Résultats et discussion

Les résultats obtenus et leur discussion sont exposés dans la publication que vous trouverez à partir de la page suivante, et que je signe en co-premier auteur :

Regulation of Phospholipase D by Arf6 during FcγR-mediated Phagocytosis.

Tanguy E.*, Tran Nguyen A. P.*, Kassas N., Bader M.-F., Grant N. J., and Vitale N. (* co-first authors) Journal of Immunology, 2019 May 15; 202(10):2971-2981

The Journal of Immunology

Regulation of Phospholipase D by Arf6 during

FcgR-Mediated Phagocytosis

Emeline Tanguy,¹ An Phu Tran Nguyen,¹ Nawal Kassas, Marie-France Bader, Nancy J. Grant, and Nicolas Vitale

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcgR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcgR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcgR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis. The Journal of Immunology, 2019, 202: 000–000.

S

Membrane trafficking is essential for phagocytosis during both the formation and maturation of the phagosome. Different in-tracellular compartments, including early and late endosomes, lysosomes (6), and the endoplasmic reticulum (7), have been proposed as membrane sources for focal exocytosis at phago-cytic sites. Formation of SNARE complexes between the plasma membrane and vesicular structures arising from these compart-ments are necessary for this focal exocytosis (8, 9). In addition, different lipids, including phosphoinositides and phosphatidic acid (PA) (10, 11), contribute to phagosome formation, internalization, and maturation. Using specific sensors, we have previously dem-onstrated that PA is produced and accumulates at phagocytic sites (12–14). Although diacylglycerol kinases may be an important source of PA during phagocytosis (15), an increase in phospho-lipase D (PLD) activity, which generates PA from phosphatidyl-choline, has also been reported during the activation of several phagocytic receptors, including the FcgR (16, 17). Both PLD1 and PLD2 isoforms seem to be necessary for efficient phagocytosis (12). In RAW 264.7 macrophages, PLD2 is present at the plasma membrane, whereas PLD1 associated with the late endosome/ lysosome compartment is recruited to phagocytic sites (12), sug-gesting a sophisticated regulation of PLD activity and PA synthesis during phagocytosis. Interestingly, PLD1 and PLD2 have also been shown to regulate different steps in FcεRI-mediated degranula-tion and anaphylactic reacdegranula-tions in mast cells (18–20), suggesting a widespread critical role for PLDs in immune cell functions. Note that immune cell activities are tightly controlled functions, which makes the issue of PLD regulation during immune responses particularly interesting.

Monomeric GTPases are master regulators of membrane traf-ficking processes, including receptor-mediated endocytosis, endo-somal recycling, and exocytosis of secretory granules (21–23). The first GTPase that has been directly implicated in phagocyto-sis, in particular in the delivery of endomembranes to forming phagosomes, was the ADP ribosylation factor (Arf) 6 (24–26). Yet, the downstream pathway by which Arf6 participates in

Centre National de la Recherche Scientifique, Universite´ de Strasbourg, Institut des Neurosciences Cellulaires et Inte´gratives, F-67000 Strasbourg, France

1E.T. and A.P.T.N. contributed equally to the work. ORCID:0000-0002-4752-4907(N.V.).

Received for publication July 23, 2018. Accepted for publication March 11, 2019. This work was supported by grants from La Ligue Contre le Cancer and from Fondation pour la Recherche Me´dicale (to N.V.).

A.P.T.N., E.T., N.K., and N.J.G. performed and analyzed experiments. N.V. and N.J.G. designed the experiments. N.V., M.-F.B., and N.J.G. wrote the manuscript. All authors revised the manuscript.

