Regulates Phagolysosome Biogenesis the Protein Tyrosine Phosphatase SHP-1

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The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis Carolina P. Gómez, Marina Tiemi Shio, Pascale Duplay, Martin Olivier and Albert Descoteaux This information is current as of September 29, 2021. J Immunol 2012; 189:2203-2210; Prepublished online 23 July 2012; doi: 10.4049/jimmunol.1103021 http://www.jimmunol.org/content/189/5/2203 Downloaded from

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

Carolina P. Go´mez,*,† Marina Tiemi Shio,†,‡,x Pascale Duplay,* Martin Olivier,†,‡,x and Albert Descoteaux*,†

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1–deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1–deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa pro- Downloaded from cathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis. The Journal of Immunology, 2012, 189: 2203–2210. http://www.jimmunol.org/

hagocytosis is the process by which cells internalize large (6). Through these interactions, phagosomes acidify and sequen- particulate materials from their milieu. In contrast to en- tially acquire an array of hydrolases culminating in the generation P docytosis, phagocytosis involves clathrin-independent mecha- of a highly hydrolytic environment (7). This process takes place nisms and requires actin polymerization. Internalization is triggered rapidly, as within a few hours newly formed phagosomes acquire by the interaction of ligands on the target particles with surface phagolysosomal functionality (8). receptors of phagocytic cells. The nascent phagosome is formed Tyrosine (Tyr) phosphorylation is a critical mechanism for the to a large extent by invagination of the plasma membrane (1). control of numerous physiological processes in eukaryotes (9), The particles are surrounded by pseudopods and, following inter- including cytoskeletal function, signal transduction, intracellular by guest on September 29, 2021 nalization, they end up in a vacuole, the phagosome. The newly traffic, and immune defense (9, 10). Phosphotyrosine signaling formed phagosome rapidly matures into a phagolysosome where is regulated by the dynamic interplay of three distinct functional the internalized material is degraded. Phagolysosome biogenesis modules, as follows: protein tyrosine kinases, protein tyrosine is a dynamic process, requiring the coordinated action of multiple phosphatases (PTP), and Src homology 2 domains (11). In met- signaling molecules that regulate the interactions between the azoans, the multiple possible combinations of these modules il- phagosome and the cytoskeleton, and the endosomal/lysosomal lustrate the complexity of phosphotyrosine signaling, which can compartments (2–5). These interactions are transient and are regulate cellular functions either positively or negatively. A recent characterized by the formation of small fusion pores between the large-scale quantitative proteomic and phosphoproteomic analysis two organelles, allowing the exchange of membrane and contents revealed that Tyr phosphorylation sites are overrepresented in phagosomes (12), as compared with global phosphoproteome (13). This finding suggested that Tyr phosphorylation plays an impor- *Institut National de la Recherche Scientifique–Institut Armand-Frappier, Laval, tant role in phagosome signaling. Given their role in controlling † Quebec H7V 1B7, Canada; Center for Host-Parasite Interactions, Laval, Quebec Tyr phosphorylation levels (10, 11), PTPs are expected to partic- H7V 1B7, Canada; ‡Department of Medicine, Centre for the Study of Host Resis- tance, Research Institute of McGill University Health Centre, Montreál, Quebec H3A ipate in the regulation of phagosome function. Interestingly, x 2B4, Canada; and Department of Microbiology and Immunology, McGill University, characterization of the nonreceptor PTP MEG2 in neutrophils ´ Montreal, Quebec H3A 2B4, Canada revealed that it is present on the cytoplasmic face of secretory Received for publication October 19, 2011. Accepted for publication June 25, 2012. vesicles, and is recruited to nascent phagosomes (14). A physio- This work was supported by the Natural Science and Engineering Research Council logical substrate for PTP-MEG2 is the vesicle fusion protein N- of Canada (to A.D.) and by a New Initiative Fund from the Center for Host-Parasite Interactions (to A.D. and M.O.). A.D. is the holder of a Canada Research Chair, and ethylmaleimide–sensitive factor (NSF), the dephosphorylation of C.P.G. was partly supported by a studentship from the Fondation