Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing Β-1,3 Glucan Nida S

Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing β-1,3 Glucan Nida S. Khan, Pia V. Kasperkovitz, Allison K. Timmons, Michael K. Mansour, Jenny M. Tam, Michael W. Seward, This information is current as Jennifer L. Reedy, Sravanthi Puranam, Marianela Feliu and of September 26, 2021. Jatin M. Vyas J Immunol published online 1 February 2016 http://www.jimmunol.org/content/early/2016/01/30/jimmun

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 1, 2016, doi:10.4049/jimmunol.1401545 The Journal of Immunology

Dectin-1 Controls TLR9 Trafﬁcking to Phagosomes Containing b-1,3 Glucan

Nida S. Khan,*,† Pia V. Kasperkovitz,‡ Allison K. Timmons,* Michael K. Mansour,*,x Jenny M. Tam,* Michael W. Seward,* Jennifer L. Reedy,*,x Sravanthi Puranam,* Marianela Feliu,*,{ and Jatin M. Vyas*,x,‖

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate b-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that b-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes con- Downloaded from taining b-1,3 glucan beads. Dectin-1–mediated spleen tyrosine kinase activation is required for TLR9 trafficking to b-1,3 glucan–, A. fumigatus–, and C. albicans–containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of b-1,3 glucan by Dectin-1 triggers TLR9 trafficking to b-1,3 glucan–containing phagosomes, which may be critical in coordinating innate antifungal defense. The Journal of Immunology, 2016, 196: 000–000. http://www.jimmunol.org/ nvasive fungal infections by Candida albicans and Asper- localization of TLRs is critical in recognition of microbial ligands, gillus fumigatus cause significant morbidity and mortality in downstream signaling, and self- versus non–self-discrimination (4). I immunocompromised patients despite the widespread use of TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed at the plasma potent antifungal medications, suggesting that the immune system membrane and recognize bacterial and fungal cell wall components, plays a critical role in determining the outcome of the infection (1, whereas TLR3, TLR7, TLR8, and TLR9 are localized to the in- 2). Advances in the treatment of invasive fungal infections require tracellular compartments and recognize bacterial and viral nucleic elucidation of the molecular mechanisms that govern the host acids (5). A role for TLR9 has been implicated in host defense immune response. against A. fumigatus and C. albicans (6, 7). Activation of TLR9 Pathogen recognition by the host requires binding of highly homodimers in response to unmethylated CpG DNA requires pro- by guest on September 26, 2021 conserved microbial pathogen-associated molecular patterns epi- teolytic cleavage of its N-terminal ectodomain to generate a func- topes by germline-encoded pattern-recognition receptors includ- tional receptor, as well as a conformational change in the cytoplasmic ing TLRs and C-type lectin receptors (CLRs) (3). Subcellular signaling domains that is required for the recruitment of adaptor molecules (8–11). This N-terminal proteolytic cleavage of TLR9 is also required for recruitment to fungal-containing phagosomes *Division of Infectious Diseases, Department of Medicine, Massachusetts General (9, 12). Unlike other TLRs, macrophages lacking TLR9 show Hospital, Boston, MA 02114; †Biomedical Engineering and Biotechnology, Univer- sity of Massachusetts, Lowell, MA 01854; ‡Alnylam Pharmaceuticals, Cambridge, enhanced antifungal immunity (13). The molecular mechanisms MA 02142; xDepartment of Medicine, Harvard Medical School, Boston, MA governing regulation of TLR9 trafficking to fungal phagosomes 02115; {Nutrition and Metabolism, Boston University, Boston, MA 02118; ‖ remain to be determined. and Program in Immunology, Harvard Medical School, Boston, MA 02115 Dectin-1, a CLR, recognizes the carbohydrate b-1,3 glucan, ORCIDs: 0000-0002-5448-8289 (N.S.K.); 0000-0002-6364-9435 (P.V.K.); 0000- 0002-8412-315X (A.K.T.); 0000-0002-4813-6308 (M.W.S.); 0000-0002-9985-9565 which constitutes the major fungal cell wall component of mul- (J.M.V.). tiple pathogenic fungi including A. fumigatus, C. albicans, and Received for publication June 17, 2014. Accepted for publication January 3, 2016. Pneumocystis jirovecii (14–16). Dectin-1 plays an essential role in This work was supported by National Institutes of Health Grants 5R01 A1 092084 and pulmonary defense against A. fumigatus and chronic mucocuta- 1R01 A1 097519 and National Institutes of Health/National Institute of Allergy and neous Candida infections (15, 17). Upon ligand recognition, the Infectious Diseases Grants 1K08AI110655 (to M.K.M.), T32 A1007061-35 (to J.L.R.), and KL2 TR001100 (to J.L.R.). cytoplasmic ITAM motif of Dectin-1 is phosphorylated by Src family kinases, resulting in the recruitment of spleen tyrosine kinase The data presented in this article have been submitted to Gene Expression Omni- bus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73474) under acces- (Syk). Syk triggers an intracellular signaling cascade resulting in sion number GSE73474. proinflammatory cytokine production and induction of Th17 cells, Address correspondence and reprint requests to Dr. Jatin M. Vyas, Division of In- central to the control of mucosal fungal infections (18–20). fectious Diseases, Department of Medicine, Massachusetts General Hospital, 55 Fruit Dectin-1 interacts with TLR2 and TLR4, leading to a specific Street, Boston, MA 02114. E-mail address: [email protected] innate immune response against C. albicans (19, 21). Dectin-1 The online version of this article contains supplemental material. controls phagosome maturation (22), but its ability to regulate Abbreviations used in this article: BafA1, bafilomycin A1; CLR, C-type lectin receptor; DIC, differential interference contrast; D1KO, Dectin-1–knockout; HK, heat- trafficking of intracellular TLRs has not been established. killed; IFI, IFN-inducible; PBS-T, PBS-0.01% Tween 20; PVDF, polyvinylidene We sought to define the role of Dectin-1 and Syk signaling on fluoride; RAW, RAW 264.7 macrophage-like cell line; Syk, spleen tyrosine kinase; TLR9 subcellular redistribution in APCs to phagosomes contain- TLR9KO, TLR9 knockout. ing pathogenic fungi. Using “fungal-like particles” that display Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 monodispersed b-1,3 glucan on a polystyrene platform (23), as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401545 2 DECTIN-1 CONTROLS TLR9 RECRUITMENT well as C. albicans and A. fumigatus, our data show that the Surface fluorescence labeling of b-1,3 glucan beads and presence of b-1,3 glucan in phagosomes is sufficient for TLR9- CpG+IgG beads GFP recruitment. We further demonstrate that Dectin-1 controls b-1,3 Glucan beads and CpG+IgG beads were labeled for live cell imaging TLR9 trafficking to C. albicans and multiple A. fumigatus spore by mixing ∼5 3 106 beads with 30 mg N-hydroxysuccinimidyl ester Alexa stages. Endolysosomal acidification is required for TLR9 re- fluor dye (Invitrogen AF658 and AF647) in dimethylformamide for 1 h at cruitment to b-1,3 glucan beads-containing phagosomes. Using room temperature followed by three washes in PBS. Then labeled beads chemical inhibitors of Syk and the signaling-incompetent Dectin- were resuspended in PBS. 1, which are incapable of activating Syk, we demonstrate that Syk Confocal microscopy activation is required for TLR9 recruitment to b-1,3 glucan beads Macrophages were plated onto eight-chambered coverglass (LabTek; and fungal-containing phagosomes. Thermo Scientific, Rochester, NY) (13, 22, 23). Cells were incubated with stimuli at 37˚C for specified times. Coverglass were then mounted on Nikon Materials and Methods Ti-E inverted microscope equipped with CSU-X1 confocal spinning-disk head (Yokogawa, Sugarland, TX). A coherent, 4-W laser (Coherent, Santa Reagents Clara, CA) was used as an excitation light source to produce excitation Bafilomycin A1 (BafA1) was purchased from A.G. Scientific (Billerica, wavelengths of 488, 568, and 647 nm using an acoustic optical tunable MA). CpG phosphorothioate oligodeoxynucleotide 1826 (59-TCCAT- tuner. To acquire high-quality fluorescence images, we used a high- GACGTTCCTGACGTT-39) was synthesized by Integrated DNA Tech- magnification, high-numerical aperture objective (Nikon, 1003, 1.49 nu- nologies (Coralville, IA). Mouse anti-dsDNA mAb (IgG) was purchased merical aperture, oil immersion). A polarizer (MEN 51941; Nikon) and from Millipore (Temecula, CA). Mouse mAb to b-1,3 glucan (IgG) was Wollaston prisms (MBH76190; Nikon) were used to acquire differential interference contrast (DIC) images. Emission light from the sample was purchased from Biosupplies Australia (VIC, Australia). Mouse monoclonal Downloaded from GFP-HRP Ab and R406, a Syk inhibitor, were purchased from Santa Cruz collected after passage through the appropriate emission filters (Sem- Biotechnology (Santa Cruz, CA). rock, Rochester, NY). Images were acquired using an EM-CCD camera (C9100-13; Hamamatsu, Bridgewater, NJ). Image acquisition was per- Cell lines and cell culture formed using MetaMorph software (Molecular Devices, Downingtown, PA). Raw image data files were processed using Adobe Photoshop CS4 and The mouse macrophage-like cell line, RAW 264.7 macrophage-like cell assembled in Adobe Illustrator, version CS4 (Adobe Systems, San Jose, CA). line (RAW), and human embryonic kidney cell line, HEK293T, were

