Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

FcγRI cross-linking on monocytes may trigger clathrin-mediated endocytosis, likely through interaction of multiple intracellular molecules that are controlled by phosphorylation and dephosphorylation events. However, the identity of phospho-proteins and their regulation are unknown. We proposed the leukocyte immunoglobulin-like receptor B4 (LILRB4) that inhibits FcγRI-mediated cytokine production via Tyr dephosphorylation of multiple kinases, may also regulate endocytosis/phagocytosis through similar mechanisms. FcγRI and/or LILRB4 were antibody-ligated on THP-1 cells, lysates immunoprecipitated using anti-pTyr antibody and peptides sequenced by mass spectrometry. Mascot Search identified 25 Tyr phosphorylated peptides with high confidence. Ingenuity Pathway Analysis revealed that the most significantly affected pathways were clathrin-mediated endocytosis and Fc-receptor dependent phagocytosis. Tyr phosphorylation of key candidate proteins in these pathways included common γ-chain of the Fc receptors, Syk, clathrin, E3 ubiquitin protein ligase Cbl, hepatocyte growth factor-regulated tyrosine kinase substrate, tripartite motif-containing 21 and heat shock protein 70. Importantly, co-ligation of LILRB4 with FcγRI caused significant dephosphorylation of these proteins and was associated with suppression of Fc receptor-dependent uptake of antibody-opsonised bacterial particles, indicating that LILRB4. These results suggest that Tyr phosphorylation may be critical in FcγRI-dependent endocytosis/phagocytosis that may be regulated by LILRB4 by triggering dephosphorylation of key signalling proteins.


Results
Increased global Tyr phosphorylation in THP-1 lysates after surface cross-linking of FcγRI. Western blotting with a pan anti-pTyr MAb showed that cross-linking of surface Fcγ RI with specific mouse mAb followed by goat anti-mouse secondary antibody in suspension markedly increased Tyr phosphorylation of multiple proteins above those seen when cells were treated with IgG1 alone. The intensity of reactivity was markedly reduced in lysates from cells that had been co-ligated with anti-FcgR1 and anti-LILRB1 (Fig. 1A). Immunoprecipitation using anti-pTyr mAb showed marked enrichment of phosphorylated proteins in lysates of Fcγ RI-cross-linked cells compared with those treated with IgG1 alone (Fig. 1B). Silver staining of SDS-PAGE gels loaded with anti-pTyr mAb-precipitated lysates from IgG1 and anti-Fcγ RI+ IgG1 cross-linked cells showed enrichment of 8 bands that separated at 100, 70, 50, 47, 43, 35, 30 and 14 kDa compared to components separated from IgG1-treated cell immunoprecipitates (Fig. 1C). The 8 bands were excised and peptides sequenced by Nano LC-MS/MS. The Mascot Search output of 3 combined experiments identified 80 hits that comprised 25 Tyrphosphorylated candidate proteins in peptides sequenced from lysates of anti-Fcγ RI+ IgG1 cross-linked cells that were not identified in IgG 1 control-treated cells (Mowse score > 50, p = 0.05; > 3 peptide matches) ( Table 1).
