremen, 10.08.2017__________

Versicherung an Eides Statt

Ich, _Wei He, Leher Heerstr. 118, 28359, Bremen. Matr.-Nr. 2778354_(Vorname, Name, Anschrift, Matr.-Nr.)

versichere an Eides Statt durch meine Unterschrift, dass ich die vorstehende Arbeit selbständig und ohne fremde Hilfe angefertigt und alle Stellen, die ich wörtlich dem Sinne nach aus Veröffentlichungen entnommen habe, als solche kenntlich gemacht habe, mich auch keiner anderen als der angegebenen Literatur oder sonstiger Hilfsmittel bedient habe.Ich versichere an Eides Statt, dass ich die vorgenannten Angaben nach bestem Wissen und Gewissen gemacht habe und dass die Angaben der Wahrheit entsprechen und ich nichts verschwiegen habe.Die Strafbarkeit einer falschen eidesstattlichen Versicherung ist mir bekannt, namentlich die Strafandrohung gemäß § 156 StGB bis zu drei Jahren Freiheitsstrafe oder Geldstrafe bei vorsätzlicher Begehung der Tat bzw. gemäß § 161 Abs. 1 StGB bis zu einem Jahr Freiheitsstrafe oder Geldstrafe bei fahrlässiger Begehung.

_________________________Ort, Datum Unterschrift

I. Summary

Type 2 Diabetes (T2D) is strongly associated with obesity and characterized by chronic insulin resistance, progressive failure of pancreatic -cells, and ultimatelyhyperglycaemia. The association of T2D with chronic sterile inflammation has been extensively demonstrated, and the elevation of inflammatory mediators can predict type 2 diabetes progression. Pro-inflammatory cytokines and chemokines can cause insulin resistance in peripheral insulin-sensing tissues like fat, liver and muscle, and also lead toprogressive -cell failure. This eventually shifts metabolism from relative insulin insufficiency - due to the greater insulin demand in obesity - to definite insulin deficiency, while on the -cell level - from compensation to decompensation.

Toll-like receptor (TLR)-4 signaling is one of the major pro-inflammatory pathways activated by exogenous pathogen-related or endogenous danger-related molecules. Its ligands, including the classical ligand LPS as well as saturated fatty acids and CXCL10,among others, are increased systemically in patients with T2D as well as in at-risk individuals. TLR4-deficiency or its pharmacological inhibition have been shown to ameliorate obesity- or lipid-induced tissue inflammation and insulin resistance in humans and in mouse models. Increasing evidence also connects TLR4 to islet inflammation and

-cell dysfunction in the context of the pathogenesis of T2D, but many underlying mechanisms remain unknown.

In the first part of this thesis, I aimed to uncover the role of TLR4 activation by lipopolysaccharide (LPS), the classical TLR4 ligand, in islet inflammation -cell function in human islets. My special focus was to identify the inflammatory mechanism in the intercellular level – the possible interplay among different cells in human islets. I found that LPS-triggered TLR4 activation in cultured human islets induced -cell dysfunction, apoptosis and a pro-inflammatory profile with markedly increased IL- , IL-6, and IL-8 production. Macrophage-depletion demonstrates that islet resident macrophages are responsible for the production of IL- -/chemokines are predominantly produced from islet endocrine cells. IL-6 is partially responsible for the LPS- -cell dysfunction, while IL-8 produced from -cells is responsible for monocyte migration to islets during TLR4-activation-induced islet inflammation. This complex inflammatory response in islets is further potentiated in obese individuals, with more IL- , IL-6 and IL-8 expression and a tendency to more islet macrophage accumulation, suggesting a possibly self-augmented inflammatory cycle involving -cells, -cells and islet macrophages, which may explain the higher susceptibility of obese individuals to the -cell damage und eventually T2D.

Ageing is known to be associated to elevated T2D risk, though the underlying mechanism remains largely undiscovered. In the second part of this thesis, I aimed to

1

find out if ageing could aggravate obesity-induced T2D, and further focus on the effects o -cell function and the role of inflammation in such processes. In a mouse model of high fat diet induced obesity, I found an adverse potentiation of impaired glucose homeostasis, -cell dysfunction and chronic tissue inflammation by the combination of obesity and aging. In contrast, TLR4-deficiency exhibited a protection against thosedeleterious effects through inhibiting pro-inflammatory cytokine expression and switchingtissue macrophage activation to a more anti-inflammatory phenotype.

In both parts of this thesis, I provide further evidence that TLR4 and inflammation play acausative role in the development of T2D, and thereby support the concept of TLR4- or inflammation-targeted therapeutic strategies.

2

II. Zusammenfassung

Typ 2 Diabetes (T2D) ist stark assoziiert mit Übergewicht und durch chronische Insulinresistenz und progressives Versagen der insulin-produzierenden -Zellen im Pankreas gekennzeichnet, das letztlich zur Hyperglykämie führt. Anzeichen chronischer steriler Entzündungen im T2D wurden weitgehend nachgewiesen, aufgrund entzündlicher Botenstoffe lässt sich ein beginnender Typ-2-Diabetes bereits im Vorstadium vorhersagen.

Pro-inflammatorische Zytokine und Chemokine können Insulinresistenz im peripheren insulinsensitiven Geweben wie Fett, Leber und Muskel verursachen und auch zu einem progressiven -Zellversagen führen. Dies verschiebt schließlich den Stoffwechsel von einer relativen Insulin-Insuffizienz - aufgrund des größeren Insulinbedarfs bei Übergewicht - auf einen absoluten Insulinmangel und in der -Zelle von Kompensation zu Dekompensation.

Der Toll-like Rezeptor (TLR)-4 Signalweg ist einer der wichtigsten entzündungsvermittelnden Wege, der durch exogene oder endogene Pathogene aktiviert wird. Die Liganden, sowohl der klassische Ligand Lipopolysacharid (LPS) als auch gesättigte Fettsäuren und CXCL10, sind bei Patienten mit T2D sowie bei Risiko-Patienten systemisch erhöht. Ein TLR4-Mangel sowie dessen pharmakologische Hemmung konnte eine Übergewicht- oder Lipid-induzierte Gewebeentzündung sowie eine Insulinresistenz in Patienten und auch in Mausmodellen lindern. Vorangegangene Studien konnten einen Zusammenhang zwischen TLR4 und -Zell-Entzündung und -Dysfunktion sowie der Entwicklung von T2D erhärten. Allerdings sind vielezugrundeliegende Mechanismen weiterhin unbekannt.

