MAJOR ARTICLE
Dectin-1–Dependent LC3 Recruitment to Phagosomes Enhances Fungicidal Activity in Macrophages Jenny M. Tam,1,6 Michael K. Mansour,1,6 Nida S. Khan,1 Michael Seward,1 Sravanthi Puranam,1 Antoine Tanne,8 Anna Sokolovska,2 Christine E. Becker,3,4,5,7 Mridu Acharya,11 Michelle A. Baird,12 Augustine M. K. Choi,9 Michael W. Davidson,12 Brahm H. Segal,10 Adam Lacy-Hulbert,11 Lynda M. Stuart,11 Ramnik J. Xavier,3,4,5,7 and Jatin M. Vyas1,6 1
Department of Medicine, Division of Infectious Diseases, 2Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital, Gastrointestinal Unit, 4Center for the Study of Inflammatory Bowel Disease, 5Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 6Department of Medicine, Harvard Medical School, Boston, and 7Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge; 8Icahn School of Medicine at Mt. Sinai, Tisch Cancer Institute, 9Weill Cornell Medical College, and 10 Roswell Park Cancer Institute, University of Buffalo School of Medicine, New York; 11Benaroya Research Institute, Seattle, Washington; and 12National High Magnetic Field Laboratory, Florida State University, Tallahassee 3
Keywords. dectin-1; autophagy; LC3; Candida albicans; ROS; NADPH oxidase. Invasive candidiasis is a leading cause of death among immunocompromised patients and has a crude mortality rate of >50% [1]. Candida albicans is recognized by phagocytes (eg, macrophages, neutrophils, and dendritic cells) through pattern-recognition receptors, including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), which recognize highly conserved microbial epitopes called pathogen-associated molecular patterns (PAMPs) on the fungal cell wall. A major PAMP of C. albicans is β-1,3-glucan [2], and this
Received 20 December 2013; accepted 30 April 2014; electronically published 19 May 2014. Presented in part: American Association of Immunologists Annual Meeting, Boston, Massachusetts, May 2012. Correspondence: Jatin M. Vyas, MD, PhD, 55 Fruit St, GRJ-5-504, Boston, MA 02114 (
[email protected]). The Journal of Infectious Diseases® 2014;210:1844–54 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected]. DOI: 10.1093/infdis/jiu290
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carbohydrate is recognized by dectin-1, a CLR highly expressed on the surface of phagocytes [3]. Dectin-1 is required for proper modulation of immune responses, and patients with mutations in dectin-1 are at higher risk for fungal infections [4, 5] and autoimmune colitis driven by C. albicans [6]. The cytoplasmic tail of dectin-1 contains an immunoreceptor tyrosine-based activation (ITAM)–like motif [7]. Upon ligation of the extracellular domain of dectin-1, the tyrosine residue within the cytoplasmic ITAM motif is phosphorylated [8]. This event results in recruitment of spleen tyrosine kinase (Syk) [9] and, ultimately, leads to production of reactive oxygen species (ROS), activation of NF-ĸB, and induction of proinflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) [10]. Autophagy has an important role in cell homeostasis, but in the context of innate immunity, it targets intracellular pathogens for engulfment within doublemembrane autophagosomes for lysosomal degradation [11, 12]. Although the double membrane is a distinctive
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Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.
XL site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) with the following primers: 5′- GAG AAT CTG GAT GAA GAT GGA TTT ACT CAA TTA GAC TTC AGC AC -3′ (forward) and 5′- GTG CTG AAG TCT AAT TGA GTA AAT CCA TCT TCA TCC AGA TTC TC -3′ (reverse). Lentiviral Transduction and Plasmids
GFP-LC3 [17] and red fluorescent protein LC3 (RFP-LC3) [18] were obtained from Addgene (item nos. 22 405 and 21 075, respectively; Cambridge, MA). GFP–dectin-1, GFP–dectin1ΔY15, GFP-LC3, or RFP-LC3 was subcloned into pHAGE2 [19]. Lentivirus was generated as described elsewhere [20, 21]. Confocal Microscopy
MATERIALS AND METHODS
Macrophages were plated onto 8-chambered coverslips (LabTek, ThermoScientific, Rochester, NY) 1 day prior to imaging. Cells were incubated with fluorophore-conjugated BG particles for 30 minutes at 37°C. Coverslips were mounted on a Nikon Ti-E inverted microscope equipped with a CSU-X1 confocal spinning-disk head (Yokogawa, Sugar Land, TX) for live cell microscopy. A Coherent 4 W continuous-wave laser (Coherent, Santa Clara, CA) excited the sample. A high-magnification, high-numerical aperture objective (Nikon, 100X, 1.49 numerical aperture, oil immersion) was used. Images were obtained using an EM-CCD camera (Hamamatsu, C9100-13). Image acquisition was performed using MetaMorph software (Molecular Devices, Downingtown, PA). Images were then cropped using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).
