AUTOPHAGY 2016, VOL. 12, NO. 9, 1440–1446 http://dx.doi.org/10.1080/15548627.2016.1191724
BASIC BRIEF REPORT
Autophagy proteins are not universally required for phagosome maturation Marija Cemmaa,b, Sergio Grinsteina,c,d,e, and John H. Brumella,b,e,f a Cell Biology Program, Hospital for Sick Children, Toronto, ON Canada; bDepartment of Molecular Genetics and Biochemistry, University of Toronto, Toronto, ON Canada; cDepartment of Biochemistry, University of Toronto, Toronto, ON Canada; dKeenan Research Center of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON Canada; eInstitute of Medical Science, University of Toronto, Toronto, ON Canada; fSick Kids IBD Center, Hospital for Sick Children, Toronto, ON Canada
ABSTRACT
ARTICLE HISTORY
Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcgR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.
Received 7 October 2015 Revised 9 May 2016 Accepted 16 May 2016
Introduction Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway that targets long-lived proteins, damaged organelles, and pathogens for lysosomal degradation in eukaryotic cells and requires more than 30 autophagy-related (ATG) proteins.1 A hallmark of canonical autophagy is that the cargo is sequestered into a double-membrane compartment (the phagophore, which matures into an autophagosome), which is decorated by MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). LC3 is a ubiquitin-like molecule that is covalently conjugated to phosphatidylethanolamine on phagophore membranes through a series of reactions similar to ubiquitin conjugation and requires a subset of ATG proteins, including ATG5 and ATG7.2 A number of ATG proteins have been implicated in having alternative (noncanonical) functions.3 A seminal study by Sanjuan et al. revealed that LC3 can be conjugated to the singlemembrane phagosome upon stimulation of TLR2 (toll-like receptor 2) and TLR4 in a process termed LC3-associated phagocytosis (LAP).4 These receptors initiate reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase, an event that is required for LAP.5,6 In fact, intraphagosomal ROS in the absence of other signals is sufficient to recruit LC3 to phagosomes.6 Other receptors implicated in LAP, such as FCGR2A/FcgR2A and CLEC7A/dectin-1, also result in CYBB/NOX2-dependent ROS production.5,7 LAP is triggered in response to particles that stimulate TLRs, FCGR2A/FcgR2A,
KEYWORDS
autophagy; ATG8/LC3; ATG5; ATG7; LC3-associated phagocytosis; phagosome maturation
CLEC7A/dectin-1, and other cargo such as apoptotic and entotic cells.4-10 In addition to ROS, LAP requires a subset of autophagy components, such as ATG5, ATG7, ATG12, ATG16L1, BECN1/Beclin 1, and PIK3C3/VPS34, whereas others, such as ULK1 (unc51-like kinase 1), ATG13, and RB1CC1/FIP200 are dispensable.4,6 LC3 conjugation to a phagosome is observed in hematopoietic cell types, including macrophages, dendritic cells, and neutrophils.4-12 It has also been observed in retinal pigment epithelial cells,13,14 which are macrophage-like and expresses some hematopoietic markers.15 The role of LAP in nonhematopoietic cell types is not yet clear. One of the major functions ascribed to LAP is to promote phagosome maturation. This conclusion is based on the observation that autophagy-deficient murine macrophages (RAW264.7 and CSF2/GM-CSF-differentiated BMDMs) fail to degrade live yeast4 and apoptotic cells,9 and phagosomes containing beads coated with the synthetic triacylated lipopeptide PAM3CSK4 or zymosan exhibit delayed phagosome fusion with the lysosome.4 However, a study in human macrophages observed quite the opposite—LC3 recruitment to zymosan-containing phagosomes was associated with delayed phagosomal fusion with the lysosome.10 Therefore, the impact of LC3 recruitment to phagosomes remains unclear. Hence, we explored how the phagosome maturation is affected in the absence of ATG proteins. We used autophagy-deficient mouse embryonic fibroblasts (MEFs), as well as CSF1/M-CSF- and CSF2-differentiated BMDMs to assess phagosome maturation of IgG-coated zymosan and sheep red blood cells (SRBC) using 3 distinct assays. We did not find a
CONTACT John H. Brumell [email protected] Cell Biology Program, Hospital for Sick Children, PGCRL, 686 Bay Street, Toronto, ON M5G0A4, Canada. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
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requisite role for Atg5 or Atg7 in phagosome maturation, challenging the notion that autophagy proteins are universally required for phagosome maturation.
