The Lipid Kinase Pikfyve Coordinates the Neutrophil Immune Response Through the Activation of the Rac Gtpase

The Lipid Kinase PIKfyve Coordinates the Neutrophil Immune Response through the Activation of the Rac GTPase

This information is current as Roya M. Dayam, Chun X. Sun, Christopher H. Choy, of September 28, 2021. Gemma Mancuso, Michael Glogauer and Roberto J. Botelho J Immunol published online 4 August 2017 http://www.jimmunol.org/content/early/2017/08/04/jimmun ol.1601466 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 4, 2017, doi:10.4049/jimmunol.1601466 The Journal of Immunology

The Lipid Kinase PIKfyve Coordinates the Neutrophil Immune Response through the Activation of the Rac GTPase

Roya M. Dayam,*,† Chun X. Sun,‡ Christopher H. Choy,*,† Gemma Mancuso,* Michael Glogauer,‡ and Roberto J. Botelho*,†

Neutrophils rapidly arrive at an infection site because of their unparalleled chemotactic ability, after which they unleash numerous attacks on pathogens through degranulation and reactive oxygen species (ROS) production, as well as by phagocytosis, which se- questers pathogens within phagosomes. Phagosomes then fuse with lysosomes and granules to kill the enclosed pathogens. A complex signaling network composed of kinases, GTPases, and lipids, such as phosphoinositides, helps to coordinate all of these processes. There are seven species of phosphoinositides that are interconverted by lipid kinases and phosphatases. PIKfyve is a lipid kinase that generates phosphatidylinositol-3,5-bisphosphate and, directly or indirectly, phosphatidylinositol-5-phosphate

[PtdIns(5)P]. PIKfyve inactivation causes massive lysosome swelling, disrupts membrane recycling, and, in macrophages, blocks Downloaded from phagosome maturation. In this study, we explored for the ﬁrst time, to our knowledge, the role of PIKfyve in human and mouse neutrophils. We show that PIKfyve inhibition in neutrophils does not affect granule morphology or degranulation, but it causes LAMP1+ lysosomes to engorge. Additionally, PIKfyve inactivation blocks phagosome–lysosome fusion in a manner that can be rescued, in part, with Ca2+ ionophores or agonists of TRPML1, a lysosomal Ca2+ channel. Strikingly, PIKfyve is necessary for chemotaxis, ROS production, and stimulation of the Rac GTPases, which control chemotaxis and ROS. This is consistent with

observations in nonleukocytes that showed that PIKfyve and PtdIns(5)P control Rac and cell migration. Overall, we demonstrate http://www.jimmunol.org/ that PIKfyve has a robust role in neutrophils and propose a model in which PIKfyve modulates phagosome maturation through phosphatidylinositol-3,5-bisphosphate–dependent activation of TRPML1, whereas chemotaxis and ROS are regulated by PtdIns (5)P-dependent activation of Rac. The Journal of Immunology, 2017, 199: 000–000.

eutrophils are the first responders to an infection and, All of these responses are coordinated through a variety of re- thus, play an essential role in coordinating the innate ceptors and intracellular signals, including small GTPases (7) and N immune response (1–4). They do this because of their the phosphoinositide (PtdInsP) lipids (1, 3, 8). Based on the phos- unmatched chemotactic ability, sensing and tracking the chemical phorylation pattern of the head group, there are seven species of by guest on September 28, 2021 trail to sites of infection (2). Once in contact with pathogens, they PtdInsPs that typically function by differentially distributing to in- unleash a variety of attacks, including degranulation to secrete tracellular membranes and then recruiting a set of protein effectors cytokines, hydrolytic enzymes, and antibacterial peptides; acti- specific to that PtdInsP species (9, 10). Through this general process, vation of the NADPH oxidase, to generate reactive oxygen species phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol- (ROS); and phagocytosis, to engulf and sequester the pathogens 3,4,5-trisphosphate help to coordinate actin and membrane dy- into phagosomes (1–3, 5). Phagosomes then mature by fusing with namics to direct chemotaxis and phagocytosis (11–14). In com- granules and lysosomes to kill and digest the pathogens (1, 6). parison, phosphatidylinositol-3-phosphate regulates endosomal membrane trafficking, phagosome maturation, and activation of the NADPH oxidase (11, 15–18). However, there is a dearth of *Department of Chemistry and Biology, Ryerson University, Toronto, Ontario M5B knowledge about the importance of phosphatidylinositol-5-phosphate 2K3, Canada; †Molecular Science Graduate Program, Ryerson University, Toronto, [PtdIns(5)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] Ontario M5B 2K3, Canada; and ‡Faculty of Dentistry, University of Toronto, Tor- onto, Ontario M5S 3E2, Canada in neutrophil function. ORCID: 0000-0002-7820-0999 (R.J.B.). PtdIns(3,5)P2 is synthesized by the lipid kinase PIKfyve by phos- phorylating phosphatidylinositol-3-phosphate (19–21). In contrast, Received for publication August 24, 2016. Accepted for publication July 11, 2017. controversy remains about the source of PtdIns(5)P (19, 22). In This work was supported by a Discovery Grant from the Natural Sciences and Research Council of Canada, a Tier II Canada Research Chair Award, an Early one model, PIKfyve synthesizes PtdIns(5)P directly by phos- Researcher Award from the Government of Ontario, and Ryerson University (to phorylating phosphatidylinositol (22, 23), whereas in another R.J.B.). R.M.D. was supported by a Doctoral Canada Graduate Scholarship awarded by the Canadian Institutes of Health Research, C.H.C. was supported by an Ontario model, PtdIns(3,5)P2 is converted to PtdIns(5)P via action of the Graduate Scholarship, and M.G. is supported by a Bone Research Team Grant from myotubularin lipid phosphatases (19, 24, 25). Regardless, loss of the Canadian Institutes of Health Research. PIKfyve function causes multiple defects, including embryonic Address correspondence and reprint requests to Dr. Roberto J. Botelho, Department lethality in PIKfyve2/2 mice (26), swollen endolysosomes (27), of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON 2+ M5B 2K3, Canada. E-mail address: [email protected] hindered membrane recycling (28, 29), impaired lysosomal Ca The online version of this article contains supplemental material. signaling (30), and defective autophagic flux (31, 32), attesting to its importance (20). Interestingly, PIKfyve has an emerging role in Abbreviations used in this article: MPO, myeloperoxidase; PBD, p21-binding domain; PFA, paraformaldehyde; PtdInsP, phosphoinositide; PtdIns(3,5)P2, phosphati- the immune response. For example, PIKfyve inhibition disrupts dylinositol-3,5-bisphosphate; PtdIns(5)P, phosphatidylinositol-5-phosphate; ROS, TLR and cytokine signaling; in fact, the PIKfyve inhibitor apili- reactive oxygen species. mod was used to suppress IL-12/IL-13 signaling before it was Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 discovered to be a selective inhibitor of PIKfyve (33, 34). In addition,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601466 2 PIKfyve LIPID KINASE AND NEUTROPHILS