Mice, Cell Lines, and Cell Culture
Phagosome Isolation
Mice were maintained in specific-pathogen-free facilities at Massachusetts General Hospital (MGH; Boston, MA). All animal studies were conducted under protocols approved by the Subcommittee on Research Animal Care at MGH. LC3β−/− and gp91−/− mice were purchased from Jackson Laboratories; p40phox−/− (NCF4−/−) mice were provided by Phillip T. Hawkins (Babraham Institute, Cambridge, United Kingdom); MyD88/TRIF-deficient mice were a gift from Douglas Golenbock (University of Massachusetts Medical School, Worcester, MA). The following cell lines were used for described experiments: RAW 264.7 (RAW), HEK293T, immortalized bone-marrowderived macrophages (BMDMs) and dectin-1–deficient macrophages. Further details of reagents, cell culture, bone marrow harvest, and C. albicans culture can be found in the Supplementary Materials.
Phagosome isolation was adopted from previously described protocols [22]. A total of 1 × 107 macrophages were cultured in 10cm plates and were stimulated with BG particles (E:T = 25) for 45 minutes, followed by addition of hypotonic lysis buffer (2 mM MgCl2, 6 mM β-mercaptoethanol, and 10 mM HEPES) with a protease inhibitor cocktail (Roche, Indianapolis, IN). Cells were then mechanically sheared by aspirating and ejecting the cell suspension through a 1-cm3 syringe fitted with a 1.3-cm, 26-gauge needle for 15 cycles. Hypotonic buffer was adjusted to isotonicity by adding 62% (w/v) sucrose solution to a final concentration of 40%. A discontinuous gradient was then constructed in highspeed ultracentrifugation tubes (Beckman Polyallomer, Brea, CA) overlayering 2 mL of 62%, lysed cell suspension at 40% (sample), 30%, 25%, and 10%. Sucrose gradients were then subjected to ultracentrifugation at 80 000 ×g for 1 hour at 4°C without brake (Beckman, SW-28 rotor, L8-M ultracentrifuge, Brea, CA). Phagosomes were isolated at the interface of the 25%/10% sucrose layers and washed in phosphate-buffered saline (PBS).
Generation of Green Fluorescent Protein (GFP)–Dectin-1ΔY15 (Syk Kinase-Signaling Mutant)
A vector encoding dectin-1 fused at the N-terminus with GFP ( pMax backbone) was a gift of Dr David Underhill (CedarsSinai Medical Center, West Hollywood, CA). A tyrosine to phenylalanine substitution at amino acid position 15 (within the intracytoplasmic domain) was introduced using QuikChange
LC3 Immunoblot
Macrophages were stimulated with either BG beads, heat-killed C. albicans, or live C. albicans (E:T = 25). Inhibitors were added 1 hour before stimulation and remained in culture for Dectin-1 Controls LC3 Recruitment
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feature of autophagy, LC3 also translocates to single phagosomal membranes containing the yeast cell wall fragment zymosan, along with TLR2 [13]. Since rearrangement of LC3 to the phagosome was not associated with the formation of doublemembrane structures, this phenomenon may be distinct from conventional autophagy pathways. Indeed, proteomic analysis of polystyrene-bead-containing phagosomes identified LC3 as linked to phagocytosis [14]. Although LC3 resides on phagosomal membranes containing bacteria [15], the role of autophagy proteins on fungal phagosomes remains poorly understood. We sought to determine whether the presence of LC3 affects macrophages’ fungicidal activity and to dissect the molecular mechanisms that regulate LC3 trafficking to the phagosomal membrane. Specifically, we show that the presence of LC3 affects both killing of C. albicans and pathogen-induced expression of proinflammatory cytokines in primary macrophages. We demonstrate that dectin-1, through Syk activation, controls LC3 recruitment to fungal phagosomes, using live, heat-killed, and chemically defined “fungal-like particles”, which consist of β-1,3-glucan covalently conjugated to polystyrene beads (BG beads) [16]. We identify ROS production by the phagosomal NADPH oxidase as a key signal for LC3 recruitment. These data support the model that dectin-1 controls recruitment of LC3 to the fungal phagosome, and that this rearrangement enhances fungicidal activity by macrophages.
