Dysfunction Cytokine Release and Pancreatic Islet Graft Polypeptide-Induced Proinflammatory IL-1 Blockade Attenuates Islet Amylo

IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinflammatory Cytokine Release and Pancreatic Islet Graft Dysfunction This information is current as of September 23, 2021. Clara Westwell-Roper, Derek L. Dai, Galina Soukhatcheva, Kathryn J. Potter, Nico van Rooijen, Jan A. Ehses and C. Bruce Verchere J Immunol 2011; 187:2755-2765; Prepublished online 3 August 2011; Downloaded from doi: 10.4049/jimmunol.1002854 http://www.jimmunol.org/content/187/5/2755 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/08/04/jimmunol.100285 Material 4.DC1 References This article cites 57 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/187/5/2755.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinﬂammatory Cytokine Release and Pancreatic Islet Graft Dysfunction

Clara Westwell-Roper,* Derek L. Dai,* Galina Soukhatcheva,* Kathryn J. Potter,* Nico van Rooijen,† Jan A. Ehses,‡ and C. Bruce Verchere*,‡

Islets from patients with type 2 diabetes exhibit b cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by

islets and induced secretion of TNF-a, IL-1a, IL-1b, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow- Downloaded from derived macrophages. hIAPP-induced TNF-a secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts http://www.jimmunol.org/ contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP- induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to b cell dysfunction in type 2 diabetes and islet transplantation. The Journal of Immunology, 2011, 187: 2755–2765.

atients with type 2 diabetes mellitus are unable to secrete tion following islet transplantation (8), and isolated islets develop sufficient insulin to compensate for increased peripheral amyloid rapidly during pretransplant culture and following en- P insulin resistance (1). Their pancreatic islets exhibit pro- graftment (9–13). Anti-inflammatory drugs such as IL-1R antag- by guest on September 23, 2021 gressive b cell loss (2), likely due in part to increased expression onists (IL-1Ra) or anti–IL-1b mAbs can preserve b cell function of proinflammatory cytokines (3, 4), macrophage infiltration (5, in type 2 diabetic patients (14, 15) and in cultured human islets 6), and islet amyloid deposition (7). Similarly, immune cell infil- (16, 17). tration and proinflammatory cytokine release impair b cell func- Islet amyloid deposits form by aggregation of human islet amyloid polypeptide (hIAPP), a 37-aa peptide cosecreted with insulin by b cells (18). Fibrils comprised of aggregated hIAPP are phago- *Department of Pathology and Laboratory Medicine, Child and Family Research cytosed but ineffectively degraded by islet macrophages (19). The Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4; †Department of Molecular Cell Biology, Vrije Universiteit Medical Cen- increased diabetes susceptibility observed in transgenic mice with ter, 1081 BT Amsterdam, The Netherlands; and ‡Department of Surgery, Child and b cell expression of hIAPP suggests that hIAPP aggregation is a Family Research Institute, University of British Columbia, Vancouver, British cause and not merely a consequence of b cell dysfunction (20– Columbia, Canada V5Z 4H4 22). Furthermore, prefibrillar aggregates are directly toxic to b Received for publication August 24, 2010. Accepted for publication July 1, 2011. cells in vitro (23, 24). hIAPP aggregation is also associated with b This work was supported by Canadian Institutes for Health Research Operating Grant cell death in cultured human islets (10, 25) and with recurrence of MOP-64427, Canadian Diabetes Association Grant OG-3-08-2640-CV, a Vancouver Coastal Health–University of British Columbia Canadian Institutes for Health Re- hyperglycemia following islet transplantation (9, 12). Rodent islet search MD/Ph.D. studentship (to C.W.-R.), a Michael Smith Foundation for Health amyloid polypeptide (rIAPP) does not share this toxicity because Research Junior Graduate studentship (to C.W.-R.), and a Michael Smith Foundation for Health Research Senior Scholar award (to C.B.V.). three proline substitutions between aa 20 and 29 disrupt the b-sheet formation required for aggregation (26). The sequences presented in this article have been submitted to Gene Expression Omnibus under accession number GSE23534. Aggregates of hIAPP share a common cross b-sheet structure Address correspondence and reprint requests to Dr. C. Bruce Verchere, Department with those of other amyloidogenic peptides known to induce a of Pathology and Laboratory Medicine, Room A4-181, Child and Family Research potent proinflammatory response. Peptides such as enterobacterial Institute, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada. E-mail address: CsgA and mammalian amyloid-b (Ab), which forms amyloid [email protected] plaques in Alzheimer’s disease, induce TLR signaling in mono- The online version of this article contains supplemental material. cytes (27), providing one potential pathway for amyloid-induced Abbreviations used in this article: Ab, amyloid-b; BMDM, bone marrow-derived b Αb b macrophage; hIAPP, human islet amyloid polypeptide; IAPP, islet amyloid polypep- pro–IL-1 synthesis (28, 29). also promotes pro–IL-1 mat- tide; IL-1Ra, IL-1R antagonist; LDH, lactate dehydrogenase; NLRP3, NACHT, LRR, uration (the second signal required for secretion of mature IL-1b) and PYD domain-containing protein 3; qRT-PCR, quantitative RT-PCR; rIAPP, ro- via activation of the NACHT, LRR, and PYD domain-containing dent IAPP. protein 3 (NLRP3) inflammasome (30). We therefore hypo- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 thesized that hIAPP may contribute to islet inflammation by www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002854 2756 REGULATION OF hIAPP-INDUCED ISLET INFLAMMATION BY IL-1 activating the same innate immune pathways. To address this hy- RPMI 1640 supplemented with 100 U/ml penicillin, 100 mg/ml strepto- pothesis, we first characterized the effects of hIAPP aggrega- mycin, 2 mM Glutamax, and 10% FBS. For analysis of cytokine and tion on cytokine release by bone marrow-derived macrophages chemokine release, islets were incubated overnight prior to the start of each experiment. Consistency in islet size and number was verified by (BMDMs) and chemokine release by islets. We next assessed the comparison of Alamar Blue reduction among treatments at the end of effects of IL-1R and TLR signaling on the macrophage response each experiment. to hIAPP. Finally, we evaluated the effects of IL-1Ra on hIAPP- induced islet dysfunction and macrophage recruitment in a rodent Chemotaxis assay model of islet transplantation. Human islets were plated at a density of 200 islets/well of a 24-well plate and treated with varying concentrations of hIAPP or rIAPP for the indicated length of time. Supernatants were collected and stored at 280˚C Materials and Methods until analysis. Thawed supernatants were diluted 1:1 with RPMI 1640, and Animals 275 ml was added to the bottom chamber of a 96-well Transwell plate 2/2 (Corning). THP-1 monocytes were suspended at a concentration of 1 3 C57BL/6J, B6.129-Tlr2tm1Kir/J (Tlr2 ), C57BL/10J, C57BL/10ScScNJ 6 2 2 10 cells/ml in RPMI 1640 containing 0.1% BSA, and 70 ml of the sus- (Tlr4 / ), FVB/N-Tg(Ins2-IAPP)RHFSoel/J (hIAPP transgenic), and pension was added to the upper chamber of the Transwell plate. After 4 h, NOD.CB17-Prkdcscid/J (NOD/SCID) mice were purchased from The 2 2 the media in the bottom wells were collected and the number of cells was Jackson Laboratory (Bar Harbor, ME). C57BL/6 Myd88 / mice were determined by analyzing each sample on a flow cytometer for 15 s and purchased from Oriental BioScience (Kyoto, Japan). Heterozygotes were interpolating from a standard curve. The chemotactic index was the ratio bred in-house to produce littermate controls. All animals were maintained of the concentration of migrated cells in the test sample to that in the in compliance with Canadian Council on Animal Care guidelines, and media control, because in our preliminary experiments IAPP alone did not studies were approved by the University of British Columbia Committee

induce monocyte chemotaxis. Downloaded from on Animal Care. Cell culture Islet macrophage depletion

