Downloaded from January 2016; Doi: 10.4049/Jimmunol.1501239

Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of This information is current as IFN-Activated STAT1 of September 23, 2021. M. Jubayer Rahman, Gwendoline Rahir, Matthew B. Dong, Yongge Zhao, Kameron B. Rodrigues, Chie Hotta-Iwamura, Ye Chen, Alan Guerrero and Kristin V. Tarbell J Immunol 2016; 196:2031-2040; Prepublished online 29 Downloaded from January 2016; doi: 10.4049/jimmunol.1501239 http://www.jimmunol.org/content/196/5/2031 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2016/01/29/jimmunol.150123 Material 9.DCSupplemental References This article cites 58 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/196/5/2031.full#ref-list-1 by guest on September 23, 2021 Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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

Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1

M. Jubayer Rahman,* Gwendoline Rahir,*,1 Matthew B. Dong,*,1 Yongge Zhao,* Kameron B. Rodrigues,* Chie Hotta-Iwamura,* Ye Chen,† Alan Guerrero,* and Kristin V. Tarbell*

Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF den- Downloaded from dritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reﬂect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed 2 2 for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1 / mice also display a defect in http://www.jimmunol.org/ CpG-induced CD86 upregulation compared with B6 Rag12/2, indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-a/b receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti–IFN-a/b receptor Ab is added. IFN-a–induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c+ cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but dimin- ished cDC type 1 IFN response. The Journal of Immunology, 2016, 196: 2031–2040.