Der erste Teil dieser Arbeit identifiziert den Effekt der LPS-induzierten TLR4-Aktivierung auf Entzündungsmechanismen und Funktionsstörungen in humanen pankreatischen Inselzellen. Mein Ziel war es hier, den entzündlichen Mechanismus auf interzellulärerEbene aufzudecken, nämlich das Zusammenspiel verschiedener Zelltypen innerhalb dermenschlichen Inseln. Ich habe festgestellt, dass die LPS-ausgelöste TLR4-Aktivierung in menschlichen Inseln ein pro-inflammatorisches Profil mit deutlich erhöhter IL-1 -, IL-6-,TNF - und IL-8-Produktion induziert und somit zur -Zell-Dysfunktion und Apoptose führt. Dabei sind die in den Inselzellen residierenden Makrophagen für die Produktion von IL-1 verantwortlich, während alle anderen Cyto- und Chemokine überwiegend vonendokrinen Inselzellen selbst produziert werden. IL-6 ist teilweise verantwortlich für die LPS-induzierte -Zell-Dysfunktion, während IL-8, das in den -Zellen produziert wird, für die Monozytenmigration in die Insel während TLR4-Aktivierung verantwortlich ist. Diese komplexen Entzündungsreaktionen in den Inseln ist bei adipösen Individuen weiter

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verstärkt, hier wird in den Inseln vermehrt IL-1 -, IL-6- und IL-8 produziert. Weiterhin gibt es eine Tendenz zur Makrophagen-Akkumulation in den Inseln, was auf einen selbstverstärkten Entzündungszyklus von - und -Zellen sowie Makrophagen hindeutet. Damit könnte auch eine höhere Anfälligkeit von übergewichtigen Personen auf die Entwicklung von -Zell-Schäden und schließlich T2D erklären werden.

Ein weiterer Risikofaktor für T2D ist das Altern. Es ist bekannt, dass mit zunehmenden Alter das Risiko, an T2D zu erkranken steigt; auch hier ist der zugrundeliegendeMechanismus noch nicht völlig erkannt. Im zweiten Teil dieser Arbeit gehe ich der Frage nach, ob mit zunehmendem Alter die Übergewicht-induzierte Entwicklung des Diabetes verstärkt wird, und ob dies weitere Auswirkungen auf -Zellfunktion und Entzündung hat. Im Mausmodell, in dem eine fett- und zuckerreicher Diät Übergewicht induziert, fand ich eine nachteilige Potenzierung beider Faktoren; eine damit verbundene stark beeinträchtigte Glukose-Homöostase, -Zell-Dysfunktion und chronische Gewebsentzündung. Dabei konnte jedoch ein TLR4-Mangel einen Schutz gegen jene schädlichen Wirkungen mittels Hemmung der pro-inflammatorischen Zytokin-Expression sowie einer Verschiebung von entzündlichen Gewebs-Makrophagen zu einem entzündungshemmenden Phänotyp bewirken.

In beiden Teilen meiner Arbeit gebe ich weitere Beweise dafür, dass TLR4 und die damit verbundenen chronischen Entzündungen eine ursächliche Rolle bei der Entwicklung von T2D spielen. Damit unterstützt meine Arbeit das Konzept der anti-TLR4- und anti-entzündlichen Therapie für den Diabetes.

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Abbreviations: AP-1, activator protein-1

ATM, adipose tissue macrophage

Arg1, Arginase 1

BAT, brown adipose tissue

CCL2, chemokine (C-C motif) ligand 2

CCR2, chemokine (C-C motif) receptor 2

CD, cluster of differentiation

CRP, C-reactive protein

CTL, cytotoxic T lymphocyte

CXCL1/10, C-X-C motif ligand 1/10

DAMPS, damage/danger-associated molecular pattern molecules

DC, dentritic cell

Emr1, EGF-like module-containing mucin-like hormone receptor-like 1

FFA, free fatty acids

GSIS, Glucose-stimulated insulin secretion

HFD, high-fat diet

IAPP, islet amyloid polypeptide

IFN, interferon

IL-1 /4/6/10/12/18, Interleukin 1 /4/6/10/12/18

iNOS, Inducible nitric oxide synthase

JNK, c-Jun N-terminal kinase

KO, knockout

LPS, Lipopolysaccharides

Ly6c1, lymphocyte antigen 6 complex, locus C1

MAPKs, mitogen-activated protein kinases

MCP1, monocyte chemoattractant protein 1

Mrc1 (CD206), Mannose receptor, C Type 1 (cluster of differentiation 206)

MyD88, myeloid differentiation primary response protein 88

5

ND, normal chow diet

NF- B, nuclear factor kappa-light-chain-enhancer of activated B cells

NO, nitric oxide

PAMPs, pathogen-associated molecular pattern molecules

PDX1, Pancreas/duodenum homeobox protein 1

ROS, reactive oxygen species

RNS, reactive nitrogen species

SFA, saturated fatty acid

TGF , Transforming growth factor

TLR, Toll-like receptor

TNF , Tumor necrosis factor

T1D, type 1 diabetes

T2D, type 2 diabetes

WAT, white adipose tissue

6

src

in vivo

In vivo

In vivo

in vitro

Ikbkb

ob/ob

Jnk2

in vitro

In vivo

Ccl2

Ccl2

in

vitro

in vitro in vivo

de novo

In

vitro

in

vivo

db/db

CCL2 CCL13

db/db

Il1 Tnf

Il10

db/db

db db

Il1b Tnfadb db

db db

db db

db/db

Pdx1

in vitro

Tlr4–/– Myd88–/–

Tnf Nos2 Nlrp3 Cnr1 Tgfb1 Il10

Arg1

Rhodobacter Oscillatoria

in vitro in vivo

Exogenous natural ligands(PAMPs)

Flavobacterium meningosepticumS. cerevisiae C. albicans

Dengue virus Rhodobacter

Oscillatoria

Endogenous ligands (DAMPs) (i) Extracellular matrix ligands

(ii) Intracellular ligands released/secreted from cells

(iii) Other ligands

in vitro in vivo

Tlr Tlr1–Tlr9)

P.

gingivalis

in vitro

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ARTICLE

TLR2/6 and TLR4-activated macrophages contributeto islet inflammation and impair beta cell insulingene expression via IL-1 and IL-6

Dominika Nackiewicz & Meixia Dan & Wei He & Rosa Kim & Anisa Salmi &Sabine Rütti & Clara Westwell-Roper & Amanda Cunningham & Madeleine Speck &