purchased from American Type Cell Culture Collection (Manassas, VA). Phagosome isolation http://www.jimmunol.org/ RAW cells were cultured in DMEM containing 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% L-glutamine (Thermo Scientific, Logan, Phagosome isolation was adopted from previous protocols (22). For cell UT) (DMEM complete media). Dectin-1–deficient immortalized mac- lysates and phagosome isolations, RAW cells were plated into six-well 6 rophages were a gift from Gordon Brown (University of Aberdeen) and dishes (Corning, Tewksbury, MA). A total of 2 3 10 RAW-TLR9-GFP 6 Stuart Levitz (University of Massachusetts Medical School). Dectin-1– cells were incubated with 4 3 10 b-1,3 glucan beads or CpG+IgG beads deficient macrophages were cultured in RPMI-GlutaMax (Life Technologies) for indicated times. Cells were washed in ice-cold PBS three times. Cells containing 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% HEPES were then mechanically sheared in a hypotonic lysis buffer (2 mM MgCl2, buffer, and 2 mM 2-ME (RPMI complete media). Puromycin (5 mg/ml) 6 mM 2-ME, 10 mM HEPES with protease inhibitors (Roche, Indian- was used for selection of transduced cells. apolis, IN) by passing through a 1-ml syringe fitted with a 1/2-inch 26-G C. albicans, SC5314, a wild-type strain, and A. fumigatus strain 293 needle (Fisher, Agawam, MA) for 15 cycles. Sixty-two percent sucrose were gifts from Eleftherios Mylonakis (Brown Medical School). A. fumi- solution was added to adjust the final solution to 40% sucrose. A dis- by guest on September 26, 2021 gatus strain 293 expressing cytosolic RFP was a gift from Michelle continuous gradient was then constructed in high-speed ultracentrifugation Momany (University of Georgia) (24). C. albicans was grown in yeast tubes (Polyallomer; Beckman, Brea, CA) overlaying 2 ml of 62%, 40% of extract peptone dextrose agar media (Sigma-Aldrich) with ampicillin lysed cell suspension, 30%, 25%, and 10%. Sucrose gradient was then overnight at 30˚C in a shaker incubator at 250 rpm. The next morning, centrifuged at 80,000 3 g for 1 h at 4˚C (SW-28 rotor, L8- M ultracen- yeast was washed three times in PBS, heat-killed (HK) at 95˚C for 15 min trifuge; Beckman). Phagosomes were isolated at the interface of the 25%/ in 500 ml PBS, and counted by hemacytometer. A. fumigatus was grown on 10% sucrose layers, washed in PBS, and counted using a hemacytometer in Sabouraud dextrose agar plates supplemented with 100 mg/ml ampi- preparation for flow cytometry and immunoblot analysis. cillin at 30˚C for 3 d. Conidia were harvested by scraping and washing three times in ice-cold PBS. Conidia were stored at 4˚C for use or im- Immunoblot analysis mediately HK at 100˚C for 20 min. A. fumigatus swollen conidia were RAW macrophages were incubated with R406 at the concentration of 10 prepared by incubation in complete RPMI media at 37˚C in water bath for mM for 30 min or 100 nM BafA1 for 15 min in complete DMEM. Cells 6 h. Spore germination stages were distinguished by visual inspection using were lysed in 1% Nonidet P-40 (American Bioanalytical, Natick, MA) m light microscopy. Resting conidia are 2–5 m and swollen conidia are 6–8 with protease inhibitors (Roche, Indianapolis, IN) for 1 h at 4˚C. A total of m . m. After 6 h, the majority ( 95%) of conidia were determined to be 1 3 106 phagosomes or cell lysate in 13 loading buffer/reducing agent swollen. (Invitrogen) were heated to 95˚C for 5 min, and proteins resolved by SDS- Viral transduction and plasmids PAGE using 4–12% gels (Invitrogen). After electrophoresis, gels were removed and methanol-activated polyvinylidene fluoride (PVDF) mem- The retroviral pMSCV vector containing murine TLR9 fused at the C brane (Perkin Elmer, Waltham, MA) was applied to the gel in transfer buffer terminus to GFP (pMSCV-TLR9-GFP) and plasmids encoding VSV-G and (0.025 M Tris, 0.192 M glycine, 20% methanol). All buffer components gag-pol were gifts from Hidde Ploegh (Whitehead Institute for Bio- were from National Diagnostics (Atlanta, GA) or Sigma-Aldrich. The gel medical Research, Cambridge, MA). Murine TLR9 fused at the C ter- and PVDF membrane were sandwiched between transfer sponge and minus to mCherry encoded in the retroviral pMSCV vector was a gift from blotting paper and were subjected to electrophoretic transfer at 100 V for Melanie Brinkmann (Helmholtz Center for Infection Research). Retroviral 1h. transduction was performed as described previously (12). GFP–Dectin-1 or For detection of GFP proteins, PVDF-immobilized gel transfers were GFP–Dectin-1DY15 was subcloned into pHAGE II (22). HEK293T cells blocked with 5% milk in PBS-0.01% Tween 20 (PBS-T; Sigma-Aldrich) were used to generate lentivirus as described previously (25). overnight at 4˚C. Blots were incubated with anti–GFP-HRP in 1% milk in PBS-T for 1 h at room temperature. After three 5-min washes in PBS-T Generation of CpG+IgG beads and PBS, the blot was visualized using the Western Lighting Plus ECL chemiluminescent substrate (Perkin Elmer, Waltham, MA) on Kodak CpG+IgG beads were generated similar to Henault et al. (26) with slight BioMax XAR Film (Sigma). Films were then scanned and cropped, and modifications. In brief, 3 mM amine-terminated polystyrene beads (Poly- contrast was adjusted evenly to the entire image using Adobe Photoshop sciences, Warrington, PA) were washed three times in PBS and incubated CS4. with 1 mM CpG in acetonitrile overnight at 4˚C. The next morning, CpG beads were washed in PBS and incubated with 0.25–1.25 mg of anti- Microarray analysis dsDNA Ab in PBS/BSA for 1 h. After incubation, CpG+IgG beads were washed and stored at 4˚C in PBS for later use. b-1,3 Glucan–conjugated A total of 4 3 106 wild-type B6 and TLR9 knockout (TLR9KO) im- beads were generated as described previously (23). mortalized murine macrophages were plated onto 10-cm cell culture dishes The Journal of Immunology 3