n autoimmune diseases such as type 1 diabetes (T1D), au- neous b cell–targeted autoimmunity found in T1D (2). Stages of by guest on September 23, 2021 toreactive T cells lend specificity to the killing of target diabetes pathogenesis have been defined by adaptive immune I tissues, but innate immune cells also contribute significantly changes: first, autoreactive T cells are activated (initiation of insu- to autoimmune pathogenesis (1). NOD mice model the sponta- litis), then these cells infiltrate the pancreas (prediabetic phase), and later these pathogenic cells overwhelm immune regulation and de- *Immune Tolerance Section, Diabetes, Endocrinology, and Obesity Branch, National stroy insulin-producing cells (overt diabetes) (3–5), but innate im- Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of mune contributions at each stage are less clear. Deficiencies in Health, Bethesda, MD 20892; and †Bioinformatics and Systems Biology Core, Na- tional Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD innate signaling lowers diabetes incidence in NOD mice, likely via 20892 early effects on initiation of insulitis (6). Dendritic cells (DCs) 1G.R. and M.B.D. contributed equally to this work. contribute to diabetes pathogenesis at this early stage (7–9), but ORCIDs: 0000-0002-4496-8385 (M.J.R.); 0000-0003-4466-2391 (Y.Z.); 0000-0002- because of their role in maintaining regulatory T cells (Tregs), in- 0270-3688 (K.B.R.); 0000-0002-5501-8553 (C.H.-I.); 0000-0001-8785-7653 (Y.C.); creasing DCs at later stages of T1D can block disease (10). Auto- 0000-0003-3738-379X (K.V.T.). immunity disrupts DC-mediated steady-state tolerance either through Received for publication June 2, 2015. Accepted for publication December 23, 2015. intrinsic genetic alterations or because of endogenous innate signals This work was supported by the intramural research programs of the National Insti- that induce some level of maturation (11, 12). We now ask, how is tute of Diabetes and Digestive and Kidney Diseases and the National Heart, Lung, and Blood Institute and by a collaborative research agreement with Janssen Research DC innate responsiveness altered in prediabetic NOD mice? and Development. To date, studies on innate stimulation of NOD DCs have focused The data presented in this article have been submitted to the National Center for on in vitro bone marrow (BM) GM-CSF cultures and have found Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih. hyporesponsiveness to LPS stimulation such as lower IL-12 but gov/geo/query/acc.cgi?acc=GSE75883) under accession number GSE75883. increased NF-kB activation (13, 14). Importantly, a recent study Address correspondence and reprint requests to Dr. Kristin V. Tarbell, Immune Tolerance Section, National Institute of Diabetes and Digestive and Kidney Diseases, 10 Center shows that BM GM-CSF cultures are a complex mix of macro- Drive, Building 10, 5-5940, Bethesda, MD 20892. E-mail address: [email protected] phages, monocytes and related cells, and conventional DCs (cDCs) The online version of this article contains supplemental material. that varies depending on exact culture conditions, making these Abbreviations used in this article: B6, C57BL/6; B6g7, B6.H2g7; BM, bone marrow; studies hard to interpret or align with any DC population in vivo cDC, conventional DC; DC, dendritic cell; DE, differentially expressed; FDR, false- (15, 16). Therefore, to understand how innate responses contribute discovery rate; Flt3L, Flt3 ligand; IFNAR, IFN-a/b receptor; IRF, IFN regulatory factor; ISGF3, IFN-stimulated gene factor 3; moDC, monocyte-derived DC; NIDDK, to diabetes pathogenesis, in vivo DC subsets need to be studied. National Institute of Diabetes and Digestive and Kidney Diseases; PCA, principal Several subsets of DCs have been described that can affect component analysis; pDC, plasmacytoid DC; PFA, paraformaldehyde; pLN, pancreatic lymph node; poly(I:C), polyinosinic-polycytidylic acid; sLN, skin LN; T1D, type autoimmune pathogenesis (17, 18). cDCs are the migratory and 1 diabetes; Treg, regulatory T cell. lymphoid resident cells that often control T cell fate. Two main www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501239 2032 LOW TLR9-DRIVEN, IFNAR-DEPENDENT cDC ACTIVATION IN NOD MICE subsets of cDCs are found in both mouse and human (19). CD8+ heim, Germany) (1 mg CpG/well with 2.5 ml DOTAP/well). Where indi- cDC1s normally contribute to tolerance via induction of Tregs or cated, wells were pretreated with anti-IFNAR Abs (clone MAR1-5A3, deletion of autoreactive cells but have tolerance defects in NOD IgG1, 20 mg/ml) (BioLegend, San Diego, CA) for 24 h before CpG was + + added. Following CpG stimulation, cells were collected and analyzed by mice (20). CD11b cDC2s can induce strong CD4 T cell stim- flow cytometry. ulation and retain more tolerogenic activity in NOD mice. Distinct from cDCs, MHC class II+ monocyte-derived DCs (moDCs) pri- In vivo CpG injection and DC isolation from lymphoid organs marily develop in response to inflammatory signals and may Where indicated, 200 mg anti-IFNAR or isotype (human IFN-gR1 mAb contribute to both Treg homeostasis and activation of pathogenic clone GIR208, mouse IgG1) was injected 24 h before CpG injection. Five responses (14, 21, 22). Plasmacytoid DCs (pDCs) are the major micrograms of CpG formulated with 30 ml DOTAP (1 mg/ml) was administered i.v., and after 12 h, spleens, pancreatic lymph nodes (pLNs), producer of type 1 IFN, which then acts on cDCs, and is required and skin LNs (sLNs) were harvested separately as indicated for DC en- for upregulation of genes that are associated with DC maturation richment. Untreated (no CpG or anti-IFNAR Ab) mice received 200 ml and Ag presentation (23, 24). PBS. DCs were isolated from individual spleens and LNs with collagenase Type 1 IFN production is critical for induction of protective III digestion (Worthington, Lakewood, NJ) as described by Guerrero et al. m innate and adaptive immunity against pathogens (25), yet dysreg- (31). Cells were then strained using 60- m filters, stained, and run by flow cytometry. ulated type 1 IFN responses can also induce inflammatory tissue damage (26). However, IFN responses can induce regulatory re- Flow cytometry sponses, such as production of IL-10 and IDO (27, 28). In fact, Cells were collected, blocked with anti-CD16/32 (Fcg R III/II; BioLegend), IFN-b is successfully used to treat multiple sclerosis, another tissue- and stained with appropriate Abs against surface proteins in PBS with 2% specific autoimmune disease. Type 1 IFN contributes to initiation of FCS as described previously (31). Downloaded from insulitis in NOD mice, and an IFN-inducible transcriptional signa- In some experiments, intracellular proteins were stained after fixation ture has been detected in diabetic-prone children that later develop and permeabilization using fixation and permeabilization buffers from eBioscience (San Diego, CA). Phosphoflow experiments were performed as T1D, suggesting that type 1 IFN is critical for the initial break in previously described but with addition of blocking with anti-mouse 16.2 tolerance for T1D (5, 7, 29, 30), Yet, it is not clear what role innate Abs (FCgRIV; BioLegend) to reduce nonspecific binding (32, 33). For responses and in particular type 1 IFN play at later stages of disease intracellular TNF-a staining, cells were fixed, permeabilized, and stained with appropriate Abs. in setting the balance of pathogenesis and regulation that can ulti- http://www.jimmunol.org/ The following Abs were used: anti-CD3 (145-2C11), anti-CD11b (M1/70), mately lead to hyperglycemia. Therefore, we set out to determine anti-CD11c (N418), anti-CD49b (DX5), anti-Ly6C (HK1.4), anti-Ly6G how cDCs respond to an IFN-inducing stimulus in the context of (1A8), anti–Siglec-H (551), anti-CD24 (M1/69), anti-CD8a (53-6.7), anti- chronic autoimmunity, focusing on the prediabetic NOD mice. CD86 (GL-1), anti-CD80 (16-10A1), anti-CD40 (3/23), anti–TNF-a (MP6- In this study, we show that, CpG-induced type 1 IFN is higher in XT22), anti-IFNAR (MAR1-5A3), and IL-12p40 (C15.6) from BioLegend; prediabetic NOD mice serum and DCs, but the overall transcrip- anti-phospho (p)ERK1/2 (T202/Y204), anti-STAT1 (pY701), and anti- STAT4 (pY693) from BD Biosciences (San Jose, CA); and anti-STAT1 tional CpG response is muted in NOD cDCs compared with (D1K9Y) and anti-STAT4 (C46B10) from Cell Signaling Technology C57BL/6 (B6) cDCs. In contrast to this overall reduced response, (Danvers, MA). Brilliant Violet 510–conjugated streptavidin (BioLegend) CD40 is higher in NOD cDCs after CpG stimulation, and moDCs and Pacific Orange–conjugated streptavidin (Life Technologies, Grand were more activated in NOD mice as evidenced by higher in- Island, NY) were used to detect biotinylated Abs, and Aqua Dead Cell Stain by guest on September 23, 2021 (Life Technologies) was used to gate on viable cells. Fluorescence minus one flammatory cytokine production. We hypothesized that NOD cDCs controls include all fluorophores, except the color being analyzed. Samples have impaired signaling downstream of the IFN-a/b receptor were collected on a BD LSRII flow cytometer with four lasers and analyzed (IFNAR). Supporting this, the DC response to CpG-treatment is using FlowJo software 9.8.2 (Tree Star, Ashland, OR). similar in NOD and B6 mice in the presence of IFNAR blocking Affymetrix microarray analysis Abs, and gene targets of the IFN-induced transcriptional complex IFN-stimulated gene factor 3 (ISGF3) are enriched more in B6 CD8+ cDCs and CD11b+ cDCs from NOD and B6 mice were sorted, and DCs after CpG stimulation. Indeed, we find that IFN-induced RNA was purified for GeneChip Mouse Genome 430 array analysis. Generation of principal component analysis (PCA) plots and heat maps nuclear localization of STAT1 is lower in NOD. Therefore, NOD were performed using Partek Genomic Suite 6.5 (Partek, St. Louis, MO). cDCs likely have impaired IFNAR responses because of impaired Differentially expressed (DE) genes that belong to each mouse strain nuclear localization of STAT1. These alterations in innate immunity (NOD or B6) following CpG treatment were determined using the samr impact pathways that may have effects on Tregs and diabetes package with 0.1 false-discovery rate (FDR) and were represented using a pathogenesis. Venn diagram. Comparison of genes that were DE before or after treatment with CpG was further analyzed by Ingenuity Pathway Analysis software (National Institutes of Health library). Normalized intensity data were log Materials and Methods base 2 transformed for all the analysis unless otherwise stated for some Mice analysis where fold changes were calculated from the untransformed values. Gene lists for gene enrichment analysis (Table I) were obtained 2/2 B6, B6.H2g7 (B6g7), NOD, and RAG1 mice in both B6 and NOD through the Qiagen Web site. The array data have been deposited in the background were purchased from The Jackson Laboratory (Bar Harbor, National Center for Biotechnology Information Gene Expression Omnibus ME) and bred in the National Institute of Diabetes and Digestive and under accession number GSE75883 (http://www.ncbi.nlm.nih.gov/geo/query/ Kidney Diseases (NIDDK) animal facility. Animals were housed in spe- acc.cgi?acc=GSE75883). cific pathogen-free conditions and handled according to the Animal Care and Use Committee of the National Institutes of Health. The B6g7 con- Cytokine measurements genic strain is on the B6 background but carries the NOD-derived H2g7 haplotype. B6g7 mice were used in the experiments where indicated. Supernatant from either Flt3L-supplemented BMDC cultures or in vitro NOD.MyD882/2 mice were received from D. Mathis (Harvard University, enriched DC stimulation was collected 12 h poststimulation with CpG at Cambridge, MA) and bred at NIDDK. Age-matched female 8- to 10-wk- day 6; serum samples were collected 6 h after in vivo CpG treatment of mice 2 old mice with 3–4 mice/group were used in all experiments. and stored at 70˚C for cytokine analysis. TNF-a and IL-12p70 were measured using eBioscience Mouse Cytokine ELISA kits, and IFN-a and Generation of Flt3 ligand BM-derived DCs in vitro and CpG IFN-b were measured using the ELISA kit from PBL assay science. In all stimulation the ELISA kits, 3,39,5,59-Tetramethylbenzidine Liquid Substrate was common for colorimetric reaction as supplied by the manufacturer, and the OD Flt3 ligand (Flt3L) BMDCs were cultured as described previously (31). was measured in a multiscan ELISA reader (Synergy H1, Winooski, VT) at Cells were stimulated on day 6 for 12 h with CpG-A (InvivoGen, San 450 nm. The concentrations were calculated from the standard curves Diego, CA) + a liposomal transfection reagent, DOTAP (Roche, Mann- established with corresponding purified recombinant mouse cytokines. The Journal of Immunology 2033