Carole Schuster-Klein & Beatrice Guardiola & Kathrin Maedler & Jan A. Ehses

Received: 28 October 2013 /Accepted: 8 April 2014 /Published online: 12 May 2014# Springer-Verlag Berlin Heidelberg 2014

AbstractAims/hypothesis Inflammation contributes to pancreatic betacell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2and -4 ligands are increased systemically in recently diag-nosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequencesof a high-fat diet. Here we investigated the role of

macrophages in TLR2/6- and TLR4-mediated effects on isletinflammation and beta cell function.Methods Genetic and pharmacological approaches were usedto determine the effects of TLR2/6 and TLR4 ligands onmouse islets, human islets and purified rat beta cells. Isletmacrophages were depleted and sorted by flow cytometryand the effects of TLR2/6- and TLR4-activated bone-marrow-derivedmacrophages (BMDMs) on beta cell functionwere assessed.Results Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expressionin mouse and human islets and IL-1β secretion fromhuman islets. TLR2/6 and TLR4 ligands also reducedinsulin gene expression; however, this occurred in anon-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretionpartly via reducing Ins1, Ins2, and Pdx1 mRNA expres-sion. Antagonism of the IL-1 receptor and neutralisationof IL-6 completely reversed the effects of activatedmacrophages on beta cell gene expression.Conclusions/interpretation We conclude that islet macro-phages are major contributors to islet IL-1β secretion inresponse to TLR2/6 and TLR4 ligands. BMDMs stimulatedwith TLR2/6 and TLR4 ligands reduce insulin secretion frompancreatic beta cells, partly via IL-1β- and IL-6-mediateddecreased insulin gene expression.

Keywords Beta cell . Diabetes . Inflammation . Pancreaticislet . Toll-like receptor 2 . Toll-like receptor 4

AbbreviationsBMDM Bone-marrow-derived macrophageCCL2 Chemokine (C-C motif) ligand 2DAMPs Danger-associated molecular patterns

Dominika Nackiewicz and Meixia Dan contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s00125-014-3249-1) contains peer-reviewed but uneditedsupplementary material, which is available to authorised users.

D. Nackiewicz :M. Dan : R. Kim :A. Salmi :A. Cunningham :M. Speck : J. A. Ehses (*)Department of Surgery, Faculty of Medicine, The University ofBritish Columbia, Child and Family Research Institute,950 W 28th Ave, Vancouver, BC, Canada V5Z 4H4e-mail: [email protected]

W. He :K. MaedlerCenter for Biomolecular Interactions, The University of Bremen,Bremen, Germany

S. RüttiDepartment of Genetic Medicine and Development, UniversityMedical Center, University of Geneva, Geneva, Switzerland

C. Westwell-RoperDepartment of Pathology and Laboratory Medicine, Faculty ofMedicine, The University of British Columbia, Child and FamilyResearch Institute, Vancouver, BC, Canada

C. Schuster-Klein : B. GuardiolaADIR – Groupe de Recherche Servier, Suresnes, France

J. A. EhsesDepartment of Cellular and Physiological Sciences, Faculty ofMedicine, The University of British Columbia, Child and FamilyResearch Institute, Vancouver, BC, Canada

Diabetologia (2014) 57:1645–1654DOI 10.1007/s00125-014-3249-1

GSIS Glucose-stimulated insulin secretionLPS LipopolysaccharidePAMPs Pathogen-associated molecular patternsqPCR Quantitative PCRTLR Toll-like receptorWT Wild-type

Introduction

Type 2 diabetes occurs when there is insufficient compensa-tion for insulin resistance by failing pancreatic beta cells,resulting in hyperglycaemia. It has long been appreciated thatchronic activation of the innate immune system is associatedwith type 2 diabetes [1, 2]; however, only recently haveclinical trials with IL-1-targeting agents confirmed that in-flammation contributes to beta cell failure in humans with thisdisease [3].

Macrophages infiltrate islets in humans with type 2diabetes [4, 5] and in numerous animal models of thisdisease [4, 6–9], leading to beta cell dysfunction [7, 8].We recently proposed that Toll-like receptor (TLR) 2signalling may drive macrophage-derived IL-1β expres-sion in an animal model of type 2 diabetes [10]. More-over, we now know that both TLR2 and TLR4 pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) are increased inthe circulation of recently diagnosed type 2 diabetespatients (HSP60, HSP70, HMBG1, endotoxin andhyaluronan) [11, 12]. Increased systemic endotoxin levelsin individuals with type 2 diabetes have been reported bya number of groups [13, 14] and are found in rodentmodels of this disease characterised by islet inflamma-tion [4, 15–17]. Indeed, both TLR2- and TLR4-deficientmice are protected from the metabolic consequences of ahigh-fat diet [10, 18–23]. Thus, TLR2 and/or TLR4activation may act as a trigger of islet inflammation intype 2 diabetes, promoting production of IL-1β andother proinflammatory stimuli and the recruitment ofmacrophages.

TLR2 and TLR4 mRNAs have been detected in rodent andhuman islets [24, 25] and TLR2/6 and TLR4 ligands increasecytokine gene expression in human islets [26]. TLR4 activa-tion also reduces insulin gene expression in rodent islets [27].However, there is a paucity of information about the effects ofTLR2 signalling on islet inflammation and beta cell function.Further, the role of macrophages in TLR2- and TLR4-inducedislet inflammation has not been explored. Here we investigat-ed the effects of TLR2/6 and TLR4 activation on islet inflam-mation and beta cell function, using both rodent and humanislets, with a focus on the role of islet macrophages in medi-ating these effects.

Methods

Animals and materials Wild-type (WT), Tlr2−/−, and Tlr4−/−

mice on a C57BL/6J background were acquired from B.Vallance [28] (Division of Gastroenterology, Department ofPediatrics, BC's Children's Hospital and Child and FamilyResearch Institute, Vancouver, BC, Canada), bred in-houseand maintained under specific pathogen-free conditions at theChild and Family Research Institute. Male mice aged8–12 weeks were used for all experiments. All animals weremaintained in compliance with Canadian Council on AnimalCare guidelines and the studies were approved by the Univer-sity of British Columbia Committee on Animal Care.