2 2 mice carrying a platelet-specific PIKfyve / genotype suffer from external beads as above; this was followed by fixation and processing for massive macrophage activation and inflammation (35). Lastly, in- immunofluorescence as described previously (43) and below. hibition of PIKfyve blocks phagosome maturation in macrophages Stimulation of degranulation (36). This likely occurs because PtdIns(3,5)P2 is needed to activate 2+ Neutrophils were plated and pretreated with 30 nM apilimod for 30 min at TRPML1, a PtdIns(3,5)P2-gated lysosomal Ca channel (30, 37). m 2+ 37˚C prior to the addition of 1 M latrunculin A (Abcam, Cambridge, When TRPML1 is silenced or Ca is chelated, phagosomes and MA) for 30 min. Degranulation was induced by adding 300 nM fMLF lysosomes dock but fail to fuse (37). It remains possible that some (Sigma-Aldrich, Oakville, ON, Canada) for 3 min, followed by fixation of these defects are due to the concomitant loss of PtdIns(5)P when with 4% paraformaldehyde (PFA) for 20 min and quenching with 100 mM PIKfyve is inhibited. Indeed, PIKfyve and MTMR3, a myotubu- glycine. To assess degranulation for primary granules, cells were stained larin, are implicated in cell migration via the PtdIns(5)P-dependent with anti-CD63 and anti-myeloperoxidase (MPO) Abs (see below). To assess degranulation of secondary and tertiary granules, cells were stained activation of the Rac GTPase, a critical coordinator of actin with anti-lactoferrin and MMP9, respectively (see below). remodeling (25, 38). Activation of the Rac GTPases is critically important for neu- Immunofluorescence trophil chemotaxis, phagocytosis, and stimulation of the NADPH After the required manipulation, neutrophils were washed and fixed with oxidase (39–41). Given all of this, we postulated that PIKfyve 4% PFA for 20 min, followed by three washes in PBS and quenching using 100 mM glycine in PBS for 20 min. For intracellular immunostaining, activity, through the synthesis of PtdIns(3,5)P2 and/or PtdIns(5)P, is essential for coordinating various neutrophil functions. Indeed, cells were permeabilized with 0.5% Triton X-100 for 10 min at room temperature to stain with rabbit anti-mouse polyclonal Abs to MPO, MMP9, we found that PIKfyve inhibition blocks phagosome maturation, lactoferrin (all used at 1:100; Bioss, Boston, MA), or CD63 (H-193, 1:200; chemotaxis, and the NADPH oxidase but not degranulation. In Santa Cruz Biotechnology, Paso Robles, CA). Alternatively, cells were Downloaded from part, PIKfyve regulates these functions by modulating the lyso- permeabilized with 100% ice-cold methanol for 5 min to stain with rat anti- somal TRPML1 Ca2+ channel and activation of the Rac GTPases. mouse LAMP1 mAbs (clone 1D4B, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA). Human neutrophils were fixed with 4% PFA and permeabilized for 3–5 min with ice-cold 100% methanol before staining with rabbit anti-human LAMP1 mAbs (1:200; Cell Signaling Materials and Methods Technology, Danvers, MA). For extracellular staining, no permeabilization Bone marrow–derived murine neutrophil isolation was performed before immunostaining. All primary Abs were incubated for http://www.jimmunol.org/ and stimulation 1 h at room temperature in 0.5% BSA, followed by three washes in PBS and a 1-h incubation with fluorescently labeled secondary Abs at 1:1000 (Jackson Bone marrow from the femur and tibia of C57BL/6 mice was extracted by ImmunoResearch Laboratories, West Grove, PA). Lastly, cells were washed flushing the bones with complete DMEM plus 10% FBS using a 27-gauge 3 every 5 min with 0.5% BSA for 30 min to remove the excess secondary needle. Cells were centrifuged at 1000 g for 5 min to yield a pellet that Abs. The coverslips were mounted with Dako mounting media and visualized. was then resuspended in 1 ml of complete DMEM and centrifuged at 1000 3 For staining ruptured cells, after the desired treatment, neutrophils were g for 30 min over a Percoll gradient containing 55, 65, and 80% Percoll. washed three times with cold 13 PBS and scraped into 1 ml of cold ho- The band of neutrophils between 80 and 65% Percoll was collected, washed mogenization buffer (20 mM Tris [pH 7.4], 1 mM AEBSF, 1 mM MgCl , with PBS, and resuspended in complete DMEM. To plate cells, glass cover- 2 1 mM CaCl2, 1 mg/ml each RNase and DNase and supplemented with slips were coated with 3% BSA at room temperature for 30 min, followed by a mammalian protease inhibitor mixture [Sigma-Aldrich]). Cells were then PBS wash. One million neutrophils were then plated by incubating for 30 min lysed by passaging six to eight times through a 25-gauge needle, followed by guest on September 28, 2021 at 37˚C and at 5% CO2. Animal handling and treatment was done according by centrifugation for 10 min at 10,000 3 g at 4˚C to collect lysed cell to guidelines established by the Institutional Animal Care Ethics Board. bodies. The pellet was then resuspended and fixed in 400 ml of 4% PFA for Blood-derived human neutrophil isolation and stimulation 20 min, followed by a 20-min incubation at room temperature with 100 mM glycine to quench PFA. Cells were then pelleted and stained with Select assays were performed with human neutrophils isolated by layering anti-LAMP1 and anti-CD63 Abs, as described above. 4.5 ml of citrate-buffered human peripheral blood onto 4 ml of 1-Step Polymorphs buffer in a 15- ml tube (Accurate Chemical & Scientific, Microscopy and image analysis Westbury, NY). This was spun for 35 min at 500 3 g at room temperature. For scoring vacuolation, live cell imaging was done using an inverted Neutrophils were recovered from the bottom layer. RBCs were lysed with Olympus IX83 microscope (Olympus, Richmond Hill, ON, Canada) with a 13 BD Pharm lyse buffer (BD Biosciences, ON, Canada). The total 3 Hamamatsu ORCA-flash 4.0 digital camera. Cells plated on coverslips were number of neutrophils was diluted in 1 PBS and counted with a Beck- placed in a chamber with HEPES-buffered RPMI 1640 medium at 37˚C and man Coulter Z2 cell counter. Isolation of human blood was done according imaged using differential interference contrast microscopy. to guidelines established by the Institutional Research Ethics Board. For fluorescence imaging, we used a Quorum spinning disk confocal PIKfyve inhibition and vacuolation microscope (Quorum, Guelph, ON, Canada) equipped with a Hamamatsu C9100-13 EM-CCD camera and a 1003 oil objective (NA 1.4) to obtain To inhibit PIKfyve, neutrophils were treated with the indicated concen- single-plane images. Images were analyzed using ImageJ (v. 1.47 bundled trations of apilimod (Toronto Research Chemicals, Toronto, ON, Canada) or with 64-bit Java) and processed with Adobe Photoshop (v. 7.0.1; Adobe YM201636 (Adooq Bioscience, Irvine, CA) for the indicated periods of Systems, San Jose, CA) without altering the relative fluorescence intensity time. To quantify vacuolation, cells were imaged live in the continuous of the images. To quantify phagosome maturation, images were converted presence of the inhibitor for no more than 20 min using differential inter- to 8-bit and into false color, as described (36, 37). Briefly, phagosome- ference contrast microscopy. Vacuoles were defined as being .1 mm. Al- associated LAMP1 fluorescent intensities were clustered into three groups ternatively, cells were stimulated and processed for live cell imaging or fixed. based on false-color range: white-yellow color indicated a high intensity of LAMP1 with grayscale intensities of 225–180, orange to red indicated a Phagocytosis and phagosome maturation assays partial intensity of LAMP1 (grayscale intensities of 180–80), and purple to blue indicated the absence of LAMP1 (grayscale intensities of 80–1) For phagocytosis and phagosome maturation, we used polymer beads with a diameter of 2.08 mm (Bangs Laboratories, Fishers, IN) opsonized with around the phagosomes (43). To quantify fluorescence associated with human IgG, as described (37). Particles were added to neutrophils and granule markers, regions of interest were drawn around individual cells, synchronized by centrifugation of the cells for 5 min at 400 3 g. Subse- and total fluorescence was acquired, followed by background correction. quently, cells were washed three times with PBS and incubated with Zigmond chamber chemotaxis assay complete DMEM at 37˚C and 5% CO2 for 15 min to allow internalization of the beads. For quantification of phagocytic index and efficiency, cells One million bone marrow neutrophils in 100 ml of HBSS with 1% gelatin were fixed with 4% PFA for 20 min at room temperature, followed by were incubated with different concentrations of apilimod for 30 min at 37˚C quenching with 100 mM glycine for 20 min. To identify external versus and then plated onto a 5% BSA–coated microscope cover glass (22 3 40 mm) internal beads, cells were incubated with fluorescently labeled goat anti- for 10 min. The cover glass was inverted onto a Zigmond chamber with 100 ml human Abs at 1:1000, as described (42). For phagosome maturation, cells of HBSS medium, and 100 ml of HBSS containing 1 mM fMLF was added were incubated for a chase time of 1 h at 37˚C and 5% CO2 before staining to the right and left chambers. Time-lapse video microscopy was used to The Journal of Immunology 3

