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1 European Journal of Cell Biology xxx (2010) xxx xxx Contents lists available at ScienceDirect European Journal of Cell Biology journal homepage: Initial receptor ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis Eik Hoffmann a,b,,1, Sabrina Marion b,2, Bibhuti Bhusan Mishra c,3, Mathias John d, Ramona Kratzke a, Syed Furquan Ahmad a, Daniela Holzer b, Paras Kumar Anand b, Dieter G. Weiss a, Gareth Griffiths b,4, Sergei A. Kuznetsov a, a Institute of Biological Sciences, Cell Biology and Biosystems Technology Unit, University of Rostock, Germany b European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany c Molecular Pathogenesis Center, Faculty of Pharmacy, University of Lisbon, Portugal d Department of Computer Science, University of Rostock, Germany article info abstract Article history: Received 8 February 2010 Received in revised form 27 April 2010 Accepted 29 April 2010 Keywords: Phagocytosis Receptor ligand interaction Macrophage Phagosome maturation Phagocytic receptor Phagosomal proteome Gene expression Signaling pathways Immune response The receptors engaged during recognition and phagocytic uptake of microorganisms and particles influence signaling events and diverse subcellular responses that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands on their surface, making it difficult to dissect the roles of individual receptors during phagocytosis. Moreover, it remains elusive to which extent receptor ligand interactions and early binding events define the subsequent intracellular fate of phagosomes. Here, we used latex beads coupled to single ligands, focusing on immunoglobulin G, mannan, bacterial lipopolysaccharides and avidin, and monitored: (1) phagocytic uptake rates, (2) fusion of phagosomes with lysosomal compartments, (3) the gene expression profile during phagocytosis, (4) the protein composition of mature phagosomes and (5) time-dependent dynamics of protein association with phagosomes in J774.A1 mouse macrophages. The differently coated latex beads were internalized at different rates and exhibited different kinetics of phagolysosomal fusion events dependent on their specific ligand. Furthermore, less than 60% of identified phagosomal proteins and only 10 15% of changes in gene expression were common to all investigated ligands. These findings demonstrate that each single ligand induced a distinct pattern of genes and a different protein composition of phagosomes. Taken together, our data argue that phagocytic receptor-specific programs of signaling events direct phagosomes to different physiological states and support the existence of a specific receptor ligand signature during the whole process of phagocytosis Elsevier GmbH. All rights reserved. Introduction Corresponding author at: Institute Curie, INSERM U932, Immunity and Cancer, 26 rue d Ulm, Paris Cedex 05, France. Tel.: ; fax: Corresponding author at: University of Rostock, Institute of Biological Sciences, Albert-Einstein-Str. 3, Rostock, Germany. Tel.: ; fax: addresses: (E. Hoffmann), (S.A. Kuznetsov). 1 Present address: Institute Curie, INSERM U932 - Immunity and Cancer, Paris, France. 2 Present address: Institute Cochin, University Paris Descartes, CNRS UMR 8104, Paris, France. 3 Present address: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA, USA. 4 Present address: Department of Molecular Biosciences, University of Oslo, Norway. Phagocytosis is a process initiated by binding of a particle usually bigger than 0.5 m in diameter to the cell surface of phagocytes followed by its ingestion into a newly formed vesicle, called the phagosome. When extracellular ligands bind to cell surface receptors they induce a receptor-specific program of signaling events, which present a particular time scheme. A well-characterized phagocytic receptor such as the Fc receptor (Fc R), which is engaged during the uptake of particles that have been opsonized with immunoglobulin G (IgG), can be cited as an example. When these particles bind to Fc R, they change the cytoplasmic domains of the receptor, which recruit and activate proteins within seconds initiating immediate responses, such as receptor tyrosine phosphorylation, PI3K/Akt and MAPK activation (Cox and Greenberg, 2001; Swanson and Hoppe, 2004). These signals induce actin polymerization and rapid extension of membrane over the opsonized surface, which engage other IgG molecules that /$ see front matter 2010 Elsevier GmbH. All rights reserved. doi: /j.ejcb

2 2 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx induce similar responses closing the phagocytic cup and forming the phagosome within a few minutes (Swanson, 2008). At a later stage, a subsequent wave of signaling activates transcription factors that turn on the expression of a receptor-specific set of genes. Therefore, even when internalization is accomplished, a specific signaling program can run for many hours or even days to modulate specific intracellular functions (Ernst, 2000; Aderem, 2003). A major challenge for the innate immune system is to discriminate between a large number of potential pathogens from self antigens, utilizing a finite number of phagocytic receptors. This challenge has been solved by the evolution of a variety of receptors that recognize conserved motifs on pathogens that are not found in higher eukaryotes (Kabelitz and Medzhitov, 2007). These receptors are called pattern-recognition receptors (PRR) and the targets of these receptors pathogen-associated molecular patterns (PAMP; Janeway, 1992). Pathogen-associated motifs include mannans in the yeast cell wall, formylated peptides, lipopolysaccharides and lipoteichoic acids on the surface of Gramnegative and Gram-positive bacteria, respectively. While direct pathogen recognition is a fundamental aspect of innate immunity, opsonization allows additional diversification of the phagocyte recognition repertoire (Stuart and Ezekowitz, 2005). Receptors bearing immunoreceptor tyrosine-based activation motifs (ITAM) or inhibitory motifs (ITIM) are crucial in the immune response (Pasquier et al., 2005). In macrophages, receptors are classified into opsonic phagocytic receptors, like members of the FcR family and complement receptors (CR), and non-opsonic phagocytic receptors, such as lectins (mannose receptor, MR; DC-SIGN, Dectin-1), integrins and scavenger receptors (SR). Many non-opsonic receptors were believed to be unable to induce inflammatory responses on their own, but some C-type lectins, such as Dectin-1 and Mincle, are able to induce gene expression in the absence of other PRR signaling (Brown, 2006; Geijtenbeek and Gringhuis, 2009). A third group contains Toll-like receptors (TLR) which often function as co-receptors in phagocytosis by their discrimination of a broad range of microbial products, including PAMP, lipopolysaccharide, peptidoglycan and bacterial lipopeptides (Underhill et al., 1999; Underhill and Ozinsky, 2002). The role of stimulated TLR in accelerating and modulating phagosome maturation and the hypothesis of TLR-mediated phagosomal autonomy are still matter of debate (Blander and Medzhitov, 2006; Russell and Yates, 2007). Finally, during phagocytosis the activation of macrophages by either pro-inflammatory or anti-inflammatory cytokines contributes to triggering, instructing and terminating the immune response (Martinez et al., 2008). As mentioned before, receptors on the surface of the phagocytic cell orchestrate a set of signaling events that are required for particle internalization. This has also been elegantly demonstrated in non-phagocytic cells, such as CHO cells, which can selectively phagocytose IgG-coated particles after transfection with constructs encoding Fc