the duration of the experiments. Whole-cell lysates were made in SDS sample buffer (Invitrogen), heated at 95°C for 10 minutes, and loaded onto precast 12% sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (BioRad), blocked with 5% milk for 1 hour at room temperature and stained with indicated antibodies. The blot was visualized using a chemiluminescent substrate (PerkinElmer, Waltham, MA) on Kodak BioMax XAR film (Sigma-Aldrich) that was then developed. Densitometry analysis was performed using LiCor Image Studio Lite densitometry software (LiCor, Lincoln, NE). Analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Enzyme-Linked Immunosorbent Assay (ELISA) and ColonyForming Units (CFU) Assay
Lucigenin-Enhanced Chemiluminescence Assay for ROS
Primary macrophages were plated at 5 × 104 cells/well in a 96well plate (Costar, Cambridge, MA) 24 hours before use. Cells were put on ice and washed 3 times with PBS. Lucigenin solution (0.9 mM CaCl2, 0.5 mM MgCl2, 20 mM dextrose, and 20 µM lucigenin) was added, and cells were incubated on ice for 10 minutes. BG beads or heat-killed C. albicans were added (E:T = 25), and then the plate was centrifuged briefly to facilitate cell-ligand contacts. An initial reading was then taken immediately after centrifugation. The plate was then incubated at 37°C and read every 10 minutes. An unpaired t test was used for statistical analysis and was performed using GraphPad Prism. RESULTS LC3 Is Required for Killing of C. albicans and Modulates TNF-α, IL-6, and IL-1β Production by Macrophages
To investigate the role of LC3 in killing C. albicans, primary macrophages from LC3β−/− and C57BL/6 (wild-type) mice were stimulated with live wild-type (strain SC5314) and yeast-locked (hyphalincompetent) C. albicans (mutant Δefg1/Δcphi) [23]. The mutant strain of C. albicans does not effectively lyse macrophages, allowing a more accurate measurement of viable intracellular yeast. Lysates from overnight stimulation of LC3β−/− macrophages showed a 1846
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Surface β-1,3-glucan Triggers LC3 Recruitment to the Fungal Phagosome
For LC3 to contribute to fungal killing, we hypothesized that it would be recruited to phagosomes containing fungal organisms. To determine whether C. albicans triggers LC3 recruitment to fungal phagosomes, we incubated RAW macrophages expressing GFP-LC3 with live C. albicans and heat-killed C. albicans (heat killing exposes more β-1,3-glucan [24, 25]; Supplementary Figure 1B). When stimulated with live C. albicans, only modest recruitment of GFP-LC3 to the fungal phagosome was observed at 45 minutes (Figure 2A). In sharp contrast, at the same time point, we observed an increased GFP-LC3 signal on the fungal phagosomal membrane containing heat-killed C. albicans (Figure 2A). We sought to determine whether β-1,3glucan alone was sufficient to trigger LC3 recruitment. Polystyrene beads covalently conjugated with pure β-1,3-glucan [16] were incubated with RAW macrophages expressing GFP-LC3. BG beads and GFP-LC3 colocalized, as shown by yellow rings in the phagosome, indicating that BG beads were sufficient to recruit LC3 to the phagosome (Figure 2B). The degree of endogenous lipidated LC3 (LC3-II) correlates with the fraction of LC3 inserted onto phagosomal or autophagosomal membranes [26]. To support the observations made by live cell imaging, immortalized macrophages from C57BL/6 mice were stimulated with live C. albicans and heat-killed C. albicans for 30 and 60 minutes, and lysates were analyzed by immunoblot. More LC3 lipidation (LC3-II) was detected upon stimulation with heat-killed C. albicans, compared with live C. albicans, at 30 minutes, but live C. albicans triggers higher LC3-II lipidation at 60 minutes, compared with heat-killed C. albicans (Figure 2C). Densitometry analysis confirmed this observation (Figure 2D).