TLR ligands (ultrapure lipopolysaccharide [LPS] was fromEscherichia coli 0111:B4 strain) and antibody (mouse anti-TLR2 IgG1, clone T2.51, used at 15 μg/ml) were fromInvivogen (San Diego, CA, USA). Isotype mouse IgG1 anti-body was from eBiosience (San Diego, CA, USA). TAK-242was synthesised by Servier (Suresnes, France) according tothe published chemical structure [29–31] and used at a con-centration of 1.5 μmol/l. L929 conditioned media was fromAbLab, University of British Columbia. Mouse cytokineswere measured using Luminex technology (EMD Millipore,Billerica, MA, USA). Human IL-1β was assessed usingan ultra-sensitive ELISA (IBL International, Hamburg,Germany) and human IL-6, IL-8 and TNF-α were mea-sured using Meso Scale Discovery technology (Rockville,MD, USA). IL-1 receptor antagonist was from Biovitrum(Stockholm, Sweden) and goat anti-mouse IL-6 neutralisingantibody and goat IgG isotype control were from R&D Sys-tems (Minneapolis, MN, USA).

Pancreatic islet and rat purified beta cell isolation and resi-dent islet macrophage depletion Mouse islets and rat betacells were isolated as described [32, 33]. Human islets wereisolated from the pancreases of seven organ donors at theUniversity of Alberta, University of British Columbia andProdo Laboratories (Irvine, CA, USA). All human islet prep-arations had >80% purity of islets, were hand-picked andplated to ensure purity and were cultured as described [4].Human islet studies were approved by the University ofBritish Columbia and University of Bremen committees onHuman Ethics.

To deplete islet macrophages, 1 mg/ml clodronate-loadedliposomes (supplied by N. van Rooijen, Vrije UniversiteitAmsterdam, the Netherlands) were added to islets in suspen-sion (mouse) or plated on extracellular matrix (human),followed by the addition of Pam2CSK4 or LPS for 24 h.PBS-containing liposomes served as a control. Based ondose–response experiments, mouse islets or rodent beta cellswere treated with maximal effective doses of Pam2CSK4(100 ng/ml) or LPS (1 μg/ml) for 24 h unless otherwise stated(see electronic supplementary material [ESM] Fig. 1a, b).

1646 Diabetologia (2014) 57:1645–1654

Similarly, human islets were treated with 100 ng/mlPam2CSK4 or 20 μg/ml LPS for 24 h unless otherwise stated.

Histology Mouse islets in suspension were fixed, sectionedand stained using anti-F4/80 and anti-insulin antibodies asdescribed [34]. TUNEL+ beta cells (positive in a terminaldeoxynucleotidyl transferase-mediated dUTP nick-end label-ling assay) were stained as described [35] and imaged using aBX61 microscope.

Analysis and isolation of resident islet macrophages by flowcytometry Dispersed islet cells were stained with CD11b-PE(eBioscience, clone M1/70), CD11c-PE-Cy7 (eBioscience,clone N418) and F4/80-FITC (eBioscience, clone BM8) anti-bodies to identify resident macrophages [36]. Others havedescribed this cell population as immature dendritic cells[36, 37]. F4/80+Cd11b+Cd11c+ cells were confirmed to be99.4 ± 0.6% (n = 4) CD45+ using CD45-eFluor450(eBioscience, clone 30-F11). The proportion of islet CD45+

cells that was F4/80+Cd11b+Cd11c+ cells was 87.2±1.6%(n=4). Dead cells were excluded using the viability dyeeFluor506 (eBioscience) or 7-AAD. Unstained, single stainsand FMO (fluorescence minus one) controls were used to setgates and compensation. Data were analysed using FlowJovX.0.7, (Tree Star, Ashland, OR, USA) and FACSDiva v6.0(BD Biosciences, Mississauga, ON, USA) software. Islet F4/

80+CD11b+CD11c+ and F4/80−CD11b−CD11c− cells weresorted and plated in 96-well tissue-culture-treated dishes torecover for 6 h and then stimulated with 10 ng/ml Pam2CSK4or 100 ng/ml LPS for 6 h.

RNA extraction and real-time PCR cDNA from mousepancreases and islets was generated using theMNNucleoSpinRNA II kit (Macherey-Nagel, Düren, Germany) followed bySuperscript II (Invitrogen, Carlsbad, CA, USA). cDNA fromsorted islet cells was generated using the RNeasy micro kit(Qiagen, Valencia, CA, USA) followed by Superscript III(Invitrogen). cDNA from human islets was generated by usingpeqGOLD Isolation Systems TriFast (PEQLAB, Erlangen,Germany) followed by RevertAid Reverse Transcriptase(Thermo Scientific, Waltham, MA, USA). Quantitative PCR(qPCR) was performed using PrimeTime primers/probes (In-tegrated DNATechnologies, Coralville, IA, USA) or TaqManGene Expression Assay (for human islets; AppliedBiosystems, Foster City, CA, USA) in a ViiA7 or StepOnereal-time PCR system (Applied Biosystems), and differential

expression was determined by the 2−ΔΔCt method [38] withRplp0 used as a reference gene.

Bone-marrow-derived macrophage-conditioned media Bone-marrow-derived macrophages (BMDMs) were prepared asdescribed [10]. To generate conditioned media, cells were

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Fig. 1 Resident mouse islet macrophage depletion decreases TLR2/6-and TLR4-mediated cytokine expression. (a, b) Following treatment withclodronate-loaded liposomes (CLOD-lip) or PBS liposomes (PBS-lip),islet macrophage depletion was confirmed by histology (a) (green, F4/80;red, insulin; scale bar, 50 μm) and flow cytometry (b). The proportion ofF4/80+Cd11b+Cd11c+ cells per total islet cells was 0.52±0.03% (n=4).(c) TUNEL+ beta cells were quantified following PBS-lip or CLOD-liptreatment. An average of 2,887±393 beta cells were counted per

experimental condition per experiment. (d–o) Islets depleted of macro-phages were treated with Pam2CSK4 (Pam2) or LPS (black bars, PBS-lip; white bars, CLOD-lip) followed by total RNA extraction and qPCRanalysis. *p<0.05, **p<0.01 vs PBS-lip; †p<0.05 vs Pam2CSK4/LPSPBS-lip, as tested by ANOVA and Newman–Keuls post hoc test; (a, b)n=2 mice performed in two independent experiments, (c) n=5 miceperformed in two independent experiments and (d–o) n=4–7 mice per-formed in at least three independent experiments

Diabetologia (2014) 57:1645–1654 1647

stimulated with maximal effective doses of Pam2CSK4(10 ng/ml) or LPS (100 ng/ml) overnight for 18 h in isletmedia, followed by removal and filtering of media. Isletmedia spiked with Pam2CSK4 (10 ng/ml) and LPS(100 ng/ml) and left overnight in 75 cm2 flasks wereused as controls.