Results Phagosomes in Atg5-deficient MEFs mature normally upon FCGR2A stimulation To examine if autophagy machinery is required for phagosome maturation in fibroblasts, we performed 2 phagosome maturation assays using wild-type (WT) and Atg5-deficient MEFs. It was previously established that heterologous transfection of fibroblastic cells with the FCGR2A receptor confers phagocytic capacity and results in phagosome maturation similar to that observed in professional phagocytes.16,17 As expected, FCGR2A receptor-transfected WT and Atg5-deficient fibroblasts were able to internalize antibody-opsonized particles and acquire phagosome maturation markers. LAMP1 is localized to lateendosomes and lysosomes and is commonly used as a phagosome maturation marker. FCGR2A-transfected MEFs were challenged with IgG-opsonized zymosan particles (OpZ) for 30, 60, 90, and 120 min, then stained for LAMP1 and OpZ. In both WT and autophagy-deficient MEFs, OpZ particles acquired LAMP1 with similar kinetics; nearly all became LAMP1-positive at 120 min after the challenge (Fig. 1A and B). No significant difference in LAMP1 acquisition was observed between WT and Atg5-deficient fibroblasts. We next explored if phagosome acidification might be impaired in Atg5-deficient fibroblasts. To visualize acidic compartments such as lysosomes and mature phagosomes, we used LysoBrite, a fluorescent indicator that permeates cell membranes and accumulates in acidic compartments. FCGR2A-transfected MEFs were incubated with LysoBrite for 30 min and then challenged with antibody-coated SRBC. Anti-SRBC antibody was used to opsonize SRBC in order to facilitate its phagocytosis via FCGR2A. Cells were imaged live at 30, 60 and 90 min following phagocytosis. We observed no significant difference in the percentage of LysoBrite-positive phagosomes between WT and Atg5-deficient fibroblasts (Fig. 1C and D). Hence, we conclude that ATG5 does not play a role in FCGR2A-mediated phagosome maturation in murine fibroblasts. Phagosomes in autophagy-deficient BMDM mature normally Next, we sought to determine the role of autophagy proteins in primary murine bone marrow-derived macrophages (BMDMs). To ensure that our findings are of relevance to most research groups, we have utilized 2 commonly used methods to differentiate macrophages, using either CSF1- or CSF2-containing media. Since conventional knockout of Atg518 or Atg719 causes neonatal lethality, we used mice where Atg5 or Atg7 was selectively deleted from monocytes/macrophages and granulocytes. This was achieved by crossing Atg5flox/flox or Atg7flox/flox (referred to here as WT) mice with mice expressing the Cre recombinase from the endogenous lysozyme 2/M locus (Lyz2creC).20,21 In all cases we confirmed gene knockout using
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RT-PCR (Fig. S1A-D). As expected, autophagy-deficient macrophages were unable to process the cytosolic form of LC3 (LC3-I) into the lipidated (and membrane-associated) form of LC3 (LC3-II) as demonstrated by the disappearance of the LC3-II band via western blot (Fig. S1E and F).22 To investigate if autophagy proteins are required for acquisition of LAMP1, we challenged Atg5- or Atg7-deficient CSF1-differentiated macrophages and respective control macrophages with OpZ and quantified the percentage of LAMP1positive phagosomes at 30, 60, 90, and 120 min post challenge. LAMP1 acquisition by phagosomes followed the same kinetics in WT and autophagy-deficient CSF1-differentiated macrophages and significant differences were not observed (Fig. 2A– C). This suggests that ATG proteins are not required for and do not play a significant role in LAMP1 acquisition during phagocytosis of OpZ by CSF1-cultured BMDMs. The same results were observed in CSF2-differentiated macrophages (Fig. S2A, B). We also assessed phagosome maturation using unopsonized zymosan particles (not coated with IgG as above) in WT and Atg5-deficient CSF1- and CSF2-differentiated BMDMs. Phagosomes containing zymosan particles matured at comparable rates in WT and autophagy-deficient macrophages (Fig. S3A and B). Next, we assessed phagosome acidification in control and autophagy-deficient macrophages. To this end, we quantified the percentage of LysoBriteC SRBC-containing phagosomes. As with LAMP1 acquisition, no significant difference was observed between the controls and Atg5- or Atg7-deficient CSF1-cultured macrophages (Fig. 2D–F). This indicates that ATG proteins do not play a significant role in acidification of SRBC-containing phagosomes in CSF1-cultured BMDMs. We have further confirmed these findings in CSF2-differentiated macrophages (Fig. S2C and D). Finally, we explored if autophagy proteins might play a role in conferring the phagosome its degradative properties in CSF1-cultured macrophages by using DQ-BSA dye. DQ-BSA is a self-quenched BODIPY dye conjugate of bovine serum albumin (BSA). The proteolytic digestion of BSA in the lysosome results in dequenching of fluorescence, enabling the visualization of proteolytic compartments. CSF1-differentiated BMDMs were pulsed with DQ-BSA for 1 h, chased for 1 h in regular medium and then challenged with antibody-coated SRBC for 30, 60 or 90 min. Phagosomes in Atg5- and Atg7-deficient BMDMs acquired the capacity to cleave DQ-BSA with similar kinetics as their respective controls (Fig. 2G–I). We have further confirmed these findings in the CSF2-differentiated macrophages and again saw no impact of ATG proteins on phagosome maturation (Fig. S2E, F). In summary, using 3 distinct phagosome maturation assays, we were unable to detect a significant difference in phagosome maturation of antibodyopsonized particles between control and autophagy-deficient CSF1- and CSF2-differentiated BMDMs.
Discussion Our findings challenge the notion that LC3 recruitment is required for phagosome maturation. The difference between our observations and previously reported data4,6,9 could be due to the fact that different phagocytic particles were used in each
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Figure 1. Phagosome maturation of IgG-coated particles is unaffected in atg5¡/¡ MEFs. WT and atg5¡/¡ MEFs were transfected FCGR2A-GFP (green) to render them phagocytic. (A, B) MEFs were challenged with OpZ and fixed at 30, 60, 90, or 120 min post challenge, then stained for LAMP1 (red) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMPC particle marked by an arrow. (B) The percentage of LAMP1C OpZ was enumerated in WT and atg5¡/¡ MEFs. At least 200 OpZ were counted per condition. (C, D) MEFs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (C). A representative image is shown at 60 min post challenge with a LysoBriteC particle marked by an arrow. (D) The percentage of LysoBriteC SRBC was enumerated in WT and atg5¡/¡ MEFs. At least 50 SRBC were counted per condition. Scale bar: 8 mm.