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A total of 1 × 106 macrophages were plated in duplicate on tissueculture-treated 48-well plates and stimulated either 3 hours or overnight with Δefg1/Δcphi1 C. albicans (multiplicity of infection [MOI], 5). Supernatants were collected for cytokine analysis (TNF-α, IL-6, and interleukin 1β [IL-1β]) with ELISA as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Cells were washed 3 times with PBS and then lysed with 0.02% Triton-X-100. Serial dilutions from each well were made in Nanopure water and plated on yeast extract peptone dextrose agar plates. CFU were determined manually after 24-hour incubation at 30°C. An unpaired t test was used for statistical analysis and was performed using GraphPad Prism.
significant increase in the survival percentage (as measured by CFU count), as compared to wild-type macrophages (Figure 1A) at MOIs of 5 and 10, indicating that LC3 is required for fungal killing. These results indicate that LC3β is necessary for fungal killing in macrophages. Supernatants from stimulated cells were also analyzed for TNF-α, IL-6, and IL-1β production. Shortterm exposure of macrophages to C. albicans led to a similar cytokine response (Figure 1B–D). However, overnight stimulation by Δefg1/Δcphi C. albicans induced increased TNF-α, IL-6, and IL-1β production in the LC3β−/− macrophages, compared with wild-type macrophages (Figure 1B–D). Although the LC3β−/− macrophages showed a modest increase in intracellular Δefg1/Δcphi yeast after overnight incubation, compared with wild-type macrophages (Supplementary Figure 1A), the amount of intracellular yeast in the LC3β−/− macrophages did not correlate with the difference in cytokine production between the 2 cell types. The higher CFU count in LC3β−/− macrophages may be due to yeast survival rather than to intracellular replication. Taken together, these results indicate that LC3β is necessary for fungal killing in macrophages.
We also determined whether LC3 and other autophagy-related proteins were recruited to the phagosomal membrane in response to dectin-1 signaling. We took advantage of the fact that polystyrene beads are buoyant in sucrose solutions and can be efficiently separated from other cellular constituents [22, 27]. Phagosomes containing BG beads from macrophages expressing GFP–dectin1 were isolated, and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting. In addition to LC3-II, beclin-1 and p62, additional components of the autophagy pathway, and LAMP-1, a phagosomal protein marker, were also present (Figure 2E). These results indicate that phagosomes containing BG beads recruit not only LC3, but also multiple proteins of the autophagy pathway. Dectin-1 Is Required for LC3 Recruitment to Fungal Phagosomes
The observation that BG beads recruited LC3 and other autophagy proteins led us to hypothesize that dectin-1 regulates the trafficking of these proteins to the fungal phagosome. To evaluate this hypothesis, we stably expressed GFP-LC3 in dectin-1 −/− macrophages and incubated them with labeled heat-killed C. albicans and live C. albicans. Despite comparable phagocytosis of heat-killed C. albicans, compared with wild-