Glucose-stimulated insulin secretion and content Insulin se-cretion and content were determined as published [4], withmouse islets plated on 804G-ECM [33] coated six-well platesat 20 islets/well. Insulin was assayed by ELISA (Alpco,Salem, NH, USA).

Statistics Data are expressed as means ± SEM unless other-wise stated, with the number of individual experiments pre-sented in the figure legends. Data were analysed using thenon-linear regression analysis program PRISM v6 (GraphPad,La Jolla, CA, USA) and significance was tested using Stu-dent’s t test and analysis of variance (ANOVA) with Dunnett’s

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Fig. 2 TLR2/6 and TLR4 activation increases cytokine expression inmouse islet resident macrophages. (a) Gating strategy for flow cytometrysorting. Viable, single islet cells (see ESM Fig. 3) were sorted into F4/80+Cd11b+Cd11c+ (blue) and F4/80−Cd11b−Cd11c− (green) cells. (b–h)Total RNA was extracted from mouse pancreas (dark grey bars), islets(light grey bars) and sorted islet F4/80+Cd11b+Cd11c+ cells (black bars)(b–d) or F4/80−Cd11b−Cd11c− cells (white bars) (e–h) followed by

qPCR. ( i–p ) F4/80+Cd11b+Cd11c+ (black bars) and F4/80−Cd11b−Cd11c− cells (white bars) were stimulated with Pam2CSK4(Pam2) or LPS, followed by qPCR (i–n) or cytokine secretion analysis (o,p). Ctrl, control. *p<0.05, **p<0.01, ***p<0.001 vs pancreas (b–h) orCtrl (i–p) as tested by ANOVA and Dunnett’s post hoc test; n=4 or 5independent sorting experiments; nd, not detected

Fig. 3 TLR2/6 activation, not TLR4 activation, increases Tnf and Ccl2mRNA expression in rodent beta cells. MIN-6 beta cells (a, b) or ratpurified beta cells (c, d) were treated with Pam2CSK4 (black bars) or LPS(grey bars) for the indicated time; white bars, control. *p<0.05, **p<0.01,***p<0.001 vs untreated control, as tested by ANOVA andDunnett’s posthoc test; n=3 independent experiments

1648 Diabetologia (2014) 57:1645–1654

or Newman–Keuls post hoc test for multiple comparisonanalysis. Significance was set at p<0.05.

Results

TLR2/6- and TLR4-induced islet IL-1β secretion is macro-phage dependent Ligands were confirmed to be specific fortheir cognate receptors; Pam2CSK4-induced cytokine mRNAexpression was absent in Tlr2−/− islets (ESM Fig. 2a) andLPS-induced cytokine mRNA expression was absent inTlr4−/− islets (ESM Fig. 2b). Pam2CSK4-induced cytokinemRNA expression was unchanged in Tlr4−/− islets (ESMFig. 2c) and LPS-induced cytokine mRNA expression wasunchanged in Tlr2− /− islets (ESM Fig. 2d). TLR2neutralisation and TAK-242 mediated inhibition of TLR4signalling completely reversed islet cytokine mRNA expres-sion induced by Pam2CSK4 and LPS, respectively (ESMFig. 2e, f).

Treatment of mouse islets with clodronate-loaded lipo-somes for 48 h resulted in 63.8±3.3% (mean ± SD, n=2)depletion of islet macrophages (Fig. 1a, b). Clodronate-loadedliposomes (CLOD-lip) did not adversely affect beta cell sur-vival (Fig. 1c). Macrophage depletion significantly decreased

Pam2CSK4-induced islet Il1a, Il1b, Tnf and Ccl2 mRNAcompared with PBS-liposome (PBS-lip) control and Il6mRNA also tended to decrease (Fig. 1e–i). Similarly, isletmacrophage depletion significantly decreased LPS-inducedislet Il1a, Il1b, and Tnf mRNA compared with PBS-lip con-trol, with Il6 and Ccl2 mRNA also tending to decrease(Fig. 1k–o). Protein secretion of IL-1α, IL-1β, TNF-α, andchemokine (C-C motif) ligand 2 (CCL2) were below the limitof detection of the assay, as published previously for rat islets[6]. However, IL-6 secretion did not differ in treated isletswithout and with macrophage depletion (PBS-lip control7.35±2.29 pg/ml, Pam2CSK4 29.80±3.28 pg/ml, Clo-lipcontrol 4.96±0.54 pg/ml, Pam2CSK4 32.44±8.85 pg/ml,n=3; PBS-lip control 2.11±0.48 pg/ml, LPS 26.55±6.35 pg/ml, Clo-lip control 0.75±0.27 pg/ml, LPS33.29±19.19 pg/ml, n=4).

We purified islet macrophages (F4/80+Cd11b+Cd11c+

cells) and compared their response with that of a mixedpopulation of all other islet cell types (F4/80−Cd11b−Cd11c−

cells; ESM Fig. 3 and Fig. 2). Cell enrichment for F4/80+Cd11b+Cd11c+ cells was confirmed, while islet F4/80–Cd11b–Cd11c– cells were a mix of islet endocrine cells(beta cells, alpha cells) and endothelial cells based onmRNA expression (Fig. 2b–h). Islet macrophages had in-creased Il1a, Il1b, Il6, Il12b, Tnf and Il12b mRNA levels in

Fig. 4 Resident human islet macrophage depletion decreases TLR2/6-and TLR4-mediated IL-1β secretion. (a, b) Pam2CSK4 (a) and LPS (b)were added to human islets at 1 ng/ml (white bars), 10 ng/ml, (light greybars), 100 ng/ml LPS only (dark grey bars), 100 ng/ml Pam2CSK4 or20 μg/ml LPS (black bars). (c–l) Islets were depleted of macrophageswith clodronate-loaded liposomes (white bars; CLOD-lip) or PBS lipo-somes (black bars; PBS-lip) as control in the absence or presence of

Pam2CSK4 or LPS and qPCR (c–i) or cytokine secretion (j, l) wasassessed. (a, b) *p<0.05, ***p<0.001 vs untreated control, as tested byANOVA using Dunnett’s post hoc test. (c) **p<0.01 vs PBS-lip, usingStudent’s t test. (d–i) *p<0.05 vs PBS-lip and †p<0.05 vs LPS PBS-lip,as tested by ANOVA and Newman–Keuls post hoc test. (a–i) n=3–6human islet preparations performed in independent experiments; (j–l)mean ± SD of duplicate samples from one human islet preparation

Diabetologia (2014) 57:1645–1654 1649

response to Pam2CSK4 or LPS (Fig. 2i–n); however, Ccl2mRNAwas not detectable. Islet F4/80–Cd11b–Cd11c– cellshad increased Il6, Tnf and Ccl2 mRNA levels in responseto Pam2CSK4, with LPS showing a similar trend for Il6and Ccl2 mRNA (Fig. 2i–n). The expression of Il1b andIl12b mRNA was not detectable in F4/80–Cd11b–Cd11c–

cells. Pam2CSK4- and LPS-stimulated IL-6 and TNF-αsecretion was higher in islet macrophages than in F4/80−Cd11b−Cd11c− cells on a per cell basis (Fig. 2o, p).IL-1α, IL-1β and CCL2 secretion were below the limit ofdetection of the assay.