FIGURE 1. Potency and kinetics of apilimod-induced vacuolation in neutrophils. (A) Neutrophils were exposed to vehicle or to the indicated apilimod concentrations for 1 h, followed by live cell imaging. (B) Neutrophils were exposed to vehicle or 20 nM apilimod for the indicated time points, before live cell imaging. Scale bar, 10 mm. (C and D) Vacuolation was quantified by scoring the number of vacuoles larger than .1 mmin diameter per cell. Shown are the mean number of vacuoles per neutrophil 6 SEM from n = 3 independent experiments. *p , 0.05 versus control (vehicle or t = 0 min), Student t test. Downloaded from record neutrophil movements in the Zigmond chambers for 15 min (one EDTA, 150 mM NaCl, 100 mg/ml lysozyme, 5 mM DTT, 1% Triton X-100, frame per 20 s). Captured images were analyzed for cell direction and speed and supplemented with bacterial protease inhibitor mixture [Bio Basic, using cell-tracking software (Retrac version 2.1.01 freeware). Data were Markham, ON, Canada]) and 1 mM PMSF. Bacteria were ground using a collected from five independent experiments. mortar and pestle with 1 g of Celite (Sigma-Aldrich). Lysates were cleared by centrifugation, and supernatant was added to reduced glutathione- Cytochrome C reduction assay Sepharose (Invitrogen, Carlsbad, CA) and incubated at 4˚C for 1 h with http://www.jimmunol.org/ agitation, followed by three washes with bacterial lysis buffer. To measure ROS generation, we used reduction of cytochrome C. One million bone marrow neutrophils in 100 ml of PBS with 10 mM D-glucose Affinity precipitation of GTP-bound Rac GTPase and were incubated with different concentrations of apilimod for 30 min. They Western blotting were then mixed with 880 ml of PiCM-G (138 mM NaCl, 2.7 mM KCl, 0.6 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM NaH2PO4/Na2HPO4 One million neutrophils were treated at 37˚C for 30 min with 50 nM [pH 7.4]) supplemented with 0.1 mM cytochrome C and incubated for an apilimod or DMSO, followed by the addition of 1 mM fMLF or vehicle additional 10 min at 37˚C. Cells were then stimulated with 1 mM fMLF or for 1 min. Cells were placed immediately on ice and lysed with 100 mlof 1 mM PMA for 30 min at 37˚C. The absorbance of reduced cytochrome C ice-cold 53 MLB lysis buffer (125 mM HEPES [pH 7.5], 25 mM EDTA, at 550 nm was recorded and background corrected (reaction lacking cell 1% Triton X-100, 750 mM NaCl, 25 mM MgCl2, and 50% glycerol, lysates). Data were collected from five independent experiments. supplemented with mammalian protease inhibitors [Sigma-Aldrich]). Cell by guest on September 28, 2021 lysates were clarified by centrifugation at 10,000 3 g for 5 min at 4˚C. Ten Preparation of recombinant fusion protein of GST and percent of cell lysates were removed to measure protein levels across each p21-binding domain protein sample. The remaining cell lysates were incubated with 50 ml of glutathione- Sepharose beads attached to GST–PBD or GST (50% suspension) and To quantify Rac GTPase activation, we used affinity chromatography and a incubated for 1 h at 4˚C with agitation. The samples were centrifuged for fusion protein of GST and the p21-binding domain (PBD) of PAK, as de- 2 min at 10,000 3 g, and the supernatant was removed. The pellets were scribed previously, with a few modifications (44). Briefly, recombinant pro- washed three times with 13 MLB lysis buffer before protein elution with teins were induced in BL21* Escherichia coli in the presence of 0.4 mM 23 Laemmli buffer containing 2-ME. Protein eluants were loaded and IPTG for 3 h at 30˚C. Fifty ODs of bacterial culture were centrifuged before separated in a 12% SDS-PAGE, transferred to a polyvinylidene difluoride the addition of 50 ml of bacterial lysis buffer (10 mM Tris [pH 8], 1 mM membrane, and processed for Western blotting with mouse anti-Rac1 Abs