Address correspondence and reprint requests to Dr. Nicolas Vitale, Institut des Neurosciences Cellulaires et Inte´gratives, 5 Rue Blaise Pascal, Strasbourg 67000, France. E-mail address: vitalen@unistra.fr

Abbreviations used in this article: Arf, ADP ribosylation factor; GGA3, Golgi-localized g-adaptin ear homology Arf binding protein 3; HA, hemagglutinin; PA, phosphatidic acid; PLD, phospholipase D; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; RNAi, RNA interference; siRNA, small interfering RNA. CopyrightÓ 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801019

Published April 3, 2019, doi:10.4049/jimmunol.1801019

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FcgR-dependent phagocytosis is still unknown. GTPases, together with protein kinase C, are also the best-characterized regulators of PLD. Initially, only PLD1 activity was thought as being regu-lated by these GTPases because of the high basal activity of PLD2. However, it was later shown that activities of both PLD isoforms are subject to regulation (27). Altogether, these findings prompt us to investigate whether Arf6 might be implicated in phagocytosis by regulating PLD activity and PA synthesis. We show, in this study, that Arf6 and PLDs partially colocalize and interact in macrophages undergoing phagocytosis. Arf6 silencing effectively reduces PLD activation and PA synthesis at the phagocytic sites, consistent with the idea that Arf6 contributes to optimal phagocy-tosis through the regulation of PLD.

Materials and Methods

Reagents and Abs

RPMI 1640 and FBS were purchased from Invitrogen. Latex beads (3 mm; Sigma-Aldrich) were coated with human IgG (Zymed). Escherichia coli coupled to Alexa Fluor 594 and zymosan particles were purchased from Molecular Probes. Rat anti-hemagglutinin (HA) affinity matrix was pur-chased from Roche. The following mAbs were used: anti-Arf6 (mouse IgG2b; Santa Cruz Biotechnology), anti-Arf1 (mouse IgG2a; Abcam), anti-CD64 (rat IgG2b; R&D Systems), anti-HA (mouse IgG1, HA.11; Covance), anti-GFP (mouse IgG1; Roche), anti–b-actin (mouse IgG1, clone AC-15; Sigma-Aldrich), and anti–b-tubulin (mouse IgG1, clone TUB 2.1; Sigma-Aldrich). Polyclonal anti-Arf6 (rabbit; Bethyl Labo-ratories) was also used for immunoblots. Goat secondary Abs coupled to Alexa Fluor 555 or 568 (Molecular Probes) or to peroxidase (Thermo Fisher Scientific) were used for immunofluorescence and immunoblots, respectively.

Cell culture

Culture conditions for the murine macrophage RAW 264.7 cell line in RPMI 1640 glutamax medium supplemented with 10% FBS were as previously described (12–14).

Plasmids and transfection

Arf6-pEGFP and small interfering RNA (siRNA)–resistant Arf6-HA-pXS and Arf6(N48I)-HA-pXS were described previously (23, 28). The plas-mids pCGN-human PLD1 (PLD1-HA) and pCGN-mouse PLD2 (PLD2-HA) were described previously (29). MT2-GFP was used to visualize the localization of the active form of Arf6 (28). Similarly, Spo20p-GFP served as a probe to detect sites of PA production (30). RAW 264.7 cells were transfected with the plasmids by electroporation (12) or using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific). For confocal experiments, RNA interference (RNAi)–treated macrophages were collected, replated on glass inserts in 35-mm Petri boxes (Maktek), and transfected with MT2-GFP or Spo20-GFP.

Stimulation of phagocytosis and phagocytosis assay

IgG-opsonized 3-mm latex particles (IgG beads) or E. coli particles, washed with PBS and resuspended in medium, were added to cells grown on glass coverslips (20 beads per cell or 50 E. coli per cell). For these assays, phagocytosis was synchronized after adding particles by

centri-fuging cells for 2 min at 1003 g at 18˚C and then initiating phagocytosis

by placing them at 37˚C as previously described (12). Briefly, phagocytosis was stopped 30 min later by washing twice in cold PBS. Fluorescence of noninternalized E. coli was bleached by trypan blue 0.4%. After fixation, external beads were labeled with goat anti-human IgG coupled to Alexa Fluor 555 (Molecular Probes). Unlabeled internalized beads were visual-ized with phase contrast optics (Axio Imager 2; ZEISS). The mean number of internalized beads or the mean fluorescence intensity (for E. coli assay) per cell was determined for randomly chosen fields (minimum of 80 cells for each field) using superimposed fluorescent and phase contrast images (Adobe Photoshop 9). The phagocytic index was normalized to 100% for cells transfected with the control RNAi.