Next, rodent beta cells were stimulated with Pam2CSK4and LPS. Pam2CSK4 robustly stimulated Tnf and Ccl2mRNA expression in MIN6 beta cells and purified rat betacells (Fig. 3). We could not detect Il1a, Il1b or Il6 mRNA inMIN6 beta cells in response to Pam2CSK4 or LPS. Similarly,no significant effects on Il1a, Il1b or Il6 expression wereobserved following Pam2CSK4 or LPS stimulation in purifiedrat beta cells (ESM Fig. 4). LPS did not significantly increasecytokine mRNA expression in either rodent beta cell type(Fig. 3 and ESM Fig. 4). We detected Tlr4 but not Cd14

mRNA in MIN6 beta cells, and could not detect Tlr4 mRNAin purified rat beta cells.

Finally, we investigated whether TLR-stimulated cytokineexpression was dependent on resident macrophages in humanislets. Both Pam2CSK4 and LPS increased islet IL1A, IL1Band IL8 mRNA expression in a dose-dependent manner(Fig. 4a, b). We also analysed CCL13 mRNA because thischemokine was found to be increased in laser-captured betacells from individuals with type 2 diabetes [39]. However, wecould not detect CCL13 mRNA. Macrophage depletion con-sistently decreased Pam2CSK4- and LPS-induced islet IL1Aand IL1B mRNA compared with control, with little effect onother cytokines (Fig. 4d–i). Secretion of IL-1β protein wasalso decreased following macrophage depletion, with the de-pletion having no effect on IL-6 or IL-8 secretion (Fig. 4j–l).TNF-α secretion was below the detection limit of the assay.Thus, resident islet macrophages contribute to TLR2/6- andTLR4-induced Il1a/IL1A and Il1b/IL1B mRNA expression inmouse and human islets, and in human islets also contribute toIL-1β secretion.

TLR2/6 and TLR4 activation decrease insulin gene expressionin a non-beta cell autonomous manner Pam2CSK4 de-creased islet Ins1, Ins2 and Pdx1 mRNA levels in aTLR2-dependent manner (Fig. 5a), while LPS decreasedislet Ins1 and Ins2 mRNA levels in a TLR4-dependentmanner (Fig. 5b). TLR2 neutralisation blocked the effectsof Pam2CSK4 on Ins1 and Pdx1 mRNA expression, whileTAK-242 reversed the effects of LPS on Ins2 mRNAexpression (Fig. 5c, d). No significant effects on Ins1,Ins2 or Pdx1 mRNA expression were observed whenMIN6 beta cells or rat purified beta cells were treatedwith Pam2CSK4 or LPS (Fig. 5e, f).

Finally, we observed a twofold and 2.4-fold increase inTUNEL+ beta cells following Pam2CSK4 and LPS treatmentof islets, respectively (ESM Fig. 5). Thus, in addition toreducing insulin gene transcription, TLR2/6 and TLR4 acti-vation can also induce beta cell apoptosis.

TLR2/6- and TLR4-activated macrophages decrease beta cellinsulin gene expression via IL-1 and IL-6 BMDMs increasedcytokine mRNA expression and secretion of IL-1β, IL-6,IL-12 and TNF-α in response to Pam2CSK4 or LPS(Fig. 6a–d). When macrophage-conditioned media wereadded to mouse islets, both Pam2CSK4- and LPS-stimulatedmacrophages impaired glucose-stimulated insulin secretion(GSIS; Fig. 6e, g). These effects were partly due to reductionsin insulin content (Fig. 6f, h), because when insulin secretionwas normalised to content, reduced GSIS was no longerobserved (not shown). Therefore, we determined the effectsof TLR2/6- and TLR4-activated macrophages on Ins1, Ins2,and Pdx1 mRNA levels in mouse islets. TLR2/6- and TLR4-activated macrophages significantly reduced Ins1, Ins2 and

Fig. 5 TLR2/6 and TLR4 activation decrease insulin gene expression inwhole islets from mice, but not in rodent beta cells. (a, b) Islets fromWT(black bars), Tlr2−/− (a) (white bars) and Tlr4−/− mice (b) (white bars)were stimulated with Pam2CSK4 (a) or LPS (b). (c, d) Islets werestimulated with Pam2CSK4 (c) (black bars) or LPS (d) (black bars) inthe presence of a TLR2 neutralising antibody (c) or TAK-242 (d). (e, f)MIN-6 beta cells (e) or rat purified beta cells (f) were treated withPam2CSK4 (black bars) or LPS (grey bars). White bars, control. (a, b)*p<0.05 vs untreated control and †p<0.05 vs WT Pam2CSK4/LPStreatment, as tested by ANOVA using Newman–Keuls post hoc test. (c,d) *p<0.05 vs ligand-treated control using Student’s t test; n=3–18 miceperformed in at least three independent experiments

1650 Diabetologia (2014) 57:1645–1654

Pdx1mRNA levels (Fig. 6i–k). TAK-242 inhibited the effectsof TLR4-activated macrophages on islet Ins1, Ins2 and Pdx1mRNA expression levels (Fig. 6l).

Finally, to determine which cytokines were responsible forthe effects of activated macrophages on insulin gene transcrip-tion, we antagonised IL-1 signalling and neutralised IL-6activity. IL-1 was antagonised because beta cells are extreme-ly sensitive to low concentrations (pg/ml) of IL-1β [40]. IL-6was neutralised because it was the most highly secreted cyto-kine in response to TLR2/6 or TLR4 activation (Fig. 6b, d).IL-1Ra almost completely reversed the effects of TLR-activated macrophages on insulin gene transcription. Indeed,IL-1Ra together with IL-6 neutralisation completely reversed

the effects of TLR-activated macrophages on Ins1, Ins2 andPdx1 mRNA expression levels (Fig. 6m–o).