type macrophages, no GFP-LC3 recruitment to heat-killed C. albicans–containing phagosomes was seen in dectin-1−/− macrophages, as determined by spinning-disk confocal microscopy (Figure 3A). To verify our observations, we compared dectin-1−/− macrophages with dectin-1−/− cells reconstituted with GFP–dectin-1 (dectin-1) after stimulation with heat-killed C. albicans and live C. albicans, using total LC3-II produced as a readout. Consistent with findings of microscopy, LC3 lipidation was observed upon stimulation with heat-killed C. albicans in dectin-1 macrophages, but in macrophages lacking dectin-1, heat-killed C. albicans stimulation did not increase LC3 lipidation to a level greater than that observed for the unstimulated control (Figure 3B). Similarly, increased LC3 lipidation was seen in dectin-1–proficient macrophages, compared with dectin-1–deficient macrophages, when stimulated with live C. albicans (Figure 3D). Densitometry was used to quantitate the total amount of LC3-II produced [28], confirming that dectin-1 macrophages produce a greater amount of LC3-II than dectin-1−/− macrophages (Figure 3C and 3E ) when stimulated with both heat-killed C. albicans and live C. albicans. These results indicate that dectin-1 is required for LC3 recruitment to phagosomes containing β-1,3-glucan. Dectin-1 Controls LC3 Recruitment
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Figure 1. LC3 is required for killing of Candida albicans and regulates proinflammatory cytokines. A, Primary wild-type (WT) or LC3β−/− bone-marrowderived macrophages (BMDMs) were infected overnight with yeast-locked C. albicans at multiplicities of infection (MOIs) of 1, 5, and 10. Lysates were plated, and C. albicans colony-forming units were counted and survival percentage was calculated on the basis of the initial inoculum. Duplicate investigations were performed, and mean values and standard deviations are shown. *P < .05 and **P < .01, by the unpaired t test , using GraphPad Prism. B–D, Primary WT or LC3β−/− BMDMs were stimulated with WT C. albicans or yeast-locked C. albicans mutant for 3 hours or overnight at a MOI of 5, and levels of tumor necrosis factor α (TNF-α; B), interleukin 6 (IL-6; C), and interleukin 1β (IL-1β; D) produced in the supernatant were measured by enzyme-linked immunosorbent assay.
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Figure 2. Heat-killed Candida albicans (HKCA) and BG beads trigger LC3 rearrangement to the fungal phagosome. A, RAW macrophages expressing green fluorescent protein LC3 (RAW-GFP-LC3) were stimulated with live C. albicans (left) and HKCA (right). Bar indicates 5 µm. B, Stimulation of RAWGFP-LC3 (green) with BG beads labeled with AF 647 (red). The merged image shows colocalization of the GFP-LC3 signal with the beads (yellow). DIC image is also shown. Bar indicates 5 µm. C, Stimulation of RAW macrophages with HKCA and analysis of LC3 lipidation by Western blot. Lipidated LC3 (LC3-II) migrates faster by sodium dodecyl sulfate polyacrylamide gel electrophoresis than its unmodified counterpart. Actin blot is shown as a loading control. D, Densitometry analysis of total LC3-II lipidation, using the immunoblot from panel C. The mean value (+SD) of the total LC3-II intensity is shown. E, Phagosome isolation from macrophages expressing GFP–dectin-1 stimulated with BG beads and analyzed by Western blot. Total lysates are compared to the phagosomal compartment and probed for LC3, beclin-1, p62, and LAMP-1.
LC3 Recruitment to the Fungal Phagosome Depends on Dectin-1 Signaling Through Syk Phosphorylation
Dectin-1 stimulation triggers both Raf- and Syk-mediated signaling cascades in response to β-1,3-glucan [29]. Dectin-1 is phosphorylated by the kinase Syk, which in turn leads to production of ROS catalyzed by the phagosome NADPH oxidase 1848
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[30]. We investigated whether LC3 recruitment to the phagosome requires dectin-1 phosphorylation. We used a signalingincompetent mutant of GFP-dectin-1 (ΔY15) that ablates Syk-mediated downstream signaling [30] but does not affect phagocytosis. Dectin-1−/− macrophages were transduced stably with RFP-LC3 and either ΔY15 or GFP–dectin-1. Upon