(Corning, Tewksbury, MA) and were either stimulated with b-1,3 glucan TLR9 to the phagosomes. However, b-1,3 glucan beads conju- beads at multiplicity of infection of 20:1 for 4 h or unstimulated. Total gated with b-1,3 glucan Ab (b-1,3 glucan + IgG) failed to enhance RNA was extracted using RNA easy mini kit (Qiagen, Valencia, CA). TLR9 recruitment in RAW macrophages compared with b-1,3 Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA using amplification kit WT Plus, followed glucan beads as shown by Western blot, flow cytometric, and live by labeling of 5.5 mg cDNA. After fragmentation, 90 ml cDNA was hy- cell imaging analyses (Supplemental Fig. 1A–C). Henceforth, the bridized for 16 h at 45˚C on GeneChip Mouse 1.0 ST Array in an Affy- CpG+IgG beads were used as a positive control for TLR9 re- metrix GeneChip Hybridization oven 645 (Santa Clara, CA). GeneChips cruitment. Because individual cells may express varying levels of were washed and stained in the Affymetrix Fluidics station 450. Gene- Chips were scanned using the Affymetrix GeneChip Scanner 3000 TLR9-GFP, we sought to compare directly phagosomes containing 7G running Affymetrix Gene Command Console ver 3.2. The data either CpG+IgG or b-1,3 glucan beads within the same cell. There were normalized using the Robust Multichip Average summary program was comparable recruitment of TLR9-GFP to phagosomes con- RMAexpress (PubMed ID: 12538238). Differentially expressed genes taining b-1,3 glucan beads or CpG+IgG beads within the same were identified using the mercury linear fit model. Transcripts with fold- macrophage (Fig. 1A, bottom panel). change .2.0 between TLR9KO and wild-type B6 macrophages with p , 0.001 were identified. Among these genes, transcripts with fold-change To quantify the amount of TLR9-GFP on the phagosomal .1.8 between macrophages stimulated with b-1,3 glucan beads and membrane, we isolated phagosomes from RAW TLR9-GFP cells without stimulation were considered differentially regulated between incubated with b-1,3 glucan beads or CpG+IgG beads for 3 h. TLR9KO and wild-type B6 macrophages. Our data were deposited at This type of analysis permits interrogation of at least 20,000 Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE73474) under accession number GSE73474. phagosomes per population. Flow cytometry analysis of isolated phagosomes revealed a comparable amount of TLR9 recruited to