Discussion

TLR2 and TLR4 ligands represent potential triggers of isletinflammation in type 2 diabetes [12]. Here we build upon ourrecent findings that TLR2/6 and TLR4 ligands increase cyto-kine gene expression in human islets [26] and that Tlr2−/−

mice were protected from elevated islet Il1b mRNA expres-sion when fed a high-fat diet [10]. We also show that residentmacrophages in both mouse and human islets contribute to

Fig. 6 TLR2/6- and TLR4-activated macrophages reduce Ins1, Ins2 andPdx1mRNA expression inmouse islets via IL-1 and IL-6. (a–d) BMDMswere stimulated with Pam2CSK4 (a, b) (black bars) or LPS (c, d) (blackbars) overnight followed by qPCR (a, c) or analysis for cytokine secretion(b, d). White bars, control. (e–k) Conditioned media (Mac) fromBMDMs stimulated with Pam2CSK4 (Pam2) (e, f) or LPS (g, h) andcontrol islet media (No Mac) spiked with the indicated ligands wereadded to islets for 48 h. Thereafter, GSIS was assessed (e, g) (white bars,2.8 mmol/l glucose; black bars, 16.7 mmol/l glucose), insulin content wasassayed (f, h) or gene expression was analysed (i–k). (l) Conditionedmedia from untreated, TAK-242-treated (white bars), LPS-treated (blackbars) and LPS + TAK-242-treatedmacrophages (grey bars) were added to

islets for 48 h. Mφ, macrophages. (m–o) Conditioned media (CM) wastreated with isotype (white bars) or IL-6 antibody (light grey bars) andadded to islets in the absence or presence of IL-1Ra (2 μg/ml) for 48 hRNA (dark grey bars, IL-1Ra; black bars, anti-IL-6 antibody + IL-1Ra),followed by qPCR analysis. (a–h) *p<0.05, ***p<0.001 vs Ctrl: Mac, astested using Student’s t test. (i–k) *p<0.05 vs Ctrl: No Mac, †p<0.05 vsCtrl: Mac, as tested by ANOVA using Newman–Keuls post hoc test. (l)*p<0.05 vs CM macrophage control, †p<0.05 vs CM macrophage +LPS, as tested by ANOVA using Newman–Keuls post hoc test. (m–o)*p<0.05, **p<0.01 vs CM macrophage isotype, as tested by ANOVAusing Dunnett’s post hoc test n=4–8 mice performed in at least threeindependent experiments

Diabetologia (2014) 57:1645–1654 1651

islet IL-1 expression in response to TLR2/6 and TLR4 li-gands. Further, we propose that TLR2/6 and TLR4 ligandsreduce insulin gene expression in a non-cell-autonomousmanner and, via effects on beta cells and other islet cells(e.g. endothelial cells, alpha cells), increase chemokine secre-tion to attract circulating monocytes. Infiltrating islet macro-phages activated by TLR2/6 and/or TLR4 ligands might thenfurther reduce beta cell insulin gene transcription and insulinsecretion via IL-1 and IL-6, contributing to beta cell dysfunc-tion during type 2 diabetes (Fig. 7).

A number of recent studies have confirmed a role formacrophages in regulating islet inflammation and beta cellfunction in rodents with type 2 diabetes [7, 8, 34], with dataindicating a prominent role for IL-1β in mediating theseeffects [7, 34]. Our experiments using macrophage-conditioned media support these findings and once againhighlight the sensitivity of pancreatic beta cells to low levelsof IL-1β [40], with effects on insulin gene expression. How-ever, the effects of macrophages on beta cells are complex,and likely also include cytokine-mediated beta cell death inaddition to impairment of insulin secretion via effects oninsulin gene expression [41]. It also remains to be determinedwhether TLR2- and/or TLR4-activated macrophages impairbeta cell insulin secretion in vivo in type 2 diabetes.

Recently published clinical studies support the initial ob-servation that IL-1 inhibition improves beta cell function andreduces HbA1c levels in humans with type 2 diabetes [42].These include industry-sponsored studies using IL-1β-specific antibodies [43–45] and a clinical study in non-diabetic individuals with the metabolic syndrome [46]. How-ever, one unpublished clinical trial using an IL-1β-specificantibody reported no effects on HbA1c [47]. Hyperglycaemiawas originally shown to induce human beta cell IL-1β ex-pression [48] but macrophages may represent an additionalcellular origin of IL-1β in type 2 diabetes [7, 8, 49]. In agree-ment with these studies, our data clearly show that TLR2 andTLR4 ligands induce islet IL1A and IL1BmRNA expression inislets and that macrophages are a major cellular source of IL-1βin response to these stimuli, despite only comprising a verysmall proportion of islet cells. Whether pancreatic beta cells arealso a cellular source of IL-1β in response to TLR2 and/orTLR4 ligands requires further investigation.

Overall, our findings are consistent with those recentlyreported by Eguchi and colleagues on the in vivo effects ofethyl palmitate on pancreatic beta cells, acting via macro-phages to reduce insulin gene expression [8]. However, incontrast to these findings [8], our present rodent beta cell dataand previous human beta cell data [26] do not support a role

TLR2/6 and TLR4 ligands

CCL2/IL-8

Infiltrating macrophages

Inflamed isletin type 2 diabetes

Insulin

IL-1/IL-6

TLR2/6

IL1a, IL1b

Macrophage

IL-8, TNF, CCl2

Beta cell

IL-8, CCL2

Non-beta cell

TLR2/6

Pancreaticislet

TLR4

TLR4TLR2/6?

Fig. 7 The role of macrophages in the regulation of islet cytokineexpression and beta cell function by TLR2/6 and TLR4 ligands. Inaddition to inducing islet IL1A/B mRNA expression via effects on isletresident macrophages, TLR2/6 increases chemokine secretion (CCL2 inmouse; IL-8 in humans) from beta cells, while TLR4 increases chemo-kine secretion mostly from non-beta cells (e.g. endothelial cells, alpha

cells) in islets. These chemokines attract circulating monocytes to islets,which when activated by TLR2/6 and/or TLR4 ligands reduce beta cellinsulin gene transcription and insulin secretion via IL-1 and IL-6, poten-tially contributing to beta cell dysfunction during type 2 diabetes. Basedon our findings and others’ [26]

1652 Diabetologia (2014) 57:1645–1654

for TLR4-induced expression of the chemokines CCL2 andIL-8 in beta cells. This is consistent with the Tlr4 messagebeing undetectable in purified rat beta cells, while in MIN6beta cells we could not detectCd14 at the mRNA level. Whilewe did not analyse TLR4 signalling in purified mouse betacells, it is unlikely that a highly conserved receptor such asTLR4would show differences in beta cell expression betweenspecies. Thus, while islet TLR4 activation is undoubtedly ableto increase islet CCL2/IL-8 expression, we propose thatnon-beta cells (e.g. endothelial cells, alpha cells) areresponsible for this effect (Fig. 7). Tissue-specific dele-tion of TLR4 in vivo will ultimately be required todetermine the role of TLR4 signalling in islet inflam-mation in type 2 diabetes.