stimulation with heat-killed C. albicans, the dectin-1 ΔY15 mutant recruited to the fungal phagosome as expected, but RFPLC3 failed to colocalize with it (Figure 4A). Macrophages expressing GFP–dectin-1 showed colocalization of both GFP– dectin-1 and RFP-LC3 (Figure 4B). GFP–dectin-1 and ΔY15 macrophages were also stimulated with heat-killed C. albicans and live C. albicans, and LC3-II conversion was examined by immunoblot and analyzed by densitometry. The ΔY15 cells did not show LC3-II conversion, compared with the unstimulated cells, whereas dectin-1 cells exhibited substantial LC3-II formation upon stimulation (Figure 4C and 4E ), again confirmed by densitometric analysis (Figure 4D and 4F ). To validate further the role of Syk phosphorylation in LC3 recruitment, we chemically inhibited Syk and observed inhibition of
LC3-II formation upon stimulation by BG glucan beads (Figure 4G) and confirmed this observation by densitometry analysis (Figure 4H). Taken together, these results indicate that dectin-1 phosphorylation is required for LC3 recruitment to the fungal phagosome. LC3 Recruitment Is Independent of Card9 Signaling but Requires ROS Generation Through NADPH Oxidase
Syk-mediated dectin-1 phosphorylation leads to activation of both Card9 [31] and ROS generation [32]. We sought to determine whether Card 9 is required for LC3 trafficking to the phagosome. Primary macrophages from Card9−/− mice and wild-type littermate controls were stimulated with BG beads or heat-killed C. albicans, and LC3-II formation was observed. Dectin-1 Controls LC3 Recruitment
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Figure 3. Dectin-1 is required for LC3 recruitment to fungal phagosomes. A, LC3 (green) fails to recruit to phagosomes containing HKCA in Dectin-1−/− GFP-LC3 macrophages as observed by spinning disk confocal microscopy. Size bar measures 5 μm. B, LC3 lipidation was assessed when Dectin-1−/− macrophages and Dectin-1−/− reconstituted with GFP-Dectin-1 macrophages (Dectin-1+/+) were exposed to HKCA for indicated times. Media only and stimulation with chloroquine serve as negative and positive controls, respectively. C, Densitometry analysis performed on experiments found in B. D, LC3 lipication was assessed in Dectin-1−/− macrophages and Dectin-1−/− reconstituted with GFP-Dectin-1 macrophages (Dectin-1+/+) exposed to live C. albicans (LvCA) for the indicated times. Media only and stimulation with chloroquine serve as negative and positive controls, respectively. E, Densitometry analysis performed on experiments found in D.
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Figure 4. LC3 recruitment requires Dectin-1-dependent Syk phosphorylation. A, Dectin-1−/− macrophages co-expressing ΔY15 (GFP-Dectin-1, Syk-signaling mutant, green) and RFP-LC3 (red) were stimulated with HKCA (blue) and imaged by live cell imaging. Size bars indicates 5 μm. B, Dectin-1−/− macrophages that co-expressed GFP-Dectin-1 (green) and RFP-LC3 (red) were stimulated with HKCA (blue) and imaged. Size bars indicates 5 μm. C, The degree of LC3 lipidation by HKCA was measured in macrophages expressing ΔY15 or Dectin-1 by Western blot analysis and quantified by densitometry D. E, LC3 lipidation by live C. albicans (LvCA) was measured in macrophages expressing ΔY15 or Dectin-1 by Western blot analysis and quantified by densitometry F. Media only and chloroquine serve as the negative and positive controls, respectively. G-H, GFP- Dectin-1 expressing cells were treated with piceatannol or DMSO for 1 hour prior to incubation with BG beads and analysis of LC3 lipidation was performed by Western blot G and quantified by densitometry H.