Phagocytosis assay b-1,3 glucan beads as compared with CpG+IgG beads as deter- Downloaded from A total of 1.8 3 105 RAW-TLR9-GFP macrophages plated onto eight- mined by emission from GFP (Fig. 1B). Thus, our results indicate chambered coverglass were stimulated with 9 3 105 CpG beads, that TLR9-GFP is specifically recruited to phagosomes containing CpG+IgG beads, b-1,3 glucan beads, b-1,3 glucan+IgG beads for 2 h at b-1,3 glucan beads and is comparable with levels achieved by 37˚C supplemented with 5% CO2. Green fluorescence around the beads CpG+IgG beads. Our previous findings indicate loss of b-1,3 was assessed by confocal microscopy. The percentage of TLR9-GFP+ phagosomes was calculated by dividing the number of engulfed beads glucan phagosomal-associated GFP–Dectin-1 within 90 min of + that were GFP by the total number of engulfed beads in ∼25 macro- phagocytosis (22). We explored the kinetics of TLR9-GFP trans- http://www.jimmunol.org/ phages per condition. Three independent experiments were performed location to b-1,3 glucan beads after phagocytosis. We stimulated per condition. RAW TLR9-GFP macrophages with b-1,3 glucan beads for up to 17 h and subjected the isolated b-1,3 glucan–containing phag- Results osomes to flow cytometry. Unlike Dectin-1, which is lost from the b Phagosomes containing -1,3 glucan beads specifically recruit phagosome within 30 min (22), TLR9-GFP was recruited within TLR9 30 min and remained on b-1,3 glucan–containing phagosomes for Our previous findings indicate that TLR9 is recruited to phag- up to 17 h (Fig. 1C). Recruitment of the cleaved fraction of TLR9 osomes containing either A. fumigatus or C. albicans (12, 13). We was confirmed by immunoblot (Supplemental Fig. 2). hypothesized that a constituent of the fungal cell wall triggered Proteolytic cleavage of TLR9 is required for its activation and by guest on September 26, 2021 redistribution and accumulation of TLR9 to the fungal-containing signaling in primary APCs (8, 9). We sought to determine whether phagosomes. The fungal cell wall is composed of a complex full-length TLR9-GFP or cleaved TLR9-GFP accumulated on mixture of carbohydrates including b-1,3 glucan polymers and b-1,3 glucan–containing phagosomes. We incubated RAW TLR9- proteins (3, 18). We took advantage of the development of poly- GFP cells with b-1,3 glucan beads for 3 h followed by phagosome styrene beads conjugated with pure, fungal-derived b-1,3 glucan isolation, and phagosomal proteins were resolved by SDS-PAGE. to probe the response of TLR9 (23). Phagocytosis of b-1,3 glucan We recovered only the cleaved fraction of TLR9-GFP from beads is Dectin-1 dependent. Although CR3, a serum complement phagosomes containing b-1,3 glucan beads as well as CpG+IgG protein, can also mediate uptake of glucan particles into cells (27), beads (Fig. 1D). In resting conditions, ∼50% of TLR9-GFP in our system used serum lacking functional CR3 (i.e., heat-inactivated unstimulated RAW macrophages localizes to endolysosomes (28), serum). Within 30 min of bead internalization, TLR9 redistributes and the lysate control confirms this finding (Fig. 1D). Incubation from the endoplasmic reticulum to endolysosomal compartments of b-1,3 glucan beads with lysates from RAW cells expressing and is enriched on phagosomes containing b-1,3 glucan beads, TLR9-GFP failed to permit sufficient TLR9-GFP to be visualized whereas an uncoated, size-matched polystyrene bead-containing by immunoblot, and phagoFACS indicating TLR9-GFP on phag- phagosome in the same cell failedtorecruitTLR9(Fig.1A,top osomes is not due to postlysis effects (Fig. 1C, Supplemental Fig. panel). Pretreatment of b-1,3 glucan beads with DNase con- 2). Our findings indicate that the proteolytically cleaved form of firmed TLR9-GFP recruitment is likely not due to any contami- TLR9 accumulates on phagosomes containing b-1,3 glucan beads. nating DNA adsorbed to the beads (data not shown). A. fumigatus conidia triggers recruitment of TLR9 to the phagosome (12). To Dectin-1 is required for TLR9 recruitment to fungal demonstrate that the recruitment of TLR9 to b-1,3 glucan beads is phagosomes comparable with Aspergillus conidia, we stimulated TLR9-GFP The b-1,3 glucan receptor, Dectin-1, synergizes with TLR2 and macrophages with both b-1,3 glucan beads and A. fumigatus.Re- TLR4 in recognition of A. fumigatus or C. albicans in innate cruitment of TLR9-GFP to phagosomes containing b-1,3 glucan immune cells and cytokine production (29, 30). Dectin-1 activa- beads was comparable with that of A. fumigatus within the same tion controls maturation of phagosome-containing b-1,3 glucan cell (Fig. 1A, middle panel). beads and C. albicans (22). Therefore, we predicted that Dectin-1 A previous study shows that CpG beads with anti-dsDNA Ab controls TLR9 recruitment to fungal phagosomes. To test this (CpG+IgG) have enhanced TLR9 recruitment compared with CpG hypothesis, we expressed TLR9-mCherry in Dectin-1–knockout beads alone (26). We sought to determine whether the addition of (D1KO) macrophages and D1KO macrophages reconstituted with anti–b-1,3 glucan Ab would enhance TLR9 recruitment to b-1,3 GFP–Dectin-1. These macrophages were incubated with HK glucan phagosomes. As expected, the CpG beads adsorbed with C. albicans for 40 min because heat killing of C. albicans exposes anti-dsDNA Ab (CpG+IgG) demonstrated potent recruitment of more b-1,3 glucan on the surface (31, 32). Confocal microscopy 4 DECTIN-1 CONTROLS TLR9 RECRUITMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 The Journal of Immunology 5 revealed that despite adequate expression of TLR9-mCherry and undergo proteolysis in the endolysosomal compartments for suc- phagocytosis of HK C. albicans, TLR9-mCherry did not recruit to cessful intracellular trafficking, subcellular compartmental reten- phagosomes in D1KO macrophages (Fig. 2A, top panel). Func- tion, and generation of a functionally competent receptor to signal tional complementation of Dectin-1–deficient macrophages re- in response to CpG (8, 9, 28). Our recent findings indicated a stored the ability of TLR9-mCherry to translocate successfully to direct correlation between Dectin-1 retention to phagosomes HK C. albicans phagosomes (Fig. 2A, bottom panel). Our data containing b-1,3 glucan and phagosomal pH (22). We hypothe- support the notion that TLR9 recruitment to phagosomes con- sized that selective accumulation of the cleaved TLR9 to b-1,3 taining b-1,3 glucan requires Dectin-1. glucan phagosomes (Fig. 1B) is dependent on phagosomal acidi- We wished to generalize our finding of Dectin-1–dependent fication. To evaluate this hypothesis, we pretreated cells with the TLR9 recruitment to other pathogenic fungi. A. fumigatus conidial vacuolar-type H+-ATPase inhibitor, BafA1. BafA1 blocks endo- spore germination starts with resting conidia and then swells and lysosomal acidification and signaling by TLR9, TLR7, and TLR3 progresses to germ-tube formation with eventual hyphal branching (38). BafA1 treatment had no effect on phagocytosis of b-1,3 (33, 34). During the A. fumigatus germination process, conidial glucan beads (22). TLR9 recruitment to b-1,3 glucan bead swelling increases surface exposure of fungal b-1,3 glucan (34). phagosomes was impaired in RAW cells expressing TLR9-GFP We have previously shown that the signal required for TLR9 re- when pretreated with BafA1 (Fig. 3A, top panel). In sharp con- cruitment appears to be continuously present throughout the dif- trast, TLR9 retained to b-1,3 glucan phagosomes when treated ferent spore stages of A. fumigatus (12). To test the dependence of with a vehicle control (DMSO) (Fig. 3A, bottom panel). To con- Dectin-1 in TLR9 recruitment to different A. fumigatus spore firm further this finding biochemically, we isolated b-1,3 glucan stages, we incubated D1KO macrophages expressing TLR9-mCherry beads containing phagosomes from RAW cells expressing TLR9- Downloaded from with live A. fumigatus resting conidia for 40 min. Despite phagocy- GFP that were treated with either BafA1 or vehicle control. tosis, we observed no TLR9 recruitment to A. fumigatus conidia Phagosomes from vehicle-treated cells retained cleaved TLR9- (Fig. 2B, top panel). In sharp contrast, reconstituted Dectin-1–defi- GFP at 3 h, whereas TLR9-GFP cells pretreated with BafA1 cient macrophages incubated with A. fumigatus resting conidia showed no TLR9-GFP (Fig. 3B). As a control, phagosome- showed recruitment of TLR9-mCherry to fungal phagosomes containing CpG+IgG beads from TLR9-GFP macrophages pre-