In contrast to LPS, TLR2/6 activation did increase Tnf andCcl2 mRNA in purified rat beta cells and MIN6 cells, andpreviously increased IL-8 expression in human beta cells [26].This suggests cell-type-specific expression of TLR2 vs TLR4in islets, with TLR2 expressed on beta cells, macrophages andpotentially non-beta cells, while TLR4 expression is mainlyconfined to non-beta cells and macrophages.

Our studies have important clinical implications. Inaddition to increased systemic TLR2 and TLR4 ligands,monocytes from individuals with type 2 diabetes showhyper-responsiveness to these ligands [50]. NeutralisingTLR2 and/or inhibiting TLR4 signalling was effective inreducing islet cytokine expression and reversing the effectsof these ligands on insulin gene expression. In addition,TAK-242 reversed the effects of TLR4-activated macro-phages on Ins1, Ins2 and Pdx1 gene expression. Theeffects of systemic TLR2 and/or TLR4 PAMPs andDAMPs on islet inflammation may be exacerbated whencombined with local inducers of islet inflammation such asislet amyloid deposits [34, 49], promoting end-stage betacell dysfunction and death in type 2 diabetes. While therelative contribution of TLR2/6 vs TLR4 activation to isletinflammation and beta cell function in type 2 diabetesremains to be determined, therapeutically targeting thesereceptors may improve hyperglycaemia in this disease.

Acknowledgements We thank L. Xu and M. Komba for technicalassistance provided by the CFRI FACS core and islet isolation corefacilities, respectively.

Funding This work was supported by funding from the Child andFamily Research Institute (JAE), the University of British Columbia(JAE), the Canadian Institutes of Health Research (PCN-110793 andPNI-120292; JAE), the European Research Council (KM), the DiabetesCompetence Network KKNDm supported by the Federal Ministry ofGermany (BMBF; KM), and a collaborative research agreement withServier (JAE, KM). JAE has salary support from a Child and FamilyResearch Institute Investigator Award and a Canadian Diabetes Associa-tion scholar award. DN is supported by a UBC Transplantation TrainingProgram and a CIHR-Vanier Canada Graduate Scholarship. CW-R issupported by a CIHR-Vanier Canada Graduate Scholarship.

Duality of interest CS-K and BG are employees of Servier, France. Allother authors declare that there is no duality of interest associated withtheir contribution to this manuscript.

Contribution statement DN, MD, WH, RK, AS, SR, CW-R, AC, MSdesigned and performed experiments, and analysed data. KM designedexperiments and CS-K and BG contributed to the conception and designof the study. All authors edited the manuscript and approved the finalversion. JAE supervised the study, designed and performed experiments,analysed data and wrote the manuscript. JAE is the guarantor of this workand, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the dataanalysis.

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1

Electronic supplementary material (ESM)

ESM Fig. 1 Dose response of Pam2CSK4 (a) and LPS (b) on cytokine expression in

mouse islets. Islets were stimulated for 6h with the indicated concentrations of

Pam2CSK4 (a) (white bars, 0.1 ng/ml; light grey bars, 1 ng/ml; dark grey bars, 10 ng/ml;

black bars, 100 ng/ml) and LPS (b) (white bars, 10 ng/ml; dark grey bars, 100 ng/ml;

black bars, 1 μg/ml). * Represents p<0.05 vs untreated control as tested by ANOVA

using Dunnett’s post-hoc test. Data represent mean ± SEM from n=3-8 mice performed in

at least three independent experiments.

2

ESM Fig. 2 Specificity of Pam2CSK4 for TLR2 and LPS for TLR4 in mouse islets. (a-d)

Islets from WT (black bars), Tlr2-/- (white bars), and Tlr4-/- (grey bars) mice were

stimulated with Pam2CSK4 (a, c) or LPS (b, d). (e-f) Islets from WT were stimulated

with Pam2CSK4 (e) (black bars) or LPS (f) (black bars) in the presence of (e) a TLR2

neutralizing antibody (white bars) or TAK-242 (f) (white bars). * Represents p<0.05,

**p<0.01 vs WT or treatment control as tested by Student’s t-test. Data represent mean ±

SEM from n=3-8 mice performed in at least three independent experiments.

3

ESM Fig. 3 Gating strategy for flow cytometry sorting of islet F4/80+Cd11b+Cd11c+ and

F4/80-Cd11b-Cd11c- cells. (a-c) Islets were dispersed and sorted by flow cytometry.

Events were gated to exclude dead cells (a), debris (b), and doublets (c).

4

ESM Fig. 4 Il1a, Il1b, and Il6 mRNA expression following TLR2 or TLR4 activation in

purified rat beta cells. Rat purified beta cells were treated with Pam2CSK4 (black bars)

or LPS (grey bars) for the indicated time followed by qPCR for Il1a (a), Il1b (b), and Il6

(c) mRNA expression. White bars, control. Data represent n=3 independent experiments.

5

ESM Fig. 5 TLR2/6 and TLR4 activation increase beta cell apoptosis. Mouse islets were

treated for 24h with 100 ng/ml Pam2CSK4 (Pam2) or 1 μg/ml LPS. (a) TUNEL+ beta

cells were quantified in images obtained from a BX61 microscope (representative images

shown in b). 0.33% ± 0.09% (n=4) TUNEL+ beta cells were detected under control

conditions. An average of 4700 ± 760 beta cells were counted per experimental condition

per experiment. Data represent mean ± SEM from n=3-4 mice performed in at least three

independent experiments.

per se

TLR4 as key player in inflammation in metabolic disease.

TLR4 and -cell failure.

Cytokines, chemokines and -cell failure.

in vitro

Escherichia coli

IL1b IL6 TNF CCL2 IL8 CD68

ITGAM TLR4PPIA

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