Control Apilimod DIC LAMPI MPO Merge DIC LAMPI MPO Merge

DIC LAMPI Lactoferrin Merge DIC LAMPI Lactoferrin Merge

DIC LAMPI MMP9 Merge DIC LAMPI MMP9 Merge

FIGURE 2. PIKfyve inhibition engorges lysosomes but not granules. Neutrophils were treated with vehicle (control) or 20 nM apilimod for 1 h before processing and staining for LAMP1, MPO, lactoferrin, and MMP9, which identify lysosomes and primary, secondary, and tertiary granules, respectively. Apilimod caused swelling of LAMP1+ structures, whereas granules remained punctate. Scale bar, 10 mm. 4 PIKfyve LIPID KINASE AND NEUTROPHILS

FIGURE 3. PIKfyve inhibition blocks phagosome- lysosome fusion. (A and B)EffectofPIKfyve inhibition on phagocytosis. Neutrophils were ex- C posed to apilimod (A) or to YM201636 (B) for 1 h before challenging with IgG-opsonized beads for 20 min. The number of macrophages with at least one engulfed particle (phagocytic efficiency) and the number of internalized particles per cell (phagocytic index) were scored and normalized to vehicle-treated cells. (C) Neutrophils were Downloaded from treated with vehicle only or with 20 nM apilimod for 1 h before being allowed to engulf IgG- coated beads and undertake maturation. Matura- tion was accompanied by no additional treatment with ionomycin, MLSA1, or BAPTA-AM. Cells were stained for LAMP1 to determine phagosome maturation. Arrows indicate LAMP1+ phagosomes, http://www.jimmunol.org/ and arrowheads indicate LAMPI2 phagosomes. D Pseudocoloring was used to facilitate quantification of LAMP1 intensity into three groups: strong LAMP1 (orange-white), intermediate LAMP1 (red-magenta), and weak LAMP1 (blue-black) intensities. (D) Quantification of LAMP1 staining of phagosomes into three groups of LAMP1 intensity, as described in Materials and Methods.

Apilimod inhibits labeling of phagosomes with by guest on September 28, 2021 LAMP1. This is partially rescued with ionomycin and MLSA1 treatment. (E) Neutrophils were treated as above with vehicle or apilimod and allowed to phagocytose beads and then rupture, before ﬁx- ation and staining for LAMP1 and CD63 (left panels). Quantiﬁcation of phagosome-associated LAMP1 and CD63 signal (right panel). Data in (A), (B), (D), and (E) are mean 6 SEM from n = 3 independent experiments. Scale bars, 10 mm. *p , 0.05 versus the respective control group, #p , 0.05 versus the apilimod-only group, Stu- E dent t test or ANOVA and the Tukey post hoc test, as appropriate.