Western blot analysis signal mimics innate signals that contribute to diabetes patho- Equal numbers of spleen lymphocytes were taken to separate cytoplasmic genesis (7, 35, 36). First, we measured serum levels of IFN-a and and nuclear protein fractions using the NE-PER Nuclear and Cytoplasmic IFN-b from NOD and B6 mice 6 h post-CpG i.v. injection. We Extraction Kit (Thermo Scientific, Waltham, MA). Fractionated protein found that CpG stimulation induced significantly higher levels of samples were run on a NuPAGE 4–12% or 12% Bis-Tris gel (Invitrogen) serum IFN-a and IFN-b in prediabetic NOD mice compared with and blotted onto a nitrocellulose membrane (Invitrogen). Western blots B6 (Fig. 1A). Furthermore, to determine how DCs directly re- were developed using an ECL system, according to the manufacturer’s + instructions (Amersham Biosciences, Pittsburgh, PA). The results were spond to CpG stimulation ex vivo, CD11c DCs were enriched visualized by ChemiDoc Touch Gel and Western blot Imaging System from spleen and stimulated with CpG for 6 h. Again, NOD DCs (Bio-Rad, Hercules, CA). Data were subjected to densitometric analysis secreted more IFN-a and IFN-b in response to CpG stimulation using Image Lab software included with the ChemiDoc. Abs used for (Fig. 1B). BM cells cultured with Flt3L differentiate into cDCs Western blotting include the following: anti-STAT1 (pY701), STAT1 (9172), and heat shock protein 90 were from Cell Signaling Technology. and pDCs, and these Flt3L BMDCs from NOD mice also pro- Lamin B was from Santa Cruz Biotechnology (Dallas, TX). duced more IFN-a in response to CpG compared with B6 Flt3L BMDCs (Fig. 1C). Similarly, type 1 IFN gene expression in Confocal microscopy and quantification of nuclear localization CD11c+ DCs taken from in vivo CpG-stimulated NOD mice was of STAT proteins higher than B6 mice (Fig. 1D). Without CpG stimulation, IFN-a Formaldehyde fixed (4%) (Electron Microscopy Sciences, Hatfield, PA) was not detected above background with the sensitivity of cur- splenocytes (6 IFN-a prestimulation for 30 min) were air-dried on the rently available assays (data not shown), but biologically relevant superfrost microscopic glass slides and blocked with 4% normal goat serum before incubating overnight with primary Abs—rabbit monoclonal responses to type 1 IFN can occur below this level (37). Taken STAT1 (D1K9Y; Cell Signaling Technology) and hamster CD11c (Bio- together, these data suggest that even long after initiation of au- Downloaded from Legend). Species-matched secondary Abs (conjugated with flurochrome toimmunity, NOD DCs produce more IFN-a and IFN-b upon CpG dye Alexa 594 or Alexa 488) were added for 2 h. The slides were washed, stimulation that may reflect the ongoing presence of low chronic air-dried, and mounted directly by Vectashield antifade mounting medium type 1 IFN. with DAPI (Vector Laboratories, Burlingame, CA). Tissue sections treated with no primary Abs were used as controls. The global response to CpG stimulation is muted in NOD cDCs Images were taken with a Zeiss confocal microscope (Nikon Instruments,

Melville, NY), and 10–15 fields/condition were analyzed with ImageJ. An To elucidate how the DCs from NOD mice respond to CpG in http://www.jimmunol.org/ algorithm was developed and used in ImageJ to quantify nuclear STAT1 relation to this increased type 1 IFN, NOD and B6 mice were intensity in cells that were positive for STAT1 and CD11c. CD11c+ cells treated with CpG for 12 h, and spleen cDC subsets (CD8+ and were defined as cells with a ring of CD11c signal, and nuclear area was + defined by DAPI signal. Total 100–150 CD11c+ cells were analyzed to CD11b ) were isolated for gene expression studies (sorting gates quantify the nuclear localization of STAT1. shown in Supplemental Fig. 1A). PCA plots showed that biolog- Quantitative gene expression analysis ical replicate samples of DCs clustered within a group, but both stimulation with CpG and strain origin separated the samples. Quantitative PCR. CD11c+ splenocytes were bead-enriched by negative selection using biotin Abs specific for CD3, CD19, NKp46, and Ly6c, with The shift in gene expression after CpG stimulation was larger in anti-biotin magnetic beads, according to the manufacturer instruction B6 DCs than NOD (Fig. 2A). A heat map generated from the (Miltenyi Biotec, San Diego, CA). RNA was then extracted immediately microarray data corresponded with PCA results, showing a dif- by guest on September 23, 2021 after enrichment using Qiagen mini prep (Molecular Research Center, ferential gene expression profile in CD8+ cDCs and CD11b+ cDCs Cincinnati, OH). RNA was reverse-transcribed into cDNA (Life Technol- between NOD and B6 mice (Supplemental Fig. 1B). To further ogies), and the cDNA was used as a template for PCR. Quantitative PCR assay was performed using The QuantStudio 6 Flex Real-Time PCR characterize the differential response, we categorized genes as up- System (Thermo Fisher Scientific) for Irf7, Oas3, Ifit1, Ifna1, Ifna2, Ifna4, or downregulated in response to CpG with an FDR of 0.1, using Ifnb, and Socs1 genes using commercially available TaqMan primers/ the unstimulated control for each strain as the baseline to focus on probes from Life Technologies. The housekeeping gene Hprt was used genes induced with CpG. As shown by the Venn diagrams, al- for normalization, and the data presented as relative gene expression. though a core response was shared by both strains, more genes NanoString. The mRNA-containing lysate from the sorted cells (pDCs) was . ∼ incubated with a custom panel of 450 bar-coded probes specific for genes were upregulated ( 500) or downregulated ( 300) uniquely in B6 associated with DC development and function (34) (NanoString Tech- cDCs compared with NOD cDCs (∼100) (Fig. 2B), showing an nologies, Seattle, WA). Samples were run by the Laboratory of Molecular overall muted response in both NOD cDCs subsets. Even within Technology at the National Cancer Institute (Frederick, MD). Raw data the set of genes induced in both strains, the induction was usually were normalized using the nSOLVER Analysis Software (NanoString lower in NOD cDCs compared with B6, as illustrated by focusing Technologies), according to company protocol. mRNA counts were nor- + malized by subtracting CodeCount = geo.mean, Background = mean.2sd, on the genes with the biggest upregulation in control B6 CD8 and SampleContent = housekeeping.geo.mean. Following normalization, cDCs: most are induced less in NOD (Fig. 2C). Next, pathway differential expression of the genes was determined using a nonparametric analysis on the set of genes induced (up or down with FDR 0.1) by Student t test. For pDC nanostring data, batch effects were removed using CpG in B6 but not NOD DCs showed that genes induced by CpG ComBat in the SVA package for R. in B6 but not NOD DCs were significantly enriched (log p . 1.3 Statistical analyses z-score . 0.5) in 14 pathways, such as downregulation of apo- Homoscedastic two-tailed t tests were performed for most analyses. The ptosis signaling and of NFAT in regulating immune response (data p values , 0.05, 0.01, and 0.001 were considered as *, **, and *** levels of not shown). Confirming the lower NOD response, a parallel significance, respectively. Error bars are represented by the SD unless noted analysis of genes uniquely induced in NOD CD8+ cDCs identified otherwise. Data that are emphasized as not significant are noted by ns. no pathways with both significant p values and z-scores. Gene expression was also assessed in CpG-stimulated pDCs. Results Because the number of pDCs decreases in the spleen after in vivo Prediabetic NOD mice and DCs produce more type 1 IFN in CpG stimulation, a custom NanoString DC panel was used that response to CpG than B6 requires fewer cells than microarray analysis (gated as shown in To learn more about type 1 IFN responses in chronic autoimmunity, Supplemental Fig. 1C). In contrast to the overall muted response we first focused on in vivo responses in 8- to 10-wk-old prediabetic to CpG in NOD cDCs, the differential response in NOD pDCs is NOD mice. For this study, CpG was chosen to study the TLR9- mixed. Among the 25 genes most induced in B6g7 pDCs, 16 mediated activation of DCs because this type 1 IFN-inducing showed differential induction in B6g7 versus NOD, but unlike the 2034 LOW TLR9-DRIVEN, IFNAR-DEPENDENT cDC ACTIVATION IN NOD MICE

A stimulation that may alter activation of both Tregs that inhibit diabetes and pathogenic T cells. Some inﬂammatory proteins are higher in NOD DCs primarily because of increased production by moDCs Despite this attenuated innate response, NOD DCs clearly exhibit strong proinﬂammatory responses in many contexts (39, 40). We