receptors (Nagarajan et al., 1995). This shows that a single type of ligand on the particle can efficiently induce phagocytic uptake. However, most pathogens possess many different ligands on their surface. Their phagocytic uptake occurs via multi-ligand interactions, which induce the engagement of many receptors at the same time. This makes it very difficult to dissect the contribution of different receptor ligand interactions into the overall process of phagocytosis and in phagosome maturation. Until now, it is unclear whether multiple receptors can signal in parallel in response to phagocytosis or whether a single receptor will dominate the signaling pathways. Here, we introduce a system that can eventually be adapted to answer such questions. We used one of the best-characterized model systems in phagocytosis studies, the latex bead phagosome system (Desjardins and Griffiths, 2003; Griffiths and Mayorga, 2007), and coated these latex beads with single ligands for a global comparison of the characteristics of receptor-mediated uptake pathways involved in phagocytosis. Latex bead phagosomes (LBP), like phagosomes generally, are able to undergo fission and fusion events with endosomes and lysosomes over long time periods (Desjardins et al., 1994; Kjeken et al., 2004), acquire lipids and phosphoinositides (Defacque et al., 2002; Corrotte et al., 2006), bind and move along microtubules (Blocker et al., 1997), assemble actin filaments and bind to them (Jahraus et al., 2001; Al-Haddad et al., 2001) and become acidified (Huynh and Grinstein, 2007). In this study, latex beads were covalently conjugated with the Fc fragment of IgG, which is recognized by Fc receptors (Swanson and Hoppe, 2004) that are expressed in high abundance on the macrophage surface and facilitate the internalization of opsonized targets. For a bead ligand entering macrophages in a non-opsonic pathway, we selected mannan that is recognized predominantly by the mannose receptor (MR; Sheng et al., 2006) and by the mannosebinding lectin (MBL; Weis et al., 1992). Latex beads were also coated with bacterial lipopolysaccharides (LPS), which are recognized by CD14 and TLR4 in macrophages (Wright et al., 1990; Poltorak et al., 1998). The fourth ligand we have selected is avidin, which has been widely used as a non-specfic ligand in phagocytosis studies (Brown et al., 1998; Jahraus et al., 1998, 2001). Avidin is believed to engage multi-ligand receptors, such as members of the scavenger receptor family and integrins (Taylor et al., 2005). Similar to other non-specific bead ligands, such as BSA, the intracellular fate of phagosomes containing this bead type is different compared to Fc R-mediated uptake (Link et al., 2010). Since the applied latex beads are identical for each ligand, we were able to compare cellular functions that are dependent on the particular ligand coupled to the bead. Moreover, LBP can be isolated by sucrose gradient flotation at a very high degree of purity (>95%) allowing one to dissect the molecular composition of these organelles (Desjardins and Griffiths, 2003; Stuart et al., 2007). In this study, our goal was to monitor downstream cellular events of phagocytosis induced by different types of interaction between a single ligand with its receptor over a broad time scale. To achieve this goal, we investigated (1) phagocytic uptake efficiency, (2) phagosomal fusion with lysosomes by confocal microscopy, (3) macrophage gene expression by a microarray approach, (4) the protein composition of phagosomes by mass spectrometry and (5) time-dependent dynamics of association of defined proteins by immunoblotting. All the results obtained indicate that the choice of a particular ligand on the bead has a significant influence on the mentioned processes and strongly supports the notion that each receptor ligand interaction induces its own signaling signature that influences the pattern of macrophage gene expression and the cellular fate of phagosomes. Materials and methods Cells, latex beads and antibodies J774.A1 mouse macrophage-like cells were obtained from the German Resource Center of Biological Material (DSMZ) Braunschweig, Germany, and were cultivated in DMEM (Invitrogen, UK) supplemented with 10% fetal bovine serum (Biochrom, Germany) at 37 C and 5% CO 2. Fluorescent carboxylated latex beads (Invitrogen, USA) and non-fluorescent carboxylated beads (Polysciences, USA), both of 1 m diameter, were conjugated to avidin (Invitrogen, USA), Fc fragment of mouse IgG (Thermo Fisher Scientific, USA), LPS from Klebsiella pneumoniae and mannan from Saccharomyces cerevisae (Sigma Aldrich, USA) as described as supplemental data accompanying this study. Primary antibodies used were anti-actin (clone C4, ICN Biomedicals, USA), anti-lamp-2 (Iowa Hybridoma Bank, USA) and anti-cathepsin-d (clone C-20, Santa Cruz, USA). Secondary anti-

3 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx 3 bodies were purchased from Dianova, Germany, and Invitrogen, USA. Immunofluorescence microscopy and analysis of phagocytic uptake rates, inhibition of uptake and phagosomal maturation J774.A1 cells were incubated with serum-free DMEM containing 0.01% latex beads (OD 600 = 1.6) for different periods of time (0, 5, 15, 30, 45 and 60 min). Cells were fixed in PBS containing 4% paraformaldehyde and 4% sucrose, ph 7.4 for 20 min followed by quenching in PBS containing 50 mm NH 4 Cl for 10 min, both at room temperature. Cells were permeabilized with 0.2% Triton X- 100 in PBS for 5 min. After blocking of non-specific binding sites with PBS containing 1% gelatin gold (Sigma Aldrich, USA) for 1 h, samples were labeled for F-actin by FITC-conjugated phalloidin (Sigma Aldrich, USA) and for the lysosomal enzyme LAMP-2. In all samples, 50 cells were analyzed for total numbers of internalized beads and the percentage of phagosomes that were positive for LAMP-2 in three independent experiments using a confocal laserscanning microscope (TCS SP2, Leica Microsystems, Germany). Alternatively, uptake of avidin beads and IgG beads was also analyzed by an inside outside staining technique. Macrophages were pre-loaded with rhodamine-conjugated nano gold of 10 nm, a fluid-phase marker that accumulates in lysosomes (Anes et al., 2006). Cells were incubated with beads and fixed as described above. Subsequently, the samples were labeled for 15 min with Alexa 405-conjugated biotin or anti-mouse IgG (Invitrogen, USA) to stain the remaining extracellular beads. After permeabilization, the samples were labeled with Alexa 488-conjugated biotin or antimouse IgG (Invitrogen, USA) to stain all beads. Analysis was carried out with a confocal microscope quantifying beads labeled by Alexa 405 and Alexa 488 (extracellular beads), by Alexa 488 alone (phagosomes before fusion with lysosomal compartments) or by Alexa 488 and rhodamine (phagosomes after fusion with lysosomal compartments). In the histogram shown as supplementary figure S1, internalized beads of 50 analyzed cells for each ligand and time point are shown. For uptake inhibition studies (shown as supplementary figure S2), cells were pre-incubated with the respective ligand in soluble form for 30 min followed by addition of the beads in presence of the soluble ligand. Increasing concentrations of the soluble ligand were tested for inhibition of uptake efficiency compared to control conditions (same bead type in absence of soluble ligand). Average data of three experiments analyzing 50 cells each are shown. Isolation of phagosomes For 2D gel electrophoresis and LC MS/MS, cells were incubated with serum-free DMEM containing 10 mm HEPES, ph 7.4, and 0.02% latex beads coupled to avidin, IgG Fc, mannan and LPS (OD 600 = 3.2) on a shaker for 1 h (pulse) followed