(Fig. 2B, bottom panel). Furthermore, metabolically active treated with BafA1 showed no TLR9, as expected (Fig. 3B). http://www.jimmunol.org/ conidia do not elicit a robust inflammatory response, because the To assess a larger number of phagosomes simultaneously, we surface hydrophobin and carbohydrate layers on the conidia pretreated TLR9-GFP–expressing macrophages with BafA1 and mask pathogen-associated molecular patterns including b-1,3 DMSO for 15 min followed by incubation with b-1,3 glucan glucan (34–36). Our previous study shows that TLR9 properly beads for 3 h. When isolated phagosomes were analyzed by recruited and did not distinguish between different A. fumigatus flow cytometry, we observed trace green fluorescence on b-1,3 spore stages (12). We examined the role of Dectin-1 in TLR9 glucan–containing phagosomes from cells pretreated with BafA1 recruitment to HK resting conidia. The process of heat killing (Fig. 3C). In contrast, phagosomes from macrophages exposed to halts spore germination and disrupts the fungal cell wall, thereby DMSO demonstrated robust green fluorescence, indicating potent increasing b-1,3 glucan exposure (37). After incubating D1KO recruitment of TLR9-GFP. Our results demonstrate that phag- by guest on September 26, 2021 macrophages expressing TLR9-mCherry with HK resting con- osomal acidification is required for retention of TLR9 to b-1,3 idia for 40 min, we observed no recruitment of TLR9-mCherry glucan phagosomes. around fungal phagosomes (Fig. 2C, top panel). In contrast, in TLR9 recruitment to b-1,3 glucan phagosomes requires Syk reconstituted D1KO macrophages expressing TLR9-mCherry, signaling HK A. fumigatus containing phagosomes recruited TLR9- mCherry (Fig. 2C, bottom panel). Syk-coupled pattern-recognition receptors collaborate with Next, we induced conidial swelling, which also unmasks b-1,3 Myd88-coupled TLRs to induce enhanced cytokine production glucan (34). Cells were incubated with live A. fumigatus swollen (39). Blockade of Syk activation arrests b-1,3 glucan–containing conidia for 40 min and TLR9 recruitment was observed. TLR9- phagosomes at an early endosomal stage (22). We hypothesized mCherry recruitment was impaired in the absence of Dectin-1, that Syk activation is required for successful TLR9 rearrangement whereas reconstituted D1KO macrophages recruited TLR9-mCherry to b-1,3 glucan phagosomes. To investigate the role of Syk acti- to swollen conidial phagosomes (Fig. 2D). Taken together, our re- vation in TLR9 trafficking, we pretreated RAW TLR9-GFP sults indicate Dectin-1 is required for TLR9 recruitment to phagosomes macrophages with a Syk inhibitor (R406) or vehicle control for containing C. albicans and A. fumigatus, independent of spore stages. 30 min and incubated these cells with b-1,3 glucan beads for 3 h. R406 treatment had no effect on phagocytosis of beads as deter- Phagosomal acidification is required for TLR9 recruitment to mined by microscopy (data not shown). Despite adequate ex- b -1,3 glucan phagosomes pression of the GFP, TLR9-GFP recruitment to phagosomes Compartmentalized proteolysis of TLR9 is an essential step for containing b-1,3 glucan beads was impaired in R406-pretreated nucleic acid recognition (8). The full-length form of TLR9 must cells. In contrast, TLR9-GFP successfully translocated to b-1,3

FIGURE 1. TLR9 is specifically recruited to b-1,3 glucan phagosomes. (A) Confocal microscopy of RAW macrophages expressing TLR9-GFP (green) incubated with Alexa Fluor 647–labeled b-1,3 glucan beads (blue) and Alexa Fluor 568–labeled polystyrene beads (red, top panel), or A. fumigatus-RFP (red, middle panel), or Alexa Fluor 568–labeled CpG-IgG beads (red, bottom panel) for 30 min. Scale bars, 5 mm. Original magnification 360 or 3100. (B) Purified phagosomes containing either b-1,3 glucan beads or CpG-IgG beads were assessed for TLR9-GFP recruitment (black line) by phagoFACS and compared with polystyrene beads control (gray shaded histogram). (C) Kinetics of TLR9-GFP recruitment (black line) to purified b-1,3 glucan–containing phagosomes were assessed by phagoFACS for the indicated times and compared with polystyrene bead-only control (gray shaded histogram). b-1,3 Glucan beads were added to cell lysates assessed for TLR9-GFP recruitment. (D) b-1,3 Glucan beads and CpG-IgG beads were incubated with 2 3 106 RAW TLR9-GFP cells for 3 h. Lysate control and 1 3 106 purified phagosomes were analyzed by immunoblot and blotted for GFP. Arrows indicate full-length- TLR9GFP (FL-TLR9-GFP) and cleaved TLR9-GFP as labeled. Data are representative of five independent experiments. 6 DECTIN-1 CONTROLS TLR9 RECRUITMENT

glucan beads in macrophages exposed to the vehicle control (Fig. 4A). To confirm our findings by microscopy, we exposed RAW-TLR9-GFP cells to either b-1,3 glucan beads or CpG+IgG beads for 3 h and performed biochemical analysis on isolated phagosomes to assess thousands of phagosomes simultaneously. Given that Fc receptors use Syk to initiate downstream signaling events (20), we used CpG-IgG beads as a control for Fc-dependent Syk activation. Whereas Syk inhibition blocks IgG/Fc receptor phagocytosis, granulin facilitates the uptake and delivery of CpG to TLR9+ compartments (40). Indeed, we observed uptake of CpG beads by macrophages treated with a Syk inhibitor. Phagosomal proteins resolved by SDS-PAGE and probed for GFP showed cleaved TLR9-GFP for CpG+IgG and b-1,3 glucan bead-containing phagosomes from vehicle-treated cells, as expected (Fig. 4B). b-1,3 Glucan bead-containing phagosomes from R406 pretreated cells showed no TLR9-GFP, whereas recruitment of TLR9-GFP to CpG+IgG phagosomes was present, although impaired (Fig. 4B). We confirmed these findings by flow cytometry and observed no GFP

signal (TLR9-GFP) to b-1,3 glucan beads in the presence of Downloaded from R406, whereas vehicle pretreatment showed TLR9 recruitment as expected (Fig. 4C). Our results indicate that Syk signaling is required for TLR9 recruitment to b-1,3 glucan phagosomes. Dectin-1–dependent Syk activation is required for TLR9 recruitment to b-1,3 glucan and fungal-containing phagosomes http://www.jimmunol.org/ To eliminate any off-target effects of chemical inhibitors, we used a molecular approach to demonstrate a direct role of Dectin-1– dependent signaling through Syk phosphorylation for TLR9 recruitment to b-1,3 glucan–containing phagosomes. To study TLR9 intracellular trafﬁcking, we stably transduced D1KO macrophages with TLR9-mCherry and GFP–Dectin-1 or GFP–Dectin- 1DY15, a signaling-incompetent mutant of Dectin-1 that ablates