Arf activation assay

Phagocytosis was initiated by adding IgG-opsonized particles to 13 107

cells in suspension at 37˚C and stopped at different times (t = 0, 5, 10, and 20 min) as previously described (31). For each time point, Arf-GTP was precipitated

from lysates (2 mg total protein) with the Arf-GTP binding domain of Golgi-localized g-adaptin ear homology Arf binding protein 3 (GGA3) linked to GST, according to the instructions in the Arf Activation Assay Kit (Thermo Fisher Scientific). Control samples with or without the addition of opsonized particles were maintained at 4˚C. Arf-GTP pre-cipitates and aliquots of total cell lysates (10–20 mg protein) were an-alyzed on immunoblots using Abs against Arf6, Arf1, and actin. Immunoprecipitation of PLD

Macrophages were transfected with Arf6-GFP and either PLD1-HA, PLD2-HA, or bPIX-PLD2-HA, and cells were collected 24 h later. Phagocytosis

was then initiated by adding IgG beads to the cell suspensions (53

10⁶cells) at 37˚C for different times, and cell lysates were then prepared

in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2,0.1 mM DTT,

0.5% Triton X-100, 0.5% DOC, and protease inhibitor mixture (Sigma-Aldrich). PLD proteins were immunoprecipitated from lysates (0.5 mg protein) using rat Anti-HA Affinity Matrix (Roche) and analyzed by immunoblots.

RNAi

Macrophages were transfected with 40 pmoles of a stealth RNAi duplex specific for mouse Arf6 (duplex A: 59-GGAACAAGGAAATGCGGATCC-TCA-39; duplex B: 59-CAGCCGGCAAGACAACGAUCCUGUA-39; and duplex C: 59-CCCAGGGUCUGAUCUUCGUGGUAGA-39) or a control nontargeted oligonucleotide (CG medium) using Lipofectamine RNAi-MAX according to the manufacturer’s instructions (Invitrogen). Using a control oligonucleotide Alexa Fluor 488 (Invitrogen), the transfection

efficiency of stealth RNAi was estimated by flow cytometry to be.85%.

After 48 h, the reduction in the Arf6 protein expression and activation was determined by immunoblots, and as a control, the expression of Arf1 was checked.

Immunoblotting

Lysates were prepared before and following FcgR activation. Total cell extracts were prepared in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1.0% Triton X-100, 0.5% deoxycholate, and a protease inhib-itor mixture (Sigma-Aldrich) and cleared by centrifugation for 10 min

at 10,0003 g at 4˚C. For subcellular fractions, cells were suspended in

50 mM Tris-HCl (pH 7.5), 2 mM EDTA, containing protease inhibitors at 4˚C and lysed in a Potter homogenizer. After an initial centrifugation at

8003 g for 10 min at 4˚C, the lysate was centrifuged at 20,000 3 g for

30 min at 4˚C to separate soluble cytoplasmic supernatant and a membrane fraction composed of organelles and large-membrane fragments. Immu-noblotting was carried out as previously described (12). Briefly, proteins were separated on 4–12% NuPAGE gradient gels in MES buffer (Invitrogen) and transferred to nitrocellulose. After immunolabeling, blots were revealed using the SuperSignal West Dura or Femto Chemiluminescent Substrate (Pierce Biotechnology, Thermo Fisher Scientific). For cell lysate samples, actin served as a control for equal protein loadings. Images were acquired using a Chemi-Smart 5000 and the Chemi-Capt program (Vilber Lourmat), and protein bands were quantified using the program Bio1D (Vilber Lourmat). Images were then processed with Photoshop 9.