by brief washing in PBS and further incubation in complete medium for 1 h (chase). For SDS- PAGE analysis of phagosomes, cells were incubated similarly for 30 min or 1 h pulse followed by a period of 1 or 3 h chase in some experiments. Phagosomes containing latex beads were isolated by sucrose gradient flotation as described elsewhere (Kühnel et al., 2006). Microarray hybridization All gene expression studies with each condition and time point were carried out in triplicates. Cells were seeded into 6-well plates 2 days prior the experiment and were grown as monolayer until they reached 75% confluency. Cells were incubated with serumfree DMEM containing 0.02% latex beads coupled to avidin, IgG Fc, LPS and mannan (OD 600 = 3.2) for 30 and 60 min. Control cells were incubated in serum-free DMEM alone. Subsequently, cells were washed three times in PBS and total RNA was isolated using the RNAeasy Mini kit (Qiagen, Germany) followed by quality control tests using a Bioanalyzer (Agilent Technologies, USA). The preparation of crna and hybridization was carried out using the CodeLink iexpress expression assay reagent kit (Applied Microarrays Inc., USA) following the manufacturers instructions. Subsequently, hybridization to CodeLink mouse whole genome bioarrays (Applied Microarrays Inc., USA) containing 36,000 mouse gene targets was performed. Ten micrograms of crna and 5 l of5 fragmentation buffer were incubated at 94 C for 20 min. Ten micrograms of fragmented crna, 78 l of hybridization buffer component A, 130 l of component B to a final volume of 260 l were incubated at 90 C for 5 min to denature the samples followed by immediate cooling on ice for 30 min to sustain annealing. The hybridization reaction mixture was loaded onto the array input port and sealed with strips to avoid dehydration. The slides were incubated for 20hat37 C on a shaker at 300 rpm. Then, bioarrays were transferred to a bioarray rack containing 0.75 TNT (0.1 M Tris HCl, 0.15 M NaCl and 0.05% Tween 20) followed by an incubation at 46 C for 1 h. Each bioarray was transferred to a chamber containing 3.4 ml of streptavidin-cy5 solution and incubated at room temperature for 30 min. The arrays were washed four times with 1 TNT followed by a brief rinse with 0.1 SSC (0.05% Tween 20). Bioarrays were dried by centrifugation at 9,000 rpm for 10 min and scanned with a GenePix 4000B (Molecular Devices, USA). The complete triplicate data set has been deposited in the GEO database with the accession number GSE17761 using the following URL: Analysis of microarray data The scanned files were analyzed using the CodeLink expression analysis software EXP 4.0. The intensity of each spot was calculated as the difference between mean signal intensity and mean local background intensity. The output CodeLink files were analyzed using GeneSpring GX 7.3 software (Agilent Technologies, USA). Analysis of fold changes in signal intensities of the latex bead samples compared to control conditions were performed using two-way ANOVA with a P-value cut-off <0.01. Genes that exhibited a change in their expression in all three replicates displaying 1.4-fold higher signal intensities were considered significantly upregulated, whereas the ones with 0.6-fold lower signal intensities were considered significantly down-regulated. For analyzing gene regulation data, we used the two-tone table lens (TTTL) visualization tool (John et al., 2008). For gene annotation enrichment analysis we were using the freely accessible database for annotation, visualization and integrated discovery (DAVID; Huang et al., 2009). Functional classification of genes was performed with gene ontology (GO) terms for the biological process using an enrichment P-value cut-off < Two-dimensional SDS-PAGE All chemicals were from Merck, USA, unless otherwise stated. Two-dimensional gel electrophoresis was performed according to the procedures reviewed previously (Görg et al., 2000). The same amount of phagosomes containing latex beads coupled to the Fc fragment of mouse IgG and mannan, respectively, isolated after 2 h, were used. After lysis in 2% Triton X-100, 10 mm DTT, 50 mm Tris, ph 8.0, and protease inhibitors, phagosomal proteins were precipitated in acetone. Precipitates were solubilized in 9 M urea, 16 mm CHAPS, 2 mm TBP, 0.25% Biolyte (BioRad, USA), protease inhibitor cocktail and applied to 11 cm IPG strips (BioRad, USA) at ph 3 10 with rehydration loading overnight as described elsewhere (Bjellqvist et al., 1982). The samples were

4 4 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx mixed with rehydration solution (8 M urea, 16 mm CHAPS, 2 mm TBP and 0.25% Biolyte) giving a final volume of 200 l. Isoelectric focusing was carried out using a Protean IEF Cell (BioRad, USA) for about 80 kv/h at a maximum voltage of 6,000 V at 20 C. After focusing, each IPG strip was emerged first for 10 min into reduction solution (6 M urea, 30% glycerol, 50 mm Tris, ph 8.8, 173 mm SDS and bromophenolblue) containing 5 mm TBP and then into alkylation solution containing 270 mm 2-iodoacetamide. Subsequently, the IPG strips were loaded on top of 12% SDS polyacrylamide gels in a Protean II xi 2-DE device (BioRad, USA). The gels were run for 45 min at 20 ma per gel and then for 600 V/h at 15 ma/gel in a second step. Visualization was achieved by using the silver staining method according to Shevchenko et al. (1996). Two-dimensional migration of phagosomal proteins was carried out in triplicates and representative silver-stained gels are shown. Liquid chromatography tandem mass spectrometry (LC MS/MS) Purified phagosomes were lysed in 2% Triton X-100, 10 mm DTT, 50 mm Tris, ph 8.0, and protease inhibitor cocktail (Roche, Switzerland) at 4 C for 15 min. The lysate was separated from the beads and then precipitated in acetone for 2 h at 20 C. The pellet was dissolved, reduced with DTT and alkylated with iodoacetamide before being digested with 1 g of Promega-modified trypsin for 24 h at room temperature. A second 1 g of trypsin was added and digestion was allowed to proceed for 24 more hours. The sample was acidified to 5% acetic acid and then desalted on a C18 column. The LC MS/MS procedure was performed by the W.M. Keck Biomedical Mass Spectrometry Laboratory (contact: Dr. Nicholas Sherman, University of Virginia, Charlottesville, USA). Approximately 25% of the digest was introduced into the mass spectrometer for analysis using two run conditions. The LC MS system consisted of a Finnigan LCQ ion trap mass spectrometer system (Thermo Fisher Scientific, USA) with a Protana nanospray ion source interfaced to a self-packed 8 cm 75 m id Phenomenex Jupiter 10 m C18 reversed-phase capillary column. The extracts ( l volumes) were injected and the peptides were eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.25 l/min. The nanospray ion source was operated at 2.8 kv. The digests were analyzed using the double play capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in sequential scans. This mode of analysis produces approximately 3000 CAD spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed by database searching using the Sequest search algorithm against mouse IPI accessions and the publicly available UniProtKB/Swiss-Prot database (version 57.13, released January 19, 2010). Identified proteins of one sample per bead ligand were analyzed using Scaffold 2 software (Proteome Software, USA) applying a minimum protein identification probability of 99% with 2 or more unique peptides per identified protein. SDS-PAGE and Western blotting Isolated phagosomes were adjusted to similar concentrations by measuring the optical density at 600 nm (OD 600 ). SDS lysates of phagosome samples were separated by vertical gel electrophoresis using 