Armand-Frappier. which stimulates the homotypic fusion of secretory vesicles (15). Address correspondence and reprint requests to Dr. Albert Descoteaux, Institut Na- Additional PTP-MEG2–interacting proteins are associated with tional de la Recherche Scientifique–Institut Armand-Frappier, 531 Boulevard des vesicle traffic, small GTPases, and lipid interaction (16). However, Prairies, Laval, QC H7V 1B7, Canada. E-mail address: [email protected] a role for PTP-MEG2 or any other PTPs in the regulation of phag- Abbreviations used in this article: BMM, bone marrow-derived macrophage; EEA1, early endosome Ag 1; LAMP-1, lysosomal-associated membrane protein 1; LT, osome maturation remains to be demonstrated. LysoTracker; NSF, N-ethylmaleimide–sensitive factor; PTP, protein tyrosine phos- The nonreceptor PTP Src homology region 2 domain-containing phatase; SHP-1, Src homology region 2 domain-containing phosphatase 1; TC-PTP, phosphatase 1 (SHP-1) is expressed at highest levels in hemopoietic T cells–PTP; Tyr, tyrosine; v-ATPase, vacuolar-type H+-ATPase; WT, wild-type. cells. In macrophages, SHP-1 participates in the regulation of Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 various processes, including proinflammatory and innate immune www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103021 2204 SHP-1 REGULATES PHAGOLYSOSOME BIOGENESIS responses (17–20). SHP-1 has previously been shown to associate Phagocytosis assays with phagosomes (21, 22), thereby suggesting a role in the bio- Zymosan (Sigma-Aldrich) and latex beads (3 mm; Polyscience) were genesis of phagolysosomes. In this study, we present evidence that opsonized with mouse serum. For synchronized phagocytosis assays, cells SHP-1 plays a role in phagosome maturation and that it is required were incubated with particles at a particle-to-cell ratio of 5:1 for 10 min at for acidification of this organelle. 4˚C. Excess particles were removed, and phagocytosis was triggered by transferring the cells to 37˚C for the indicated time points before pro- ceeding for microscopy. Materials and Methods Microscopy and immunofluorescence Cell culture Macrophages were fixed and permeabilized using 0.1% Triton X-100, and All animals were handled in strict accordance with good animal practice as nonspecific surface FcgR binding was blocked using 1% BSA, 2% goat defined by the Canadian Council on Animal Care, and all animal work was serum, 6% milk, and 50% FBS (26). Particle internalization was quantified approved by the Comite´ Institutionel de Protection des Animaux of Institut by immunofluorescence microscopy. Quantification of phagosomal re- National de la Recherche Scientifique–Institut Armand-Frappier. Bone cruitment was performed as follows. Slides were prepared with three marrow-derived macrophages (BMM) were obtained by extracting the coverslips per time point for each experiment, and 100 phagosomes were femur and tibias from 8- to 10-wk-old female BALB/c mice (Charles River counted per coverslip for a total of 300 phagosomes per experiment. Laboratories, QC, Canada) at 37˚C in 5% CO2 for 7 d in DMEM with Experiments were repeated a minimum of three times. For distribution and L-glutamine (Life Technologies) supplemented with 10% heat-inactivated colocalization experiments, anti-mouse AlexaFluor 488, anti-rabbit Alex- FBS (Hyclone, Logan, UT), 10 mM HEPES (pH 7.4), and antibiotics aFluor 488, anti-rat AlexaFluor 488, or anti-rat AlexaFluor 568 (Molecu- (complete medium) in the presence of 15% (v/v) L929 cell-conditioned lar Probes) was used. DRAQ5 (Biostatus, Leicestershire, U.K.) was used medium as a source of CSF-1. SHP-1–deficient (C3HeBFeJ me/me, me-3, to visualize cells’ nuclei. All coverslips were mounted on slides with and me-5) and wild-type (WT) immortalized BMM were previously de- Fluoromount-G (Southern Biotechnology Associates). Detailed analysis of Downloaded from scribed (23) and were grown in complete medium at 37˚C in 5% CO2. protein presence and localization on the phagosome was performed using an oil immersion Nikon Plan Apo 100 (N.A. 1.4) objective mounted on Phagosome preparation and isolation a Nikon Eclipse E800 microscope equipped with a Bio-Rad Radiance 2000 confocal imaging system (Bio-Rad, Zeiss). Images were obtained using Adherent macrophages (2 3 107 per 150 3 20-mm tissue culture dishes) appropriate filters with a Kalman filter of at least 3. To quantify the in- were incubated with magnetic beads (3 mm average diameter, 2.5% w/v tensity of SHP-1 recruitment to phagosomes, a line spanning representative suspension; Spherotech) diluted in 1:50 in 10 ml complete