study. We used antibody-coated particles to engage the FCGR2A and also unopsonized zymosan particles. Previous studies used zymosan, PAM3CSK4- and LPS-coated beads, Escherichia coli4 and dead cells.9 Another difference is that our macrophages do not overexpress GFP-LC3, as was the case in some prior studies.4,9 Variability in specific reagents during the macrophage differentiation protocol may also underlie the observed differences. We do not regard the LAP model as false, but rather make the point that it is not universally applicable; phagosome maturation, a complex and dynamic process, may have a requirement for ATG proteins under specific conditions. An important consideration when interpreting these discordant findings may involve the manner whereby the CYBB NADPH oxidase affects phagosome maturation. We previously showed that ROS production by the CYBB NADPH oxidase is required for LC3 recruitment to phagosomes,5 an observation confirmed by others.6,10 In the present study we examined phagosome maturation in the
absence of CYBB-dependent ROS production. Despite the fact that CYBB is required for LAP,5,6,10 OpZ phagosomes matured faster in the absence of intraphagosomal ROS than in the WT counterparts (Fig. S3C). This suggests 2 things: first, phagosome maturation does not always require LAP and, second, CYBB NADPH oxidase may actually impair phagosome maturation under these conditions. Indeed, CYBB has a controversial role in modulating phagosome maturation; some investigators find that it delays phagosomal maturation,23,24 whereas others do not.25-27 Recent studies showed that the maturation status of macrophages dictates the effects that the CYBB NADPH oxidase has on phagosome maturation.28 Based on these findings, we hypothesize that both CYBB and ATG proteins, which are linked in phagosomal development, may have effects on phagosome maturation that are condition-dependent and possibly dynamic—altered by status of the macrophages, e.g., in classically activated macrophages (M1) vs.
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Figure 2. Phagosome maturation of IgG-coated particles is unaffected in autophagy-deficient CSF1-differentiated BMDMs. (A, B, C) CSF1-derived BMDMs were challenged with OpZ and fixed at 30, 60, 90, or 120 min, then stained for LAMP1 (green) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMPC particle marked by an arrow. The percentage of LAMP1C OpZ was enumerated in WT and atg5¡/¡ (B) and WT and atg7¡/¡ (C) BMDMs. At least 300 OpZ were counted per condition. (D, E, F) CSF1-derived BMDMs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (D). A representative image is shown at 60 min post challenge with a LysoBriteC particle marked by an arrow. The percentage of LysoBriteC SRBC was enumerated in WT and atg5¡/¡ (E) and WT and atg7¡/¡ (F) BMDMs. At least 50 SRBC were counted per condition. (G, H, I) CSF1-derived BMDMs were pulsed with DQ-BSA (red) for 1 h, chased for 1 h in regular medium, then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (G). A representative image is shown at 60 min post challenge with a DQ-BSAC particle marked by an arrow. The percentage of DQ-BSAC SRBC was enumerated in WT and atg5¡/¡ (H) and WT and atg7¡/¡ (I) BMDMs. At least 50 SRBC were counted per condition. Scale bar: 8 mm.
alternatively activated macrophages (M2). An additional layer of complexity is added by the expression of CYBB NADPH oxidase regulators, such as RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1interacting protein). RUBCN is required for LAP6 and is upregulated in response to TLR2 activation.29 Thus, the choice of the phagocytic particle also affects the amount of ROS produced and the destiny of the phagosome. It is
possible that other positive and negative regulators of the CYBB NADPH oxidase also play a role in LAP.30-33 Further studies are required to reveal the reason why ATG proteins are important in phagosome maturation under some conditions, but not others. Regardless of the specific reason, we have shown that in our system ATG proteins are not required for conventional phagosome maturation. Hence, we posit that LAP is not universally required for phagosome fusion with
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endosomes and lysosomes. Instead the relationship between ATG proteins and phagosome maturation is more complex and likely involves other players.
Materials and methods Cells Mouse embryonic fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (HyClone, SH3024301) supplemented with 10% fetal bovine serum (FBS; Wisent, 090-510) at 37 C in 5% CO2 without antibiotics. Atg5flox/flox (WT), Atg5flox/ flox -Lyz2creC (atg5¡/¡), Atg7flox/flox (WT) and Atg7flox/floxLyz2creC (atg7¡/–) mice were previously described.20,21 Bone marrow-derived macrophages were extracted as previously described.34 CSF1-differentiated BMDMs were cultured in RPMI-1640 medium (Wisent, SH3002701) supplemented with 10% FBS, 5% sodium pyruvate (Invitrogen, 11360-070), 5% penicillin/strep (Invitrogen, 15140122), 5% nonessential amino acids (Invitrogen, 11140050), 0.5 mM 2-ME (Invitrogen, 21985023) and 10% CSF1-conditioned medium from NIH3T3 cells. CSF2-differentiated BMDM were cultured in RPMI supplemented with 10% FBS, 5% penicillin/strep, and nonessential amino acids in the presence of 20 ng/ml murine CSF2 (R&D Systems, 415-ML-010). Phagocytic particles were added in RPMI-1640, cells were washed 3 times with phosphate-buffered saline (Multicell, 311-420-CL) at a 10-min time point after the beginning of phagocytosis, and incubated in medium without antibiotics. Images were taken in RPMI-1640 supplemented with 10 mM HEPES buffer (Life Technologies, 15630-080).
LysoBrite assay, cells were incubated with LysoBrite (AAT Bioquest, 22659) for 30 min, as per the manufacturers’ instructions. For the DQ-BSA assay, cells were incubated with 10 mg/ ml of DQ-BSA (Life Technologies, D12051) for 1 h, followed by 1-h incubation in complete medium. Phagocytosis was synchronized by spinning sRBC at 170 £ g for 5 min onto cells. The cells were imaged live at 30, 60, and 90 min time points after beginning of phagocytosis. Imaging system Images were acquired using a Wave-FX-X1 Spinning Disc Confocal, Leica DM16000B inverted research microscope with Hamamatsu ImagEMx2 (EMCCD) camera using a 63x objective. Western blotting Western blotting was performed as previously described5 using LC3 (Novus, NB600-1384) and GAPDH (Millipore, MAB374) antibodies. RNA isolation and quantitative PCR RNA was isolated using the RNeasy kit (QIAGEN, 74104), and cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708840). Ten nanograms per reaction were used for quantitative RT-PCR using predesigned Taqman probes for target genes and Hprt as housekeeping reference (Life Technologies, 4448489).