10% polyacrylamide gels according to the method of Laemmli (1970). After separation, proteins were transferred onto nitrocellulose membranes (Whatman, USA) at constant 30 V overnight using a tank blotting device (BioRad, USA), blocked in 5% non-fat dry milk (Nestlé Co., USA) and labeled according to the method of Towbin et al. (1979). Chemiluminescent signals were developed and visualized on Kodak MR films (Sigma Aldrich, USA). Results Receptor ligand interactions modulate phagocytic particle uptake We used latex beads covalently coated with different ligands to trigger the specific uptake via different receptors. Coating of latex beads with the Fc fragment of mouse IgG (subsequently referred to as IgG Fc beads) induced Fc receptor-mediated uptake, with mannan (mannan beads) induced an internalization predominantly via the mannose receptor, whereas latex beads coated with LPS (LPS beads) entered cells via established CD14- and TLR4-dependent pathways. These specific internalization routes were compared to the non-selective uptake of latex beads coupled to avidin (subsequently referred to as avidin beads). First, we estimated the internalization rates of latex beads coated to different ligands over time. J774.A1 mouse macrophages were incubated with the same numbers of 1 m latex beads coated with IgG Fc, mannan, LPS and avidin and were fixed at different time points. Internalization was studied on cross-sections of images taken by confocal microscopy using FITC-labelled phalloidin to stain the actin cortex in order to confirm full particle enclosure (Fig. 1B). Latex beads only bound to the surface of macrophages or incompletely taken up were not included in this analysis. We observed that all ligands induced an almost linear increase of bead uptake over a time course of 60 min (Fig. 1A), but the rates of uptake were significantly different and dependent on the type of ligand. Mannan beads were internalized at the highest rate and to the highest extent, followed by LPS beads and IgG Fc beads (Fig. 1A). The entry of avidin beads displayed the lowest uptake rate (Fig. 1A). We validated our approach analyzing engulfed beads by labeled actin using an alternative method applying an inside outside staining technique. Macrophages were incubated with IgG Fc beads and avidin beads over a time course of 60 min, respectively. Also by this method, IgG Fc beads were internalized more efficiently compared to avidin beads (Fig. S1, supplementary data) at rates comparable to our previous approach labeling the actin cortex. When avidin beads were applied at 5-times higher concentration, J774.A1 cells internalized these beads much more efficiently (Fig. S1, supplementary data) demonstrating that no defects in phagocytic uptake occurred when lower concentrations were used in previous experiments. In order to confirm that the different ligands are recognized by their expected receptor, competition experiments were performed where cells were first pre-incubated with increasing concentrations of the ligand in soluble form for 30 min followed by a co-incubation of ligand-coated beads together with the soluble ligand (Fig. S2, supplementary data). We found that different concentrations of a given soluble ligand efficiently competed for binding of the same ligand-coated bead at the cell surface. This resulted in a decrease of the corresponding bead uptake to less than 36% of the phagocytosis rate of control conditions for IgG Fc, 31% for mannan, 20% for LPS and 23% for avidin (Fig. S2, supplementary data). In contrast, when cells were co-incubated with beads together with another ligand than the one present on the bead surface, phagocytic uptake rates were not significantly affected (data not shown). This proves that all the studied ligands bound to the latex beads interacted specifically to their expected receptors. Receptor ligand interactions influence phagosome maturation The findings described above showed that phagocytic uptake is regulated efficiently by the specific receptor ligand interaction. This led us to study whether subsequent stages of the phagocytic process are also influenced by the early step of receptor ligand interactions. After engulfment, particles enclosed in phagosomes undergo sequential fusion and fission events with endosomal and

5 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx 5 amount of phagosomes that acquired this marker protein over a time course of 60 min is shown in Fig. 1C. Strikingly, phagosomes containing mannan beads acquired the marker very rapidly, with 90% already positive for LAMP-2 after 5 min of uptake (Fig. 1C). In contrast, the frequency of fusion events for phagosomes containing IgG Fc beads, LPS beads, and in particular avidin beads, was significantly slower. After 5 min 52% of IgG Fc bead-, 68% of LPS beadand only 9% of avidin bead-containing phagosomes were positive for LAMP-2. After 30 min uptake, phagosomes containing IgG Fc and LPS beads acquired LAMP-2 at rates similar to phagosomes containing mannan beads, whereas 67% of phagosomes containing avidin beads were positive for LAMP-2. These results indicate that the type of ligand on the latex bead surface plays a significant role not only for their uptake efficiency, but also for later stages of phagosome maturation. Gene expression is dependent on the type of receptor ligand interaction during phagocytosis Fig. 1. Latex beads are internalized and acquire lysosomal markers in dependence of their surface ligand. (A) Internalization rates of 1 m latex beads coupled to avidin, to the Fc fragment of IgG (IgG Fc), to lipopolysaccharides (LPS) and to mannan in J774.A1 macrophages over a time course of 60 min. Cells were incubated with the same concentration of beads and 50 cells per time point were analyzed. Shown are averages of three independent experiments. (B) Image of a fixed macrophage incubated with mannan-coated latex beads (blue) for 60 min obtained by confocal microscopy. Maturation of phagosomes was analyzed by co-localization with the late endosomal/lysosomal marker LAMP-2 (red). The actin cytoskeleton was stained with fluorescein-labeled phalloidin (green). Inset shows a detailed view of phagosomes that have fused with late endocytic compartments and lysosomes indicated by their acquisition of LAMP-2. (C) Histogram showing the acquisition kinetics of phagosomes for LAMP-2. Shown are averages of phagosomes that were positive for LAMP-2 analyzed in 50 cells for differently coated beads and different time points in three independent experiments. Bar: 5 m. lysosomal compartments and mature into phagolysosomes. This process is characterized by phagosomal acidification and the acquisition of hydrolases. We analyzed the extent of phagolysosomal fusion by detection on phagosomes of a marker for late endosomes and lysosomes. Macrophages were allowed to internalize latex beads coated with different ligands, then fixed at different time points and labeled using an antibody against the lysosomeassociated membrane protein 2 (LAMP-2; Fig. 1B). The relative We next monitored the pattern of macrophage gene expression in response to the different beads. For this purpose, J774 mouse macrophages were incubated with latex beads conjugated to avidin, IgG Fc, LPS and mannan for 30 and 60 min and total macrophage RNA was isolated and subjected to mouse whole genome bioarrays. This experiment was performed in triplicate for each studied ligand. As a control, macrophages were incubated with the phagocytosis medium in absence of latex beads for 30 and 60 min. Changes in gene expression were considered significant, when they appeared in all three replicates compared to the basal level at control conditions, after performing a two-way ANOVA analysis with a P-value