B A CD8+ CD11b+ DC DC

CD Downloaded from

B6 PBS NOD PBS B6 CpG NOD CpG B Genes up Genes down with CpG with CpG

FIGURE 1. NOD mice show higher expression of type 1 IFN in re- http://www.jimmunol.org/ sponse to CpG. Type 1 IFN levels were measured 6 h post-CpG stimulation by ELISA in serum samples of NOD and B6 mice 6 h post CpG treatment CD8+ B6 NOD B6 NOD 337 447 (A), culture supernatant of enriched-splenic DC culture (B), and culture DC 629 88 292 119 supernatant of Flt3L-BMDC (C). (D) Type 1 IFN gene expression in CD11c+-enriched population measured by quantitative PCR. Data presented in this paper are from one of at least two to four independent experiments. Comparisons were made between NOD and B6 (A and B)or B6g7 (C and D). *p , 0.05, **p , 0.01. CD11b+ B6 NOD B6 NOD

135 413 by guest on September 23, 2021 DC 568 82 345 107 cDCs (microarray analysis), some of these genes are induced more in NOD (Supplemental Fig. 1D, Supplemental Table I). We hypothesized that cDC response to CpG is dominated by responses downstream of the IFNAR, but pDC CpG responses will likely C represent more of a mix between the direct TLR9 response and 70 IFNAR response, which may account for a more complex pDC 60 * response. Therefore, despite increased type 1 IFN protein, the * B6 50 response to CpG in NOD cDCs is reduced compared with B6 NOD cDCs, but the pDC response is mixed. 40 * * Impaired innate responsiveness in NOD DCs inhibits induction 30 * * of costimulatory proteins 20 * * * Fitting with this overall lower gene induction, expression of costimulatory genes such as CD80 and CD86 were lower in NOD F.C. (CpG-induced) 10 DCs after CpG stimulation (fold-change NOD/B6: 21.9, p = 2.5 3 0 1027 and 21.4, p = 6.0 3 1023, respectively). Although expression of CD80 and CD86 are needed for full activation of effector Ifit3 Ifit1 Il2ra Rtp4 Ifit3b Slfn4 Oas3 Stat4 Oasl2 Pydc4 Rsad2 Ddx60 T cells, they are also necessary for Treg maintenance, and NOD Ddx58 mice lacking CD80 and CD86 have accelerated diabetes devel- FIGURE 2. The global response of cDCs to CpG stimulation is muted in opment (38). Changes in DC activation were evaluated at the NOD. NOD or B6 mice were treated with CpG i.v. After 12 h, splenic DCs protein level by measuring expression of CD86 and CD80 in cDCs were isolated and sorted into CD8+ cDC, and CD11b+ cDC. Sorted DC + 12 h after in vivo CpG stimulation in NOD and B6 mice. CD86 subsets were analyzed by Affymetrix gene array. (A) PCA plot for CD8 and + expression was upregulated less following CpG stimulation in CD11b cDCs. (B) Venn diagrams show numbers of genes that were up- NOD DCs compared with the B6 DCs. This lower level of induced regulated and/or downregulated in DC subsets after CpG stimulation. Genes were categorized as up- or downregulated in response to CpG with an FDR CD86 expression in NOD mice was observed in both cDC subsets of 0.1, using the unstimulated control for each strain as the baseline to focus and pDCs isolated from spleen, sLNs, or pLNs (Fig. 3A). We C + on genes induced with CpG. ( ) Fold-change comparison of gene expression observed similar results for CD80, especially in CD8 cDCs and (top 13 genes selected on the B6 strain) after CpG treatment between B6 and pDCs (Fig. 3B). Therefore, CD86 protein expression is one key NOD CD8+ cDC. Each sample was pooled from three mice, and each group example of the overall muted NOD DC CpG response. These data contained three samples for comparison. Comparison between NOD and B6 highlight the inability of NOD DCs to respond fully to CpG in (C)wasperformedbyStudentt test. *p , 0.05. The Journal of Immunology 2035

FIGURE 3. The costimulatory molecules CD86/CD80 are induced less in NOD DCs. NOD or B6 mice were treated with CpG i.v., and 12 h posttreatment, cells from the indicated lymphoid organs (spleen, pLNs, and sLNs) were stained for costimulatory molecule expression and DC subset markers Downloaded from (CD8+ cDC, CD11b+ cDC, and pDC subsets). CD86 expression (A) and CD80 expression (B) in DC subsets. Data presented as geometric mean ﬂuo- rescence intensity (MFI). Data were pooled from three independent experiments. NOD and B6 strains after CpG stimulation were compared by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. previously showed that CD40 expression is higher on NOD cDCs Next, to investigate whether the impaired response to CpG

(20). In this tudy, we find that CpG-induced CD40 expression was stimulation in NOD mice occurs as a result of ongoing adaptive http://www.jimmunol.org/ significantly higher in NOD DC subsets compared with B6 DCs, inflammation driven by T and B cells that directly induce diabetes indicating the less tolerogenic DC function in NOD mice (Fig. 4A). pathogenesis, we examined costimulatory molecule expression Therefore, we were specifically interested in measuring other in age-matched NOD.Rag12/2 and B6.Rag12/2 following CpG proinflammatory proteins to see how NOD DCs respond to stimu- stimulation in vivo. We found that CD86 expression in CpG- lation at the protein level. To measure cytokine production, culture treated NOD.Rag12/2 was significantly lower than CpG-treated supernatants collected from splenic CD11c+ DC culture and serum B6.Rag12/2, matching the CD86 expression pattern in CpG- samples of NOD and B6 mice after CpG-stimulation were tested for treated NOD or B6 mice (Fig. 5B, left panel). However, unlike TNF-a, and IL-12p70 by ELISA. NOD mice produced higher CD40 expression in NOD, NOD.Rag12/2 and B6.Rag12/2 amount of TNFa but less IL-12p70 than B6 DCs (Fig. 4B). showed comparable levels of CD40 in DC subsets (Fig. 5B, right by guest on September 23, 2021 Therefore, although the majority of the CpG response is lower in panel), suggesting that the level of CD40 protein expression on NOD, including CD86, CD80, and IL-12p70, some key proteins are NOD DCs may be dependent on the pathogenic T and B cell induced more in NOD DCs, including IFN-a, TNF-a,andCD40. environment. Therefore, although some proinflammatory costim- To determine which cells were making these proinflammatory ulation, such as CD40, is dependent on the adaptive autoimmune cytokines, a more specific gating scheme was used to separate the inflammation in NOD, the overall muted CpG response, as ex- CD11b+ cDCs from the moDCs, inflammatory monocytes that emplified by CD86 expression, is intrinsic to the DCs and inde- have upregulated MHC class II (32). Most IL-12 was produced by pendent of the autoimmune process. cDCs, with NOD cDCs making less than B6 (Fig. 4B). However, CD86 expression in NOD cDCs is more dependent on type 1 moDCs from NOD but not B6 were positive for IL-12p40, even IFN pathway than CD40 without activation (Fig. 4C). TNF-a is one signature cytokine of activated moDCs (41), and indeed, intracellular cytokine staining Because NOD DCs make more IFN-a in response to CpG but of DCs with this gating identified the moDCs as the primary have an overall lower response, this lower induction of gene producers of TNF-a (Fig. 4D). NOD moDCs have higher levels of expression may be downstream of the IFNAR rather than be- TNF-a staining compared with B6 moDCs. Therefore, although cause of lower TLR9 signal induction. To assess this, first, Flt3L NOD cDCs show a muted CpG response, NOD moDCs make BMDCs were treated with anti-IFNAR Ab, and then, the ex- more proinflammatory cytokines. pression of CD86 and CD40 was measured 12 h after CpG stimulation. As shown in Fig. 6A (left panel), IFNAR blocking CD40 and CD86 induction in NOD cDCs is MyD88 dependent, greatlyreducedtheexpressionofCD86inbothNODandB6 but impaired NOD CD86 induction is independent of mice. In contrast, CD40 expression was only partially reduced autoimmune B and T cell responses by IFNAR blocking and was still higher in NOD compared with Because CpG can signal via receptors other than TLR9, such as B6 DCs (Fig. 6A, right panel). Next, response of spleen cDCs to cytoplasmic receptors that signal via STING, we tested whether in vivo CpG stimulation and to acute blocking of IFNAR in vivo the response was dependent on TLR signaling adaptor MyD88. was tested. As in vitro, CD40 expression remained high in NOD CD86 was used to represent the important lower NOD DC gene cDCs compared with B6 cDCs, whereas CD86 expression was responses to CpG that dominated the gene expression analysis. reduced close to basal levels in both strains of mice (Fig. 6B). CD40 was used to represent the smaller subset of genes that are Therefore, CD40 expression may be associated with direct TLR more upregulated after CpG treatment in NOD compared with B6. signaling and interaction with activated B and T cells, whereas The upregulation of both markers as well as IFN-a production blocking IFNAR yields similar CD86 expression in NOD and B6 (data not shown) in response to CpG was MyD88 dependent and DCs, suggesting NOD mice have a deficiency in type 1 IFN therefore likely TLR9 mediated (Fig. 5A). signaling. 2036 LOW TLR9-DRIVEN, IFNAR-DEPENDENT cDC ACTIVATION IN NOD MICE