(clone 23A8, 1:2500; GeneTex, Irvine, CA), rabbit polyclonal anti-Rac2 Abs single-parameter experiments or using ANOVA and the Tukey post hoc test (EMD Millipore, ON, Canada), and HRP-linked goat anti-mouse or rabbit for multiparameter experiments. Statistical signiﬁcant was drawn at p , 0.05. secondary Abs used at 1:10,000 (CEDARLANE, ON, Canada). ECL was detected and analyzed by band densitometry using a Gel Documentation Results System (Bio-Rad, Mississauga, ON, Canada). Lysosomes, but not granules, vacuolate in PIKfyve-inhibited Statistical analyses neutrophils All experiments were repeated at least three times, and all data were The importance of PIKfyve activity in neutrophils has not been ex- subjected to statistical analysis using an unpaired or paired Student t test for amined previously. To investigate this, we used a pharmacological ap- The Journal of Immunology 5

FIGURE 4. PIKfyve activity does not affect degranulation. (A) Latrunculin-treated neutrophils were exposed to the indicated conditions, followed by fixation and staining of external CD63. (B) Fluorescence intensity of external CD63 in neutrophils treated as indicated. *p , 0.05 versus control. (C) Latrunculin-treated neutrophils were exposed to the indicated conditions, followed by fixation and staining of total cellular MPO, lactoferrin, and MMP9. (D) Quantification of cell-associated fluorescence for MPO, lacto- Downloaded from ferrin and MMP9. *p , 0.05 versus control, ANOVA and the Tukey post hoc test. Data in (B) and (D) are mean 6 SEM from n = 3 independent experiments. Scale bars, 10 mm. http://www.jimmunol.org/

proach to acutely block PIKfyve activity by apilimod or YM201636, are also professional phagocytes, we assessed the role of PIKfyve by guest on September 28, 2021 two selective inhibitors of PIKfyve (34, 45). In fact, apilimod was in phagocytosis and phagosome maturation. First, we evaluated recently shown to have exquisite selectivity for PIKfyve (46). First, the ability of neutrophils to engulf IgG-coated beads by measuring we examined the sensitivity of murine neutrophils to apilimod or the phagocytic index and efficiency before and after treatment YM201636 by testing different concentrations and incubation times with apilimod or YM201636. We observed a significant reduction and scoring the number of vacuoles larger than 1 mmindiameter. in phagocytic appetite, as measured by index and efficiency in By incubating cells for 1 h, we noted a gradual rise in the number of neutrophils treated with $30 nM apilimod or $10 nM YM201636 vacuoles in neutrophils exposed to increasing amounts of the in- (Fig. 3A, 3B), which was reminiscent of our prior observations hibitors (Fig. 1A, 1C, Supplemental Fig. 1A, 1C). We then used an with macrophages (36). intermediate concentration of 20 nM for apilimod and 10 nM for To investigate the impact on phagosome maturation, we then YM201636 to examine the rate of vacuolation. Neutrophils began treated murine neutrophils with 20 nM apilimod or 5 nM YM201636, to vacuolate significantly within 30 min of drug exposure and be- which is sufficient to vacuolate neutrophils but is permissive for came highly vacuolated at 90 min of exposure (Fig. 1B, 1D, phagocytosis. After waiting 1 h to elicit phagosome maturation, Supplemental Fig. 1B, 1D). Thus, to minimize off-target and indirect we processed, stained, and quantified phagosomal acquisition of effects of PIKfyve inhibition, we generally treated neutrophils for LAMP1 to track phagosome–lysosome fusion, as previously de- , , 1hat 50 nM apilimod or YM201636, unless otherwise noted. scribed (36, 37, 49). We observed a remarkable inhibition of We next attempted to identify the nature of the vacuoles in neu- phagosome maturation in cells blocked for PIKfyve. In vector- trophils. Neutrophils are not only equipped with lysosomes, they treated murine neutrophils, ∼60% of phagosomes labeled strongly also possess lysosome-related primary granules (or azurophilic with LAMP1 (LAMP1+), whereas only ∼10% were negative granules), secondary granules, and tertiary granules (2, 47, 48), which (LAMP12, Fig. 3C, 3D). In striking comparison, neutrophils can be labeled with Abs to LAMP1, MPO, lactoferrin, and MMP9, + + inhibited for PIKfyve had ,5% LAMP phagosomes, whereas respectively. Although we clearly observed vacuolation of LAMP1 ∼ 2 lysosomes in neutrophils treated for 1 h with 20 nM apilimod, we 70% were LAMP1 (Fig. 3C, 3D). Similar trends were ob- did not discern vacuolation of organelles that labeled with the other tained in murine neutrophils treated with 5 nM YM201636 markers (Fig. 2). This suggests that LAMP1+ lysosomes are suscep- (Supplemental Fig. 2). In contrast, control and PIKfyve-inhibited tible to swelling, whereas primary, secondary, and tertiary granules neutrophils had similar levels of CD63 and MPO associated with resist enlargement in neutrophils acutely inhibited for PIKfyve. phagosomes, suggesting that PIKfyve activity is not necessary for phagosome fusion with primary granules (Fig. 3E, 3F, Supplemental PIKfyve controls phagosome–lysosome fusion in neutrophils Fig. 3). We previously showed that PIKfyve has an important role in We then assessed the mechanism by which PIKfyve might phagosome maturation in macrophages (36). Because neutrophils control phagosome maturation in neutrophils. Given our prior work 6 PIKfyve LIPID KINASE AND NEUTROPHILS

FIGURE 6. PIKfyve activity is essential for ROS generation in neutrophils. Neutrophils were treated with the indicated apilimod levels before being exposed or not to fMLF or PMA. Reduction of cytochrome C was measured by spectrophotometry as an indication of ROS production. Data are mean 6 SEM from n = 5 independent experiments. *p , 0.05,

ANOVA and the Tukey post hoc test. Downloaded from

rophages (37), as well as a previously observed periphagosomal increase in cytosolic Ca2+ in neutrophils (50, 51). PIKfyve is not necessary for fMLF-induced degranulation