All incubations were performed at 37˚C with 5% CO2. THP-1 monocytes Prior to evaluation of hIAPP-induced islet CCL2 release, isolated islets were (ATCC TIB-202) were maintained in RPMI 1640 medium supplemented allowed to recover overnight and incubated for 24 h with 1 mg/ml clodro- with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM Glutamax, 10 mM nate delivered in liposomes to deplete islets of resident macrophages. HEPES, 1 mM sodium pyruvate, and 10% FBS (all from Invitrogen, Liposomes were prepared, as described previously (33), using phosphati- Burlington, ON, Canada). To generate BMDMs, bone marrow was flushed dylcholine (LIPOID E PC; Lipoid, Ludwigshafen, Germany) and choles- http://www.jimmunol.org/ out of mouse femurs and plated in DMEM supplemented with 100 U/ml terol (Sigma-Aldrich). Cl2MDP (or clodronate) was a gift of Roche Diag- penicillin, 100 mg/ml streptomycin, 2 mM Glutamax, 1 mM sodium py- nostics (Mannheim, Germany). Islets were washed prior to subsequent ruvate, 10% FBS, and 15% L929-conditioned media (AbLab, Biomedical experiments, and depletion of macrophages was verified by quantitative Research Centre, University of British Columbia). After 24 h, nonadherent RT-PCR (qRT-PCR). cells were transferred to tissue culture-treated plates and incubated for 7 d, with addition of fresh media on days 3 and 6. Adherent BMDMs were Global gene expression detached by gentle scraping in PBS and plated at a density of 100,000 cells/well of a 96-well plate. After 24 h, cells were starved of L929 media C57BL/6 BMDMs were treated with hIAPP or rIAPP for 12 h. RNA was for 10–12 h prior to each experiment. isolated using an RNeasy mini kit (Qiagen, Mississauga, ON, Canada), and quality was assessed using an Agilent 2100 BioAnalyzer (Agilent Tech- Peptides and inhibitors nologies, Santa Clara, CA). All samples had RNA integrity number scores by guest on September 23, 2021 .9.2. Global gene expression was evaluated with an Illumina MouseRef-8 hIAPP and rIAPP (Bachem, Torrance, CA) were dissolved in hexafluoro-2- v2 Expression Bead Chip with an Illumina BeadStation 500GX BeadArray propanol for 3 h, lyophilized, and stored at 220˚C until use. Immediately Reader (Illumina, San Diego, CA). Data were analyzed with Bead Studio prior to each experiment, islet amyloid polypeptide (IAPP) was dissolved software using rank-invariant normalization. Only transcripts with detec- in 0.1 M acetic acid, which was diluted in culture media to a final max- tion and differential expression p values ,0.05 were included in the anal- imum concentration of 595 nM acetic acid. Inhibitors were added to ysis. Differentially expressed genes (absolute fold difference .1.5) were BMDMs as indicated, including polymyxin B (Sigma-Aldrich, Oakville, uploaded to the Database for Annotation, Visualization, and Integrated ON, Canada), MyD88 homodimerization inhibitory peptide (Imgenex, San Discovery functional clustering tool (34). Functional annotation analysis Diego, CA), the caspase-1 inhibitor Z-YVAD-FMK (Sigma-Aldrich), the was used to evaluate overrepresentation of biological pathways based on actin polymerization inhibitor cytochalasin D (Sigma-Aldrich), the ca- Gene Ontology terms. The significance of overrepresented terms was de- thepsin B inhibitor CA-074-Me (EMD Chemicals, Gibbstown, NJ), the termined by a Benjamini p value to correct for multiple comparisons. NLRP3 inhibitors glibenclamide and Bay 11-8072 (Sigma-Aldrich), and Array data were deposited in National Center for Biotechnology Informa- the IL-1Ra anakinra (Kineret; Biovitrum AB, Stockholm, Sweden). Inhi- tion’s Gene Expression Omnibus database as accession number GSE23534 bitors at the concentrations shown had no effect on cell viability, as de- (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23534). termined by Alamar Blue reduction. LPS was extracted from Salmonella typhimurium, as previously described (31), and was a gift from A. Plesner qRT-PCR (University of British Columbia, Vancouver, BC, Canada). cDNAwas synthesized using 100 ng RNA and a qScript cDNA synthesis kit, Cell viability according to the manufacturer’s instructions (Quanta Biosciences, Gai- thersburg, MD). qRT-PCR was performed using Power SYBR Green PCR To evaluate mitochondrial activity, supernatants were removed at the in- Master Mix (Applied Biosystems, Warrington, U.K.), and reactions were dicated time points and replaced with fresh media containing 9% Alamar run on an ABI 7500 Fast Real-Time PCR System. Most primer sequences Blue (Invitrogen). BMDMs were incubated for 4–6 h, and fluorescence was and cycle protocols were obtained from the public database PrimerBank quantified on a Fluoroskan Ascent Reader (excitation 530 nm; emission (35). Sequences were as follows (all mouse specific): Tnf (PrimerBank ID 590 nm; Thermo Fisher Scientific, Hudson, NH). To evaluate apoptotic 7305585a1) forward 59-CCCTCACACTCAGATCATCTTCT-39, reverse death, BMDMs were fixed in 10% buffered zinc formalin (Anatech, Battle 59-GCTACGACGTGGGCTACAG-39; Il1b (PrimerBank ID 6680415a1) Creek, MI) and stained with TUNEL (Roche Diagnostics, Laval, QC, forward 59-GCAACTGTTCCTGAACTCAACT-39, reverse 59-ATCTTTT- Canada), according to the manufacturer’s instructions. TUNEL positivity 9 9 3 GGGGTCCGTCAACT-3 ; Il1a (36) forward 5 -CAGGGCAGAGAGG- and the number of cells per 10 objective field were determined using GAGTCAAC-39, reverse 59-CAGGAACTTTGGCCATCTTGAT-39; Emr1 an ArrayScan VTI HCS reader (Thermo Fisher Scientific, Pittsburgh, PA). (PrimerBank ID 2078512a1) forward 59-GCCCTGAACATGCAACCTG- To assess loss of cell membrane integrity, lactate dehydrogenase (LDH) in 39, reverse 59-CTCCTCCACTAGATTCAAGTCCT-39; Itgam (PrimerBank cell and islet supernatants was quantified with a colorimetric LDH assay, ac- ID 132626288b1) forward 59-CCATGACCTTCCAAGAGAATGC-39,reverse cording to the manufacturer’s instructions (G-Biosciences, St. Louis, MO). 59-ACCGGCTTGTGCTGTAGTC-39; Ccl2 (PrimerBank ID 6755430a1) Islet culture forward 59-TTAAAAACCTGGATCGGAACCAA-39, reverse 59-GCATT- AGCTTCAGATTTACGGGT-39; GAPDH (PrimerBank ID 6679937a1) Mouse islets were isolated by ductal collagenase injection and filtration, as forward 59-AGGTCGGTGTGAACGGATTTG-39, reverse 59-TGTAGAC- described previously (32). Islets were maintained at 37˚C with 5% CO2 in CATGTAGTTGAGGTCA-39; and Rplp0 (PrimerBank ID 6671569a1) The Journal of Immunology 2757 forward 59-AGATTCGGGATATGCTGTTGGC-39, reverse 59-TCGGGT- other experiments showed the same statistically significant differences, CCTAGACCAGTGTTC-39. Differential expression was determined by the but with different absolute values due to variation in IAPP activity among 22DDCT method (37) using GAPDH (macrophages) or Rplp0 (islets) as the lots of peptide. A p value ,0.05 was considered significant. internal control.