Impaired response to CpG in NOD cDCs is consistent with altered IFNAR signaling The impaired CpG response in NOD cDCs could be due to either defects in TLR9 signaling or IFNAR signaling. To determine whether the observed muted CpG response was specific to TLR9 activation, mice were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 agonist that also induces type 1 IFN. The poly(I:C)-induced response is very similar to the CpG response: NOD mice produced more IFN-b, but cDCs expressed less CD86 protein, and gene expression of IFN response genes was lower (Supplemental Fig. 2). The common element in the CpG and poly(I:C)-induced responses is type 1 IFN because TLR3 signaling is TRIF-dependent and distinct from TLR9. Pathway analysis of genes up/downregulated with CpG in CD8+ cDCs from our microarray data demonstrate higher enrichment of several pathways in B6 DCs compared with NOD DCs, including the Ingenuity Pathway Analysis JAK/STAT cluster. The lower signal in NOD DCs for this pathway primarily was due to lack of MEK activation and ERK-induced NF-kB activation; both are found in Downloaded from the same arm of the pathway, which has been shown to enhance transcription of ISGF3-dependent genes (Supplemental Fig. 3A) (42–44). To determine the contribution of this MEK-mediated pathway in the differential IFN DC response observed between NOD and B6, we first stimulated spleen cells in vitro with CpG and measured pERK levels by flow cytometry in different DC subsets. http://www.jimmunol.org/ pERK was significantly higher in B6 CD8+ cDCs compared with NOD (Supplemental Fig. 3B). Interestingly, when MEK inhibitor was used, IFN-a–induced CD86 expression was significantly reduced as observed in the Flt3L BMDCs (Supplemental Fig. 3C), consistent with a role for this pathway in augmenting IFN gene expression. Because these proteins are also activated directly downstream of TLR9, reduced MEK/ ERK activation in NOD cDCs may contribute to the impaired response to CpG and IFN. by guest on September 23, 2021 To focus more on transcription downstream of IFNAR, we were interested in gene expression induced by ISGF3, a complex of activated STAT1, STAT2, and IFN regulatory factor (IRF)9 that is the main transcription factor for IFN-induced genes (45). We found transcripts associated with ISGF3 binding sites are significantly enriched within the set of genes induced in B6 cDCs but not NOD cDCs, consistent with impaired type 1 IFN signal in NOD DCs (p = 0.001; Table I). Taken together, these data suggest that the reduced NOD cDC response to CpG may be due to impaired IFNAR response. Altered IFNAR signaling in NOD cDCs because of decreased nuclear localization of STAT1 Next, IFNAR expression was measured on the two cDC pop- ulations, pDCs and moDCs, to assess this pathway at the protein level. Both NOD CD8+ and CD11b+ cDCs expressed slightly lower levels of IFNAR, whereas NOD pDCs and moDCs expressed similar levels as B6 DCs (Fig. 7A). Despite this, cDCs from NOD and B6g7 mice showed similar IFN-a–induced levels FIGURE 4. NOD cDCs express higher CD40, and NOD moDCs are the of pSTAT1 measured by flow cytometry, and moDCs and pDCs source of increased IL-12 and TNF. NOD or B6 mice were treated with from NOD mice expressed more sustained pSTAT1 (Fig. 7B). CpG i.v., and 12 h posttreatment, cells from the indicated lymphoid organs Although canonical IFNAR signal is via phosphorylation of (spleen, pLNs, and sLNs) were stained for CD40, TNFR superfamily STAT1 and STAT2, STAT4 can also be activated by IFN (46). member, and DC subset markers (CD8+ cDC, CD11b+ cDC, and pDC subsets) (A). Spleen CD11c+ DCs were bead-enriched from NOD and B6 pSTAT2 induction was similar in B6 and NOD cDCs (data not mice and stimulated with the indicated concentration of CpG in vitro. IL- shown), but pSTAT4 was lower in NOD cDCs (Fig 7B). There- 12p70 and TNF-a were measured in the culture supernatant by ELISA fore, the lower type 1 IFN response in NOD cDCs is not simply 12 h post-CpG stimulation (B). Six hours post-CpG injection, serum because of the pSTAT1 levels observed by flow cytometry. The samples were collected for IL-12p70 and TNF-a analysis (B). Intracellular cytokine levels (IL-12p40 and TNF-a) were measured in CD8+ cDC, CD11b+ cDC, and moDC flow cytometry 12 h post-CpG stimulation (C ments (A and B) or presented one representative experiment from three and D). Data were either pooled from two to three independent experi- repeated experiments (C and D). *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 2037

FIGURE 5. Impaired CD86 induction in NOD mice is MyD88 dependent but independent of autoimmune B and T cell responses. CpG was given i.v. to age-matched wild-type (WT) and MyD882/2 (A) or Rag12/2 NOD and B6 mice (B), and 12 h posttreatment, splenic CD8+ and CD11b+ cDCs were

analyzed for CD86 and CD40 expression by ﬂow Downloaded from cytometry. Data represent one independent experiment repeated two to three times with three mice per group. Each dot represents an individual mouse. Values are geometric mean ﬂuorescence intensity (MFI). *p , 0.05. http://www.jimmunol.org/ by guest on September 23, 2021

lack of enrichment of ISGF3-induced genes suggests the impair- response to CpG including lower CD80 and CD86 compared with ment is due to signals downstream of STAT1 and STAT2 activa- cDCs from B6 mice. This lower CD86 in the NOD background is tion but prior to ISGF3 binding. dependent on IFNAR signaling but not adaptive autoimmune in- A key step in the elicitation of signal from IFNAR is the nuclear flammation as blocking IFNAR equalizes CpG-induced CD86 localization of the ISGF3 complex, which is mediated primarily by expression between the strains, but NOD.Rag12/2 mice still have pSTAT1 (26). Lower ISGF3 transcriptional activity in NOD DCs lower CD86 expression on cDCs compared with B6.Rag12/2.In could result from inability of activated pSTAT1 and the other contrast, CD40 differential expression was dependent on the au- components of ISGF3 to localize to or be retained in the nucleus. toimmune B and T cell responses. Therefore, some innate acti- To test this, NOD and B6g7 spleen cells were activated with IFN-a vation is secondary to adaptive autoimmunity, but some innate for 30 min, and cell nuclei were separated from the cytoplasmic alterations may be intrinsic to the APCs. fraction. Western blots analysis for pSTAT1 and total STAT1 was Although initially counterintuitive for autoimmune NOD mice performed in both nuclear and cytoplasmic fractions. We observed to display a lower innate immune response, cDCs contribute both decreased pSTAT1 in the nuclear fraction in NOD compared with to pathogenesis and tolerance induction in NOD mice, and many B6g7 (Fig. 7C, 7D). Similarly, nuclear localization of STAT1 was tolerance mechanisms require some intermediate level of matu- quantified in NOD or B6g7 CD11c+ cells following IFN-a stim- ration (47). For example, maintenance of Tregs is optimal with ulation for 30 min using confocal microscopy (Fig. 7E). Among semimature cDCs, and expression of CD80 and CD86 is necessary the CD11c+ cells, B6g7 mice showed significantly higher per- for DC-mediated expansion of Tregs, and NOD mice lacking centage of cells with STAT1 in the nuclei than NOD mice, and the CD80 and CD86 develop diabetes more rapidly (38, 48, 49). amount of STAT1 signal that overlapped with nuclear DAPI signal Reduced DC inflammatory tone could impair some immune tol- was lower in NOD CD11c+ cells (Fig. 7F). Therefore, these ob- erance mechanisms. This fits with the hygiene hypothesis, which servations at the single-cell level suggest that NOD DCs make suggests a lack of early exposure to pathogens may lead to more more type 1 IFN but display decreased IFNAR signal because of autoimmunity. Indeed, administration of TLR ligands such as LPS impaired nuclear localization of pSTAT1. or poly(I:C) can block diabetes in NOD mice (50, 51). In contrast, CD40 is a pathogenic signal that is dependent on the adaptive Discussion inflammatory response. DCs expressing low levels of CD40 in- In this study, we demonstrate differential innate activation in NOD duce Treg generation but with high level of CD40 suppress Treg cDCs characterized by higher IFN-a and CD40, but overall lower induction (52). Previously, we have shown blocking CD40 restores 2038 LOW TLR9-DRIVEN, IFNAR-DEPENDENT cDC ACTIVATION IN NOD MICE