cut-off <0.01. The complete data set of the microarray analysis was deposited at the NCBI GEO database (GSE17761). In Fig. 2A the total number of up-regulated and down-regulated genes for each examined ligand are shown for 30 and 60 min after phagocytic uptake. Strikingly, in all the cases except for IgG Fc beads at 30 min, the number of down-regulated genes was higher than the number of up-regulated genes (Fig. 2A). This result suggests that, independently of the ligand, the process of phagocytosis in macrophages induces a down-regulation of cellular functions normally active in resting cells. This is further supported by the observation that the common and overlapping genes found affected in all ligands or in two or three of the four ligand samples were mostly down-regulated at both time points (64% of all downregulated genes at 30 min and 65% at 60 min; Fig. 2B). In contrast, genes that were uniquely changed for a given ligand were mostly up-regulated (65% of all up-regulated genes at 30 min and 62% at 60 min; Fig. 2B). IgG Fc beads induced the highest number of upregulated genes after 30 min uptake (n = 1009), which decreases to 465 at 60 min. Mannan beads showed an opposite regulation, inducing the up-regulation of a smaller amount of specific genes at 30 min (n = 461), which doubled at 60 min (n = 913). The response induced by LPS beads and avidin beads exhibited less dynamic changes with about 500 unique up-regulated genes at both time points. The finding that the majority of up-regulated genes were specific to a given ligand suggests that binding and internalization of the ligand-coated latex beads induced a unique cellular response rather than stimulating a common pool of genes that would be required for the phagocytic process to proceed irrespective of the ligand. For both the 30 and 60 min time points, the listed genes common to all ligands, or unique to only one of them including their fold change values can be found as supplementary material (Table SI, supplementary data). To gain further insight into the biological function of the genes that were affected during phagocytosis of ligand-coated beads, we mapped genes to their functional annotation according to Gene

6 6 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx Fig. 2. Gene regulation of J774.A1 macrophages during phagocytosis of coated latex beads analyzed by mouse whole genome bioarrays. Cells were incubated with latex beads coupled to avidin, IgG Fc, LPS and mannan for 30 and 60 min. Gene changes that occurred in all three replicates with a P-value cut-off <0.01 were considered significant. (A) Summary of microarray results showing numbers of up-regulated and down-regulated genes at 30 and 60 min in comparison to untreated control cells at the same time points. (B) Pie charts displaying gene expression changes that were unique to either one ligand (colored), common to all ligands (black) or exhibited overlapping appearance for 2 or 3 ligands (grey). Numbers of up-regulated and down-regulated genes for all categories as well as the overall number of gene changes are shown. Ontology (GO) terms of biological process using the DAVID analysis tool (Huang et al., 2009). In Fig. 3 biological process terms of genes that were changed after 30 min of phagocytic uptake are shown to identify early responses of macrophages during phagocytosis. As expected, genes, which were found affected commonly in all the examined latex bead ligands, mainly belonged to GO terms of metabolic and developmental processes as well as the regulation of cellular and biological processes (Fig. 3A). For example, the inositol polyphosphate-4-phosphatase type I and II were down-regulated 0.04-fold and 0.21-fold, respectively (Table SI, supplementary data). The analysis of functional annotations of genes that were unique to a given ligand revealed several interesting differences (Fig. 3B). First, dominant GO term groups that we found commonly regulated by all ligand types, such as developmental, metabolic and biological processes (Fig. 3A), displayed low percentages of changes (Fig. 3B). Second, we found main differences in the functional groups affected uniquely by a given ligand (Fig. 3B). Avidin beads mainly induced the up-regulation of genes belonging to GO terms of morphogenesis (e.g. laminin: 7.3-fold; Table SI) and signaling pathways (e.g. kinase suppressor of Ras 1: 2.5-fold; Table SI), but also genes involved in cytoskeleton and organelle organization (e.g. scinderin: 4.0-fold; Table SI). In contrast, IgG Fc and LPS beads up-regulated mainly genes important for transcription (e.g. STAT5B by IgG Fc: 1.9-fold; growth factor independent 1 by LPS: 2.5-fold; Table SI) and localization (e.g. syntaxin 18 by IgG Fc: 1.8-fold; epsin 2 by LPS: 3.8-fold; Table SI), the latter including molecules which transport and/or maintain the specific location of organelles. Mannan beads triggered several genes involved in apoptosis (e.g. cell death activator CIDE-A: 2.7-fold; Table SI), intracellular transport (e.g. phosphatidylinositol transfer protein 1: 2.3-fold; Table SI) and localization (e.g. murinoglobulin 1: 2.5-fold; Table SI). These findings indicate once more that specific receptor ligand interactions induce specific changes in gene expression that lead to a unique signature depending on the ligand. Proteomic analysis of mature phagosomes formed after internalization of IgG Fc and mannan beads reveals differences on the organelle level Based on the results obtained so far, we provided evidence that specific interactions between a particular ligand and its receptor at the cell surface are able to modulate phagocytic internalization and maturation kinetics, as well as the gene expression and signaling in macrophages. To couple these findings to functional characteristics at the organelle level, we decided to study the protein composition of mature phagosomes to investigate the impact of receptor ligand interactions at late stages of phagocytosis. Given that each receptor induced to a large extent its own specific set of genes, it was important to test their impact on the protein composition of phagosomes containing different beads. LBP are ideal for these purposes, because they can be purified at very high purities and were applied successfully to proteomic studies in different phagocyte models (Griffiths and Mayorga, 2007). To compare the protein composition of phagosomes during late stages of their maturation and to find out whether initial receptor ligand interactions have an impact on these late characteristics in phagocytosis, we concentrated on phagosomes