Syk-mediated downstream signaling (19, 22). We then stimulated by guest on September 26, 2021 these cells with b-1,3 glucan beads for 2 h. As expected, we observed loss of phagosomal-associated GFP–Dectin-1 within 2 h of stimulation (22), but TLR9-mCherry was retained on b-1,3 glucan–containing phagosomes (Fig. 5A, top panel). In contrast, recruitment of TLR9-mCherry was impaired but not completely absent on b-1,3 glucan bead-containing phagosomes in the presence of GFP–Dectin-1DY15 (Fig. 5A, bottom panel). In the absence of Dectin-1–dependent Syk signaling, phagosomal maturation and acidification of b-1,3 glucan is blocked (22). Our data consistently show that the form of TLR9 recruited to b-1,3 glucan beads phagosomes is cleaved. To assess the size of the TLR9-mCherry on b-1,3 glucan beads in the presence of GFP–Dectin-1DY15, we performed immunoblot analysis on isolated phagosomes. Our data show that cleaved TLR9-mCherry was recruited as early as 1 h, suggesting TLR9 N-terminal proteolytic cleavage occurs either before or immediately after its trafficking to b-1,3 glucan bead-containing phagosomes (data not shown). To demonstrate that the requirement of Dectin-1–dependent Syk activation for TLR9 recruitment is specific for b-1,3 glucan, we incubated macrophages with CpG+IgG beads for 2 h and showed TLR9 recruitment in both GFP–Dectin-1– and GFP– Dectin-1DY15–expressing macrophages (Fig. 5B). We then fed CpG+IgG and b-1,3 glucan beads to GFP–Dectin-1DY15– and TLR9-mCherry–expressing macrophages and observed TLR9- FIGURE 2. TLR9 trafficking to fungal phagosomes requires Dectin-1. (A) mCherry recruitment to CpG+IgG beads. Specifically, TLR9- Confocal microscopy of Dectin-1 knockout macrophages expressing TLR9- b mcherry (red) in the absence (top panel)orpresence(bottom panel)of mCherry recruited to -1,3 glucan bead in the same cell Dectin-1. Cells were incubated with HK C. albicans (A), live A. fumigatus (Supplemental Fig. 3). We extended our observation of Dectin-1– resting conidia (B), HK A. fumigatus resting conidia (C), or live A. fumigatus dependent Syk signaling of TLR9 recruitment to fungal organ- swollen conidia (D) for 40 min. Scale bars, 5 mm. Original magnification isms. We incubated GFP–Dectin-1 and TLR9-mCherry–coex- 3100. (Top panels) Data are representative of five independent experiments. pressing D1KO macrophages with HK A. fumigatus for 40 min The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. TLR9 recruitment to b-1,3 glucan phagosomes requires acidification. (A) RAW TLR9-GFP cells pretreated with BafA1 or vehicle control for 15 min and incubated with either b-1,3 glucan beads and CpG-IgG beads for 3 h. TLR9-GFP recruitment to b-1,3 glucan phagosomes was assessed by confocal microscopy. Original magnification 360. Scale bars, 5mm. (B) Purified phagosomes and lysate control were analyzed by immunoblot and blotted for TLR9-GFP. Lysate controls indicate FL-TLR9-GFP and cleaved TLR9-GFP. (C) TLR9-GFP recruitment (black line) to purified b-1,3 glucan phagosomes in the presence of BafA1 or vehicle control was assessed by phagoFACS and compared with bead-only control (gray shaded histogram). Data are representative of three independent experiments. and observed robust TLR9-mCherry recruitment at the fungal tion, whereas TLR9-mCherry failed to traffic on fungal phag- phagosomal surface with weak GFP–Dectin-1 signal (Fig. 5C, top osomal compartments (Fig. 5D, bottom panel). Our results panel). In the TLR9-mCherry–expressing GFP–Dectin-1DY15 indicate Dectin-1–dependent Syk activation is critical for trig- macrophages, HK conidia failed to recruit TLR9-mCherry with gering TLR9-mCherry accumulation on b-1,3 glucan bead and moderate levels of GFP–Dectin-1DY15 around the fungal phag- fungal-containing phagosomes. osome (Fig. 5C, bottom panel). Next, to demonstrate this observation is not only specific to A. fumigatus, we exposed HK Dectin-1 regulates TLR9-dependent gene expression upon b C. albicans to the D1KO macrophages expressing TLR9-mCherry stimulation by -1,3 glucan beads and GFP–Dectin-1, and observed robust TLR9-mCherry recruit- Our data indicate that Dectin-1 controls the recruitment of TLR9 ment to phagosomes (Fig. 5D, top panel). In agreement with to phagosomes containing b-1,3 glucan. To establish whether previous findings, we observed normal decay of GFP–Dectin-1 this recruitment is functionally relevant, we sought to determine signal from the phagosomal membrane of C. albicans within whether the absence of TLR9 affects the transcriptome in b-1,3 20 min of phagocytosis (22). In contrast, GFP–Dectin-1DY15 glucan–stimulated macrophages. We stimulated wild-type and retained to HK C. albicans phagosomes after 40 min of incuba- TLR9KO immortalized macrophages with b-1,3 glucan beads for 8 DECTIN-1 CONTROLS TLR9 RECRUITMENT

FIGURE 4. TLR9 recruitment to b-1,3 glucan phagosomes requires Syk activation. (A–C) RAW macrophages expressing TLR9-GFP were pretreated with R406 or vehicle control for 30 min. Cells were stimulated with either b-1,3 glucan beads or CpG+IgG beads for 3 h. (A) Confocal microscopy of RAW TLR9-GFP recruitment to b-1,3 glucan phagosomes in the presence Downloaded from of R406 or vehicle control. Original magniﬁcation 3100. Scale bar, 5 mm. (B) TLR9-GFP recruitment to puriﬁed b-1,3 glucan or CpG-IgG phagosomes in the presence of R406 or vehicle were analyzed by immu-

noblot. Lysate control of indicated http://www.jimmunol.org/ cell type was used to indicate FL- TLR9-GFP and cleaved TLR9-GFP. (C) TLR9-GFP recruitment (black line) to puriﬁed b-1,3 glucan phagosomes in the presence of R406 or vehicle control was assessed by phagoFACS and compared with bead-only control (gray shaded histogram). Data are representative of three independent experiments. by guest on September 26, 2021

4 h, harvested RNA, and conducted a microarray analysis to identified two requirements for redistribution of TLR9 to these identify differentially expressed genes using the cutoff 1.8-fold phagosomes: phagosomal acidification and Dectin-1–dependent change in expression levels. Gene expression values from unsti- Syk activation. Finally, we show that TLR9 modulates gene ex- mulated wild-type and TLR9KO macrophages were used to de- pression in a Dectin-1–dependent manner, suggesting a functional termine the baseline differences in expression levels. Analysis of role for TLR9 in regulating the immune response to b-1,3 glucan. microarray data revealed 32 differentially expressed genes that are In this study, we demonstrate that TLR9 recruitment to phagosomes dependent on TLR9 in b-1,3 glucan–stimulated macrophages containing b-1,3 glucan is controlled by Dectin-1. Furthermore, our (Fig. 6, Table I). Of the 32 differentially expressed genes, the IFN- data indicate that b-1,3 glucan in phagosomes is sufficient for TLR9 inducible (IFI) family genes, IFI203, Mnda, and Ifih1 (MDA5), recruitment. Our experiments rely on the exclusive use of TLR9-GFP and Trim30a are directly implicated in the regulation of immune to report on the activity of TLR9. It remains possible that TLR9-GFP signaling (41–44). Trim30a is also a negative regulator of TLR9 may not faithfully replicate all features of endogenous TLR9. Unlike signaling (44). Together, these data indicate Dectin-1 modulates Dectin-1, retention of TLR9 on the phagosomes is long-lived and TLR9-dependent gene expression in response to b-1,3 glucan remains present for .16 h. To our knowledge, this observation is the beads in macrophages (Fig. 7). first to demonstrate control of TLR trafficking by a CLR. The redistribution of TLR9 to phagosomes requires phagosomal Discussion acidification and Dectin-1–dependent Syk activation. Lysosomal Our data support a model in which phagosomes containing b-1,3 cysteine protease, cathepsin L, and cathepsin S are required for glucan are recognized by Dectin-1 and trigger Syk activation, TLR9 cleavage (8). Inhibition of Dectin-1–dependent phagosomal which leads to TLR9 recruitment and retention of the proteolyti- acidification (22) may also block activation of lysosomal cathep- cally cleaved TLR9 to the phagosome (Fig. 7). In addition, we sins, which, in turn, inhibit cleavage of TLR9 (8, 9). Thus, it is The Journal of Immunology 9 Downloaded from