Islet transplantation Results Islets were isolated from 12- to 16-wk-old FVB/N-Tg(Ins2-IAPP)RHFSoel/ Prosurvival effects of IAPP in BMDMs J mice or wild-type littermate controls and allowed to recover overnight prior to transplantation. Recipient 11-wk-old NOD/SCID mice were ren- To evaluate the possible effects of hIAPP on macrophage viabil- dered hyperglycemic by a single i.p. injection of 200 mg/kg streptozotocin ity, BMDMs were treated with hIAPP or rIAPP (15 mM) in the (Sigma-Aldrich) in citrate buffer. One day prior to transplantation, recip- absence of M-CSF for 12 h. Aggregated peptide was observed by ient mice were implanted with an ALZET mini-osmotic pump (Durect, Thioflavin S staining of hIAPP-treated cells, often in association Cupertino, CA) containing PBS or IL-1Ra. Pumps were replaced every with cell membranes (Fig. 1A). Interestingly, both hIAPP and 2 wk for the duration of the experiment. Recipient mice were transplanted with 150 islets into the left renal subcapsular space 4 d after streptozotocin rIAPP inhibited LDH release associated with M-CSF deprivation, injection, when blood glucose levels were .20 mmol/l. Animals were with a reduction to 83 6 7% of the untreated control for rIAPP monitored twice weekly for nonfasting tail vein blood glucose levels us- and 62 6 3% for hIAPP after 12 h (Fig. 1B). Similarly, IAPP ing a glucometer (OneTouch, Burnaby, BC, Canada). After 8 wk, glucose protected against macrophage loss, with an average cell number tolerance was evaluated by measurement of glucose at 0, 15, 30, 60, and 6 6 120 min following i.p. injection of 1.5 g dextrose per kg body weight. Re- per field of 175 26 for hIAPP and 80 14 for rIAPP compared moval of the islet graft-bearing kidney was performed to rule out effects with 57 6 2 in the untreated control after 12 h (Fig. 1C). These of residual pancreatic islet function, and all mice included in our analyses data suggest that IAPP can provide a prosurvival signal to mac- Downloaded from returned to hyperglycemia within 24 h. Grafts were fixed in 10% formalin rophages even in the absence of aggregation; however, the more and processed for histology. dramatic effect of hIAPP compared with rIAPP suggests that Thioflavin assays peptide aggregation provides an additional survival stimulus, sim- ilar to proinflammatory agents such as LPS. hIAPP aggregation was monitored by Thioflavin T fluorescence, as described previously (38). In brief, peptide was dissolved in phenol red-free Proinflammatory cytokine release by macrophages in response m http://www.jimmunol.org/ DMEM containing 10 M Thioflavin T. Fluorescence emission was evalu- to hIAPP ated on a Fluoroskan Ascent reader (excitation 444 nm; emission 485 nm). For detection of hIAPP aggregates in cultured cells, BMDMs were grown To determine whether hIAPP induces expression of proinflam- on chamber slides coated with poly-L-lysine (Sigma-Aldrich). Cells were matory genes, we performed global gene expression analysis to fixed in 10% formalin for 20 min, permeabilized with 0.02% v/v Triton X- 100 at room temperature for 5 min, and stained with 0.01% w/v Thioflavin compare expression profiles between hIAPP- and rIAPP-treated S for 10 min at room temperature, followed by three washes in 70% v/v BMDMs after 12 h. Functional annotation analysis revealed sig- ethanol. Cells were wet mounted using Vectashield mounting medium with nificant enrichment of Gene Ontology terms among genes that DAPI (Vector Laboratories, Burlingame, CA) and visualized on a BX61 were differentially expressed in response to hIAPP (Supplemental microscope (Olympus, Center Valley, PA). All image analyses, including Table I). Overrepresented pathways among the upregulated genes deconvolution of z-stacks, were performed using Image-Pro software by guest on September 23, 2021 (MediaCybernetics, Bethesda, MD). included those associated with actin polymerization; angiogenesis and wounding; and regulation of the immune response, including Immunohistochemistry cytokine and chemokine expression (Table I). Downregulated Paraffin-embedded sections (5 mm) from two different regions of each groups included genes associated with nucleosomes, membrane- islet graft were deparaffinized and rehydrated. Ag retrieval was performed enclosed lumens, regulation of cell death, and protein serine/thre- by incubation in Target Retrieval Solution (Dako, Carpinteria, CA) in a onine kinase activity. steamer for 20 min. Sections were blocked with 2% normal goat serum To explore further the cytokine profile of hIAPP-treated cells, (Vector Laboratories) for 30 min, then incubated with guinea pig anti- we analyzed supernatants from BMDM cultures treated with IAPP insulin Ab (1:100; Dako) and rat anti-F4/80 Ab (1:100; Cedarlane) in 0.1% BSA/PBS for 1 h at room temperature. Sections were incubated with Alexa for 12 h (Fig. 2A). hIAPP, but not rIAPP, induced significant lev- 594 goat anti-guinea pig and Alexa 488 goat anti-rat secondary Abs (both els of IL-1a (11.7 6 2.3 versus ,2.9 pg/ml), IL-1b (27.2 6 2.7 1:100; Molecular Probes, Invitrogen) for 1 h at room temperature. Slides versus 2.6 6 1.4 pg/ml), TNF-a (134 6 23 versus 5.9 6 0.4 pg/ were mounted and imaged as for Thioflavin S staining. ml), CCL2 (169 6 8 versus 18 6 2 pg/ml), CXCL1 (522 6 110 versus 91 6 23 pg/ml), CCL3 (6.96 6 2.21 versus 0.10 6 0.01 ng/ Cytokine and chemokine analyses ml), CCL4 (4.97 6 1.41 versus 0.08 6 0.01 ng/ml), CCL5 (7.6 6 Supernatants were stored at 280˚C prior to analysis. An array of 12 cy- 5.9 versus ,3.0 pg/ml), and CXCL2 (5.95 6 0.64 versus 1.92 6 tokines and chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, a b a 0.25 ng/ml). A trend toward increased IL-6 was observed but did CXCL10, IL-1 , IL-1 , IL-6, TNF- , IL-12p70) was assessed by multi- 6 6 plex assay (Millipore, Bedford, MA) and analyzed on a Luminex-100 sys- not reach statistical significance (31.0 19.1 versus 14.4 10.1 tem, according to the manufacturer’s instructions (Luminex, Austin, TX). pg/ml). Induction of mRNA encoding IL-1 a, IL-1b, and TNF-a For inhibitor studies, TNF-a,IL-1b, and CCL2 levels were deter- was also confirmed by qRT-PCR (Fig. 2B). mined by ELISA (eBioscience, San Diego, CA, or BioLegend, San Because the combination of TNF-a and IL-1b has well-char- Diego, CA). acterized inhibitory effects on insulin secretion and at high con- b Statistical analyses centrations promotes cell apoptosis (39), we chose these cytokines for further mechanistic studies. TNF-a induction in BMDMs 6 Data are expressed as mean SD of the indicated number of replicates. All after 24 h was hIAPP concentration dependent (Fig. 3A) and as- cell culture experiments were performed with three to four replicate wells per treatment. Data were analyzed using a nonlinear regression analysis sociated with increased cell viability relative to rIAPP-treated program (GraphPad Prism, La Jolla, CA). Differences between two groups cells (181 6 5 versus 142 6 5% of the untreated control, respec- were evaluated with a two-tailed t test. Differences among three or more tively; Fig. 3B). No apoptotic death of hIAPP-treated BMDMs groups were evaluated with a one-way ANOVA and Bonferroni posttests to was observed at concentrations up to 15 mM after 24 h (Fig. 3C). compare all replicate means or Dunnett posttests to compare all means to a single control. Differences among three or more treatments from multi- The LPS inhibitor polymyxin-B had no effect on hIAPP-induced ple groups were evaluated with a two-way ANOVA and Bonferroni post- TNF-a release (Fig. 3D), suggesting that these effects were not tests. For figures in which representative experiments are shown, data from due to endotoxin contamination. 2758 REGULATION OF hIAPP-INDUCED ISLET INFLAMMATION BY IL-1 Downloaded from http://www.jimmunol.org/