In addition to phagocytosis and phagosome maturation, neutrophils http://www.jimmunol.org/ rely on degranulation to eliminate pathogens (5). Because PIKfyve and TRPML1 are linked to regulated exocytosis (52–54), we postulated that PIKfyve activity might govern some aspects of degranulation. Given that primary granules are lysosome-related organelles, and their secretion can depend on Ca2+ (5, 55–57), we examined the appearance of CD63 on the cell surface and the disappearance of MPO from within cells. We also tested the secretion of secondary and tertiary granules by quantifying deple- tion of lactoferrin and MMP9 from within cells after stimulation by guest on September 28, 2021 FIGURE 5. PIKfyve activity is important for neutrophil chemotaxis. (A) Neutrophils were exposed to an fMLF gradient in a Zigmond chamber in with fMLF. First, we observed that resting neutrophils and those the absence or presence of apilimod, as indicated. Shown is the positional treated with only apilimod had comparable surface levels of CD63 displacement of each cell after 15 min relative to the starting point. Cells (Fig. 4A, 4B) and similar cell-associated levels of MPO, lacto- found in the rightmost quadrants moved toward the fMLF source. (B). ferrin, and MMP9, suggesting that PIKfyve does not affect basal Neutrophil migration speed was calculated by tracking each neutrophil levels of degranulation (Fig. 4). Second, we showed that fMLF over 15 min. Data are shown as the mean 6 SEM from n = 5 independent exposure enhanced the levels of cell surface CD63 and abated experiments. *p , 0.05, ANOVA and the Tukey post hoc test. cell-associated MPO, lactoferrin, and MM9 relative to resting neutrophils (Fig. 4). Lastly, the fMLF-induced degranulation was unabated in neutrophils pretreated with apilimod (Fig. 4). Overall, in macrophages, we postulated that PIKfyve is necessary to these data show that acute loss of PIKfyve activity does not impair 2+ stimulate TRPML1 to release lysosomal Ca and trigger phag- fMLF-induced exocytosis of granules, although we cannot rule out osome–lysosome fusion. To test this, we exposed apilimod-treated an effect during chronic PIKfyve loss. cells to ionomycin, a Ca2+ ionophore, or to MLSA1, a TRPML1 agonist, to determine whether these agents could rescue phag- PIKfyve activity is necessary for fMLF-directed chemotaxis osome–lysosome fusion in PIKfyve-inhibited cells. First, and as a PIKfyve and MTMR3 cooperate to synthesize PtdIns(5)P, which, control, we showed that ionomycin or MLSA1 alone did not im- together, activate the Rac GTPase to coordinate cell migration (25, pact LAMP1 staining of phagosomes (Fig. 3C, 3D). Second, and 38, 58). Hence, we postulated that PIKfyve activity might regulate most important, ionomycin and MLSA1 were able to decrease the neutrophil chemotaxis. To test this hypothesis, we examined the number of phagosomes devoid of LAMP1 staining; it decreased ability of murine neutrophils to move toward an fMLF chemical from ∼70% in apilimod-alone neutrophils to ∼30% in apilimod- gradient by quantifying their speed and directionality toward the treated cells exposed to ionomycin or MLSA1 (Fig. 3C, 3D). fMLF source at different concentrations of apilimod. As expected, However, the rescue was partial, and most phagosomes became vector-exposed neutrophils exhibited a remarkable capacity to partially stained with LAMP1, as deﬁned in Materials and orient and move toward the fMLF gradient. By mapping the po- Methods. Consistent with a role for TRPML1 and lysosomal Ca2+ sition of neutrophils relative to their starting position, we found in phagosome–lysosome fusion in neutrophils, we showed that that ∼80% had moved toward the fMLF gradient (within the Ca2+ chelation with BAPTA-AM potently hindered LAMP1 la- rightmost quadrants in Fig. 5A) and traveled at an average speed beling of phagosomes (Fig. 3C, 3D). Overall, these data suggest of 8.8 6 0.2 mm/min (Fig. 5B). Strikingly, at apilimod concen- that PIKfyve controls phagosome–lysosome fusion in neutrophils, trations as low as 10 nM, neutrophils became disoriented, with in part by stimulating TRPML1 and releasing lysosomal Ca2+ to only ∼50% of the cells moving toward the gradient (Fig. 5A). At trigger fusion. This is consistent with our previous work in mac- 35 and 70 nM apilimod, neutrophils were effectively spread The Journal of Immunology 7