medium at 37˚C phagosomes and the surrounding cytoplasm was manually traced with http://www.jimmunol.org/ for 1, 2, or 4 h. Cells were then washed with cold PBS at 4˚C and scraped a one-pixel width, and fluorescence intensity profile was represented in in cold PBS. Cells were resuspended in purification buffer (5 mM EDTA a graph for each phagocytosis time point. Intensity of the signal was [pH 8.0] and PBS, with a final pH of 7.2), lysed by aspirations through a 22 measured using the MetaMorph software package. G needle, and then transferred into microfuge tubes. Phagosomes were isolated with a magnet. Phagosome acidification Western blots Cells were preloaded with the lysosomotropic agent LysoTracker (LT) Red (Molecular Probes) diluted in DMEM (1:1000) for 2 h at 37˚C. Cells were Cells and purified phagosomes were lysed in ice-cold lysis buffer (20 mM washed, and synchronized phagocytosis was performed for indicated time Tris-HCl [pH 7.5], 150 mM NaCl, and 1% Nonidet P-40) containing points at 37˚C, as described above. Cells were then rinsed, fixed with 2% protease (cOmplete EASYpacks; Roche Diagnostics, Mannheim, Ger- paraformaldehyde for 10 min, washed, and directly mounted for confocal by guest on September 29, 2021 many) and phosphatase inhibitors (Na3VO4 and NaF). Phagosomal and analysis. total cell lysate proteins were separated by SDS-PAGE and transferred onto Hybond-ECL membranes (Amersham Biosciences, QC, Canada), and Statistical analyses immunodetection was achieved by chemiluminescence (Amersham Bio- sciences). Unless specified otherwise, Western blots were performed with Statistical analyses were performed using the statistical tools provided in 15 mg total cell lysates and phagosome lysates. GraphPad prism software. SDs were calculated from three independent experiments. Antibodies Results The rat mAb against lysosomal-associated membrane protein 1 (LAMP-1) developed by J. August (1D4B) was obtained through the Developmental SHP-1 is recruited to nascent phagosomes and remains Studies Hybridoma Bank at the University of Iowa and the National Institute throughout the maturation process of Child Health and Human Development. The rabbit Ab against flotillin-2 SHP-1 has previously been shown to associate with phagosomes, was from Cell Signaling; the mouse Ab against SHP-1 was from Millipore; and the mouse Ab against early endosome Ag 1 (EEA1) was from BD raising the possibility that it participates in the biogenesis of Transduction Laboratories. The Abs against PTP-1B and T cells–PTP (TC- phagolysosomes (21, 22). To investigate a potential role for SHP-1 PTP) (rabbit and mouse, respectively) were donated by M. Tremblay in this process, we first performed confocal immunofluorescence (McGill University, QC, Canada). The rabbit Ab against Rab7 was from experiments to visualize SHP-1 redistribution in murine BMM Santa Cruz Laboratories. Rabbit Abs against phospho–Dok-1 (Y362) were upon phagocytosis of serum-opsonized zymosan particles (27– from ECM Biosciences. Abs directed against the C-terminal region of murine Dok-1 were produced in rabbits using a GST fusion protein 29). As shown in Fig. 1A, SHP-1 was detectable on nascent encompassing aa 252–482 of murine Dok-1 (a gift of J. Nunes, Institut phagosomes (15 min) and remained associated to the phagosome Paoli-Calmettes, Marseille, France). Anti–Dok-1 Abs were affinity purified throughout the maturation process (up to 4 h after the initiation on GST columns. The rabbit antiserum against cathepsin D was from of phagocytosis). Quantification of the kinetics of SHP-1 phag- Upstate. Finally, the rabbit Ab against the 16-kDa c subunit from the + osomal association (Fig. 1B) revealed that, by 30 min, 50% of V0 sector of the vacuolar-type H -ATPase (v-ATPase) was donated by M. Skinner (University of Guelph, ON, Canada) (24). phagosomes were positive for SHP-1. From 1 h postphagocytosis, SHP-1 was present on .80% of phagosomes (Fig. 1B). These In-gel PTP assay results confirmed the early recruitment and presence of SHP-1 Macrophages were lysed, as described previously, for phagosome isolation throughout the maturation process. To further analyze the phag- without addition of sodium orthovanadate to the lysis buffer. Cell lysate osomal association/recruitment of SHP-1, we purified phagosome controls (25 mg) were obtained using a PTP lysis buffer (50 mM Tris [pH extracts using magnetic beads at different time points after the 7.0], 0.1 mM EDTA, 0.1 mM EGTA, 0.1% 2-ME, 1% Nonidet P-40, 1 mM initiation of phagocytosis (26, 30). To this end, we used immor- Na3VO4, and protease inhibitors). Samples