FIGURE 1. IAPP preserve BMDM viability in the absence of M-CSF. A, BMDMs were deprived of M-CSF for 12 h, cultured with hIAPP or rIAPP (15 mM) for the indicated time period, fixed in formalin, and stained with Thioflavin S to detect amyloid fibrils (green fluorescence, overlaid on brightfield). Scale bars, 5 mm. B, LDH content in culture supernatants was analyzed by a colorimetric assay as a measure of cell death. C, The number of cells per field was determined at each time point for cells treated with IAPP or LPS (100 ng/ml) using a Thermo Fisher ArrayScan VTI HCS instrument. Statistical significance is shown relative to the untreated control. Data are representative of three independent experiments with three or four replicates per treatment. **p , 0.01, ***p , 0.001. by guest on September 23, 2021 Effect of hIAPP aggregation state on TNF-a release (Fig. 4D) was observed with freshly dissolved peptide presumably To determine which state of hIAPP fibrillogenesis is responsible containing primarily prefibrillar aggregates, whereas hIAPP that for TNF-a and IL-1b induction, IAPP was dissolved in media had been allowed to aggregate for 6 h prior to addition to BMDMs induced much smaller increases in cytokine levels (TNF-a, 260 6 and aggregation was monitored by Thioflavin T fluorescence (Fig. 43 versus 968 6 235 pg/ml; IL-1a, ,3.2 pg/ml versus 199 6 68; 4A). Thioflavins undergo a red shift in their emission spectra upon IL-1b, 160 6 22 versus 451 6 127 pg/ml). Following 7 d of b binding to -sheet motifs present in fibrillar hIAPP aggregates aggregation, induction of both IL-1a and IL-1b by hIAPP fibrils (40). BMDMs were treated with freshly dissolved peptide or pep- was even further reduced (Fig. 4C,4D). Consistent with these ob- tide that had been allowed to aggregate prior to the experiment. servations, TNF-a secretion induced by freshly dissolved hIAPP Maximal release of TNF-a (Fig. 4B), IL-1a (Fig. 4C), and IL-1b did not follow the kinetics of LPS-induced release (Fig. 4E), sug-

Table I. Functional annotation analysis of hIAPP-induced gene expression in BMDMs

Pathway FE p Value Differentially-Expressed Genes Actin ﬁlament-based 3.2 0.011 MYL6, MTSS1, LIMA1, PDGFB, PDLIM7, PDGFA, GM8034, SRF, PFN1, GM5915, EZR, process (GO:0030029) LOC100044675, BCL2, LOC100044177, GM3787, PLS3, FMNL3, GM3655, ACTN4, ACTN1, TMSB10, GM10080, GM8894, ELMO1, LOC100048142, NRAP, APBB2, GM9844, LCP1 Vasculature development 2.9 0.003 GNA13, TNFRSF12A, PDGFA, EDN1, ANPEP, SRF, CITED2, ARHGAP22, LOC100048867, (GO:0001944) HMOX1, ITGAV, TGM2, IL1B, RHOB, LOX, PPAP2B, RAPGEF1, COL18A1, PDPN, SPHK1, MMP14, JUNB, ANXA2, VEGFC, JMJD6, HBEGF Response to wounding 2.7 0.002 GNA13, CXCL1, CCL3, GM8762, TNF, CCL2, NFKBID, TOLLIP, PPARG, CXCL2, SGMS1, (GO:0009611) CCL4, CCL7, MIF, CD44, BCL2, MTPN, IL1B, GM16379, IL1A, KLF6, GM3655, F10, PLEK, PIK3CB, GM6097, GM10169, MAP2K3, TLR13, SPHK1, ANXA5, NLRP3, CD180, PLAUR, HBEGF, LOC100044948, CD14, LCP1 Immune response 2.3 0.005 CXCL1, CCL3, GM8762, TNF, CCL2, TOLLIP, CXCL2, RSAD2, IL7R, CCL4, CCL7, MIF, (GO:0006955) KLHL6, CLEC4E, SQSTM1, GP49A, LOC641240, OASL2, IL1B, GM16379, IL1A, RMCS2, GM6097, H2-M2, GM10169, TLR13, H2AB1, MALT1, TNFSF9, NLRP3, VAV1, CD180, LOC100046097, LAT2, FCGR2B, GADD45G, LILRB4, OAS1A, LOC100044948, OAS1G, CLEC5A, CD14, LCP1, TNFAIP1, CD300LD Cells were treated with hIAPP or rIAPP for 12 h (n = 4 per treatment). Global gene expression was analyzed by mouse 8-chip Illumina array. Pathway overrepresentation analysis was performed on genes upregulated by at least 1.5-fold using the Database for Annotation, Visualization, and Integrated Discovery functional annotation tool. Enriched Gene Ontology terms with a Benjamini p value ,0.01 were considered highly signiﬁcant. FE, fold enrichment. The Journal of Immunology 2759 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. hIAPP induces proinflammatory cytokine release by BMDMs. A, Cells were incubated with hIAPP or rIAPP (15 mM) for 12 h. Supernatants were assessed for proinflammatory cytokines and chemokines with a multiplex assay. B, Gene expression was evaluated by qRT-PCR. Data are representative of three independent experiments with four replicates per treatment. **p , 0.01, ***p , 0.001. gesting a mechanism distinct from immediate cell surface TLR of IAPP fibril formation (41). Taken together, these data suggest activation and a delay in response until the proinflammatory spe- that exposure of macrophages to intermediate hIAPP species, cies was formed. Furthermore, hIAPP-induced TNF-a release was rather than preformed fibrils, is required for maximal cytokine almost completely inhibited by Congo red (Fig. 4F), an inhibitor release.