Regulation of FcγRI-mediated Tyr phosphorylation of Fc receptor γ chain, clathrin, Cbl, HGS, HSP70, TRIM21 and Syk by LILRB4. Immunoprecipitation of cell lysates using anti-pTyr mAb followed by Western blotting using antibodies against the FcRγ chain, clatherin, Cbl, HGS, HSP70 and TRIM21 showed Tyr-phosphorylation of all these proteins in cells activated through Fcγ RI cross-linking (Fig. 3A) (supplementary Fig. 1), validating the LC-MS/MS data. Importantly, phosphorylation of all proteins except HSP70 was markedly suppressed upon co-ligation of Fcγ RI with LILRB4 (Fig. 3A). Semi-quantitative analysis indicated that LILRB4 significantly reduced Tyr phosphorylation of HGS by an average of 80.2%, Cbl by 51.3%, clathrin by 45.5% and TRIM21 by 36.9% ( Fig. 3B; n = 3). In contrast, co-ligation of Fcγ RI with LILRB4 significantly enhanced HSP70 phosphorylation by 36.4%, suggesting selective suppressive effects by LILRB4. Similarly, Western blotting of cell lysates from Fcγ RI cross-linked cells caused Tyr-phosphorylation of Syk that was markedly reduced up on co-ligation with LILRB4 ( Fig. 3E), further validating the LC-MS/MS data and confirming our previous finding 19 . As expected, the brief cross-linking/co-ligation protocols used in this study did not affect the total amounts of any of the above proteins ( LILRB4 suppressed uptake of antibody-opsonised bacterial particles. For this experiment we used PMA-differentiated THP-1 cells because these have superior phagocytic activity. The mean percentage of these cells that took up antibody-opsonised fluorescent DH5α E-coli particles identified by flow cytometry, was 32.9 ± 2.3%. Uptake was significantly reduced to 3.5 ± 0.74% when surface Fcγ RI was blocked using a specific anti-Fcγ RI mAb (blocked by ~90%; p = 0.0005, n = 4; Fig. 4B), but not when cells were treated with an irrelevant negative control mAb (36.7 ± 4.6%) confirming involvement of Fcγ RI-dependent endocytosis/phagocytosis. Ligation of surface LILRB4 using an anti-LILRB4 mAb, followed by goat anti-mouse secondary antibody, significantly suppressed uptake of opsonised DH5α E-coli by up to 55% (p = 0.004, n = 4; Fig. 4B). In contrast, co-?ligation of surface MHC-I using anti-MHC-I mAb did not significantly alter uptake of opsonised DH5α E-coli particles when compared with non-ligated cells (28.9 ± 1.7% versus 32.9 ± 2.3% Fig. 4B), suggesting specific suppression of endocytosis/phagocytosis by LILRB4.

Discussion
Reversible Tyr phosphorylation of proteins in eukaryotes is critical in regulating intracellular signalling pathways involved in cellular activation, growth, proliferation, differentiation migration and gene transcription 27,28 . Immune-complex mediated activation of Fcγ RI on innate immune cells is essential for protection against bacterial infection. Activation of Tyr phosphorylation of selected upstream protein tyrosine kinases and downstream mitogen activated protein kinases result in production of pro-inflammatory cytokines, generation of an oxidative burst, and/or triggering of endocytosis/phagocytosis 1,5 . In this study we immunoprecipitated total Tyr phosphorylated proteins from lysates of THP-1 that had been cross-linked with specific anti-Fcγ RI or control IgG mAb and found marked enrichment of phosphorylated proteins in cells activated with the specific mAb. Peptide sequencing by Nano LC-MS/MS identified 80 candidate peptides that were significantly modified, representing 25 Tyr-phosphorylated proteins. Pathway analysis predicted that Ty-phosphorylation of proteins mediating clathrin-mediated endocytosis and Fc receptor-mediated phagocytosis were the most affected. The most  Table 1). prominent phosphorylated proteins included Fc receptor γ chain, Syk, clathrin, Cbl, HGS, STAM1/2, HSP70, TRIM21, actin, actin-related proteins, actinin-4, tubulin and actin binding proteins. This is the first study to demonstrate the simultaneous Tyr phosphorylation of these proteins during Fcγ RI-mediated monocyte activation. This is particularly novel for clathrin, Cbl, HGS and HSP70, key molecules involved in the clathrin-mediated endocytosis 10,13,14 and for TRIM21, a ligase recently identified as high affinity