were loaded on a gel containing a g-[32P]–labeled poly(Glu4Tyr) peptide (Sigma-Aldrich, ON, Canada), talized bone marrow macrophages derived from SHP-1–deficient and the SHP-1 band was observed by in-gel PTP assay, as previously motheaten mice (me-3) and their control WT littermates (25). described (25). Purified phagosome extracts of various ages (15 min, 1 h, and 2 h) The Journal of Immunology 2205 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Active PTPs are present in phagosome extracts. (A)SHP-1 is present in WT phagosome extracts isolated from WT cells at various A FIGURE 1. SHP-1 is recruited to phagosomes. ( ) BMM were allowed time points after the start of internalization. Western blot analysis was to internalize serum-opsonized zymosan, fixed, and stained for SHP-1 at performed on total cell and magnetic bead–phagosome lysates. SHP-1– different time points after the initiation of internalization. Scale bar, 3 mm. deficient me-3 cell lysates were used as a control. The arrow shows SHP- A line spanning representative phagosomes and the surrounding cytoplasm 1. For total cell lysates, 15 mg protein represents an equivalent of 105 was manually traced with a one-pixel width, and the fluorescence intensity cells. For purified phagosome lysates, 15 mg protein represents an profile of SHP-1 was represented in a graph for each phagocytosis time equivalent of 1.5 3 106 cells. (B) Active SHP-1, TC-PTP, and PTP-1B B point. Arrows show the position of the phagosomes. ( ) SHP-1 acquisition are present in phagosome extracts. In-gel PTP assay analysis was per- was determined on at least 300 phagosomes for each condition and formed on phagosome lysates isolated from WT and SHP-1–deficient expressed as a percentage of recruitment. Three independent experiments me-3 macrophages at various time points after the start of internalization. were performed, and the bars show the SDs of these experiments. Top arrow, Indicates the position of SHP-1; middle arrow, indicates the position of PTP-1B; and bottom arrow, indicates the position of TC-PTP. (C) TC-PTP and PTP-1B are present in phagosome extracts. Western blot were isolated from WT macrophage cells and were analyzed by analysis was performed on total cell and phagosomes lysates isolated Western blot to detect the presence of SHP-1. In agreement with from WT and SHP-1–deficient me-3 macrophages at various time points our confocal immunofluorescence data, we detected a progressive after the start of internalization. (D) Increased tyrosine phosphorylation increase in SHP-1 levels in phagosome extracts from 15 min to 2 h in phagosome extracts from SHP-1–deficient macrophages. Western blot after the initiation of phagocytosis (Fig. 2A). As expected, no analysis was performed on total cell and phagosome lysates isolated SHP-1 was detected in SHP-1–deficient me-3 macrophage extracts from WT and SHP-1–deficient me-3 macrophages at various time points (Fig. 2A), confirming the specificity of the Ab against SHP-1. after the start of internalization. Similar results were obtained in three E 362 Next, we performed an in-gel PTP activity assay (31) to monitor independent experiments. ( ) Dok-1 is not phosphorylated on Tyr in phagosome extracts from WT and SHP-1–deficient macrophages. West- the presence of PTPs in magnetic bead–phagosome extracts iso- ern blot analysis was performed on total cell and phagosome lysates lated from WT and SHP-1–deficient me-3 macrophages at 1, 2, isolated from WT and SHP-1–deficient me-3 macrophages at various and 4 h after the initiation of phagocytosis (Fig. 2B). Active SHP- time points after the start of internalization. Similar results were obtained 1 was present in phagosome extracts from WT cells at all time in three independent experiments. points tested and, as expected, was not detected in phagosome extracts from SHP-1–deficient me-3 macrophages (upper band). 2206 SHP-1 REGULATES PHAGOLYSOSOME BIOGENESIS