FIGURE 3. hIAPP-induced TNF-a release is dose dependent and not due to cell death or endotoxin contamination. A, TNF-a production in response to treatment with 5–20 mM IAPP for 24 h was determined by ELISA. B, Viability was assessed by Alamar Blue fluorescence. C, Cells were fixed after 24 h, and cell death was evaluated by ArrayScan VTI HCS analysis of TUNEL staining. D, hIAPP-induced TNF-a release was evaluated in the presence of the cationic peptide polymyxin-B. Data are representative of five independent experiments with three replicates per treatment. ***p , 0.001. 2760 REGULATION OF hIAPP-INDUCED ISLET INFLAMMATION BY IL-1

FIGURE 4. hIAPP aggregation inter- mediates induce maximal TNF-a and IL-1 release. A, hIAPP or rIAPP (15 mM) was dissolved in DMEM, and fibril formation was monitored by Thioflavin T fluorescence. Peptide was removed at the indicated time points and added to BMDM cultures for 24 h. Levels of TNF-a (B), IL-1a (C), and IL-1b (D)in cell supernatants were determined by multiplex assay. E, Freshly dissolved a hIAPP was evaluated for TNF- in- Downloaded from duction after 6, 12, and 24 h and compared with the kinetics of induction by LPS (100 ng/ml). F, TNF-a levels were assessed following coincubation with Congo red (200 mM, CR), an inhibitor of hIAPP fibrillogenesis. Data are representative of three independent experi- http://www.jimmunol.org/ ments with three replicates per treatment. **p , 0.01, ***p , 0.001. UT, untreated. by guest on September 23, 2021

Role of MyD88 and IL-1R signaling in hIAPP-induced TNF-a that also blocks NLRP3 activation (44). Cytokine release was release dependent on actin polymerization, because it was blocked by To determine whether TLR pathways are involved in the macro- cytochalasin D. The potential requirement for phagocytosis is phage response to hIAPP, as described for other amyloidogenic further emphasized by the significant overrepresentation of genes peptides, we evaluated TNF-a induction in BMDMs from mice related to actin filament-based processes in our microarray study lacking TLR signaling pathway components. hIAPP-induced (Table I). As described for other inflammasome activators, cyto- TNF-a release was markedly attenuated in BMDMs lacking kine release was blocked by CA-074-Me, an inhibitor of the ly- MyD88 (0.13 6 0.01 versus 5.27 6 0.69 ng/ml; Fig. 5A), but not sosomal enzyme cathepsin B. Thus, hIAPP can provide both b TLR2 (Fig. 5C) or TLR4 (Fig. 5D). A specific peptide inhibitor signals required for IL-1 secretion, and autocrine or paracrine signalingbyIL-1a or IL-1b appears to be at least partially re- of MyD88 homodimerization also reduced hIAPP-elicited TNF-a sponsible for amplification of TNF-a release. release in wild-type BMDMs from 3.66 6 0.28 to 1.68 6 0.53 ng/ ml (Fig. 5B). Thus, the MyD88 dependency of this cytokine can- Effect of hIAPP on islet chemokine release and monocyte not be attributed to nonredundant TLR2 or TLR4 signaling. recruitment ex vivo Because the IL-1R also uses the adaptor protein MyD88, and IL- To determine whether hIAPP aggregation causes chemokine re- 1 is known to induce TNF-a (42), we evaluated the effect of IL-1 lease from islets, we measured CCL2 and CXCL1 release by whole signaling inhibition on hIAPP-induced TNF-a release. Inhibition islets treated with rIAPP or hIAPP for 24 h. We found that both of pro–IL-1b cleavage with a caspase-1 inhibitor and blockade chemokines were markedly upregulated in response to hIAPP, but of IL-1 signaling with IL-1Ra significantly attenuated hIAPP-in- not rIAPP (Fig. 6A). This effect was preserved following macro- duced TNF-a release (Fig. 5E). To determine whether NRLP3 phage depletion of islets with clodronate-liposomes, verified by inflammasome signaling might be involved in caspase-1 activation decreased expression of genes encoding F4/80 and CD11b with no leading to IL-1b secretion (a process that requires both synthesis significant effect on Ins2 or Ccl2 expression (Supplemental Fig. 1). of pro–IL-1b and cleavage of the propeptide to its mature form), To assess the effect of endogenously produced hIAPP on CCL2 we evaluated the effects of NLRP3 inflammasome inhibitors on and CXCL1 release, we cultured islets from transgenic mice ex- hIAPP-induced IL-1b and TNF-a secretion (Fig. 5F,5G). Cyto- pressing hIAPP under the control of a b cell-specific promoter. kine induction was significantly reduced by Bay 11-8072, an in- Male homozygous FVB/N-Tg(Ins2-IAPP)RHFSoel/J mice spon- hibitor of both NF-kB and the NLRP3 inflammasome (43), and by taneously develop diabetes by 8 wk of age (20), whereas the glibenclamide, an inhibitor of ATP-sensitive potassium channels hemizygous animals are phenotypically normal and do not The Journal of Immunology 2761 Downloaded from http://www.jimmunol.org/

FIGURE 5. hIAPP-induced TNF-a release by BMDMs is dependent on MyD88, but not TLR2 or TLR4. C57BL/6 wild-type or Tlr4+/2 and Myd882/2 (A), Tlr22/2 (C), or Tlr42/2 (D) BMDMs were evaluated for TNF-a release by ELISA after 24-h incubation with IAPP (15 mM) or the TLR agonists polyI:C

(10 mg/ml), Pam3CSK4 (1 mg/ml), and LPS (100 ng/ml). B, Wild-type BMDMs were pretreated with MyD88 homodimerization inhibitory peptide or control peptide (both 100 mM) for 12 h prior to addition of IAPP (15 mM) or LPS (10 ng/ml) for 24 h. TNF-a levels were determined by ELISA. E, The