LAMP1 phagosome maturation assay
Statistical analysis
Zymosan particles (Sigma, Z4250) were opsonized by overnight incubation in 6 mg/mL human IgG (Meridian Life Sciences, A50170H) at 4 C. For unopsonized particles, Zymosan A, S. cerevisiae BioParticles, Texas Red conjugate (Molecular Probes, Z2843) were used. On d 1, MEFs were seeded on glass coverslips in 24-well tissue culture plates at 2.5 £ 104 cells/well, and, on d 2, transfected with FCGR2A-GFP construct using GeneJuice (Novagen, 70967-3) as per the manufacturers’ instructions. On d 3, phagocytosis was synchronized by spinning opsonized zymosan (OpZ) at 170 £ g for 5 min onto cells. BMDMs were seeded on glass coverslips in 24-well tissue culture plate at 2.0 £ 105 cells/well, and, on the following day, phagocytosis was synchronized by spinning OpZ at 170 £ g for 5 min. Cells were then fixed with 2.5% paraformaldehyde (EMS, 15710) at 30, 60, 90, or 120 min and stained for OpZ and LAMP1 (Developmental Studies Hybridoma Bank at the University of Iowa, 1D4B).
For quantification studies, at least 50 particles were enumerated for the LysoBrite or DQ-BSA assays and at least 300 particles were enumerated for the LAMP1 phagosome maturation assay for each condition in each experiment, unless otherwise indicated. At least 3 independent experiments were performed for each graph, unless otherwise indicated. The mean § SEM is shown in the figures. Statistical significance was determined using a 2-tailed, 2-sample, unequal variance Student t test with an a level of 0.05.
LysoBrite and DQ-BSA phagosome maturation assays SRBC (MP Biomedicals, 55876) were opsonized by 1-h incubation with rabbit anti-sRBC antibody (MP Biologicals, 55806) at room temperature. Cells were seeded on Ibidi microscopy chambers (80827). MEFs were seeded at 1.0 £ 104 cells/chamber and transfected with FCGR2A-GFP using GeneJuice. BMDMs were seeded at 6.0 £ 104 cells/chamber. For the
Abbreviations Atg ATG5 ATG7 BMDM CLEC7A CSF1/M-CSF CSF2/GM-CSF CYBB/NOX2 DQ-BSA GFP LAMP1 LAP LPS
autophagy related autophagy-related 5 autophagy-related 7 bone marrow-derived macrophages C-type lectin domain family 7 member A colony stimulating factor 1 (macrophage) colony stimulating factor 2 (granulocytemacrophage) cytochrome b-245, beta polypeptide dye quenched-bovine serum albumin green fluorescent protein lysosomal-associated membrane protein 1 LC3-associated phagocytosis lipopolysaccharide
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Lyz2cre
Cre recombinase from the endogenous lysozyme 2/M locus MAP1LC3/LC3 microtubule-associated protein 1 light chain 3 MEFs mouse embryonic fibroblasts NADPH nicotinamide adenine dinucleotide phosphate-oxidase OpZ opsonized zymosan PIK3C3/VPS34 phosphoinositide-3-kinase, class 3 RB1CC1 RB1-inducible coiled-coil 1 ROS reactive oxygen species RUBCN RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein SRBC sheep red blood cells TLR toll-like receptor ULK1 unc51-like kinase 1
[8]
[9]
[10]
[11]
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
[12]
Acknowledgments We are grateful to Herbert Virgin, Donna MacDuff and Yating Wang for generously supplying bones from Atg5flox/flox-Lyz2creC and Atg7flox/floxLyz2creC mice and their respective controls for these studies. We thank members of the Brumell laboratory for discussions and Ju Huang for critical review of the manuscript. We also thank Helen Sarantis for help with LysoBright assay, Nicolas Pillon for help with real time PCR, Michael Woodside and Paul Paroutis for help with confocal microscopy.
[13]
[14]
Funding This work was supported by an operating grant from the Arthritis Society of Canada (to J.H.B) and NSERC PGS-D scholarship and a CIHR Training Fellowship (TGF-53877) (to M.C.).