Interestingly, our in-gel PTP activity assay revealed the presence of other PTPs in phagosome extracts that, based on their apparent molecular masses, corresponded to TC-PTP (Fig. 2B, lower band) and PTP-1B (Fig. 2B, middle band). The presence of both TC-PTP and PTP-1B was confirmed by Western blot analysis (Fig. 2C). Of note, we consistently observed a reduction in the activity of TC- PTP and PTP-1B in phagosome extracts from SHP-1–deficient macrophages, despite that the levels of both phosphatases were comparable in phagosome extracts from WT and SHP-1–deficient cells. Comparison of the patterns of tyrosine-phosphorylated proteins present in phagosome extracts from WT and SHP-1–deficient me-3 macrophages revealed an increased tyrosine phosphorylation in the absence of SHP-1 (Fig. 2D). To determine whether known SHP-1 substrates were differentially phosphorylated and recruited to phagosomes in the absence of SHP-1, we focused on Dok-1 (p62Dok), a major SHP-1 substrate (32) present in the phagosome proteome (22). Western blot analyses revealed that Dok-1 levels in phagosome lysates increased progressively over time, independently of the presence of SHP-1 (Fig. 2E). Downloaded from Consistent with previous observations (32), we found a significant FIGURE 3. Recruitment of early and late phagosome markers in SHP- increase in the phosphorylation of Dok-1 (Tyr362) in total lysates 1–deficient cells. WT and SHP-1–deficient me-3 macrophages were from SHP-1–deficient macrophages with respect to lysates from allowed to internalize latex beads, and, at various time points after the start A WT macrophages (Fig. 2E). Surprisingly, phosphorylation of Dok- of internalization, were fixed and stained for either EEA1 or Rab7. ( )WT and SHP-1–deficient me-3 macrophages were allowed to internalize latex 1(Tyr362) was below detectable levels in phagosome lysates pu- beads, and recruitment of EEA1 to phagosomes was assessed by confocal rified from both WT and SHP-1–deficient macrophages (Fig. 2E). immunofluorescence microscopy. Scale bar, 2 mm. (B) WT and SHP-1– http://www.jimmunol.org/ Together, these results indicate that SHP-1 is recruited early and deficient me-3 macrophages were allowed to internalize latex beads, and remains associated to phagosomes, and suggest that it may par- recruitment of Rab7 to phagosomes was assessed by confocal immuno- ticipate in phagolysosome biogenesis by modulating phosphotyr- fluorescence microscopy. Arrowheads (A, B) show the recruited protein osine signaling in phagosomes. around the phagosome. The magnification in (B) is the same as in (A). (C) Presence of EEA1 was determined on at least 300 phagosomes for each Impaired recruitment of lysosomal markers in the absence of condition and expressed as a percentage of recruitment. Three independent SHP-1 experiments were performed, and the bars show the SDs of these experiments. No significant difference was found in the phagosomal recruitment As they mature, phagosomes interact with various endosomal D

of EEA1 between WT and SHP-1–deficient macrophages. ( ) Presence of by guest on September 29, 2021 populations to acquire lysosomal features (2). To evaluate the po- Rab7 was determined on at least 300 phagosomes for each condition and tential impact of SHP-1 on the maturation process, we fed WT and expressed as a percentage of recruitment. Three independent experiments SHP-1–deficient me-3 macrophages with serum-opsonized zymo- were performed, and the bars show the SDs of these experiments. Sig- san, and at the indicated time points we assessed the phagosomal nificant difference was found in the phagosomal recruitment of Rab7 be- association of endosomal markers by confocal immunofluores- tween WT and SHP-1–deficient macrophages. *p , 0.005; p values cence microscopy. In the case of the early endosomal marker compare the percentage of Rab7-positive phagosomes in WT macrophages EEA1, phagosomal recruitment occurred with similar kinetics in versus SHP-1–deficient macrophages. WT and SHP-1–deficient me-3 macrophages (Fig. 3A). In the case of the late endosomal marker Rab7 (33, 34), we observed no major difference in its recruitment at early time points postphagocytosis slightly reduced in phagosome extracts isolated from SHP-1–defi- (Fig. 3B). However, at 1 h postphagocytosis, whereas ∼60% of cient macrophages with respect to WT macrophages. phagosomes in WT cells were positive for Rab7, ,40% of phag- The aspartic protease cathepsin D is delivered to phagosomes as osomes were positive in SHP-1–deficient macrophages. For a 51-kDa procathepsin D and is processed into an active 46-kDa LAMP-1, ∼90% of phagosomes were positive at 2 h after the ini- form (36, 37). As shown in Fig. 4C, lower panel, the 51-kDa tiation of phagocytosis by WT cells, which is very similar to the procathepsin D was present in purified phagosome lysates isolated kinetics of LAMP-1 phagosomal recruitment observed in BMM at 1, 2, and 4 h from both WT and SHP-1–deficient macrophages. (Fig. 4A, 4B). In contrast, the phagosomal recruitment of LAMP-1 In contrast, the 46-kDa mature form of cathepsin D, which grad- was reduced and delayed in SHP-1–deficient macrophages, with ually appeared in purified phagosome lysates from WT macro- ,60% of phagosomes being positive at the