caspase-1 inhibitor Z-YVAD-FMK (40 mM) and IL-1Ra (4 mg/ml) were added to BMDM cultures 2 h prior to treatment with hIAPP for 24 h. TNF-a levels by guest on September 23, 2021 were determined by ELISA. TNF-a (F) and IL-1b (G) levels were determined by multiplex assay following 24-h culture with hIAPP in the presence of the NLRP3 inhibitors Bay 11-8072 (Bay, 5 mM), glibenclamide (GLB, 200 mM), cytochalasin D (CD, 5 mM), or CA-074-Me (CA-074, 80 mM). Statistical significance is shown relative to the hIAPP-treated control. Data are representative of two (C, D, F, G), three (B, E), or six (A) independent experiments. **p , 0.01, ***p , 0.001. UT, untreated. develop detectable amyloid deposits (45). However, isolated These results are consistent with the hypothesis that chemokines hemizygous islets form extracellular amyloid deposits after produced by resident islet cells attract immune cells to sites of 8 d of culture with high glucose (46). In our experiments, a small hIAPP aggregation. amount of Thioflavin S-positive material was also observed in islets cultured with 11 mM glucose for 7 d, but not in freshly Effect of IL-1Ra on islet function and macrophage recruitment isolated islets (Supplemental Fig. 2). After 96 h in culture, we in vivo observed higher levels of CCL2 (288 6 45 versus 170 6 34 pg/ To determine whether hIAPP expression is associated with im- ml) and CXCL1 (1294 6 135 versus 558 6 116 pg/ml) in paired islet function in vivo, we transplanted islets from hIAPP supernatants from transgenic islets compared with wild-type transgenic and wild-type littermate control mice into diabetic littermate controls (p , 0.05; Fig. 6C). Consistent with the NOD/SCID recipients treated with 50 mg/kg/d IL-1Ra or PBS. macrophage depletion experiments, immunostaining of cultured Recipients of transgenic islet grafts displayed impaired glucose islets for CCL2 revealed colocalization of this chemokine with tolerance 8 wk following transplantation compared with recipients insulin (Fig. 6D). These data suggest that hIAPP could trigger of wild-type grafts (area under the curve = 1940 6 80 versus 1260 6 recruitment of macrophages to the site of aggregation by induction 60, p , 0.001). Administration of IL-1Ra reduced nonfasting hy- of b cell chemokines. Addition of IL-1Ra to islet cultures reduced perglycemia associated with islet hIAPP expression (Supplemental both basal and hIAPP-induced release of CCL2 and CXCL1 (Fig. Fig. 3) and significantly improved glucose tolerance in recipients 6E), suggesting that blockade of IL-1 signaling may not only in- of transgenic grafts (area under the curve = 1470 6 160 versus hibit hIAPP-induced macrophage cytokine release, but may also 1940 6 80, p , 0.05), but not wild-type grafts (Fig. 7A,7B). No limit macrophage recruitment to islets. differences in body weight were observed among transplant reci- To evaluate directly monocyte chemotaxis in vitro, we assessed pients (Supplemental Fig. 3). Quantification of intragraft staining THP-1 migration in response to conditioned media from human for the macrophage glycoprotein F4/80 demonstrated that hIAPP- islets treated with varying concentrations of synthetic IAPP. Media expressing grafts contained 50% more macrophages than wild- from hIAPP-, but not rIAPP-treated human islets induced THP-1 type grafts, an effect that was significantly inhibited by IL-1Ra chemotaxis, with a chemotactic index of 3.3 6 0.7 versus 1.7 6 (Fig. 7C). Furthermore, a 5-fold reduction in amyloid area was 0.3 for media from islets treated with 20 mM hIAPP (Fig. 6F). observed in transgenic grafts from IL-1Ra–treated recipients (Fig. 2762 REGULATION OF hIAPP-INDUCED ISLET INFLAMMATION BY IL-1 Downloaded from http://www.jimmunol.org/

FIGURE 6. hIAPP promotes islet chemokine release. A, Pancreatic islets were isolated from 12-wk-old C57BL/6 mice, allowed to recover overnight, and treated with IAPP (15 mM), or LPS (100 ng/ml) for 24 h. Supernatants were collected and analyzed for the chemokines CXCL1 and CCL2 by multiplex assay. B, Prior to treatment with IAPP or LPS, islets were incubated with clodronate-containing liposomes (CLO-lip) or control liposomes (PBS-lip) for 24 h to determine the effect of macrophage depletion on hIAPP-induced islet CCL2 release. C, Islets were isolated from 12- to 20-wk-old hemizygous hIAPP by guest on September 23, 2021 transgenic mice and wild-type FVB littermate controls (islets from four mice per genotype were pooled into five wells). After 7-d culture, supernatants were analyzed for CCL2 and CXCL1 by multiplex assay, and D, formalin-fixed, paraffin-embedded islet sections were stained for insulin (red), CCL2 (green), and DAPI (blue). Scale bars, 25 mm. E, CXCL1 and CCL2 release by wild-type and hIAPP transgenic islets were evaluated after 7-d culture in the presence or absence of 4 mg/ml IL-1Ra. F, Human islets from a single cadaveric donor were treated with varying concentrations of synthetic IAPP for 24 h. THP-1 cell chemotaxis in response to islet-conditioned media was evaluated in triplicate in a Transwell assay. Data are representative of two (A, B, E) or three (C, D, F) independent experiments, each with three to five donor mice per group. *p , 0.05, **p , 0.01, ***p , 0.001.

7D). Interestingly, most Thioflavin S-positive amyloid deposits in hIAPP induced IL-6 and IL-8 secretion by human astrocytoma transgenic grafts were associated with F4/80-expressing macro- cells (48) and release of IL-1b,TNF-a, IL-6, IL-8, CCL3, and phages, both extra- and intracellularly (Fig. 7E). CCL4 by LPS-activated THP-1 monocytes, a model for human microglia (29). Our data suggest that prefibrillar hIAPP induces Discussion proinflammatory cytokine release by both macrophages and islets hIAPP fibrils are present within the lysosomes of pancreatic in the absence of priming by a distinct TLR ligand, consistent with macrophages in type 2 diabetic humans, monkeys, and hIAPP- previously reported increases in monocyte IL-1b and TNF-a expressing transgenic mice (47). Autopsy studies have also dem- mRNA in response to hIAPP alone (29) and in response to Ab onstrated more islet macrophages together with increased IL-1b (30). Unlike monocytes, macrophages do not express constitu- expression in type 2 diabetic islets compared with nondiabetic tively active caspase-1 (49), and we found that hIAPP mediates controls (3, 5, 6, 17). Our data suggest a possible link between IL-1b secretion in a caspase-1–dependent manner. hIAPP-induced hIAPP aggregation and islet inflammation, with four novel find- NLRP3 activation in dendritic cells was recently described by ings, as follows: 1) prefibrillar hIAPP species induce proinflam- Masters et al. (50), although the lack of significant TNF-a induc- matory cytokine and chemokine release by BMDMs, an effect tion reported by these authors may be explained by peptide han- that is blocked by inhibition of fibrillogenesis; 2) hIAPP-induced dling affecting the aggregation state of the peptide or by cell type- TNF-a release is almost entirely dependent on MyD88, an effect specific effects. that is at least partially explained by autocrine/paracrine effects TLR4 signaling is required for efficient microglial clearance of of IL-1a and/or IL-1b; 3) hIAPP expression is associated with Ab deposits in the mouse brain (51) and is also involved in Ab- chemokine release by islets ex vivo and increased islet macro- induced neuronal cell death (52). Both CD36-induced TLR4– phage content in vivo; and 4) inhibition of IL-1R signaling im- TLR6 heterodimer formation (53) and TLR2 activation (27, 54) proves hIAPP-induced pancreatic islet graft dysfunction. have also been implicated in the response to Ab. We found Our observation that hIAPP aggregation induces release of hIAPP-induced TNF-a release from macrophages to be largely multiple proinflammatory cytokines and chemokines by BMDMs MyD88 dependent. TNF-a levels in MyD88-deficient BMDMs is consistent with previous studies in neurons and glia. Like Ab, were nevertheless significantly increased compared with untreated The Journal of Immunology 2763 Downloaded from http://www.jimmunol.org/