7 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx 7 Fig. 3. Mapping of gene expression changes 30 min after internalization of latex beads coupled to avidin, IgG Fc, LPS and mannan according to their gene ontology (GO) functional annotation. Shown are biological process terms of the most prominent functional groups of up- and down-regulated genes that were common to all ligands (A) as well as the ones, which were unique to only one of the bead ligands (B). containing IgG Fc- and mannan-coated latex beads (subsequently referred to as IgG Fc LBP and mannan LBP). We were interested into these two phagosome types because IgG Fc beads are internalized into macrophages via Fc receptors in opsonic pathways, whereas mannan beads trigger predominantly a non-opsonic uptake via the mannose receptor. Phagosomes made in response to these beads were isolated 2 h after their internalization (1 h pulse followed by 1 h chase). This time point was selected because it has been the most-commonly used time point for many assays investigating mature phagosomes containing latex beads including proteomics (Garin et al., 2001; Jutras et al., 2008). Therefore, we were able to minimize the influence of time-dependent dynamics of proteins involved in phagosome maturation during phagosomal fusion and fission events with endosomal and lysomal compartments and were able to analyze more ligand-dependent differences between the two phagosome types. Equal amounts of phagosomes were lysed and their proteins separated by two-dimensional gel electrophoresis. Silver-stained gels of phagosome extracts were carried out in triplicates and displayed qualitative as well as quantitative differences. Representative examples of IgG Fc and mannan LBP are shown in Fig. 4A. Although many polypeptide spots appeared similar in both samples, a number of other proteins were unique to a given sample (arrows in Fig. 4A), or found in different amounts (arrowheads in Fig. 4A). For a more global analysis of phagosomal proteins we next used LC MS/MS applied to same amounts of IgG Fc LBP and mannan LBP to identify their protein composition. Using a quite stringent cut-off of protein identification probability (99%), we carried out a restrictive analysis of the phagosomal proteomes. Interestingly, only 58% of all 265 identified proteins were common to both phagosome types. Reminiscent of the gene expression results, several proteins in the phagosomal proteomes were unique to either IgG Fc LBP or mannan LBP (Fig. 4B). A selection of identified proteins categorized into functional groups is shown in Table 1, whereas a comprehensive list of all proteins identified by LC MS/MS including numbers of their unique spectra and sequence coverage can be found as supplementary material (Table SII, supplementary data). Many proteins known to be involved in phagosome maturation were found in both LBP types, such as proton ATPase subunits, LAMP-1 and LAMP-2, cathepsin-b and cathepsin-d as well as Rab5 and Rab7 (Table 1). However, many proteins involved in signaling as well as receptor subunits were also present on phagosome membranes and exhibited remarkable differences between IgG Fc LBP and mannan LBP. For example, in IgG Fc LBP we were able to identify the IgG-1 chain as well as the Fc receptor of IgG (Table 1), which provide evidence that IgG Fc beads were internalized by Fc receptor-dependent phagocytosis. In mannan LBP, but not in IgG Fc LBP samples, several signaling molecules were identified, such as beta 2-microglobulin, CD68, CD180, lymphocyte antigen 86, programmed cell death 6-interacting protein, TLR13 and the tyrosine protein kinase Lyn (Table 1). These results suggest that mature

8 8 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx Fig. 4. Proteomic analysis of phagosomes formed after internalization of latex beads coated with IgG Fc (IgG Fc LBP) and mannan (Mannan LBP) isolated from J774.A1 macrophages 2 h after uptake. (A) Images of phagosomes after two-dimensional gel electrophoresis and silver staining showing proteins, which are unique to IgG Fc and mannan LBP (arrows) as well as proteins that appeared at different quantities (arrowheads). (B) Diagram representing the relative distribution of phagosomal proteins identified by LC MS/MS with a protein identification probability of 99%. 265 proteins were identified in total. phagosomes containing mannan beads, which were formed after non-opsonic internalization, possess the capability to modulate diverse signaling pathways. Since phagosomes rely on intracellular trafficking and interactions with the cytoskeleton to acquire and sustain their degradative properties (Araki, 2006), we also analyzed the presence of small GTPases of the Rab family and cytoskeletal proteins. Interestingly, among the identified Rab proteins found in both phagosome types, such as Rab-1A, -2A, -5B, -5C, -7, -14 and -33B, some were found associated specifically with IgG Fc LBP (Rab-5A, -31) or mannan LBP (Rab-10, -18, -22A, -27A) after 2 h internalization (Table 1). The finding of the association of different isoforms of Rab proteins to different types of phagosomes at a particular time point emphasizes the different composition of these organelles. Additionally, we were also able to detect proteins of all cytoskeleton filament networks on phagosome membranes (cytoplasmic actin, tubulin subunits and vimentin, a member of the intermediate filament system) as well as a high number of identified actin-associated proteins (Table 1), which is further supported by immunoblotting results of phagosomal lysates (see below). The association of cytoskeletal proteins with mature phagosomes did not show many differences between the analyzed LBP types, but points out the importance of intracellular trafficking during phagosome maturation. Receptor ligand interactions also modulate the time-dependent association of proteins with mature phagosomes Since phagosomes acquire proteins during their maturation by fusion with endosomes and lysosomes in a very dynamic way, we finally isolated phagosomes at different time points after uptake to analyze them for their association with different proteins. The same amount of LBP lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes and analyzed by immunoblotting. Our data obtained by confocal microscopy indicated the presence of LAMP-2 at different times depending on the latex bead type (Fig. 1C). We analyzed lysates of phagosomes containing IgG Fc and mannan beads isolated 30, 60, 120 and 240 min after uptake for the presence of LAMP-2, cathepsin-d and actin. As shown in Fig. 5, LAMP-2 was associated with all phagosome types at all analyzed time points with increasing concentration without significant differences between IgG Fc and mannan LBP lysates (Fig. 5). However, when phagosomes were studied for the presence of cathepsin-d and actin, differences were obvious between the two phagosome types. At 60 and 120 min, we detected the immature form of cathepsin-d (52 kd) associated at higher amounts with IgG Fc LBP compared to mannan LBP, whereas it was not present on early phagosomes (30 min). Additionally, we observed a rather striking pattern for actin associated with phagosomes (Fig. 5). Both phagosomes types displayed similar amounts of associated actin early after uptake (30 min), which was absent at 60 min and associates again with mature phagosomes (120 and 240 min). However, actin was much more prominent on mannan LBP at 120 min compared to IgG Fc LBP (Fig. 5). In conclusion, our results show that, in macrophages, receptor ligand interactions during the initial step of phagocytosis dictate gene expression patterns and modulate signaling pathways. Moreover, they also lead to differences on the protein level evident for protein composition, maturation kinetics and dynamics of associated proteins of the analyzed phagosome types. Discussion It is well established that the initial contact of a pathogen with cell surface receptors initiates an immediate signaling response. Since pathogens possess a variety of ligands on their surface, they cause a high complexity of multi-ligand interactions with host receptors inducing extensive overlap and crosstalk between different pathways. It is also clear that a given receptor on phagocytes such as macrophages induce a specific pattern of signaling that show significant differences from other receptors. The question we posed in this study was How do professional phagocytes, such Fig. 5. Receptor ligand interactions modulate the time-dependent association of proteins with mature phagosomes. Phagosomes containing beads coated with IgG Fc (IgG Fc LBP) and mannan (Mannan LBP) were isolated from J774.A1 macrophages after different time points (30, 60, 120 and 240 min). Phagosomal lysates were separated by SDS-PAGE and tested by immunoblotting using specific antibodies against LAMP-2, cathepsin-d and actin.