intracellular Fc receptor 17,29 which is critical for ubiquitination and degradation of antibody-opsonised viruses 17 . We validated the data generated by LC-MS/MS using a combination of Western blotting and immunoprecipitation and confirmed that these molecules were indeed Tyr phosphorylated following cross-linking of Fcγ RI on the surface of THP-1 cells. Moreover, we confirmed our previous finding 19 of Tyr phosphorylation of Syk in response to Fcγ RI cross-linking and identified Tyr phosphorylation of the intracellular tyrosine-based activating motifs (ITAMs) of the common γ chain of Fc-receptors that is upstream of Syk, directly linking for the first time, these two critical signalling events. Functionally, Tyr phosphorylation of Syk after Fcγ RI cross-linking on monocytes promotes cytokine production 19,21,22 and Syk phosphorylation has been associated with increased phagocytosis of opsonised pathogens 30 and polybeads, 31 and enhanced endocytosis of immune complexes 32 . Here, we found that co-ligation of Fcγ RI with LILRB4 that contains intracellular tyrosine-based inhibitory motifs (ITIMs), significantly reduced phosphorylation of the ITAMs of the common γ chain, and of Syk. Suppression of Syk phosphorylation is consistent with our previous report showing that LILRB4, through recruitment of SHP-1-like phosphatase, dephosphorylated Syk and multiple downstream protein tyrosine kinases including Lck, LAT and Erk in THP-1 cells leading to suppressed cytokine production 19 . Importantly, we show here that LILRB4 ligation significantly inhibited Fcγ RI-dependent uptake of antibody-opsonised E. coli particles. One mechanism may involve dephosphorylation (inactivation) of the common γ chain ITAMs and Syk, similar to its inhibitory effects on Fcγ RI-mediated cytokine production 19,21,22 . Clathrin-mediated endocytosis is an important energy efficient pathway of pathogen/antigen clearance by innate immune cells as an alternate to phagocystosis 7,9,33,9,33 . It is also an important mechanism of endogenous surface ligand/receptor internalisation, ubiquitination and recycling/degradation 7,9,33 . Clathrin-mediated endocytosis may share some upstream signalling molecules with phagocytosis but unlike to phagocytosis, it is associated with internalisation of small particles (< 0.2 μ m in diameter) and soluble aggregated molecules 10 . During endocytosis, receptor-ligand or antibody-antigen complexes are first ubiquitinated and internalised into clathrin-coated pits assembled within AP2, dynamin, epsin and related molecules followed by HSP70-mediated un-coating of the pits and their endosomal sorting 10,13,14 . Receptors and ligands in the endosome are then either ubiquitinated and directly degraded by Cbl [10][11][12]17 or the ubiquitinated molecules are delivered to lysosome by HGS/STAM1/2 complexes for lysosomal degradation 15,16 . Alternatively, the receptor in the endosomes is rapidly recycled while the ligands undergo endo-lysosomal degradation 10 . Although the involvement of clatherin, Cbl, HSP70, HGS and STAM in this pathway is generally accepted, whether Tyr phosphorylation/dephosphorylation regulate their functions, particularly during Fc-receptors mediated monocyte activation remain unexplored. There is limited evidence that Tyr phosphorylation of clathrin heavy chain promotes bacterial internalisation 34 and that phosphorylation of Cbl is associated with receptor/ligand ubiquitination after receptor clustering in antigen presenting cells, T cells and B cells 35,36 . Tyr phosphorylation of HGS in Hela cells during epidermal growth factor (EGF)-mediated activation may be involved in intracellular receptor sorting and vesicle formation 37 . C-terminal Tyr phosphorylation of HSP70 is described as a switch that regulates co-chaperon binding in cancer cells and determines whether it facilitates protein folding, or directs proteins for ubiquitin-mediated degradation 38 . These functional observations collectively suggest that Tyr phosphorylation of these particular molecules may play critical roles in Fc receptor-dependent endocytosis of immune complexes. Here we show that co-ligation of LILRB4 with Fcγ RI significantly