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No difference in LC3-II formation was seen between the wildtype and Card9−/− macrophages, indicating that LC3 lipidation is independent of Card9 (Figure 5A). As LC3 recruitment was independent of Card9, we hypothesized that generation of ROS by the NADPH oxidase [33–36] was required to recruit LC3 to the phagosome [30]. We used macrophages deficient in the membrane-bound gp91 subunit of the NADPH oxidase (gp91−/−) [37] and stimulated them with heat-killed C. albicans and BG beads. Macrophages from gp91−/− mice showed no LC3-II lipidation when stimulated with either heat-killed C. albicans or BG beads. However, wild-type macrophages produced LC3-II when incubated with the same stimuli. Upon incubation with diphenyleneiodonium (DPI; a nonspecific chemical inhibitor of ROS production), LC3-II production decreased upon stimulation with either heat-killed C. albicans or BG beads (Figure 5B). We monitored ROS generation upon uptake of the heat-killed C. albicans or
BG beads. As expected, wild-type macrophages produced a pronounced burst of ROS, while mutant cells showed drastically reduced levels, as expected (Figure 5C). To confirm these findings, we also investigated the role of the cytosolic p40phox subunit of the NADPH oxidase on LC3 lipidation. NCF4−/− macrophages also failed to produce LC3-II when stimulated with either heat-killed C. albicans or β-1,3glucan beads. Furthermore, while wild-type macrophages produced LC3-II in response to these stimuli, this response was potently inhibited by DPI (Figure 5D). We verified that NCF4−/− primary macrophages were deficient in ROS production by stimulating them with heat-killed C. albicans and BG beads (Figure 5E ). Wild-type macrophages generated ROS within 10 minutes of stimulation, whereas NCF4−/− cells showed drastically reduced levels. These results demonstrate that ROS generation through dectin-1 signaling and NADPH oxidase is required for LC3 recruitment to the fungal phagosome. Dectin-1 Controls LC3 Recruitment
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Figure 5. LC3 recruitment is independent Card9 signaling but requires reactive oxygen species (ROS) generation through NADPH oxidase. A, Primary macrophages from Card9−/− or wild-type (WT) littermates were stimulated with either BG beads or heat-killed Candida albicans (HKCA) for the indicated times. Cell lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by Western blot analysis, to examine the ratio of lipidated LC3 compared with the unmodified form. Medium only and chloroquine were used as negative and positive controls, respectively. Actin blot is shown as a loading control. B, Western blot of WT and gp91−/− bone-marrow-derived macrophages (BMDMs) stimulated with HKCA or BG beads for 45 minutes in the presence or absence of diphenyleneiodonium (DPI). C, Levels of ROS produced by primary macrophages from WT and gp91−/− macrophages stimulated with HKCA or BG beads for 1 hour. ROS production was measured at 10-minute intervals. D, Western blot of WT and NCF4−/− primary macrophages. Primary macrophages were stimulated with HKCA or BG beads for 45 minutes in the presence or absence of DPI. Chloroquine and medium only served as the positive and negative controls, respectively, for LC3 lipidation. E, Levels of ROS produced by BMDM from NCF4−/− and WT macrophages stimulated with HKCA or BG beads for 1 hour. ROS production was measured at 10-minute intervals. RLU, relative luminescence units.
DISCUSSION Mammalian autophagy proteins, including Atg5 and Atg7, are known to generally participate in host-defense activities against fungal pathogens, such as the phagocytosis of fungal organisms [13, 38, 39]. Knockdown of Atg5 decreases recruitment of GFPLC3 to fungal phagosomes in RAW cells [13], and J774.16 macrophages transfected with Atg5 small-interfering RNA phagocytose less. Mice lacking Atg5 demonstrated increased susceptibility upon challenge to intravenous C. albicans [39]. Atg5 and Atg7 function to produce LC3-II, the lipidated and membrane-anchored form of LC3. LC3 does not recruit to phagosomes in Atg7−/− macrophages [13]. However, a direct role for LC3 recruitment to the fungal phagosome has not been shown. We sought to determine the role of LC3 in C. albicans infection and the molecular mechanism by which LC3 is recruited to the fungal phagosome. We have shown that LC3-II 1852
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Figure 6. Proposed model of LC3 recruitment to the fungal phagosome. β-1,3-glucan on the fungal surface engages dectin-1, leading to the phosphorylation of the tyrosine in the hemi- immunoreceptor tyrosine-based activation motif present in the cytoplasmic tail. Syk is then recruited to the phosphorylated tail of dectin-1 and activates reactive oxygen species (ROS) production by the NADPH oxidase on the phagosome. ROS production then leads to the processing of cytosolic LC3-I to the formation of membranebound lapidated form of LC-II, which affects Candida albicans killing by the macrophage.