[15]
[16]
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[22] Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007; 3:542-5; PMID:17611390; http://dx.doi.org/ 10.4161/auto.4600 [23] Savina A, Jancic C, Hugues S, Guermonprez P, Vargas P, Moura IC, Lennon-Dumenil AM, Seabra MC, Raposo G, Amigorena S. NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 2006; 126:205-18; PMID:16839887; http://dx.doi.org/10.1016/j.cell.2006.05.035 [24] Mantegazza AR, Savina A, Vermeulen M, Perez L, Geffner J, Hermine O, Rosenzweig SD, Faure F, Amigorena S. NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells. Blood 2008; 112:4712-22; PMID:18682599; http://dx. doi.org/10.1182/blood-2008-01-134791 [25] Rybicka JM, Balce DR, Khan MF, Krohn RM, Yates RM. NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes. Proc Natl Acad Sci U S A 2010; 107:10496-501; PMID:20498052; http://dx.doi.org/10.1073/pnas.0914867107 [26] Rybicka JM, Balce DR, Chaudhuri S, Allan ER, Yates RM. Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner. EMBO J 2012; 31:932-44; PMID:22157818; http://dx.doi.org/10.1038/emboj.2011.440 [27] Allan ER, Tailor P, Balce DR, Pirzadeh P, McKenna NT, Renaux B, Warren AL, Jirik FR, Yates RM. NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing. J Immunol 2014; 192:4989-5001; PMID:24778444; http://dx.doi.org/10.4049/jimmunol.1302896 [28] Canton J, Khezri R, Glogauer M, Grinstein S. Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages. Mol Biol Cell 2014; 25:3330-41; PMID:25165138; http://dx. doi.org/10.1091/mbc.E14-05-0967
[29] Yang CS, Lee JS, Rodgers M, Min CK, Lee JY, Kim HJ, Lee KH, Kim CJ, Oh B, Zandi E, et al. Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation. Cell Host Microbe 2012; 11:264-76; PMID:22423966; http://dx.doi.org/10.1016/j. chom.2012.01.018 [30] Sokolovska A, Becker CE, Ip WK, Rathinam VA, Brudner M, Paquette N, Tanne A, Vanaja SK, Moore KJ, Fitzgerald KA, et al. Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function. Nat Immunol 2013; 14:543-53; PMID:23644505; http://dx.doi.org/10.1038/ ni.2595 [31] Noubade R, Wong K, Ota N, Rutz S, Eidenschenk C, Valdez PA, Ding J, Peng I, Sebrell A, Caplazi P, et al. NRROS negatively regulates reactive oxygen species during host defence and autoimmunity. Nature 2014; 509:235-9; PMID:24739962; http://dx.doi.org/10.1038/ nature13152 [32] Kim BH, Shenoy AR, Kumar P, Das R, Tiwari S, MacMicking JD. A family of IFN-gamma-inducible 65-kD GTPases protects against bacterial infection. Science 2011; 332:717-21; PMID:21551061; http://dx.doi.org/10.1126/science.1201711 [33] Gardet A, Benita Y, Li C, Sands BE, Ballester I, Stevens C, Korzenik JR, Rioux JD, Daly MJ, Xavier RJ, et al. LRRK2 is involved in the IFN-gamma response and host response to pathogens. J Immunol 2010; 185:5577-85; PMID:20921534; http://dx.doi.org/10.4049/ jimmunol.1000548 [34] Lam GY, Cemma M, Muise AM, Higgins DE, Brumell JH. Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection. Autophagy 2013; 9:985-95; PMID:23584039; http://dx.doi.org/ 10.4161/auto.24406
BASIC BRIEF REPORT
Autophagy proteins are not universally required for phagosome maturation Marija Cemmaa,b, Sergio Grinsteina,c,d,e, and John H. Brumella,b,e,f a Cell Biology Program, Hospital for Sick Children, Toronto, ON Canada; bDepartment of Molecular Genetics and Biochemistry, University of Toronto, Toronto, ON Canada; cDepartment of Biochemistry, University of Toronto, Toronto, ON Canada; dKeenan Research Center of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON Canada; eInstitute of Medical Science, University of Toronto, Toronto, ON Canada; fSick Kids IBD Center, Hospital for Sick Children, Toronto, ON Canada
ABSTRACT
ARTICLE HISTORY
Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcgR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.
Received 7 October 2015 Revised 9 May 2016 Accepted 16 May 2016
Introduction Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway that targets long-lived proteins, damaged organelles, and pathogens for lysosomal degradation in eukaryotic cells and requires more than 30 autophagy-related (ATG) proteins.1 A hallmark of canonical autophagy is that the cargo is sequestered into a double-membrane compartment (the phagophore, which matures into an autophagosome), which is decorated by MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). LC3 is a ubiquitin-like molecule that is covalently conjugated to phosphatidylethanolamine on phagophore membranes through a series of reactions similar to ubiquitin conjugation and requires a subset of ATG proteins, including ATG5 and ATG7.2 A number of ATG proteins have been implicated in having alternative (noncanonical) functions.3 A seminal study by Sanjuan et al. revealed that LC3 can be conjugated to the singlemembrane phagosome upon stimulation of TLR2 (toll-like receptor 2) and TLR4 in a process termed LC3-associated phagocytosis (LAP).4 These receptors initiate reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase, an event that is required for LAP.5,6 In fact, intraphagosomal ROS in the absence of other signals is sufficient to recruit LC3 to phagosomes.6 Other receptors implicated in LAP, such as FCGR2A/FcgR2A and CLEC7A/dectin-1, also result in CYBB/NOX2-dependent ROS production.5,7 LAP is triggered in response to particles that stimulate TLRs, FCGR2A/FcgR2A,
KEYWORDS
autophagy; ATG8/LC3; ATG5; ATG7; LC3-associated phagocytosis; phagosome maturation
CLEC7A/dectin-1, and other cargo such as apoptotic and entotic cells.4-10 In addition to ROS, LAP requires a subset of autophagy components, such as ATG5, ATG7, ATG12, ATG16L1, BECN1/Beclin 1, and PIK3C3/VPS34, whereas others, such as ULK1 (unc51-like kinase 1), ATG13, and RB1CC1/FIP200 are dispensable.4,6 LC3 conjugation to a phagosome is observed in hematopoietic cell types, including macrophages, dendritic cells, and neutrophils.4-12 It has also been observed in retinal pigment epithelial cells,13,14 which are macrophage-like and expresses some hematopoietic markers.15 The role of LAP in nonhematopoietic cell types is not yet clear. One of the major functions ascribed to LAP is to promote phagosome maturation. This conclusion is based on the observation that autophagy-deficient murine macrophages (RAW264.7 and CSF2/GM-CSF-differentiated BMDMs) fail to degrade live yeast4 and apoptotic cells,9 and phagosomes containing beads coated with the synthetic triacylated lipopeptide PAM3CSK4 or zymosan exhibit delayed phagosome fusion with the lysosome.4 However, a study in human macrophages observed quite the opposite—LC3 recruitment to zymosan-containing phagosomes was associated with delayed phagosomal fusion with the lysosome.10 Therefore, the impact of LC3 recruitment to phagosomes remains unclear. Hence, we explored how the phagosome maturation is affected in the absence of ATG proteins. We used autophagy-deficient mouse embryonic fibroblasts (MEFs), as well as CSF1/M-CSF- and CSF2-differentiated BMDMs to assess phagosome maturation of IgG-coated zymosan and sheep red blood cells (SRBC) using 3 distinct assays. We did not find a
CONTACT John H. Brumell [email protected] Cell Biology Program, Hospital for Sick Children, PGCRL, 686 Bay Street, Toronto, ON M5G0A4, Canada. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
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requisite role for Atg5 or Atg7 in phagosome maturation, challenging the notion that autophagy proteins are universally required for phagosome maturation.