reduced Fcγ RI-mediated Tyr phosphorylation (activation) of clathrin, Cbl, HGS and STAM1/2 (Fig. 5). We propose that LILRB4 may inhibit Fc-receptor-dependent endocytosis of antigen-antibody complexes by promoting Tyr dephosphorylation (deactivation) of these key molecules. This proposal is consistent with its reported anti-inflammatory and immunosuppressive properties 19,39-41 and our demonstration that it significantly suppressed Fcγ RI-dependent endocytosis/phagocytosis of antibody-opsonised E. coli particles (Fig. 4). In contrast to these dephosphoryating events, co-ligation of LILRB4 with Fcγ RI significantly enhanced Fcγ RI-mediated HSP70 Tyr phosphorylation by 36.4%, indicating selective effects. Whether enhanced HSP70 phosphorylation by LILRB4 regulates ubiquitin-mediated degradation of antibody-opsonised bacterial, particles and/or the sorting of the internalised Fc-receptors, requires further investigation. TRIM21 is described as an important high affinity intracellular Fc receptor implicated in elimination of antibody-bound intracellular viruses 17 , although little is known about interactions that link extracellular antibody-bound pathogen to intracellular TRIM21. Here we found that surface cross-linking of Fcγ RI that uses a method that mimics antibody-antigen complexes, promoted strong phosphorylation of TRIM21. Thus Fcγ RI might be the missing link between activation by extracellular antibody-bound pathogens (antigen) and their intracellular Fc receptor (TRIM21) that may also function as a novel downstream pathway in Fc receptor-dependent endocytosis of immune complexes. Interestingly, TRIM21 is Tyr phosphorylated in TLR3 or TLR4-stimulated monocytes and macrophages and is suggested to activate downstream TLR-mediated signalling 42 . The significant dephosphorylation of TRIM21 caused by LILRB4 ligation shown by us, may therefore indicate functional deactivation of this molecule when Fcγ RI-induced monocyte activation is moderated by LILRB4.
In conclusion, results presented here suggest that Tyr phosphorylation of the upstream common γ -chain, Syk, and clathrin and the downstream molecules such as TRIM21 may be critical in Fc-receptor (Fcγ RI)-dependent endocytosis/phagocytosis of antibody-opsonised particles. Importantly, LILRB4 may regulate this important innate immune function by promoting mechanisms that dephosphorylate these proteins.

Materials and Methods
Cells and antibodies. Human monocytic leukemic THP-1 cells (ATCC clone TIB-202, Manassas, VA, USA) were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μ g/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 0.1% β -mercaptoethanol (all from Life Technologies) and 20 mM sodium bicarbonate (Sigma-Aldrich) at 37 °C with 5% CO 2 19 . The following antibodies were used for flow cytometry and/or cross-linking/co-ligation experiments; anti-LILRB4 (kindly donated by Dr. Luis Borges, Amgen Inc), anti-Fcγ RI (R&D System, Minneapolis, MN,  experiments showed significant reduction of Fcγ Rs, clatherin, Cbl, HGS and TRIM21 phosphorylation, but not HSP70, in THP-1 cells co-ligated with anti-Fcγ RI and anti-LILRB4 mAbs, compared to cells co-ligated with anti-Fcγ RI and negative control mAb (n = 3, **p < 0.01; ***p < 0.001). (C) Representative Western blotting of total cell lysates showed that co-ligation of Fcγ RI with LILRB4 did not alter the total amounts of any of the above proteins when compared to co-ligation of Fcγ RI+ IgG 1 control, ligation of LILRB4 alone or treatment with IgG 1 control alone; the lower panel is the same membrane stripped and reprobed with anti-β actin Ab, confirming comparable protein loading. (D) Summary of densitometry analysis of 3 independent experiments showed no significant differences in total Fcγ Rs, clatherin, HSP70, Cbl, HGS and TRIM21 in THP-1 cells within the 4 different treatment groups (n = 3). Full image of the Western blots is shown in Supplementary Fig. 1

. (E) Western blotting of cell lysates from Fcγ RI cross-linked cells showing increased
Tyr-phosphorylated Syk that was markedly reduced upon co-ligation with LILRB4, confirming our earlier finding 21 and validating current LC-MS/MS data (Fig. 2) (n = 1).
containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA and 1% NP-40, freshly-made protease inhibitors (2 mg/ml; Roche Applied Science) and 10 μ M sodium pervanadate (Sigma-Aldrich). After vortexing for 1 min, samples were incubated on ice for 30 min then supernatants collected by centrifugation at 20,000xg for 10 min at 4 °C. Tyr-phosphorylated proteins were immunoprecipitated using 5 μ g/ml anti-pTyr mAb (clone 4G10) at overnight 4 °C then incubated with goat anti-mouse secondary antibody conjugated to Sepharose beads (10 μ g/ml; Zymed Laboratories Inc., San Francisco, CA, USA) for 2 hrs at 4 °C. Bead-bound proteins were washed once with 1 ml cold dilution buffer (0.1% Triton X-100 in TSA buffer pH 8.0; 0.01 M Tris buffer, 0.14 M NaCl, 0.025% NaNa 3 ), two washes with TSA and a single wash with 50 mM Tris buffer pH 6.9. Beads were then resuspended in Tricine gel loading buffer containing 10 mM dithiothreitol, heated for 5 min at 100 °C and supernatants resolved in 10% Tris-Tricine SDS-PAGE gels under reducing conditions then silver-stained. Specific silver-stained bands were excised and Tyr-phosphorylated proteins were identified by Nano Liquid Chromatography tandem Mass Spectrometry (Nano LC-MS/MS) as described 43,44 . Bands excised from lanes loaded with immunoprecipitates of irrelevant IgG 1 -cross-linked THP-1 cells were used as negative controls. Peak lists of MS/MS data were generated using Mascot Daemon/extract_msn (Matrix Science, London, England, Thermo) were interrogated using Mascot version 2.1 (http://www.matrixscience.com) and searched against Homo sapiens proteins in the Swissprot protein database (version 80). Precursor tolerances were 4.0 ppm and product ion tolerances were ± 0.4 Da and acceptable cut-off scores for individual MS/MS spectra were set to 20. Specific phosphorylated peptides identified in Fcγ RI cross-linked cells, but not in cells treated with control IgG 1 from 3 independent experiments, were combined and uploaded onto Ingenuity Pathway Analysis software version 24718999, used to predict the most significantly-enriched pathways (IPA ® ; www.qiagen.com/ingenuity, QIAGEN, Redwood City, CA). Alternatively, proteins were transferred onto PVDF membranes (0.2 μ m pore size; Millipore, Bayswater, VIC, Australia) for Western blotting using 1 μ g/ml biotinylated α -pTyr-100 mAb.

Validation of enriched Tyr phosphorylated proteins by immunoprecipitation and Western blotting and regulation by LILRB4.
Six proteins including the common γ chain of the Fc receptor, clathrin, Cbl, HGS, TRIM21 and HSP70 had multiple Tyr phosphorylated peptides with high Mascot scores following Fcγ RI cross-linking. Four of these are reported to be involved in clathrin-mediated receptor endocytosis, although this was the first demonstration of their simultaneous phosphorylation. Hence, results were validated, and their regulation by LILRB4 examined using a combination of immunoprecipitation and Western blotting. In brief, 2 × 10 7 THP-1 cells were activated via Fcγ RI cross-linking with or without LILRB4 co-ligation [21], lysates immunoprecipitated using anti-pTyr mAb (4G10) followed by serial Western blots using antibodies against FcR common γ chain, clathrin, Cbl, HGS, TRIM21 or HSP70. In separate experiments, Western blotting using anti-pTyr (4G10) was performed using 20 μ g total lysates to detect global protein phosphorylation; membranes were re-probed with 1 μ g/ml mouse anti-β -actin mAb to confirm equal protein loading. For detection of Syk and p-Syk proteins, 20 μ g of total lysates were serially Western blotted using rabbit anti-pSyk Ab followed by rabbit anti-Syk Ab and mouse anti-β -actin mAb.