FIGURE 7. PIKfyve is essential to Rac GTPase stimulation in neutrophils. Western blots against Rac1 (A and C) and Rac2 (B and D) GTPases after affinity precipitation for GTP-bound Rac GTPases (upper panels) from neutrophils treated as indicated and as described in Materials and Methods. GST alone is a negative control showing that Rac1 and Rac2 did not interact with the matrix or GST itself. Total Rac1 and Rac2 (10% of the input for the pull- down) were used to normalize for differences in loading for each sample (lower Downloaded from panels). Note that images shown in (D) were acquired after stripping and reprobing the same polyvinylidene difluoride membrane used in (C). GTP-bound Rac1 (A and C) and Rac2 (B and D) were quantified by normal- izing to the respective total Rac GTPases. Data are mean 6 SD from n =3experiments. http://www.jimmunol.org/ *p , 0.05 versus control (resting cells), Student t test. by guest on September 28, 2021 equally across each quadrant and traveled a shorter overall dis- depend on the activation of the Rac GTPases (16, 17, 40, 41, 59). tance (Fig. 5A), which effectively suggests randomized and slower Moreover, PIKfyve activity, through the action of MTMR3, is movement by neutrophils. Indeed, neutrophil speed was signifi- linked to Rac GTPase activation to catalyze migration of non- cantly abated at 10 nM apilimod (5.1 6 0.5 mm/min) and brought leukocytes (25, 38, 58). Finally, PtdIns(5)P was shown to bind and to a near standstill at 70 nM (Fig. 5B). Overall, these experiments stimulate Tiam1, a guanyl exchange factor for Rac (58). Given all reveal an important function for PIKfyve activity in neutrophil of this, we postulated that PIKfyve activity is necessary to stim- chemotaxis. ulate Rac GTPases in neutrophils. To test this hypothesis, we used PIKfyve activity is necessary for fMLF-induced ROS production an affinity chromatography assay that uses a GST–chimeric protein of the PBD of p21-activated kinase to precipitate GTP-bound We then considered the possibility that PIKfyve activity may be Rac, followed by Western blotting against the Rac1 and Rac2 necessary for ROS synthesis, which primarily occurs through GTPases. As expected, GST alone did not recover any Rac1 activation of the NADPH oxidase (4). To test this, we measured or Rac2 from cells stimulated with fMLF (Fig. 7). In addition, ROS production in response to fMLF in control and apilimod- treated neutrophils using a cytochrome reduction assay. Resting GST–PBD recovered little Rac1 or Rac2 in resting cells or cells neutrophils and those treated with apilimod alone (10, 35, or 70 treated with only apilimod or YM201636. In striking contrast, nM) had similar levels of ROS production (Fig. 6). As expected, fMLF treatment led to a strong recovery of both Rac GTPases, neutrophils exposed to stimulants like fMLF or phorbol esters which was abolished by pretreatment with apilimod or YM201636 exhibited a large increase in ROS (Fig. 6). Strikingly, even with (Fig. 7). Overall, these results support a model in which PIKfyve pretreatment with 10 nM apilimod, ROS production was signifi- activity is necessary for Rac GTPase activation during neutro- cantly subdued in neutrophils exposed to fMLF or PMA (Fig. 6). phil stimulation to control chemotaxis, ROS production, and At 35 and 70 nM apilimod, ROS synthesis was essentially phagocytosis. thwarted in stimulant-exposed neutrophils (Fig. 6). Overall, this PIKfyve activity is necessary for human neutrophil function suggests that PIKfyve activity is important for ROS generation, likely by stimulating the NADPH oxidase. Lastly, we tested whether the role of PIKfyve in neutrophil function was applicable to human neutrophils by examining two specific PIKfyve activity is necessary for fMLF-induced Rac activation aspects: phagosome maturation and chemotaxis. Using interme- Our observations so far indicate that PIKfyve activity affects diate concentrations of apilimod and YM201636, we showed that chemotaxis and activation of the NADPH oxidase and can impact phagosomes in human neutrophils were impaired for acquisition phagocytosis. A common factor in all of these processes is that they of LAMP1, suggesting a defect in phagosome–lysosome fusion 8 PIKfyve LIPID KINASE AND NEUTROPHILS Downloaded from http://www.jimmunol.org/ FIGURE 8. PIKfyve inhibition blocks phagosome maturation and chemotaxis in human neutrophils. (A) Human neutrophils were treated with vehicle or PIKfyve inhibitors, followed by phagocytosis, maturation, and staining for LAMP1, as before. Arrows indicate LAMP1+ phagosomes, and arrowheads point to negative or weakly stained phagosomes. Scale bar, 10 mm. (B) Quantification of LAMP1 staining of phagosomes in human neutrophils, as described before. (C and D) Quantification of human neutrophil direction (D) and speed toward (C) an fMLF gradient, as described previously. Data are mean 6 SEM from n =3(B) and n =5(C and D) independent experiments. *p , 0.05, ANOVA and the Tukey post hoc test.