FIGURE 5. Dectin-1–dependent Syk acti- http://www.jimmunol.org/ vation is required for TLR9 recruitment to b-1,3 glucan and fungal phagosomes. (A–D) Confocal microscopy of D1KO macrophages expressing TLR9-mCherry and GFP–Dectin-1 or GFP–Dectin-1DY15 incubated with b-1,3 glucan beads for 3 h (A), CpG+IgG beads for 2h(B), HK A. fumigatus (C), or HK C. albicans (D). Original magniﬁcation 360 and 3100. Scale bars, 5 mm. Data are representative of ﬁve independent experiments. by guest on September 26, 2021

formally possible that TLR9 may still be recruited to the phag- transduction machinery and subcellular distribution of signaling osome, but the failure to undergo cleavage may prevent its re- components (4, 5). Considerably less is known about mechanisms tention to b-1,3-glucan–containing phagosomes. that regulate TLR signaling from endosomes. Defects in these Receptor signaling by TLRs at the plasma membrane is regulated mechanisms can have profound effects including increased sus- precisely by mechanisms that control receptor coupling to signal ceptibility to infections or immunodeﬁciency (45). Unc93B1, an 10 DECTIN-1 CONTROLS TLR9 RECRUITMENT

FIGURE 6. Dectin-1 regulates

TLR9-dependent gene expression. Downloaded from (A and B) Microarray data analysis of TLR9KO and wild-type B6 immortalized macrophages in response to b-1,3 glucan beads. (A) A scat- terplot indicating a ratio of fold- changes in gene expression between wild-type and TLR9KO macrophages. http://www.jimmunol.org/ Off-diagonal dots (red) indicate differentially regulated, TLR9-dependent genes that are fold-change .2.0 between macrophages stimulated with b-1,3 glucan beads and unstimulated. (B) A heat map of the 32 genes differentially expressed (fold change .1.8) between TLR9KO and wild- type macrophages in response to b-1,3 glucan beads. by guest on September 26, 2021

ER resident protein, controls the egress of TLR9 and other endo- concept that Dectin-1 induces an immunomodulatory pathway has somal TLRs from the ER to the endosomal compartments (46, 47). been previously established. SOCS-1 is induced by Dectin-1 in TLR9 must be localized in AP-3+ compartments to become fully mouse bone marrow–derived dendritic cells and macrophages, which competent in IFN signaling (48). Proteolytically cleaved TLR9 re- leads to decreased and abbreviated NF-kB activation in DCs and cruits MyD88 in response to CpG (8). It remains to be determined macrophages triggered by TLR9 (51). It is noteworthy that IL-12 and whether TLR9 that is recruited to b-1,3-glucan phagosomes assem- IL-10 secretion were inhibited by SOCS-1. The precise role of bles a functional myddosome for signaling. TLR9 signaling by SOCS-1 in this model of fungal infection remains to be determined. A. fumigatus phagosomes can bypass MydD88 (49). Therefore, it is In this study, we extend the role of Dectin-1 in triggering in- possible that Dectin-1–dependent Syk activation modulates TLR9 flammatory responses to regulating recruitment of TLR9 to fungal signaling independent of myddosome formation. phagosomes. We have previously shown that TLR9 serves to Dectin-1 signaling provides a powerful activating signal and triggers modulate inflammatory response (13). Thus, the Dectin-1–TLR9 potent inflammatory cytokines in response to b-1,3 glucan (50). The axis may represent a feedback loop to control the activation of The Journal of Immunology 11

Table I. List of differentially expressed genes in TLR9KO and wild-type immortalized macrophages

Gene Symbol Gene Description Delta_LogFC Gene Function Olfml3 Olfactomedin-like 3 1.42 Multicellular organismal development Rnf213 Ring finger protein 213 1.30 Ubiquitin-transferase activity Pde7b Phosphodiesterase 7B 1.27 Signal transduction Hmgn5 High-mobility group nucleosome binding 1.20 Regulation of transcription domain 5 Nt5e 59 nucleotidase, ecto 1.17 Negative regulator of inflammatory response Gm8989 Predicted gene 8989 1.14 Unknown function Trim30a Tripartite motif-containing 30A 1.09 Negative regulator of immune signaling Mnda Myeloid cell nuclear differentiation Ag 1.09 Type I IFN induced genes Cd72 CD72 Ag 1.07 Carbohydrate binding Jag1 Jagged 1 1.01 Cell differentiation Ifi203 IFN-induced gene 203 0.99 Type I IFN induced genes Phf11d PHD finger protein 11D 0.99 Regulation of transcription Zfp125 Zinc finger protein 125 0.98 Unknown function Phf11a PHD finger protein 11A 0.97 Regulation of immune response Nat6 N-acetyltransferase 6 0.96 Transferase activity mt-Tt Mitochondrially encoded tRNA threonine 0.95 Translation Fam49a Family with sequence similarity 49, member A 0.93 Unknown function Ms4a6b Membrane-spanning 4-domains, subfamily 0.92 Unknown function Downloaded from A, member 6B Mical2 Microtubule-associated monooxygenase, 0.89 Actin filament depolarization calponin and LIM domain containing 2 Slc44a1 Solute carrier family 44, member 1 0.88 Choline transporter Lphn2 Latrophilin-2 0.88 Unknown function Gm8995 Predicted gene 8995 0.87 Unknown function S1pr1 Sphingosine-1-phosphate receptor 1 0.85 Cell adhesion http://www.jimmunol.org/ Pstpip2 Proline-serine-threonine phosphatase- 0.84 Actin binding interacting protein 2 Gm22866 Gm22866 0.84 Unknown function Ifih1a IFN induced with helicase C domain 1 0.84 Type I IFN induced genes Ptplad2 Protein tyrosine phosphatase-like A domain 0.84 Fatty acid elongation containing 2 Anp32-ps Acidic nuclear phosphoprotein 32 family, 0.84 Regulation of transcription member A Ddx26b DEAD/H (Asp-Glu-Ala-Asp/His) box 0.84 Unknown function polypeptide 26B

Gm11545 Predicted gene 11545 0.83 Unknown function by guest on September 26, 2021 Maf Avian musculoaponeurotic fibrosarcoma 0.82 Regulation of transcription (v-maf) AS42 oncogene homolog Hpgd HydroxyPG dehydrogenase 15 (NAD) 0.80 Oxidoreductase activity List of TLR9-dependent genes in response to b-1,3 glucan beads is determined by microarray gene analysis. Transcripts with fold-change .2.0 between TLR9KO and wild-type B6 macrophages with p , 0.001 were identified. Among these genes, 32 transcripts with fold-change .1.8 between macrophages stimulated with b-1,3 glucan beads and without stimulation were considered differentially regulated between TLR9KO and wild-type B6 macrophages. aIfih1 encodes for MDA5 previously described as host defense against Candida infections.