9 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx 9 Table 1 Selection of proteins identified by LC MS/MS in phagosome preparations containing IgG Fc-coated or Mannan-coated latex beads isolated from J774.A1 macrophages after 1 h pulse/1 h chase divided into functional categories. Proteins IgG Fc Mannan Phagosomal/lysosomal markers ATPase, H+ transporting, various subunits + + ATP synthase, vacuolar, various subunits + + Calreticulin + + Calnexin + + Cathepsin-B, -D, -L, -Z + + Early endosome antigen Lysosomal acid lipase + + Lysosomal acid phosphatase + + Lysosome-associated membrane protein (LAMP) 1 and Syntaxin Transferrin receptor protein 1 + Vesicle-associated membrane protein (VAMP) 4 + Vesicle-associated membrane protein (VAMP) Signaling molecules Annexin-1 + Annexin-2, -4, -5, Annexin-7 + Beta 2-microglobulin + CD68 (Macrosialin) + CD180 (Toll-like receptor protein RP-105) + + Fc receptor, IgG, IIb isoform + G protein-coupled receptor Heat shock protein 70 (HSP70) + + IgG-1 chain + Lymphocyte antigen 86 + Programmed cell death 6-interacting protein + Toll-like receptor Toll-like receptor 13 + Tyrosine protein kinase Lyn + Rab proteins Rab-1A + + Rab-2A + + Rab-5A + Rab-5B, -5C + + Rab Rab-10 + Rab Rab-18 + Rab-22A + Rab-27A + Rab-31 + Rab-33B + + Cytoskeletal proteins Actin, cytoplasmic + + ADP-ribosylation factor 6 + Arp2/3 complex + + Coronin-1A + Elongation factor 1 alpha + + Elongation factor Moesin + + Profilin Rac Tubulin, alpha and beta chain + + Vimentin + + as macrophages, respond to the binding and phagocytic uptake of latex beads coated with a specific ligand during early and late events of phagocytosis?. The latex bead phagosome offers a system of multivalent interaction of a defined ligand with a defined receptor on the macrophage surface. Here, using different methods we compared the impact of beads coated with the Fc fragment of mouse IgG, mannan, LPS and avidin with their specific receptors on different stages of the phagocytic process and on gene expression. Irrespective of the ligand, J774.A1 cells were able to bind and internalize the beads in a morphologically indistinguishable manner. However, although macrophages were incubated with same numbers of beads, the ones coated with mannan were internalized at faster rates and in higher numbers compared to beads coated with other ligands. In macrophages, mannan and terminal mannose residues are predominantly recognized by the MR, a prototypic pattern-recognition receptor and C-type lectin (Wileman et al., 1986). The MR binds a broad spectrum of pathogens including Candida albicans and Pneumocystis carinii (Ezekowitz et al., 1990, 1991). Although it was shown that serum opsonization enhances the internalization of some pathogens (Balagopal et al., 2006), our observation confirms previous findings in COS cells transfected with different chimeric receptor constructs showing a higher phagocytic index for the MR compared to Fc RI (Kruskal et al., 1992). We also observed that phagosomes containing mannan beads acquired the late endosomal/lysosomal protein LAMP-2 more rapidly than the other phagosome types. In fact, within only 5 min after adding these beads, around 90% of their newly formed phagosomes already exhibited fusion events with late endosomes/lysosomes. Although we did not address this issue, it seems unlikely that receptor-induced newly synthesized proteins are relevant during this short time scale. In murine and rabbit macrophages, several studies support the idea that the MR is coupled to bactericidal functions, such as the production of reactive oxygen intermediates (Klegeris et al., 1996) and cytokines (Shibata et al., 1997). However, in human cells, the data argue that this receptor activates anti-inflammatory signaling (Nigou et al., 2001; Chieppa et al., 2003) and delays the maturation of phagosomes (Astarie-Dequeker et al., 1999). The mechanistic differences between rodents and humans for the same receptor (Paul et al., 1996) remain to be elucidated. Together these data support the idea that interactions of mannan beads with the MR induce a specific signaling, different from the other ligands, which leads to a more efficient bead uptake and more rapid fusion events with lysosomes. The precise nature of these signaling pathways as well as the possible involvement of co-receptors, such as Toll-like receptors, remain to be elucidated. Our study of the gene expression response upon bead binding and uptake showed that the majority of affected genes are down-regulated, independent of the bead ligand, suggesting a general down-regulation of cellular functions during phagocytosis. A similar result was found during phagocytosis of Mycobacterium smegmatis (Gutierrez et al., 2008) and Aspergillus fumigatus (Cortez et al., 2006). We observed that the down-regulated genes common to all ligands encode proteins involved in metabolism, energy homeostasis and development suggesting a decrease of these processes during phagocytosis compared to resting cells. In contrast, around two-third of the up-regulated genes at both investigated time points, 30 and 60 min, were unique to one of the bead ligands. Our analysis mapping altered genes at 30 min to GO functional annotation terms strengthen the observation that specific receptor ligand interactions induce particular cellular responses early after uptake, which differ broadly between the investigated ligands. We found that IgG Fc and LPS beads upregulated many genes involved in transcription and processes of intracellular localization, whereas avidin beads induced predominantly signaling pathways and genes important for cytoskeleton organization. Mannan beads up-regulated genes important for intracellular transport, but also genes involved in apoptotic processes. These also include molecules involved in the recognition of apoptotic cells. Previous findings have demonstrated that particular C-type lectins, such as macrophage galactose-type C-type lectin-1 (MGL-1; Yuita et al., 2005) sense products from dying cells and modulate immunogenic cell death (Cambi and Figdor, 2009). Further analysis of single genes will be necessary to understand the impact of receptor ligand interactions, and coated latex beads provide a useful tool to investigate these processes in more detail.