FIGURE 6. CD86 expression in NOD mice is more dependent on signaling downstream of IFNAR than CD40. (A) BMDCs were cultured with Flt3L, and cultures were blocked with anti-IFNAR Ab for 24 h before stimulation with CpG in vitro. CD86 (left panel) and CD40 (right panel) expression were measured by ﬂow cytometry on cells gated on CD11c+ DCs. (B) CpG was given i.v., and anti-IFNAR Ab was injected i.p. in mice 24 h before i.v. CpG stimulation. Twelve hours post-CpG treatment, spleen cells from NOD and B6g7 mice were gated on CD8+ cDC and CD11b+ cDC subsets, and CD86 and CD40 were measured by ﬂow cytometry. Data were pooled from three independent

experiments. Data represent fold change compared Downloaded from with CpG-B6g7. Each dot represents an individual mouse. Values are geometric mean ﬂuorescence intensity (MFI). *p , 0.05, **p , 0.01. http://www.jimmunol.org/

tolerance by lowering the number of effector T cells and IFN-g even in the presence of high levels of type 1 IFN (53). Viral in- production (20). Therefore, the particular combination of lower fections may precipitate development of T1D in islet autoantibody– intrinsic responsiveness paired with higher levels of select path- positive (prediabetic) individuals, which could account for the ways, including CD40, could contribute to the imbalance between seasonal differences in onset of T1D (54, 55). CpG-induced changes effector and Tregs. in prediabetic NOD cDCs might model viral-induced changes in We measured parameters associated with IFNAR signal, in- APC function that are relevant for the failure of immune regulation by guest on September 23, 2021 cluding IFNAR, and levels of the activated forms of STAT1 and that precipitates b cell destruction and diabetes onset. STAT2, after IFN-a stimulation, and found that NOD mice express Previous studies on NOD mice have shown several alterations in lower IFNAR on cDCs, but pSTAT1 and pSTAT2, as measured by DC phenotype, including increased NF-kB signaling (14). How- phosphoflow, are similar to B6 DCs. Yet, despite induction of ever, most functional studies of NOD DCs use BM GM-CSF– pSTAT1 and pSTAT2, gene transcripts associated with ISGF3 induced DCs that may be similar to moDCs, which our data binding sites are not significantly enriched in NOD cDCs, con- suggest function quite differently from cDCs (56). Consistent with sistent with a poor induction of downstream gene expression as- these prior studies using in vitro–derived moDCs (41, 57–59), we sociated with IFNAR pathway in NOD mice. We show that this is find moDCs directly from the spleen of NOD mice are hyperac- due to impaired nuclear localization of IFN-a–activated STAT1, a tive, with higher TNF-a production. Other studies focus on pri- component of ISGF3. The observed reduction of MEK activation mary cDCs from the pancreas or pLN that are measuring effects of and pSTAT4 activation in NOD cDCs could contribute to this the local autoimmune inflammation, which is clearly relevant to defect as these pathways may normally amplify ISGF3 activation disease pathogenesis, but in this study, we focus on cDCs distal to independent of STAT1 activation (44). the autoimmune target that would likely present Ag in the context It is possible that impaired IFNAR signal in NOD DCs may be of immunotherapy in which exogenous self Ag is administered. a result of low-level chronic type 1 IFN in NOD mice (46). The Therefore, measuring the NOD spleen DC phenotype and function sensitivity of assays to detect IFN-a are not able to measure basal is relevant for designing Ag-specific immunotherapy for autoim- levels in sera, but CpG-stimulated IFN-a and IFN-b is higher in munity, and to our knowledge, this study is the first to compre- NOD than B6. Consistent with our results, a recent study in hu- hensively measure innate responses of cDCs in the context of mans with T1D reported lower expression of IFN-inducible genes prediabetic NOD mice.

Table I. Genes increased by CpG stimulation are enriched for genes containing binding sites of key IFN pathway regulators in B6 but not NOD DCs

Gene Groups IRF-7A STAT1 ISGF3 STAT3 STAT4 CREB NF-kB Induced more with CpG 0.597 0.791 0.724 0.889 0.918 0.75 0.549 in NOD CD8+ DCs Induced more with CpG 0.038* 0.001** 0.001** 0.001** 0.001** 0.001** 0.003* in B6 CD8+ DCs Kolmogorov–Smirnov test was used to evaluate the difference between the p value proﬁles of genes in the seven binding site gene lists against that of DE genes in comparing different mouse strains. *p , 0.05, **p , 0.01. The Journal of Immunology 2039 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. NOD DCs have altered IFN-a–induced signaling. (A) Comparison between NOD and B6 (A) or B6g7 (B–F) is shown at different levels of IFNAR signaling pathway. Expression of IFNAR on DC subsets was determined by flow cytometry. Data show mean fluorescence intensity (MFI) of cell surface IFNAR. (B) Intracellular levels of pSTAT1 and pSTAT4 expression in the indicated DC subsets (MHC Class IIhi gating applied) were measured by flow cytometry after IFN-a stimulation of collagenase-digested spleen cells. Data presented in this paper showed geometric mean MFI as the percentage of total STAT1 or STAT4. (C and D) Representative Western blot showing levels of pSTAT1 and STAT1 in the indicated cell fraction with and without IFN-a stimulation. Quantification of pSTAT1 was normalized to the respective fraction loading control (Lamin B for nucleus [N], heat shock protein 90 [HSP90] for cytoplasm [C], and total [T]). (E) Confocal microscopy of spleen cells with DAPI (blue), STAT1 (red), and CD11c+ DC (green). (F) Percentage of CD11c+ cells with nuclear STAT1 (left panel) and quantification of the amount of nuclear STAT1 within CD11c+ cells (right panel). Data represent one independent experiment of two to three repeat experiments. Mean + SEM is represented in (F). *p , 0.05.