Results Phagosomes in Atg5-deficient MEFs mature normally upon FCGR2A stimulation To examine if autophagy machinery is required for phagosome maturation in fibroblasts, we performed 2 phagosome maturation assays using wild-type (WT) and Atg5-deficient MEFs. It was previously established that heterologous transfection of fibroblastic cells with the FCGR2A receptor confers phagocytic capacity and results in phagosome maturation similar to that observed in professional phagocytes.16,17 As expected, FCGR2A receptor-transfected WT and Atg5-deficient fibroblasts were able to internalize antibody-opsonized particles and acquire phagosome maturation markers. LAMP1 is localized to lateendosomes and lysosomes and is commonly used as a phagosome maturation marker. FCGR2A-transfected MEFs were challenged with IgG-opsonized zymosan particles (OpZ) for 30, 60, 90, and 120 min, then stained for LAMP1 and OpZ. In both WT and autophagy-deficient MEFs, OpZ particles acquired LAMP1 with similar kinetics; nearly all became LAMP1-positive at 120 min after the challenge (Fig. 1A and B). No significant difference in LAMP1 acquisition was observed between WT and Atg5-deficient fibroblasts. We next explored if phagosome acidification might be impaired in Atg5-deficient fibroblasts. To visualize acidic compartments such as lysosomes and mature phagosomes, we used LysoBrite, a fluorescent indicator that permeates cell membranes and accumulates in acidic compartments. FCGR2A-transfected MEFs were incubated with LysoBrite for 30 min and then challenged with antibody-coated SRBC. Anti-SRBC antibody was used to opsonize SRBC in order to facilitate its phagocytosis via FCGR2A. Cells were imaged live at 30, 60 and 90 min following phagocytosis. We observed no significant difference in the percentage of LysoBrite-positive phagosomes between WT and Atg5-deficient fibroblasts (Fig. 1C and D). Hence, we conclude that ATG5 does not play a role in FCGR2A-mediated phagosome maturation in murine fibroblasts. Phagosomes in autophagy-deficient BMDM mature normally Next, we sought to determine the role of autophagy proteins in primary murine bone marrow-derived macrophages (BMDMs). To ensure that our findings are of relevance to most research groups, we have utilized 2 commonly used methods to differentiate macrophages, using either CSF1- or CSF2-containing media. Since conventional knockout of Atg518 or Atg719 causes neonatal lethality, we used mice where Atg5 or Atg7 was selectively deleted from monocytes/macrophages and granulocytes. This was achieved by crossing Atg5flox/flox or Atg7flox/flox (referred to here as WT) mice with mice expressing the Cre recombinase from the endogenous lysozyme 2/M locus (Lyz2creC).20,21 In all cases we confirmed gene knockout using
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RT-PCR (Fig. S1A-D). As expected, autophagy-deficient macrophages were unable to process the cytosolic form of LC3 (LC3-I) into the lipidated (and membrane-associated) form of LC3 (LC3-II) as demonstrated by the disappearance of the LC3-II band via western blot (Fig. S1E and F).22 To investigate if autophagy proteins are required for acquisition of LAMP1, we challenged Atg5- or Atg7-deficient CSF1-differentiated macrophages and respective control macrophages with OpZ and quantified the percentage of LAMP1positive phagosomes at 30, 60, 90, and 120 min post challenge. LAMP1 acquisition by phagosomes followed the same kinetics in WT and autophagy-deficient CSF1-differentiated macrophages and significant differences were not observed (Fig. 2A– C). This suggests that ATG proteins are not required for and do not play a significant role in LAMP1 acquisition during phagocytosis of OpZ by CSF1-cultured BMDMs. The same results were observed in CSF2-differentiated macrophages (Fig. S2A, B). We also assessed phagosome maturation using unopsonized zymosan particles (not coated with IgG as above) in WT and Atg5-deficient CSF1- and CSF2-differentiated BMDMs. Phagosomes containing zymosan particles matured at comparable rates in WT and autophagy-deficient macrophages (Fig. S3A and B). Next, we assessed phagosome acidification in control and autophagy-deficient macrophages. To this end, we quantified the percentage of LysoBriteC SRBC-containing phagosomes. As with LAMP1 acquisition, no significant difference was observed between the controls and Atg5- or Atg7-deficient CSF1-cultured macrophages (Fig. 2D–F). This indicates that ATG proteins do not play a significant role in acidification of SRBC-containing phagosomes in CSF1-cultured BMDMs. We have further confirmed these findings in CSF2-differentiated macrophages (Fig. S2C and D). Finally, we explored if autophagy proteins might play a role in conferring the phagosome its degradative properties in CSF1-cultured macrophages by using DQ-BSA dye. DQ-BSA is a self-quenched BODIPY dye conjugate of bovine serum albumin (BSA). The proteolytic digestion of BSA in the lysosome results in dequenching of fluorescence, enabling the visualization of proteolytic compartments. CSF1-differentiated BMDMs were pulsed with DQ-BSA for 1 h, chased for 1 h in regular medium and then challenged with antibody-coated SRBC for 30, 60 or 90 min. Phagosomes in Atg5- and Atg7-deficient BMDMs acquired the capacity to cleave DQ-BSA with similar kinetics as their respective controls (Fig. 2G–I). We have further confirmed these findings in the CSF2-differentiated macrophages and again saw no impact of ATG proteins on phagosome maturation (Fig. S2E, F). In summary, using 3 distinct phagosome maturation assays, we were unable to detect a significant difference in phagosome maturation of antibodyopsonized particles between control and autophagy-deficient CSF1- and CSF2-differentiated BMDMs.