(Fig. 8A, 8B). Lastly, human neutrophils treated with apilimod PIKfyve activity. In this article, we show that neutrophils contain became disoriented and migrated more slowly than vector-treated a distinct LAMP1 compartment that becomes engorged by acute cells, showing a defect in chemotaxis (Fig. 8C, 8D). Overall, these impairment of PIKfyve activity. In contrast, none of the neutrophil by guest on September 28, 2021 data suggest that PIKfyve also plays an important role in the granules that we examined underwent perceptible changes in mor- immune response of human neutrophils. phology under acute inhibition of PIKfyve. In addition, we showed that PIKfyve-inhibited neutrophils were still competent for Discussion fMLF-induced secretion of primary, secondary, and tertiary gran- Neutrophils are exceptionally important for the immune response, ules, as well as for phagosome–primary granule fusion. The de- rapidly targeting sites of infection by chemotaxis and unleashing a granulation of primary granules in PIKfyve-inhibited cells is series of attacks on pathogens, including ROS generation, secretion perplexing because fMLF-induced Rac2 activation is blunted by 2 2 of antibacterial peptides, and engulfment and digestion of patho- PIKfyve inhibitors but Rac2 / murine neutrophils are defective gens by phagocytosis (1, 2, 4). These processes are dependent on for chemoattractant-induced primary granule secretion (60). We and coordinated by a complex signaling network that uses small speculate that this inconsistency may be caused by differences in GTPases and PtdInsP signals. However, the importance of PIK- chronic (genetic) versus acute (pharmacological) disruption of fyve activity, which synthesizes PtdIns(3,5)P2 and directly or in- Rac2 and PIKfyve function. directly, PtdIns(5)P, for neutrophil function remained unexplored. Overall, these results suggest that PIKfyve has little direct We investigated that in this study and found that PIKfyve ac- control of granule function in neutrophils and seem consistent with tivity is critical for neutrophils to perform chemotaxis, generate the normal biogenesis and stimulus-dependent secretion of alpha- ROS, and undertake phagosome fusion with lysosomes, but not granules and dense granules found in platelets in mice carrying a for degranulation. Moreover, we propose a model by which platelet-specific PIKfyve gene deletion (35). Nevertheless, it re- phagosome maturation in neutrophils uses the PtdIns(3,5)P2 ef- mains possible that chronic deficiency of PIKfyve in neutrophils fector, TRPML1, to mediate lysosomal Ca2+ release and trigger could impair biogenesis and function of the various neutrophil phagosome–lysosome fusion, whereas chemotaxis and ROS pro- granules; this will require the generation of mice carrying a ceed through PIKfyve-dependent activation of the Rac GTPases, neutrophil-specific PIKfyve gene deletion. likely through PtdIns(5)P. PIKfyve in phagosome maturation Impact of PIKfyve on neutrophil lysosomes and granules Neutrophils and macrophages have distinct phagosome-matura- PIKfyve is well established to regulate lysosome morphology. In tion processes. Although both use phagosome–lysosome fusion, the absence of its activity, lysosomes in a multitude of cells, in- phagosomes in neutrophils also merge with primary granules, cluding the yeast vacuole, undergo massive enlargement (19, 20). acquire a high concentration of ROS, and do not acidify signifi- However, to our knowledge and with the exception of platelet cantly (1, 57). In this article, we showed that phagosomes dense granules (35), lysosome-related organelles have not been enclosing IgG-coated particles require PIKfyve activity and Ca2+ examined for their susceptibility to enlarge in the absence of to fuse with LAMP1+ lysosomes in neutrophils. Importantly, The Journal of Immunology 9 ionomycin or the TRPML1 agonist MLSA1 partially rescued Disclosures phagosome acquisition of LAMP1 in PIKfyve-inhibited cells. The authors have no financial conflicts of interest. This suggests that a PtdIns(3,5)P2-dependent activation of TRPML1 releases lysosomal Ca2+ and triggers phagosome–lysosome fusion. These observations are consistent with our earlier findings in References 2+ 1. Nordenfelt, P., and H. Tapper. 2011. Phagosome dynamics during phagocytosis macrophages, which also require the PIKfyve–TRPML1–Ca by neutrophils. J. Leukoc. Biol. 90: 271–284. axis to trigger fusion between docked phagosomes and lysosomes 2. Mayadas, T. N., X. Cullere, and C. A. Lowell. 2014. The multifaceted functions (36, 37). In contrast, PIKfyve was not necessary for phagosome of neutrophils. Annu. Rev. Pathol. 9: 181–218. 3. Lee, W. L., R. E. Harrison, and S. Grinstein. 2003. Phagocytosis by neutrophils. fusion with primary granules, suggesting a different mechanism Microbes Infect. 5: 1299–1306. for this process. 4. Nauseef, W. M., and N. Borregaard. 2014. Neutrophils at work. Nat. Immunol. 15: 602–611. PIKfyve activity is necessary for Rac activation in neutrophils 5. 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Average # of 1 vacuoles/cell 0.5 0

1 nM 0 1 5 10 20 50

15 min YM 201636 concentration (nM)

D 4 * 3.5 5 nM 3 * 2.5 2 * 1.5 *

Average # of 1 vacuoles/cell 60 min 30 min 10 nM 0.5 0 0 15 30 60 90 Time (min) 90 min 50 nM 20 nM

Supplemental Figure 1: Potency and kinetics of YM201636-induced vacuolation in neutrophils. A. Neutrophils were exposed* to vehicle or to the indicated YM201636 concentrations for 1 h, followed by live- cell imaging. C. Neutrophils were exposed to vehicle or 10 nM YM201636 for the indicated time points, before live-cell imaging. B and D. Vacuolation was quantified by scoring the number of vacuoles greater than 1 µm in diameter per cell. Shown are the mean number of vacuoles per neutrophil ± SEM from n= 3 independent experiments. Asterisk (*) indicates statistically difference (p<0.05) relative to control (vehicle or t = 0 min) using Student’s t-test. A Control YM B 60 Control YM 50

DIC 40 30 20 10 Mean Percentage of Phagosomes * LAMPI 0 LAMPI LAMPI LAMPI positive partial negative Pseudo-color

Supplemental Figure S2: YM201636-dependent inhibition of PIKfyve blocks phagosome maturation in neutrophils. A. Neutrophils were treated with vehicle-only or with 5 nM YM201636 for 1 h before being allowed to engulf IgG-coated beads and undertake maturation. Cells were then stained for LAMP1 to determine phagosome maturation. Arrows indicate LAMPI-positive phagosomes and arrowheads indicate LAMPI-negative phagosomes. Pseudo-colouring was employed to facilitate quantification of LAMP1 intensity into three groups: strong LAMP1 (orange-white), intermediated LAMP1 (red-magenta) and weak LAMP1 (blue-black) intensities. B. Quantification of LAMP1 staining of phagosomes into three groups of LAMP1 intensity as described in Methods section. YM201636 inhibits labelling of phagosomes with LAMP1. Data are shown as the mean ± SEM from n = 3 independent experiments, followed by analysis with Student’s t-test or with ANOVA and Tukey's post-hoc test. Asterisk indicates statistically significant difference to control group (p<0.05). A B 70 DIC LAMPI MPO 60 50

Control 40 30 of MPO 20 10 Apilimod Average total phagosome fluorescence association 0 Control AP

Supplemental Figure S3: PIKfyve inhibition does not block phagosome acquisition of MPO, a primary granule marker. A. Neutrophils were treated with vehicle (control) or 20 nM apilimod, followed by phagocytosis of IgG-coated beads and a chase of 1 h. Cells were then stained for LAMP1 and MPO to detect lysosome and primary granule fusion with phagosomes. Scale bar = 10 µm (add arrows). B. Quantification of phagosome-associated MPO fluorescence signal as described in Methods. Shown is the mean ± SEM from n= 3 experiments. Using Student’s t-test, there was no apparent difference between control and apilimod-treated cells.