Defective DC activation in NOD mice because of a lack of Weiping Chen and Chithra Keembiyehetty, for gene array support and proper regulation of IFN pathway may be sufficient for inducing microarray data analysis. We also thank Dr. Giorgio Trinchieri and Dr. Brian pathogenic responses but may not be enough for optimal devel- Kelsall for feedback and critical reading of the manuscript. opment of tolerogenic responses. Taken together, these data offer new insights into the state of innate immunity and key APCs in Disclosures prediabetic NOD mice, a potential intervention time for Ag- The authors have no financial conflicts of interest. specific immunotherapy that would use these APCs. References Acknowledgments 1. Grieco, F. A., F. Vendrame, I. Spagnuolo, and F. Dotta. 2011. Innate immunity We thank Alice Franks (Diabetes, Endocrinology, and Obesity Branch, and the pathogenesis of type 1 diabetes. Semin. Immunopathol. 33: 57–66. NIDDK) for help with mouse husbandry and NIDDK/National Heart, Lung, 2. Pearson, J. A., F. S. Wong, and L. Wen. 2016. The importance of the non obese and Blood Institute flow core and Dr. Phil McCoy for flow cytometry sup- diabetic (NOD) mouse model in autoimmune diabetes. J. Autoimmun. 66: 76– 88. port. We thank the National Heart, Lung, and Blood Institute Bioinformatics 3. van Belle, T. L., K. T. Coppieters, and M. G. von Herrath. 2011. Type 1 diabetes: Core Facility including Xujing Wang, and the NIDDK Genomics Core, etiology, immunology, and therapeutic strategies. Physiol. Rev. 91: 79–118. 2040 LOW TLR9-DRIVEN, IFNAR-DEPENDENT cDC ACTIVATION IN NOD MICE

4. Lieberman, S. M., and T. P. DiLorenzo. 2003. A comprehensive guide to anti- decreased CD135 expression and increased monocyte/macrophage precursors. body and T-cell responses in type 1 diabetes. Tissue Antigens 62: 359–377. Immunology 143: 640–650. 5. Andre´, I., A. Gonzalez, B. Wang, J. Katz, C. Benoist, and D. Mathis. 1996. 32. Dong, M. B., M. J. Rahman, and K. V. Tarbell. Flow cytometric gating for spleen Checkpoints in the progression of autoimmune disease: lessons from diabetes monocyte and DC subsets: differences in autoimmune NOD mice and with acute models. Proc. Natl. Acad. Sci. USA 93: 2260–2263. inflammation. J. Immunol. Methods In press. 6. Wen, L., R. E. Ley, P. Y. Volchkov, P. B. Stranges, L. Avanesyan, 33. Biburger, M., I. Trenkwald, and F. Nimmerjahn. 2015. Three blocks are not A. C. Stonebraker, C. Hu, F. S. Wong, G. L. Szot, J. A. Bluestone, et al. 2008. enough—blocking of the murine IgG receptor FcgRIV is crucial for proper Innate immunity and intestinal microbiota in the development of type 1 diabetes. characterization of cells by FACS analysis. Eur. J. Immunol. 45: 2694–2697. Nature 455: 1109–1113. 34. Fonseca, D. M., T. W. Hand, S. J. Han, M. Y. Gerner, A. Glatman Zaretsky, 7. Diana, J., Y. Simoni, L. Furio, L. Beaudoin, B. Agerberth, F. Barrat, and A. L. Byrd, O. J. Harrison, A. M. Ortiz, M. Quinones, G. Trinchieri, et al. 2015. A. Lehuen. 2013. Crosstalk between neutrophils, B-1a cells and plasmacytoid Microbiota-dependent sequelae of acute infection compromise tissue-specific dendritic cells initiates autoimmune diabetes. Nat. Med. 19: 65–73. immunity. Cell 163: 354–366. 8. Calderon, B., J. A. Carrero, M. J. Miller, and E. R. Unanue. 2011. Entry of 35. Xia, C. Q., R. Peng, A. V. Chernatynskaya, L. Yuan, C. Carter, J. Valentine, diabetogenic T cells into islets induces changes that lead to amplification of the E. Sobel, M. A. Atkinson, and M. J. Clare-Salzler. 2014. Increased IFN- cellular response. Proc. Natl. Acad. Sci. USA 108: 1567–1572. a‑producing plasmacytoid dendritic cells (pDCs) in human Th1-mediated type 1 9. Ferris, S. T., J. A. Carrero, J. F. Mohan, B. Calderon, K. M. Murphy, and diabetes: pDCs augment Th1 responses through IFN-a production. J. Immunol. E. R. Unanue. 2014. A minor subset of Batf3-dependent antigen-presenting cells 193: 1024–1034. in islets of Langerhans is essential for the development of autoimmune diabetes. 36. Filippi, C. M., and M. G. von Herrath. 2008. Viral trigger for type 1 diabetes: Immunity 41: 657–669. pros and cons. Diabetes 57: 2863–2871. 10. Darrasse-Je`ze, G., S. Deroubaix, H. Mouquet, G. D. Victora, T. Eisenreich, 37. Gough, D. J., N. L. Messina, C. J. Clarke, R. W. Johnstone, and D. E. Levy. K. H. Yao, R. F. Masilamani, M. L. Dustin, A. Rudensky, K. Liu, and 2012. Constitutive type I interferon modulates homeostatic balance through tonic M. C. Nussenzweig. 2009. Feedback control of regulatory T cell homeostasis by signaling. Immunity 36: 166–174. dendritic cells in vivo. J. Exp. Med. 206: 1853–1862. 38. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, and 11. Ro¨nnblom, L., M. L. Eloranta, and G. V. Alm. 2006. The type I interferon system J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of in systemic lupus erythematosus. Arthritis Rheum. 54: 408–420. the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. 12. Turley, S., L. Poirot, M. Hattori, C. Benoist, and D. Mathis. 2003. Physiological Immunity 12: 431–440. Downloaded from b cell death triggers priming of self-reactive T cells by dendritic cells in a type-1 39. Padgett, L. E., K. A. Broniowska, P. A. Hansen, J. A. Corbett, and H. M. Tse. diabetes model. J. Exp. Med. 198: 1527–1537. 2013. The role of reactive oxygen species and proinflammatory cytokines in type 13. Feili-Hariri, M., and P. A. Morel. 2001. Phenotypic and functional characteristics 1 diabetes pathogenesis. Ann. N. Y. Acad. Sci. 1281: 16–35. of BM-derived DC from NOD and non-diabetes-prone strains. Clin. Immunol. 40. Welzen-Coppens, J. M., C. G. van Helden-Meeuwsen, P. J. Leenen, 98: 133–142. H. A. Drexhage, and M. A. Versnel. 2012. Reduced numbers of dendritic cells 14. Poligone, B., D. J. Weaver, Jr., P. Sen, A. S. Baldwin, Jr., and R. Tisch. 2002. with a tolerogenic phenotype in the prediabetic pancreas of NOD mice. J. Elevated NF-kB activation in nonobese diabetic mouse dendritic cells results in Leukoc. Biol. 92: 1207–1213.

enhanced APC function. J. Immunol. 168: 188–196. 41. Hespel, C., and M. Moser. 2012. Role of inﬂammatory dendritic cells in innate http://www.jimmunol.org/ 15. Helft, J., J. Bo¨ttcher, P. Chakravarty, S. Zelenay, J. Huotari, B. U. Schraml, and adaptive immunity. Eur. J. Immunol. 42: 2535–2543. D. Goubau, and C. Reis e Sousa. 2015. GM-CSF mouse bone marrow cultures 42. David, M., E. Petricoin, III, C. Benjamin, R. Pine, M. J. Weber, and A. C. Larner. comprise a heterogeneous population of CD11c+MHCII+ macrophages and 1995. Requirement for MAP kinase (ERK2) activity in interferon a‑ and interferon dendritic cells. Immunity 42: 1197–1211. b‑stimulated gene expression through STAT proteins. Science 269: 1721–1723. 16. Guilliams, M., and B. Malissen. 2015. A death notice for in-vitro‑generated GM- 43. Goh, K. C., S. J. Haque, and B. R. Williams. 1999. p38 MAP kinase is required CSF dendritic cells? Immunity 42: 988–990. for STAT1 serine phosphorylation and transcriptional activation induced by in- 17. Prasad, S. J., and C. C. Goodnow. 2002. Cell-intrinsic effects of non-MHC NOD terferons. EMBO J. 18: 5601–5608. genes on dendritic cell generation in vivo. Int. Immunol. 14: 677–684. 44. Uddin, S., B. Majchrzak, J. Woodson, P. Arunkumar, Y. Alsayed, R. Pine, 18. Saxena, V., J. K. Ondr, A. F. Magnusen, D. H. Munn, and J. D. Katz. 2007. The P. R. Young, E. N. Fish, and L. C. Platanias. 1999. Activation of the p38 mitogen- countervailing actions of myeloid and plasmacytoid dendritic cells control au- activated protein kinase by type I interferons. J. Biol. Chem. 274: 30127–30131. toimmune diabetes in the nonobese diabetic mouse. J. Immunol. 179: 5041– 45. Schindler, C., D. E. Levy, and T. Decker. 2007. JAK-STAT signaling: from in-