same time point. At 4 h phages, was absent from SHP-1–deficient phagosome lysates. We after the start of phagocytosis, we observed that ∼80% of phag- ensured that the effect of SHP-1 deficiency on phagosome matu- osomes were positive for LAMP-1 in SHP-1–deficient macro- ration was not the consequence of a defect in particle internali- phages (Fig. 4A, 4B). We next assessed by Western blot the zation. As shown in Fig. 5, WT and SHP-1–deficient macrophages presence of LAMP-1 in purified magnetic bead–phagosome internalized zymosan and latex beads to the same extent. Col- extracts isolated at various time points from WT and SHP-1–defi- lectively, these results indicate that phagosomal acquisition and cient macrophages (Fig. 4C, upper panel). Consistent with the processing of late endosomal/lysosomal markers are impaired in results obtained by confocal immunofluorescence microscopy, we cells lacking SHP-1, suggesting that this PTP participates in the found reduced levels of LAMP-1 in phagosome extracts from SHP- regulation of signaling or membrane fusion events involved in 1 deficient at all time points tested. Flotillin is recruited to phag- phagolysosome biogenesis. Impaired processing of procathepsin osomes from compartments distinct from those bearing LAMP-1 D also raised the possibility that phagosomal acidification may be (35). As shown in Fig. 4C (middle panel), flotillin-2 levels were defective in the absence of SHP-1 (37). The Journal of Immunology 2207 Downloaded from

FIGURE 4. Acquisition of phagolysosomal features is impaired in SHP-1–defective macrophages. (A) BMM, and WT and SHP-1–deﬁcient me-3 macrophages were allowed to internalize zymosan, and recruitment of LAMP-1 to phagosomes was assessed by confocal immunoﬂuorescence microscopy.

Scale bar, 2 mm. (B) Presence of LAMP-1 was determined on at least 300 phagosomes for each condition and expressed as a percentage of recruitment. http://www.jimmunol.org/ Three independent experiments were performed, and the bars show the SDs of these experiments. Significant difference was found in the recruitment of LAMP1 between WT and SHP-1–deficient macrophages. *p , 0.001, **p , 0.0001. The p values compare the percentage of LAMP-1–positive phagosomes in WT macrophages versus SHP-1–deficient macrophages. (C) Magnetic bead–phagosomes were isolated from WT and SHP-1–deficient macrophages at the indicated time points after the initiation of internalization, and presence of LAMP1, flotillin, and cathepsin D in phagosome extracts was assessed by Western blot analyses. Similar results were obtained in three independent experiments.

SHP-1 regulates phagosomal acidification taining latex beads were positive for LT even 4 h after the initi- Phagosome acidification is essential for the acquisition of micro- ation of phagocytosis. In the case of SHP-1–deficient me-5 , by guest on September 29, 2021 bicidal properties, as most lysosomal hydrolases are optimally macrophages, 20% of phagosomes were positive for LT. We active at acidic pH (38, 39). To assess the impact of SHP-1 defi- obtained similar results when phagocytosis was performed with ciency on acidification, we used the lysosomotropic agent LT Red zymosan (Fig. 6C). The absence of SHP-1 did not affect the as an indicator of phagosome acidity. WT macrophages and two overall staining and distribution of LT (Fig. 6A), indicating that SHP-1–deficient clones (me-3 and me-5) were preloaded with LT the defective accumulation of LT to phagosomes is not the con- prior to being fed with either latex beads or zymosan for various sequence of a generalized lysosomal acidification defect in SHP- time points (Fig. 6A). Quantification of the number of phag- 1–deficient macrophages. To further investigate the potential role osomes positive for LT showed that, in WT macrophages, ∼80% of SHP-1 in phagosomal acidification, we performed confocal im- of phagosomes containing latex beads were acidified after 1 h. In munofluorescence experiments to visualize the distribution of the absence of SHP-1, there was a significant reduction in the the c subunit of the v-ATPase in WT and SHP-1–deficient me-3 percentage of phagosomes positive for LT (Fig. 6A–C). Hence, in macrophages following the phagocytosis of latex beads. As shown SHP-1–deficient me-3 macrophages, 35% of phagosomes con- in Fig. 6D, the c subunit of the v-ATPase was present on a ma- jority of phagosomes in WT macrophages. In contrast, barely 20% of phagosomes were positive for the c subunit of the v-ATPase in me-3 macrophages. These data suggest that SHP-1 is involved in the phagosome acidification process.