FIGURE 7. IL-1Ra attenuates hIAPP-induced islet graft dysfunction. A total of 150 islets from 12- to 16-wk-old wild-type or hIAPP transgenic mice was transplanted into NOD/SCID recipients implanted with a mini-osmotic pump releasing PBS or IL-1Ra (50 mg/kg/d) for the duration of the experiment (wild-type PBS and IL-1Ra, n = 8; transgenic PBS, n = 9; transgenic IL-1Ra, n = 7). A, Glucose tolerance was assessed 8 wk following transplantation by by guest on September 23, 2021 monitoring tail vein glucose after i.p. injection of 0.75 U/kg glucose. B, Statistical significance was determined by comparing areas under the glycemia curves. C, Grafts were stained for insulin (red), the macrophage marker F4/80 (green), and DAPI (blue). The number of F4/80-positive cells was expressed relative to the insulin-positive area in each graft. D, Grafts were stained for insulin (red), amyloid (Thioflavin S, green), and DAPI (blue). The amyloid area was expressed relative to the insulin-positive area in each graft. E, Grafts were stained for F4/80 (red), amyloid (Thioflavin S, green), and DAPI (blue). Scale bars, 25 mm(C, D)or10mm(E). *p , 0.05, ***p , 0.001. controls and reached ∼3% of the levels observed in wild-type sosomal dysfunction, endosomal TLR signaling, and initial cells, which may implicate other, as yet undefined mechanisms. activation of the IL-1R by hIAPP-induced IL-1a release. In any case, because TLR2 and TLR4 deficiency had no detectable The toxicity of synthetic hIAPP toward b cells depends on the effect on hIAPP-induced TNF-a release, these pathways do not peptide’s aggregation state and on the association of growing appear to represent necessary mechanisms of innate immune ac- fibrils with the plasma membrane (23). Our data support the hy- tivation by all amyloidogenic peptides. pothesis that prefibrillar hIAPP aggregates also act as the principal That amplification of hIAPP-induced TNF-a release was in- proinflammatory stimulus, because the ability of hIAPP to induce hibited not only by MyD88 deficiency, but also by blockade of both TNF-a and IL-1b release declined when the peptide was caspase-1 and IL-1R suggests a role for IL-1a or IL-1b release in added to macrophages in a more aggregated form. This finding is the autocrine or paracrine amplification of other cytokines. Recent consistent with a previous report of maximal TNF-a induction by data suggest that hIAPP-induced NLRP3 activation, like that of prefibrillar Ab species (55). Although TNF-a induction by hIAPP Ab, is associated with lysosomal damage in dendritic cells (50), was significantly reduced in the presence of the dye Congo red, and hIAPP fibrils condense within macrophage lysosomes to form an inhibitor of amyloid formation (41), this dye could also act proteolysis-resistant protofilaments (19). Consistent with these by inhibiting interactions between hIAPP and the cell membrane. reports, we have shown that hIAPP-induced IL-1b secretion in Nevertheless, inhibitors of hIAPP aggregation, which protect cul- macrophages is blocked by the cathepsin B inhibitor CA-074-Me. tured human islets from hIAPP-induced cell death (10), may Because CA-074-Me and the inhibitor of actin polymerization achieve this effect in part by limiting IL-1b secretion. cytochalasin D also attenuated TNF-a release, lysosomal damage Hemizygous expression of the hIAPP transgene in islet b cells may be a major mechanism mediating the proinflammatory ac- induces islet amyloid formation under conditions that increase b tivity of hIAPP. However, additional studies are required to cell secretory demand and dysfunction, including high fat feeding identify the initial stimulus for pro–IL-1b gene transcription, (21), ex vivo culture (46), and islet transplantation (12). In this which presumably may also contribute to Tnf transcriptional ac- study, we have shown that both synthetic and endogenously pro- tivation. Possible mechanisms include events downstream of ly- duced hIAPP potentiate islet chemokine release ex vivo and are 2764 REGULATION OF hIAPP-INDUCED ISLET INFLAMMATION BY IL-1 associated with increased numbers of islet macrophages following chemokines such as CCL2 by whole islets, including b cells, and transplantation. Migration of monocytes toward media condi- that this effect is preserved upon macrophage depletion. Consis- tioned by hIAPP-treated human islets also suggests that the che- tent with hIAPP-induced monocyte recruitment to the islet, we mokine release observed in transgenic islets has functional observed more macrophages within hIAPP transgenic grafts, and implications. One previous study reported no difference in mac- these cells were present in association with amyloid plaques. rophage density between amyloid-containing and amyloid-free Whether IL-1Ra–mediated improvements in islet function are due human and monkey islets (47); however, immune cell recruit- to inhibition of b cell chemokine release, attenuation of macro- ment may occur prior to formation of extensive fibrillar deposits, phage cytokine secretion, blockade of IL-1R–induced b cell dys- which most likely accompany resolution of the inflammatory function, or a combination of these effects remains to be ad- response. This hypothesis is consistent with our finding of dressed. maximal proinflammatory activity of prefibrillar hIAPP in BMDM In conclusion, this study suggests that prefibrillar hIAPP aggre- cultures and with chemokine release by transgenic islets in the gates promote islet dysfunction not only via direct toxicity to b absence of widespread amyloid deposition. cells, but also by triggering a localized inflammatory response Recruitment of macrophages to islet grafts may result directly mediated by IL-1. Thus, strategies aimed at reducing hIAPP ex- from hIAPP-induced islet chemokine release, or could be an in- pression and aggregation may not only protect b cells from apo- direct consequence of sterile inflammation caused by hIAPP- ptosis, but also ameliorate deficits in insulin secretion associated induced cell death. We found no significant differences in the with proinflammatory cytokine release. Because IL-1R signaling number of TUNEL-positive b cells among the treatment groups 8 contributes to the amplification of hIAPP-induced cytokine and wk following transplantation (data not shown), suggesting that chemokine release by islets and macrophages, these data suggest Downloaded from extensive hIAPP-induced cell death is not a requirement for graft a possible mechanism by which blockade of IL-1 signaling or inflammation. Furthermore, we have demonstrated hIAPP-induced hIAPP aggregation may significantly alter the inflammatory mi- chemokine release from transgenic mouse islets ex vivo in the lieu of the pancreatic islet to improve b cell function in both type absence of significant cell death, as determined by LDH release 2 diabetes and islet transplantation. and Alamar Blue reduction. Although our in vitro studies suggest that interaction of hIAPP with macrophages induces release of Acknowledgments http://www.jimmunol.org/ cytokines that at high local concentrations are known to cause b We thank K. Hu for assistance with ArrayScan VTI HCS data acquisition. cell dysfunction (4), we cannot rule out the possibility that mac- We are grateful to the University of Chicago Medical Centre and the Ike rophages play an important role in regulating islet homeostasis. Barber Human Transplant Laboratory for providing human islets. We also Indeed, our microarray data suggest hIAPP-induced upregulation thank P.C. Orban and A. Plesner for critical review of the manuscript. of pathways involved in angiogenesis and wound healing, consistent with the proangiogenic role of islet macrophages described Disclosures in a mouse model of chronic pancreatitis (56). Nevertheless, im- The authors have no financial conflicts of interest. proved glucose tolerance was associated with a reduction in the number of F4/80-positive cells within transgenic grafts from re- by guest on September 23, 2021 cipients treated with IL-1Ra. Further phenotypic and functional References characterization of these cells will help to elucidate their poten- 1. Kahn, S. E., R. L. Hull, and K. M. Utzschneider. 2006. Mechanisms linking tial effects on islet function. obesity to insulin resistance and type 2 diabetes. Nature 444: 840–846. 2. Butler, A. E., J. Janson, S. Bonner-Weir, R. Ritzel, R. A. Rizza, and P. C. Butler. The improved glucose tolerance in IL-1Ra–treated recipients of 2003. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 hIAPP transgenic islets suggests a common mechanism of hIAPP- diabetes. Diabetes 52: 102–110. b 3. Bo¨ni-Schnetzler, M., J. Thorne, G. Parnaud, L. Marselli, J. A. Ehses, J. 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Table SI. Complete functional annotation analysis of differentially expressed genes in BMDMs1. Expression Pathway FE p Genes Upregulated ATP synthesis coupled proton 6.4 0.036 TCIRG1,ATP6V1C1,ATP6V0E,ATP6V1E1,ATP6V1H,ATP6V0D1,ATP transport 6V1D,ATP6V0B GO:0015986 Regulation of cytokine 3.4 0.020 CEBPB,TNF,GM12141,ADORA2B,MAP2K3,SPHK1,PPARG,CD40,N production LRP3,MAST2,REL,HMOX1,IL1B,HSPD1,IL1A,SRGN,CD14 GO:0001817 Actin filament-based process 3.2 0.011 MYL6,MTSS1,LIMA1,PDGFB,PDLIM7,PDGFA,GM8034,SRF,PFN1,G GO:0030029 M5915,EZR,LOC100044675,BCL2,LOC100044177,GM3787,PLS3,F MNL3,GM3655,ACTN4,ACTN1,TMSB10,GM10080,GM8894,ELMO1, LOC100048142,NRAP,APBB2,GM9844,LCP1 Vasculature development 2.9 0.003 GNA13,TNFRSF12A,PDGFA,EDN1,ANPEP,SRF,CITED2,ARHGAP2 GO:0001944 2,LOC100048867,HMOX1,ITGAV,TGM2,IL1B,RHOB,LOX,PPAP2B,R APGEF1,COL18A1,PDPN,SPHK1,MMP14,JUNB,ANXA2,VEGFC,JMJ D6,HBEGF Inflammatory response 2.8 0.020 CXCL1,CCL3,CCL2,TNF,GM8762,NFKBID,TOLLIP,PPARG,CXCL2,S GO:0006954 GMS1,CCL4,CCL7,MIF,CD44,IL1B,GM16379,IL1A,GM6097,MAP2K3 ,GM10169,TLR13,SPHK1,NLRP3,CD180,LOC100044948,CD14 Response to wounding 2.7 0.002 GNA13,CXCL1,CCL3,GM8762,TNF,CCL2,NFKBID,TOLLIP,PPARG,C GO:0009611 XCL2,SGMS1,CCL4,CCL7,MIF,CD44,BCL2,MTPN,IL1B,GM16379,IL 1A,KLF6,GM3655,F10,PLEK,PIK3CB,GM6097,GM10169,MAP2K3,TL R13,SPHK1,ANXA5,NLRP3,CD180,PLAUR,HBEGF,LOC100044948, CD14,LCP1 Small GTPase mediated signal 2.5 0.035 GNA13,5430435G22RIK,RAP2A,RASGEF1B,LOC100044232,ARF6, transduction RHOV,RALGDS,RGL1,IQGAP1,ELMO1,DOK2,RAB1,HMOX1,RASSF GO:0007264 1,RAPGEF5,RHOB,RAB15,ARL8A,RHOC,ARL8B,RAPGEF1,BCAR3 Immune response 2.3 0.005 CXCL1,CCL3,GM8762,TNF,CCL2,TOLLIP,CXCL2,RSAD2,IL7R,CCL GO:0006955 4,CCL7,MIF,KLHL6,CLEC4E,SQSTM1,GP49A,LOC641240,OASL2,IL 1B,GM16379,IL1A,RMCS2,GM6097,H2-M2,GM10169,TLR13,H2- AB1,MALT1,TNFSF9,NLRP3,VAV1,CD180,LOC100046097,LAT2,FC GR2B,GADD45G,LILRB4,OAS1A,LOC100044948,OAS1G,CLEC5A,C D14,LCP1,TNFAIP1,CD300LD Nucleolus 2.3 0.038 MTDH,GAR1,2610029G23RIK,MKI67IP,WDR74,MAK16,LOC674157, GO:0005730 HMOX1,GM11448,DDX21,CIRH1A,IMP4,PHLDA1,GNL3,GTPBP4,R RP12,NIP7,GM5243,NOP14,RSL1D1,ZFP655,NOL10,MDM2,NOP58, NOP56,MPHOSPH6,WDR43 Downregulated Nucleosome 6.0 0.010 HIST1H2AB,HIST1H2AC,HIST1H2AF,HIST1H2AG,HIST1H2AD,HIST GO:0000786 1H2AE,GM13646,GM11277,GM11276,HIST1H2BM,HIST1H2BN,HIS T1H2BL,HIST1H2BJ,H2AFZ,H2AFY,GM6722,GM8203,HIST1H2BC,H IST1H1C,HIST1H2BE,LOC100046213,HIST1H2BF,HIST1H2BG,HIST 1H2BH,HIST2H2BB,HIST1H2AI,HIST1H2AH,HIST1H2AK,HIST1H2A O,HIST1H2AN Positive regulation of 3.0 0.005 MSH6,ING4,TGFBR1,IL18,STK17B,FCGR1,IKBIP,SERINC3,CASP6, programmed cell death XPA,NOTCH1,EI24,NOD1,PRKRA,PYCARD,TRP53INP1,BCL3,FAS,I GO:0043068 D3,PHLDA3,CASP2,TEX261,RAB27A