Confocal microscopy

Live cells were observed at 37˚C 18–24 h after transfection in the absence or presence of IgG beads. Videos were obtained by acquiring images for GFP and phase contrast every minute for 20 min. Cells destined for im-munofluorescent labeling were fixed for 10 min at 4˚C with 4% parafor-maldehyde in 0.125 M phosphate buffer, and for intracellular labeling, this was followed by a 10-min permeabilization step in fixative containing 0.2% Triton X-100. Samples were then blocked with 10% goat serum, and PLD was visualized using anti-HA Abs followed by goat anti-mouse Alexa Fluor 555 or 568. Images were obtained using a Zeiss LSM 510 or a Leica SP5 II inverted microscope equipped with a Plan APO oil

(633) immersion lens (numerical aperture = 1.4). Images were recorded

with the same parameters and optimal pinhole and processed using Adobe Photoshop 9.

Using the Zeiss CLSM software 2.8, masks of double-labeled pixels were generated. The proportion of Arf6-GFP colocalized with PLD1-HA or PLD2-HA was determined in nonstimulated cells and in cells during particle ingestion using the weighted colocalization percentages gener-ated for double-labeled pixels. Quantification of Arf6-GFP signal, or beads positive for GFP signal (MT2-GFP or Spo20p-GFP) were performed using

Icy software. For each cell, a fixed region of interest of 6 pixels3 6 pixels

were randomly selected at the plasma membrane or at the periphery of

2 Arf6 AND PA PRODUCTION DURING PHAGOCYTOSIS

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beads, and the fluorescence signal was measured and compared with the fluorescence signal obtained from five distinct region of interests of the same size randomly selected in the cytosol. Beads were considered as positive for staining if the bead fluorescence level was above 1.2-fold of averaged cytosolic signal.

Measurement of PLD activity

Forty-eight hours after siRNA transfection, RAW 264.7 macrophages

(63 106

cells) were washed and then incubated at 37˚C in the absence (resting) or presence (stimulated) of IgG-opsonized zymosan particles. Cell lysates were prepared in 600 ml of ice-cold 50 mM Tris-HCl (pH 8) by three freeze and thaw cycles. Aliquots of the lysates (corresponding

to 13 106

cells) were mixed with an equal amount of the Amplex Red reaction buffer (Amplex Red Phospholipase D Assay Kit; Molecular Probes), and the PLD activity was estimated after 1-h incubation at 37˚C with a Mithras fluorometer (Berthold Technologies) as described previ-ously (32). A standard curve was established with purified PLD from Streptomyces chromofuscus (Sigma-Aldrich), and an average activity (milliunits per milliliter) was calculated from four determinations made for each condition.

Statistical analysis

Number of experiments and repeats are indicated in figure legends. Nor-mality of the data distribution was verified with ANOVA test, and statistical analysis was performed with t tests relative to the indicated control, except for Fig. 3A, for which ANOVA test was used for the analysis.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

Results

Silencing of Arf6 by RNAi reduces phagocytosis

Based on the expression of constitutively active and dominant-negative GTP binding–deficient mutants, Arf6 was the first mo-nomeric GTPase described to play a role in phagocytosis (24–26). To investigate the downstream effector of Arf6 in phagocytosis, we decided to use an RNAi strategy to decrease the expression level of the endogenous Arf6 protein in RAW 264.7 macrophages. Three Arf6-RNAi were compared. As seen by Western blot and densitometric scan analyses (Fig. 1A), the three Arf6-RNAi specif-ically reduced endogenous Arf6 levels by 60–80% without affecting the expression levels of actin or the related isoform Arf1. Decrease of endogenous Arf6 in RAW 264.7 macrophages reduced phago-cytosis of IgG beads by ∼25% (Fig. 1B). Interestingly, Arf6 silencing inhibited phagocytosis of E. coli (Fig. 1B) and of non-opsonized beads (data not shown) as well. Thus, Arf6 seems to be involved in both FcgR-mediated and FcgR-independent phagocy-tosis. It is of note that Arf6 silencing did not alter CD64 receptor expression level (Fig. 1C), indicating that the inhibition of FcgR-dependent phagocytosis by reducing endogenous Arf6 is not the consequence of an eventual FcgR downregulation.