Discussion The present study was aimed at investigating the role of the PTP SHP-1 in the regulation of phagolysosome biogenesis. We report that SHP-1 is recruited early to phagosomes and that it is required for the acquisition of lysosomal features and acidification. Previous studies established that particle internalization is accompanied by the tyrosine phosphorylation of numerous proteins, through the FIGURE 5. Particle internalization in SHP-1–deficient macrophages. action of Syk and Src family kinases (40–44). Investigation by WT and SHP-1–deficient me-3 macrophages were incubated with either Strzelecka-Kiliszek et al. (21) showed that binding of IgG-coated zymosan or latex beads at a particle-to-cell ratio of 5:1 for 10 min at 4˚C. After removing unbound particles, cells were incubated for 1 h at 37˚C and particles to the surface of macrophages induces rapid and transient Giemsa stained, and the number of internalized particles was determined. tyrosine phosphorylation of several proteins. In contrast, when For each condition, the phagocytic index was calculated for at least 100 particle internalization starts, most proteins are dephosphorylated. cells in triplicate, and two independent experiments were performed. Error The observation that tyrosine dephosphorylation coincides with bars show the SDs of these experiments. the phagosomal association of SHP-1 is consistent with this PTP 2208 SHP-1 REGULATES PHAGOLYSOSOME BIOGENESIS Downloaded from http://www.jimmunol.org/

FIGURE 6. Phagosome acidification is impaired in SHP-1–deficient macrophages. WT and SHP-1–deficient me-3 and me-5 macrophages were incubated 2 h with LT prior to internalization of either latex beads (A, B) or serum-opsonized zymosan (C) for the indicated time points and then fixed. (A) Presence of LT in latex bead–phagosomes was assessed by confocal immunofluorescence microscopy. Scale bar, 2 mm. Arrowheads show phagosomes. (B and C) Presence of LT was determined on at least 300 phagosomes for each condition and expressed as a percentage of recruitment. Three independent experiments were performed, and the bars show the SDs of these experiments. Significant difference was found in the acidification of phagosomes between WT and SHP-1–deficient macrophages. **p , 0.009; p values compare the percentage of LT-positive phagosomes in WT macrophages versus SHP-1–deficient macrophages. (D) WT and SHP-1–deficient me-3 macrophages were allowed to internalize latex beads for the indicated time points, fixed, and stained for by guest on September 29, 2021 the v-ATPase. v-ATPase acquisition was determined on at least 300 phagosomes for each condition and expressed as a percentage of recruitment. Three independent experiments were performed, and the error bars show the SDs of these experiments. Significant difference was found in the recruitment of v-ATPase between WT and SHP-1–deficient macrophages. **p , 0.0001; p values compare the percentage of v-ATPase–positive phagosomes in WT macrophages versus SHP-1–deficient macrophages. being an important modulator of tyrosine phosphorylation-dependent Phagosomal acidification is mediated by the vacuolar proton- signaling during FcgR-mediated phagocytosis (20, 45). ATPase (v-ATPase), which is present on various endocytic organ- Our observation that active SHP-1 is present on phagosomes at elles (51). The v-ATPase is a multimeric complex consisting in the all time points analyzed suggested that the role of this PTP extends multisubunit cytoplasmic V1 sector that is responsible for ATP beyond the modulation of tyrosine phosphorylation during the hydrolysis, and of the multisubunit transmembrane V0 sector that internalization process. Indeed, characterization of the phagosome pumps protons across the bilayer (52). Little is known concerning phosphoproteome and molecular analysis of the evolution of the mechanism(s) regulating the phagosomal recruitment of the phagosomes highlighted the importance of phosphorylation in the v-ATPase as well as the regulation of its activity. Previous studies regulation of phagolysosome biogenesis (4, 12, 22). This process is revealed that phagosomal acidification starts early during phag- accomplished through transient, highly regulated interactions be- osome formation, before acquisition of lysosomal features such tween the phagosome and the cytoskeleton, as well as the early, as LAMP-1 and cathepsins (8, 53, 54), which are recruited to late, and recycling endocytic compartments (46–48). Through phagosomes through distinct kinetics and mechanisms (7, 35, 55). these interactions, phagosomes acidify and sequentially acquire an Recently, it was shown that early recruitment of the a3 subunit of array of hydrolases, culminating in the generation of a hydrolytic, the v-ATPase to young phagosomes occurs via tubular structures microbicidal environment (37, 49, 50). Our results unveiled an extending from the perinuclear lysosomes along microtubules important role for SHP-1 in this process, as acquisition of lyso- (56). This finding is consistent with previous