Discussion Our findings challenge the notion that LC3 recruitment is required for phagosome maturation. The difference between our observations and previously reported data4,6,9 could be due to the fact that different phagocytic particles were used in each
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Figure 1. Phagosome maturation of IgG-coated particles is unaffected in atg5¡/¡ MEFs. WT and atg5¡/¡ MEFs were transfected FCGR2A-GFP (green) to render them phagocytic. (A, B) MEFs were challenged with OpZ and fixed at 30, 60, 90, or 120 min post challenge, then stained for LAMP1 (red) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMPC particle marked by an arrow. (B) The percentage of LAMP1C OpZ was enumerated in WT and atg5¡/¡ MEFs. At least 200 OpZ were counted per condition. (C, D) MEFs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (C). A representative image is shown at 60 min post challenge with a LysoBriteC particle marked by an arrow. (D) The percentage of LysoBriteC SRBC was enumerated in WT and atg5¡/¡ MEFs. At least 50 SRBC were counted per condition. Scale bar: 8 mm.
study. We used antibody-coated particles to engage the FCGR2A and also unopsonized zymosan particles. Previous studies used zymosan, PAM3CSK4- and LPS-coated beads, Escherichia coli4 and dead cells.9 Another difference is that our macrophages do not overexpress GFP-LC3, as was the case in some prior studies.4,9 Variability in specific reagents during the macrophage differentiation protocol may also underlie the observed differences. We do not regard the LAP model as false, but rather make the point that it is not universally applicable; phagosome maturation, a complex and dynamic process, may have a requirement for ATG proteins under specific conditions. An important consideration when interpreting these discordant findings may involve the manner whereby the CYBB NADPH oxidase affects phagosome maturation. We previously showed that ROS production by the CYBB NADPH oxidase is required for LC3 recruitment to phagosomes,5 an observation confirmed by others.6,10 In the present study we examined phagosome maturation in the
absence of CYBB-dependent ROS production. Despite the fact that CYBB is required for LAP,5,6,10 OpZ phagosomes matured faster in the absence of intraphagosomal ROS than in the WT counterparts (Fig. S3C). This suggests 2 things: first, phagosome maturation does not always require LAP and, second, CYBB NADPH oxidase may actually impair phagosome maturation under these conditions. Indeed, CYBB has a controversial role in modulating phagosome maturation; some investigators find that it delays phagosomal maturation,23,24 whereas others do not.25-27 Recent studies showed that the maturation status of macrophages dictates the effects that the CYBB NADPH oxidase has on phagosome maturation.28 Based on these findings, we hypothesize that both CYBB and ATG proteins, which are linked in phagosomal development, may have effects on phagosome maturation that are condition-dependent and possibly dynamic—altered by status of the macrophages, e.g., in classically activated macrophages (M1) vs.
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Figure 2. Phagosome maturation of IgG-coated particles is unaffected in autophagy-deficient CSF1-differentiated BMDMs. (A, B, C) CSF1-derived BMDMs were challenged with OpZ and fixed at 30, 60, 90, or 120 min, then stained for LAMP1 (green) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMPC particle marked by an arrow. The percentage of LAMP1C OpZ was enumerated in WT and atg5¡/¡ (B) and WT and atg7¡/¡ (C) BMDMs. At least 300 OpZ were counted per condition. (D, E, F) CSF1-derived BMDMs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (D). A representative image is shown at 60 min post challenge with a LysoBriteC particle marked by an arrow. The percentage of LysoBriteC SRBC was enumerated in WT and atg5¡/¡ (E) and WT and atg7¡/¡ (F) BMDMs. At least 50 SRBC were counted per condition. (G, H, I) CSF1-derived BMDMs were pulsed with DQ-BSA (red) for 1 h, chased for 1 h in regular medium, then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (G). A representative image is shown at 60 min post challenge with a DQ-BSAC particle marked by an arrow. The percentage of DQ-BSAC SRBC was enumerated in WT and atg5¡/¡ (H) and WT and atg7¡/¡ (I) BMDMs. At least 50 SRBC were counted per condition. Scale bar: 8 mm.
alternatively activated macrophages (M2). An additional layer of complexity is added by the expression of CYBB NADPH oxidase regulators, such as RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1interacting protein). RUBCN is required for LAP6 and is upregulated in response to TLR2 activation.29 Thus, the choice of the phagocytic particle also affects the amount of ROS produced and the destiny of the phagosome. It is
possible that other positive and negative regulators of the CYBB NADPH oxidase also play a role in LAP.30-33 Further studies are required to reveal the reason why ATG proteins are important in phagosome maturation under some conditions, but not others. Regardless of the specific reason, we have shown that in our system ATG proteins are not required for conventional phagosome maturation. Hence, we posit that LAP is not universally required for phagosome fusion with
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endosomes and lysosomes. Instead the relationship between ATG proteins and phagosome maturation is more complex and likely involves other players.
Materials and methods Cells Mouse embryonic fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (HyClone, SH3024301) supplemented with 10% fetal bovine serum (FBS; Wisent, 090-510) at 37 C in 5% CO2 without antibiotics. Atg5flox/flox (WT), Atg5flox/ flox -Lyz2creC (atg5¡/¡), Atg7flox/flox (WT) and Atg7flox/floxLyz2creC (atg7¡/–) mice were previously described.20,21 Bone marrow-derived macrophages were extracted as previously described.34 CSF1-differentiated BMDMs were cultured in RPMI-1640 medium (Wisent, SH3002701) supplemented with 10% FBS, 5% sodium pyruvate (Invitrogen, 11360-070), 5% penicillin/strep (Invitrogen, 15140122), 5% nonessential amino acids (Invitrogen, 11140050), 0.5 mM 2-ME (Invitrogen, 21985023) and 10% CSF1-conditioned medium from NIH3T3 cells. CSF2-differentiated BMDM were cultured in RPMI supplemented with 10% FBS, 5% penicillin/strep, and nonessential amino acids in the presence of 20 ng/ml murine CSF2 (R&D Systems, 415-ML-010). Phagocytic particles were added in RPMI-1640, cells were washed 3 times with phosphate-buffered saline (Multicell, 311-420-CL) at a 10-min time point after the beginning of phagocytosis, and incubated in medium without antibiotics. Images were taken in RPMI-1640 supplemented with 10 mM HEPES buffer (Life Technologies, 15630-080).