Protein serine/threonine kinase 2.6 0.002 CDK19,NUAK1,STK11,PRKAG2,STK17B,LOC100044493,CASK,ADR activity BK1,CAMKK2,SBK1,SNRK,MAP3K3,MAP3K1,MAP3K8,DYRK3,GM3 GO:0004674 193,STK24,NLK,TGFBR1,STRADB,RPS6KA5,1700028N14RIK,ICK,A CVR2B,EIF2AK1,RPS6KA1,ULK1,MAPK14,ULK2,NEK9,GRK5,MAP3 K14,LRRK1,LOC100044468,MAP3K11 Membrane-enclosed lumen 1.7 0.006 MRPS35,ZMAT3,ASCC1,RP9,MED22,GM2810,ERLEC1,SETX,ACO GO:0031974 X3,FOS,FXC1,ANP32A,4930526A20RIK,TGS1,DCAF17,LBR,DEDD2 ,INO80B,GM5605,ELP3,ACADM,TLE1,RB1,GM13803,EP300,NRM,PI AS3,H6PD,MED16,PPM1K,KTELC1,CPSF1,ING4,LOC100045720,FA RS2,ETHE1,YPEL3,ECHS1,GM5117,ZFP768,KDELC1,TCP11,NUMA 1,CIR1,IVD,ALDH4A1,TRP53INP1,TCF4,SDF4,OLFM1,TSEN34,BCK DHA,LOC100044089,AKAP8L,AK3,DNHD1,CASC3,USF1,ITPR1,ATX N1,LOC100048029,GMCL1,TBL1X,PBX2

1Cells were treated with hIAPP or rIAPP for 12 h (n=4 per treatment). Global gene expression was analyzed by Illumina microarray. Pathway over-representation analysis was performed on genes up- and downregulated by at least 1.5-fold using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional annotation tool. Enriched Gene Ontology (GO) terms with a Benjamini p-value < 0.05 are shown. FE: fold enrichment.