on the phagosome enhances the fungicidal activity in macrophages, and we observed increased LC3 lipidation and more LC3 recruitment to heat-killed C. albicans, compared with live C. albicans in immortalized macrophages. We hypothesize that the increase in LC3 lipidation and recruitment to heatkilled C. albicans is due to more-exposed β-1,3-glucan on the surface, which is shielded initially by mannan on live C. albicans. Upon binding of β-1,3-glucan, the hemi-ITAM motif in the cytoplasmic tail of dectin-1 is phosphorylated by Syk, resulting in phagocytosis and ROS production [30, 37]. ROS produced by the phagosomal NADPH oxidase is a signal to catalyze the conversion of cytosolic LC3-I to lipidated LC3-II (Figure 6). Interestingly, there is precedent for ROS signaling in starvation-induced autophagy, as the LC3 lipidation factor Atg4 is regulated by ROS [40, 41] The role of ROS in LC3 lipidation was also examined in LC3β−/− and beclin-1+/− (another autophagy-related protein) macrophages, whereby NALP3 inflammasome activation by lipopolysaccharide (LPS) and adenosine triphosphate produced more ROS. In turn, this ROS production leads to higher caspase-1 activation and IL-1β secretion. Furthermore, autophagic regulation of caspase-1 is necessary in murine septic shock models; LPS was more lethal LC3β −/− mice than to wild-type controls [42]. In our studies of fungal infection, we also saw increased production of ROS in LC3β−/− macrophages upon stimulation with heat-killed C. albicans (Supplementary Figure 1C), and similarly, we found elevated IL-1β production upon incubation with C. albicans. Although we used an in vitro system, we showed that lack of LC3β decreases fungicidal activity against C. albicans in primary BMDMs, establishing a critical role in fungal host defense. These observations in 2 different infection models show that LC3 participates not only in bacterial killing, but in fungal killing, as well, and represents a general mechanism of host defense. A recent report by Ma et al demonstrated no difference in TNF-α and IL-6 production and no difference in C. albicans killing between wild-type and LC3β-deficient bone-marrowderived dendritic cells (DCs) [38]. This observation is perhaps not surprising in light of the differences that have been observed between phagosomal maturation in macrophages DCs [43]. Nox2 activity in macrophages generates a respiratory burst for effective microbicidal activity, while in DCs ROS serves to control the redox potential and pH such that relevant antigens are not rapidly degraded and can be loaded onto class I major histocompatibility complex (MHC) molecules (cross-presentation) and class II MHC molecules (in the endolysosomal compartment) [44]. The difference between our observation of significant C. albicans killing in macrophages and inefficient killing in DCs and cytokine differences could be due to difference in phagosome acidification and ROS production between DCs and macrophages, a testable hypothesis. There are also
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Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases (grants 1R01AI092084 and 1R01AI097519; grant R01AI079253 to B. H. S.) and the National Heart, Lung, and Blood Institute (grant R01HL055330 to A. M. K. C.). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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additional differences in Nox2 assembly, as well as in the Rab factors, which direct recycling in these cell types [45, 46]. LC3 is recruited to phagosomes containing the yeast cell wall fragment zymosan [13]. Zymosan, derived from Saccharomyces cerevisiae, is biochemically heterogeneous and can activate multiple cell surface receptors, including TLR2 and dectin-1 [47], making the individual contribution of TLRs and CLRs difficult to ascertain. To determine the specific ligands that induce LC3 production, we used uniform monodisperse polystyrene beads coated homogenously with β-1,3-glucan [27, 48]. These fungal-like particles are similar in shape and size to C. albicans and are capable of inducing an inflammatory response in vitro [16]. This ligand triggers dectin-1 exclusively and permits interrogation of dectin-1 pathways. Moreover, experiments with MyD88/TRIF−/− macrophages showed intact LC3 lipidation upon stimulation by heat-killed C. albicans, indicating that TLR signaling is dispensable for this process (Supplementary Figure 1D). Our data also support the conclusion that dectin1, a cell surface protein, controls downstream recruitment of autophagy-related proteins, in particular LC3, to the maturing fungal phagosome. We have shown that, without LC3, higher cytokine responses are produced, which correlate with higher survival rates of fungal organisms. Without the presence of LC3, fungicidal activity decreases in macrophages, confirming yet another link between the ancient and ubiquitous autophagic machinery and the functions of multicellular immunity.
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