LysoBrite assay, cells were incubated with LysoBrite (AAT Bioquest, 22659) for 30 min, as per the manufacturers’ instructions. For the DQ-BSA assay, cells were incubated with 10 mg/ ml of DQ-BSA (Life Technologies, D12051) for 1 h, followed by 1-h incubation in complete medium. Phagocytosis was synchronized by spinning sRBC at 170 £ g for 5 min onto cells. The cells were imaged live at 30, 60, and 90 min time points after beginning of phagocytosis. Imaging system Images were acquired using a Wave-FX-X1 Spinning Disc Confocal, Leica DM16000B inverted research microscope with Hamamatsu ImagEMx2 (EMCCD) camera using a 63x objective. Western blotting Western blotting was performed as previously described5 using LC3 (Novus, NB600-1384) and GAPDH (Millipore, MAB374) antibodies. RNA isolation and quantitative PCR RNA was isolated using the RNeasy kit (QIAGEN, 74104), and cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708840). Ten nanograms per reaction were used for quantitative RT-PCR using predesigned Taqman probes for target genes and Hprt as housekeeping reference (Life Technologies, 4448489).
LAMP1 phagosome maturation assay
Statistical analysis
Zymosan particles (Sigma, Z4250) were opsonized by overnight incubation in 6 mg/mL human IgG (Meridian Life Sciences, A50170H) at 4 C. For unopsonized particles, Zymosan A, S. cerevisiae BioParticles, Texas Red conjugate (Molecular Probes, Z2843) were used. On d 1, MEFs were seeded on glass coverslips in 24-well tissue culture plates at 2.5 £ 104 cells/well, and, on d 2, transfected with FCGR2A-GFP construct using GeneJuice (Novagen, 70967-3) as per the manufacturers’ instructions. On d 3, phagocytosis was synchronized by spinning opsonized zymosan (OpZ) at 170 £ g for 5 min onto cells. BMDMs were seeded on glass coverslips in 24-well tissue culture plate at 2.0 £ 105 cells/well, and, on the following day, phagocytosis was synchronized by spinning OpZ at 170 £ g for 5 min. Cells were then fixed with 2.5% paraformaldehyde (EMS, 15710) at 30, 60, 90, or 120 min and stained for OpZ and LAMP1 (Developmental Studies Hybridoma Bank at the University of Iowa, 1D4B).
For quantification studies, at least 50 particles were enumerated for the LysoBrite or DQ-BSA assays and at least 300 particles were enumerated for the LAMP1 phagosome maturation assay for each condition in each experiment, unless otherwise indicated. At least 3 independent experiments were performed for each graph, unless otherwise indicated. The mean § SEM is shown in the figures. Statistical significance was determined using a 2-tailed, 2-sample, unequal variance Student t test with an a level of 0.05.
LysoBrite and DQ-BSA phagosome maturation assays SRBC (MP Biomedicals, 55876) were opsonized by 1-h incubation with rabbit anti-sRBC antibody (MP Biologicals, 55806) at room temperature. Cells were seeded on Ibidi microscopy chambers (80827). MEFs were seeded at 1.0 £ 104 cells/chamber and transfected with FCGR2A-GFP using GeneJuice. BMDMs were seeded at 6.0 £ 104 cells/chamber. For the
Abbreviations Atg ATG5 ATG7 BMDM CLEC7A CSF1/M-CSF CSF2/GM-CSF CYBB/NOX2 DQ-BSA GFP LAMP1 LAP LPS
autophagy related autophagy-related 5 autophagy-related 7 bone marrow-derived macrophages C-type lectin domain family 7 member A colony stimulating factor 1 (macrophage) colony stimulating factor 2 (granulocytemacrophage) cytochrome b-245, beta polypeptide dye quenched-bovine serum albumin green fluorescent protein lysosomal-associated membrane protein 1 LC3-associated phagocytosis lipopolysaccharide
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Lyz2cre
Cre recombinase from the endogenous lysozyme 2/M locus MAP1LC3/LC3 microtubule-associated protein 1 light chain 3 MEFs mouse embryonic fibroblasts NADPH nicotinamide adenine dinucleotide phosphate-oxidase OpZ opsonized zymosan PIK3C3/VPS34 phosphoinositide-3-kinase, class 3 RB1CC1 RB1-inducible coiled-coil 1 ROS reactive oxygen species RUBCN RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein SRBC sheep red blood cells TLR toll-like receptor ULK1 unc51-like kinase 1
[8]
[9]
[10]
[11]
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
[12]
Acknowledgments We are grateful to Herbert Virgin, Donna MacDuff and Yating Wang for generously supplying bones from Atg5flox/flox-Lyz2creC and Atg7flox/floxLyz2creC mice and their respective controls for these studies. We thank members of the Brumell laboratory for discussions and Ju Huang for critical review of the manuscript. We also thank Helen Sarantis for help with LysoBright assay, Nicolas Pillon for help with real time PCR, Michael Woodside and Paul Paroutis for help with confocal microscopy.
[13]
[14]
Funding This work was supported by an operating grant from the Arthritis Society of Canada (to J.H.B) and NSERC PGS-D scholarship and a CIHR Training Fellowship (TGF-53877) (to M.C.).
[15]
[16]
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