innate immune cells. Upon ligation with a fungal pathogen that subset of 32 genes that are differentially expressed in TLR9KO contains b-1,3 glucan, Dectin-1 activates Src, which, in turn, macrophages compared with wild-type macrophages when stim- activates Syk (19). Card9 activation and ROS production are ulated with b-1,3 glucan beads (Fig. 6, Table I). Because Dectin-1 downstream events from Dectin-1–dependent Syk activation that is the receptor for b-1,3 glucan, these data demonstrate that TLR9 direct the elaboration of inflammatory cytokines (52). Dectin-1– modulates gene expression in a Dectin-1–dependent manner and dependent Syk activation triggers acidification of the phagosome suggest a functional role for TLR9 in regulating the immune re- and permits trafficking and retention of TLR9 to these compart- sponse to fungal pathogens. The effects of b-1,3 glucan on TLR9- ments. TLR9 signaling thus serves to modulate the inflammatory dependent gene expression may be direct or indirect and do not response by downregulating cytokine production (13). Topologi- suggest TLR9 is directly binding b-1,3 glucan. Comparison of our cally, b-1,3 glucan is closer to the surface of fungal cell walls differentially expressed genes to previously published data prob- when compared with chitin (34). After sufficient exposure within ing macrophages with CpG (54) identified four common genes, the phagolysosome, deeper structures of the fungi such as chitin suggesting that TLR9 signaling in response to b-1,3 glucan beads may be exposed or fungal DNA may be released. Our data in- shares minor overlap with CpG-induced changes in gene expres- dicate that the N-terminal cleaved TLR9 is the predominant form sion. Genes that are differentially regulated in response to CpG found on these phagosomes, which is capable of signaling. Al- would not necessarily be influenced by b-1,3 glucan because CpG though the precise identity of the TLR9 ligand is not known on is a direct TLR9 ligand. Our study identified TLR9-dependent fungal organisms, it is interesting to note that the recognition changes in gene expression that are specifically regulated by of fungal chitin appears to trigger IL-10 production in a TLR9- Dectin-1 (Fig. 6, Table I). dependent manner (53), thereby providing the final step in this Along with CLRs, TLRs orchestrate antifungal innate immune feedback loop. responses and generate an effective host defense against clinically Importantly, recruitment of TLR9 to phagosomes is not relevant fungal pathogens (6, 29). Bacterial and viral DNA con- equivalent to TLR9 signaling. Microarray analysis revealed a taining unmethylated CpG motifs are well-known ligands of 12 DECTIN-1 CONTROLS TLR9 RECRUITMENT Downloaded from

FIGURE 7. Schematic representation of Dectin-1–dependent TLR9 recruitment to fungal- and b-1,3 glucan–containing phagosomes. (A) Recognition and ligation of Dectin-1 with A. fumigatus, C. albicans,orb-1,3 glucan bead results in phagocytosis and Syk activation triggering the recruitment of TLR9 to the phagosomes and retention of the proteolytically cleaved version of TLR9. (B) Signaling-incompetent Dectin-1DY15 can mediate phagocytosis of fungi or b-1,3 glucan beads but is incapable of activating Syk, resulting in failed TLR9 recruitment to the phagosome. http://www.jimmunol.org/ TLR9. The specificity of TLR9 is not only restricted to microbial TLR9 in the cellular and molecular mechanisms that govern the DNA but can also directly bind malarial hemozoin, resulting in innate immune response to fungal pathogens. TLR9 conformational changes (55). For fungal organisms, TLR9 can be triggered by fungal DNA (7, 56, 57). TLR9 can recognize Acknowledgments fungal species of various groups including C. albicans, A. fumi- We thank Dr. Gordon Brown, Dr. Stuart Levitz, Dr. Eleftherios Mylonakis, gatus, Cryptococcus neoformans, Saccharomyces cerevisiae, and Dr. Michelle Momany for reagents and Arch Macinnes for assistance Paracoccidioides brasiliensis, and Malassezia furfur (12, 13, 56). with the artwork. We also thank Fei Ji and Ruslan Sadreyev from the De- TLR9 plays an essential and protective role in P. brasiliensis in- partment of Molecular Biology at Massachusetts General Hospital for per- fection as TLR92/2 mice succumb to death, yet had higher TNF-a forming the microarray analysis. by guest on September 26, 2021 and IL-6 expression (56). In contrast, TLR92/2 mice had a survival advantage compared with wild-type mice when challenged Disclosures with C. albicans and A. fumigatus (6). Also, our previous study The authors have no financial conflicts of interest. demonstrated TLR9 deficiency increases macrophage fungicidal activity and enhanced antifungal effector response against C. References albicans and S. cerevisiae (13). Thus, the precise role of TLR9 in 1. Miceli, M. H., J. A. Dıáz, and S. A. Lee. 2011. Emerging opportunistic yeast the host defense against invasive fungal infections is not com- infections. Lancet Infect. Dis. 11: 142–151. 2. Lanternier, F., S. Cypowyj, C. Picard, J. Bustamante, O. Lortholary, pletely understood, but it appears to be distinct and pathogen J. L. Casanova, and A. Puel. 2013. Primary immunodeficiencies underlying dependent. fungal infections. Curr. Opin. Pediatr. 25: 736–747. Interestingly, we identified several immunity-related genes that 3. Bozza, S., C. Clavaud, G. Giovannini, T. Fontaine, A. Beauvais, J. Sarfati, C. D’Angelo, K. Perruccio, P. Bonifazi, S. Zagarella, et al. 2009. Immune were upregulated in TLR9KO macrophages when stimulated with sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and b-1,3 glucan beads (Fig. 6, Table I). For example, the IFI genes the impact on Th immunity and vaccination. J. Immunol. 183: 2407–2414. 4. Barton, G. M., J. C. Kagan, and R. Medzhitov. 2006. Intracellular localization of (Ifih1, IFI203, and Mnda) play a role in inflammation (41, 42, 58). Toll-like receptor 9 prevents recognition of self DNA but facilitates access to Ifih1, which encodes MDA5, an RIG-I–like receptor, directly viral DNA. Nat. Immunol. 7: 49–56. participates in antifungal host defense (41). Type I IFN signaling 5. Barton, G. M., and J. C. Kagan. 2009. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nat. Rev. Immunol. 9: 535–542. confers a protective role in mice with C. albicans infection (59, 6. Bellocchio, S., C. Montagnoli, S. Bozza, R. Gaziano, G. Rossi, S. S. Mambula, 60). Therefore, upregulation of IFI genes in the absence of TLR9 A. Vecchi, A. Mantovani, S. M. Levitz, and L. Romani. 2004. The contribution could provide a possible explanation for why mice that lack TLR9 of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J. Immunol. 172: 3059–3069. are more resistant to C. albicans and A. fumigatus infections (6). 7. Ramirez-Ortiz, Z. G., C. A. Specht, J. P. Wang, C. K. Lee, D. C. Bartholomeu, IFI family proteins also recognize cytoplasmic dsDNA and reg- R. T. Gazzinelli, and S. M. Levitz. 2008. Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA. Infect. ulate gene expression (61). Therefore, we speculate that IFI genes Immun. 76: 2123–2129. could be upregulated in the absence of TLR9 to participate in 8. Ewald, S. E., B. L. Lee, L. Lau, K. E. Wickliffe, G. P. Shi, H. A. Chapman, and nucleic acid sensing pathways. G. M. Barton. 2008. 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