5053. terferons to cytokines. J. Biol. Chem. 282: 20059–20063. by guest on September 23, 2021 19. Guilliams, M., F. Ginhoux, C. Jakubzick, S. H. Naik, N. Onai, B. U. Schraml, 46. Ivashkiv, L. B., and L. T. Donlin. 2014. Regulation of type I interferon responses. E. Segura, R. Tussiwand, and S. Yona. 2014. Dendritic cells, monocytes and Nat. Rev. Immunol. 14: 36–49. macrophages: a unified nomenclature based on ontogeny. Nat. Rev. Immunol. 14: 47. Tarbell, K. V., S. Yamazaki, and R. M. Steinman. 2006. The interactions of 571–578. dendritic cells with antigen-specific, regulatory T cells that suppress autoim- 20. Price, J. D., N. M. Beauchamp, G. Rahir, Y. Zhao, C. C. Rieger, A. W. Lau- munity. Semin. Immunol. 18: 93–102. Kilby, and K. V. Tarbell. 2014. CD8+ dendritic cell‑mediated tolerance of au- 48. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, and toreactive CD4+ T cells is deficient in NOD mice and can be corrected by R. M. Steinman. 2003. Direct expansion of functional CD25+CD4+ regulatory blocking CD40L. J. Leukoc. Biol. 95: 325–336. T cells by antigen-processing dendritic cells. J. Exp. Med. 198: 235–247. 21. Nikolic, T., G. Bouma, H. A. Drexhage, and P. J. Leenen. 2005. Diabetes-prone 49. Lesage, S., and C. C. Goodnow. 2001. Organ-specific autoimmune disease: a NOD mice show an expanded subpopulation of mature circulating monocytes, deficiency of tolerogenic stimulation. J. Exp. Med. 194: F31–F36. which preferentially develop into macrophage-like cells in vitro. J. Leukoc. Biol. 50. Caramalho, I., L. Rodrigues-Duarte, A. Perez, S. Zelenay, C. Penha-Gonçalves, and 78: 70–79. J. Demengeot. 2011. Regulatory T cells contribute to diabetes protection in 22. Tarbell, K. V., S. Yamazaki, K. Olson, P. Toy, and R. M. Steinman. 2004. CD25+ lipopolysaccharide-treated non-obese diabetic mice. Scand. J. Immunol. 74: 585–595. CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic 51. Serreze, D. V., K. Hamaguchi, and E. H. Leiter. 1989. Immunostimulation cir- peptide, suppress autoimmune diabetes. J. Exp. Med. 199: 1467–1477. cumvents diabetes in NOD/Lt mice. J. Autoimmun. 2: 759–776. 23. Baccala, R., K. Hoebe, D. H. Kono, B. Beutler, and A. N. Theofilopoulos. 2007. 52. Martin, S., R. Agarwal, G. Murugaiyan, and B. Saha. 2010. CD40 expression TLR-dependent and TLR-independent pathways of type I interferon induction in levels modulate regulatory T cells in Leishmania donovani infection. J. Immunol. systemic autoimmunity. Nat. Med. 13: 543–551. 185: 551–559. 24. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, 53. Panarina, M., K. Kisand, K. Alnek, K. Heilman, A. Peet, and R. Uibo. 2014. S. Ho, S. Antonenko, and Y. J. Liu. 1999. The nature of the principal type 1 Interferon and interferon-inducible gene activation in patients with type 1 dia- interferon-producing cells in human blood. Science 284: 1835–1837. betes. Scand. J. Immunol. 80: 283–292. 25. Ng, D., and J. L. Gommerman. 2013. The regulation of immune responses by 54. Kimpima¨ki, T., A. Kupila, A. M. Ha¨ma¨laïnen, M. Kukko, P. Kulmala, K. Savola, DC derived type I IFN. Front. Immunol. 4: 94. T. Simell, P. Keskinen, J. Ilonen, O. Simell, and M. Knip. 2001. The first signs of 26. Li, X., S. Leung, S. Qureshi, J. E. Darnell, Jr., and G. R. Stark. 1996. Formation b-cell autoimmunity appear in infancy in genetically susceptible children from of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene the general population: the Finnish Type 1 Diabetes Prediction and Prevention transcription by interferon-a. J. Biol. Chem. 271: 5790–5794. Study. J. Clin. Endocrinol. Metab. 86: 4782–4788. 27. Hata, N., M. Sato, A. Takaoka, M. Asagiri, N. Tanaka, and T. Taniguchi. 2001. 55. Lo¨nnrot, M., K. Korpela, M. Knip, J. Ilonen, O. Simell, S. Korhonen, K. Savola, Constitutive IFN-a/b signal for efficient IFN-a/b gene induction by virus. P. Muona, T. Simell, P. Koskela, and H. Hyo¨ty. 2000. Enterovirus infection as a Biochem. Biophys. Res. Commun. 285: 518–525. risk factor for b-cell autoimmunity in a prospectively observed birth cohort: the 28. Scheler, M., J. Wenzel, T. Tuting,€ O. Takikawa, T. Bieber, and D. von Bubnoff. Finnish Diabetes Prediction and Prevention Study. Diabetes 49: 1314–1318. 2007. Indoleamine 2,3-dioxygenase (IDO): the antagonist of type I interferon- 56. Boudaly, S., J. Morin, R. Berthier, P. Marche, and C. Boitard. 2002. Altered driven skin inflammation? Am. J. Pathol. 171: 1936–1943. dendritic cells (DC) might be responsible for regulatory T cell imbalance and 29. Ferreira, R. C., H. Guo, R. M. Coulson, D. J. Smyth, M. L. Pekalski, autoimmunity in nonobese diabetic (NOD) mice. Eur. Cytokine Netw. 13: 29–37. O. S. Burren, A. J. Cutler, J. D. Doecke, S. Flint, E. F. McKinney, et al. 2014. A 57. Morel, P. A., A. C. Vasquez, and M. Feili-Hariri. 1999. Immunobiology of DC in type I interferon transcriptional signature precedes autoimmunity in children NOD mice. J. Leukoc. Biol. 66: 276–280. genetically at risk for type 1 diabetes. Diabetes 63: 2538–2550. 58. Palucka, K. A., N. Taquet, F. Sanchez-Chapuis, and J. C. Gluckman. 1998. 30. Li, Q., B. Xu, S. A. Michie, K. H. Rubins, R. D. Schreriber, and H. O. McDevitt. Dendritic cells as the terminal stage of monocyte differentiation. J. Immunol. 2008. Interferon-a initiates type 1 diabetes in nonobese diabetic mice. Proc. 160: 4587–4595. Natl. Acad. Sci. USA 105: 12439–12444. 59. Moser, E. K., M. M. Hufford, and T. J. Braciale. 2014. Late engagement of CD86 31. Guerrero, A. D., M. B. Dong, Y. Zhao, A. Lau-Kilby, and K. V. Tarbell. 2014. after influenza virus clearance promotes recovery in a FoxP3+ regulatory T cell Interleukin-2‑mediated inhibition of dendritic cell development correlates with dependent manner. PLoS Pathog. 10: e1004315.