Arf6 is recruited to nascent phagosomes

We then investigated the subcellular localization of Arf6 in RAW 264.7 macrophages undergoing FcgR-mediated phagocytosis. Dis-tribution of endogenous Arf6 was first compared with the distri-bution of expressed Arf6-GFP in macrophage subcellular fractions by Western blot analysis (Fig. 2A). In both resting cells and cells stimulated with IgG beads, Arf6 was found to be concentrated in the crude membrane fraction containing the plasma membrane and organelles. Of note, expressed Arf6-GFP behaved like en-dogenous Arf6 regarding its subcellular localization (Fig. 2A). Arf6-GFP was then used to study the distribution of Arf6 during phagocytosis in living cells by time-lapse confocal imaging (Fig. 2B). Incubation with IgG beads triggered the rapid recruitment of Arf6-GFP to the cell periphery, especially at sites of phagocytosis (arrow, Fig. 2B). Arf6-GFP was found to accumulate on pseudo-pods and membrane ruffles formed around the beads (Fig. 2B,

times 0–5 min). Note, however, that Arf6-GFP was not detected on fully internalized beads (Fig. 2B, times 7–20 min). Quantifi-cation of the GFP fluorescence confirmed the preferential accu-mulation of Arf6 at the phagocytic cup (nascent phagosomes) and, to some extent, on the plasma membrane but not on internalized beads (Fig. 2C), suggesting that Arf6 is mainly involved in the early stages of the internalization process.

Arf6 silencing reduces the amount of activated Arf6 present at the nascent phagosome

Like all GTPases, Arf6 cycles between inactive GDP-bound and active GTP-bound forms. The level of endogenous activated Arf6 in resting and IgG-stimulated RAW 264.7 macrophages was assessed by pull-down experiments using GST–GGA3 as bait for Arf6-GTP. Whereas the total level of Arf6 did not significantly change upon stimulation, the level of active Arf6 increased by more than 2.5-fold after 20 min of incubation with IgG beads (Fig. 3A). Based on the total amount of Arf6 and Arf6-GTP levels, we estimated the ratio of activated Arf6 and found an increase from 0.3% in control conditions to 0.75% following 20 min of IgG stimulation. This is probably an underestimation of the actual levels of Arf6-GTP. As the pull-down efficiency in our assay ranges from 50 to 95%, the actual Arf6-GTP levels might range from 0.3–0.6% in resting cells to 0.75–1.5% in cells stimulated for 20 min with IgG beads.

Because the amount of activated Arf6-GTP remains rela-tively low in many cell types (33, 34) including macrophages, the residual endogenous Arf6 expressed in Arf6-RNAi–transfected cells might be sufficient to provide levels of active Arf6 similar to that found in control cells. To probe this possibility, we used the GST–GGA3 probe to pull down the GTP-bound forms of Arf6 and Arf1 in Arf6-RNAi–expressing cells stimulated for phago-cytosis. As observed for the endogenous Arf6 protein, the amount of Arf6-GTP was significantly reduced in cells expressing an Arf6-RNAi (Fig. 3B). Thus, Arf6-RNAi not only reduced the total level of endogenous Arf6 but also the amount of acti-vated Arf6 detected in macrophages stimulated for phagocy-tosis. Surprisingly, Arf6-RNAi also significantly increased the level of activated Arf1 (Fig. 3B). Because Arf1 and Arf6 share several common downstream effector pathways, it is therefore quite possible that in cells expressing an Arf6-RNAi, active Arf1 may, to some extent, compensate for the reduced level of active Arf6, reducing the inhibitory impact of the Arf6-RNAi on phagocytosis.

To visualize the distribution of activated Arf6 in RAW 264.7 macrophages, MT2 fused to GFP was used as a specific sensor for Arf6-GTP because it does not recognize other members of