studies showing that somal features was clearly impaired in the absence of this PTP. phagosomes acquire lysosomal features through a microtubule- Hence, whereas the absence of SHP-1 had no noticeable effect on dependent mechanism (47, 50). The identification of Syt V as an the phagosomal association of the early endosomal marker EEA1, important regulator of the recruitment of the v-ATPase to the it impaired the recruitment of the late endosomal marker Rab7. phagosome membrane (26) provided additional insights into the We also found that phagosomal association of both flotillin and mechanism regulating phagosome acidification. How SHP-1 LAMP-1 was reduced in the absence of SHP-1. Most noticeable contributes to the phagosomal acidification process remains to was the impairment of phagosome acidification in SHP-1–defi- be established. The observation that there was no generalized de- cient macrophages. Collectively, those results indicated that SHP- fect in lysosomal acidification in the absence of SHP-1 suggests 1 is involved in the regulation of phagolysosome biogenesis. that the defect in phagosome acidification in SHP-1–deficient cells The Journal of Immunology 2209 is not due to an impaired activity of the v-ATPase. Rather, one crucial to understand how SHP-1 modulates phagolysosomal bio- may envision that SHP-1 is involved in the phagosomal recruit- genesis. Importantly, our results were obtained with immortalized ment of the v-ATPase, by regulating membrane fusion events, a BMM clones, which are very useful to maximize reproducibility. function previously reported for other protein phosphatases. This Although those clones bear the characteristics of the macrophage is the case for the yeast protein phosphatase 1, which was shown population from which they originate (23), macrophage popula- to control the terminal step of membrane fusion, after the SNARE tions in vivo are heterogeneous. Future studies using primary docking stage and the early priming reaction involving NSFa macrophages from motheaten mice may provide additional in- S-nitrosoacetylpenicillamine (57). This finding led the authors formation on the impact of SHP-1 deficiency on phagosome of that study to propose that protein phosphatase 1 may be part maturation in various macrophage populations. In addition to of a system other than SNARE complexes and NSFa S-nitro- SHP-1, we found that two other PTPs, namely TC-PTP and PTP- soacetylpenicillamine, which catalyzes bilayer mixing. Similarly, 1B, were recruited to phagosomes. PTP-1B and TC-PTP are both PTP-MEG2 was shown to dephosphorylate NSF, thereby stimu- localized in the endoplasmic reticulum (66, 67), an intracellular lating the homotypic fusion of secretory vesicles (15). PTPs can compartment known to contribute to the phagosome proteome also negatively regulate phagosome acidification and recruitment (68). Interestingly, we observed a reduction in the activity of these of the v-ATPase by acting on the membrane fusion machinery. two PTPs in phagosome extracts from SHP-1–deficient cells. The Hence, the mycobacterial PTP PtpA was recently shown to de- relationship between SHP-1 and the activity of these PTPs re- phosphorylate the macrophage protein VPS33B, a key regulator of mains to be established. Previous studies indicated that TC-PTP membrane fusion, leading to the inhibition of phagosome matu- and PTP-1B play a cooperative but nonredundant role in signaling ration and acidification (58). pathways associated with macrophage development and activation Downloaded from Exclusion of the v-ATPase from phagosomes may have an im- (69, 70). The possibility that these PTPs regulate both phagolyso- portant consequence on the maturation process, because earlier some biogenesis and/or phagosome function is a scenario that studies revealed that phagosomal acidification is an important remains to be experimentally addressed. element in controlling phagosome maturation (8, 37, 59). Indeed, further studies revealed that the a1 subunit of the V0 sector is Acknowledgments

a crucial factor in vacuole fusion, acting downstream of trans- We are grateful to Marcel Desrosiers for expert assistance with confocal http://www.jimmunol.org/ SNARE pairing (60, 61). Consistently, the V0 sector was involved immunofluorescence microscopy and to Dr. Michel L. Tremblay (McGill in vesicle fusion during exocytosis in neurons (62, 63). More re- University) and Dr. Mhairi Skinner (University of Guelph) for kindly pro- cently, studies with zebrafish microglia established that the a1 viding the Abs to PTP-1B and TC-PTP, and a rabbit antiserum against subunit mediates fusion independently of its proton pump activity the c subunit of the v-ATPase, respectively. (64). Further studies will be required to determine whether the lack of phagosome acidification observed in SHP-1–deficient Disclosures macrophages is the consequence of a defective phagosome mat- The authors have no financial conflicts of interest. uration process, or one of the contributing factors. 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