Detection of uptake of antibody opsonised bacteria particles by differentiated THP-1 cells and modulation by LILRB4.
To determine uptake of antibody-opsonised bacteria by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells a modified Fc-receptor dependent endocytosis/phagocytosis assay was developed. In brief, desired numbers of THP-1 cells were cultured in RPMI complete medium containing 100 ng/ml PMA (Sigma-Aldrich) at 37 °C in 5% CO 2 for 3 days. Differentiation was confirmed by assessing morphological changes; larger, partially adherent cells, vesicular with ruffled edges and non-replicating. Enhanced green fluorescent protein (EGFP) expressing DH5α E. coli (Addgene, Cambridge, MA, USA) (10 7 cells/ml PBS) were killed by freeze-thawing twice and pellets incubated with 5 μ g/ml goat anti-DH5α E. coli in PBS (Abcam 25823, Melbourne, VIC, Australia) for 2 hrs at 37 °C. The opsonised particles were added to 2 × 10 5 PMA-differentiated THP-1 cells in 400 μ l CLB, at an estimated bacteria to cell ratio of 10:1, and incubated at 37 °C for 4 hrs. Cells were then washed twice in 1.5 ml cold PBS containing 0.05% NaN 3 and 1% bovine serum albumin, and re-suspended in 0.5 ml of 1% paraformaldehyde in PBS. The percentage of cells that took up bacterial particles was determined by flow cytometry. To confirm Fc-dependent uptake, Fcγ RI (the primary Fc receptor expressed on THP-1cells 19 ) function was blocked by pre-incubating cells with 20 μ g/ml anti-Fcγ RI mAb for 15 min at RT followed by a single CLB wash prior to addition of opsonised bacteria particles; 20 μ g/ml irrelevant mouse Cross-linking of Fcγ RI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and binding of pSyk transduces activating signals. This simultaneously initiates phosphorylation of clatherin that causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, immune complexes that escape the endosome are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of Fcγ RI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that subsequently dephosphorylate (deactivate) the key molecules including clathrin (1), Fcγ RI and Syk (2), Cbl (3), HGS and STAM 1/2 (4) and TRIM21 (5). These effects may reduce cellular activation and/or suppress receptor/ligand endocytosis. *New Tyr phosphorylated and dephosphorylated proteins identified in this study. negative control IgG 1 mAb (Sigma-Aldrich) was used as a control. A total of 2 × 10 4 events were acquired using BD FACSCalibur TM , and data analysed using Cell Quest software (BD Biosciences, Mountain View, CA, USA).
To assess the effect of LILRB4 ligation on the uptake of the opsonised DH5α E. coli particles, 2 × 10 5 differentiated THP-1 cells were resuspended in 100 μ l CLB and incubated with 10 μ g/ml anti-LILRB4 mAb for 20 min at RT, followed by ligation using 15 μ g/ml goat anti-mouse IgG 1 (Fc-specific) secondary Ab at RT for 10 min, and a single CLB wash prior to addition of the E. coli particles. Mouse anti-MHC-I mAb (anti-HLA-ABC) was used as a relevant surface-binding control Ab.

Statistical analysis.
Fisher's exact test was used to determine the most significantly enriched pathways as predicted by Ingenuity Pathway Analysis software. Western blots were semi-quantified by densitometry using ImageJ software (http://rsbweb.nih.gov/ij) and compared using one-way ANOVA with Dunnett post-test for multiple comparisons. The mean percentages of differentiated THP-1 cells that took up bacterial particles without LILRB4 ligation were compared with cells pre-ligated with anti-LILRB4, or treated with control anti-MHC-I mAb using two-tailed unpaired t-test. P values < 0.05 were considered statistically significant.