10 10 E. Hoffmann et al. / European Journal of Cell Biology xxx (2010) xxx xxx The high number of up-regulated genes in response to IgG beads at 30 min and to mannan beads at 60 min also emphasize the differences in time and specificity of the utilized pathways. Signaling mediated through immunoreceptor tyrosine-based activation motifs (ITAM)-bearing Fc receptors during phagocytosis has been investigated in detail (Swanson and Hoppe, 2004) and is known to have an impact on phagosome maturation (Tian et al., 2008), and the immune response (Pasquier et al., 2005). On the other hand, the mannose receptor has a single tyrosine residue in its cytoplasmic tail and different signal transduction pathways are engaged (Kerrigan and Brown, 2009). For some members of the newly emerging family of Syk-coupled C-type lectins, such as Dectin-1, the recognition of PAMP as well as an induction of genes important for an innate immune response have been demonstrated (Rogers et al., 2005; Robinson et al., 2006). We found that 30 min after incubation with mannan beads, macrophages are characterized by a down-regulation of the pro-inflammatory cytokine TNF, whereas the interleukin-13 receptor subunit 1 (IL-13R 1) was up-regulated. IL-13R 1 is known to form a type II receptor after association with IL-4R (Junttila et al., 2008) inducing a cytoplasmic signaling cascade that involves the Janus kinase family and the phosphoinositide 3-kinase (PI3K) pathway (Keegan et al., 1995). Macrophages utilize these pathways to undergo alternative activation pathways that trigger different phenotypes during the immune response to parasites and pathogens (Martinez et al., 2009). Next, our study aimed to analyze the impact of receptor ligand interactions on the organelle level to gain insight into early as well as late events during phagocytosis. Focusing on IgG Fc and mannan LBP to analyze opsonin-dependent and opsonin-independent phagocytic events, we found that around 60% of proteins were common to both LBP types 2 h after internalization. As expected, lysosomal hydrolases important for proteolytic degradation (Brix et al., 2008) as well as cytoskeletal proteins involved in trafficking during phagosome maturation (Araki, 2006) were found at this time point. Previously, a quantitative proteomic analysis of phagosomes containing IgG-coupled latex beads in RAW macrophages has been published (Rogers and Foster, 2007). These data are in good agreement with our proteomic analysis, since 78% of our identified proteins in IgG Fc LBP in J774.A1 macrophages were also identified in that study. Additionally, we also confirmed the presence of many proteins in IgG Fc and mannan LBP in comparison with other proteomic studies of phagosomes containing latex beads (Garin et al., 2001; Jutras et al., 2008; Trost et al., 2009), Francisella tularensis (Kovarova et al., 2002) and Mycobacterium bovis BCG (Lee et al., 2010). Interestingly, some of our identified proteins were specifically found in M. bovis BCG phagosomes but not in phagosomes containing non-coated latex beads in the same study (Lee et al., 2010), such as alpha-mannosidase, Rab-14 and the G protein beta 1 (Gnb1) involved in trans-membrane signaling. Around 40% of the identified proteins from the proteomic analysis were specific for IgG Fc or mannan LBP, which mainly included signaling molecules and Rab proteins, regulators of intracellular trafficking that exhibited an isoform-dependent association with the different phagosome types. Together with our Western blot analysis of phagosomal proteins showing that a lysosomal protein, cathepsin-d, as well as the cytoskeletal protein actin are associated with phagosomes differently during their maturation, it demonstrates that the protein composition of these organelles is also altered by initial receptor ligand interactions occurring upon binding of beads to the macrophage surface. Especially Rab GTPases are reversibly associated with organelle membranes and are responsible for a close interrelationship between receptor signaling and membrane trafficking (Sorkin and von Zastrow, 2009). They function as molecular switches after specific signaling events and effector coupling to determine the type of interactions between phagosomes and other organelles (Kinchen and Ravichandran, 2008). In this respect, Rab-5 and Rab-7 are the key GTPases responsible for the recycling of proteins back to the plasma membrane and as entry points for the acquisition of lysosomal proteases during phagosome maturation. It has been elegantly demonstrated that Rab-5 positive early endocytic organelles switch to Rab-7 positive organelles after a certain threshold of acquisition, a mechanism called Rab conversion (Rink et al., 2005). Many other Rab GTPases are involved during phagosome maturation and organelle trafficking (Stenmark, 2009), but our findings also indicate that their transient appearance on phagosomes is also coupled to the type of phagosome content and signaling during the initial contact between receptor and ligand. Our findings support the idea of the individuality of phagosomes to a certain degree, which allows them to regulate their intracellular fate locally and function as independent entities (Griffiths, 2004). Recent proteomic studies of the response of RAW macrophages upon infection with Candida albicans (Martinez-Solano et al., 2009) and Salmonella enterica (Shi et al., 2009) showed that these cells varied their expression level of proteins involved in cytoskeletal organization and signal transduction resulting in the induction of an anti-inflammatory immune response. In the future, more detailed and connected approaches are necessary to gain insight into the impact of receptor ligand interactions on the inflammatory response of the cell. Our experiments provide proof of the principle that the use of latex beads conjugated to specific ligands can be a powerful tool to follow the fate of different kinds of phagosomes in a phagocytic cell. This system can be adapted to analyze the response of a phagocyte when two, three or more ligands are conjugated to the same bead, or in contrast, when different beads coated with a single ligand are applied simultaneously to these cells. The outcome of such experiments will help to understand, which receptor ligand interactions might dominate signaling events and what their impact on the cellular immune response will be. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; KU1528/2), the EU commission Marie Curie Research Training Network (MRTN-CT ) and the AXA Research Fund. We are very grateful to the support of Sebastian Amigorena (Institute Curie, Paris, France) and the Genomics core facility and the Proteomics core facility (EMBL Heidelberg, Germany). We acknowledge Nicholas Sherman and the W.M. Keck Biomedical Mass Spectrometry Laboratory, funded by the Pratt Committee of the University of Virginia, USA, for LC MS/MS and help in data analysis. 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