Proteomics Reveals a Role for Attachment in Monocyte Differentiation

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Journal of Proteome Research

Proteomics reveals a role for attachment in monocyte differentiation into efficient proinflammatory macrophages Nataliya K. Tarasova,a A. Jimmy Ytterberg,a,b Karin Lundberg, b,d Xing-Mei Zhang,c,d Robert A. Harris,c,d and Roman A. Zubarev*,a a

Department of Medical Biochemistry and Biophysics, bDepartment of Medicine, Solna, c

Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, d

Centre for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden

KEYWORDS Label-free proteomics, THP-1 cell line, tumor immunotherapy

ACS Paragon Plus Environment

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ABSTRACT Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into pro-inflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a proinflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications.


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INTRODUCTION Monocytes are bone marrow-derived cells of the innate immune system that circulate in the blood for a few days and that can migrate into tissues and differentiate into macrophages.1,2 Macrophages are professional phagocytes and antigen presenting cells that can be polarized into a range of pro- and anti-inflammatory functional states.3 Pro-inflammatory macrophages might be employed in personalized tumor immune therapy as they may directly kill tumor cells,4 alter the cytokine balance in the tumor microenvironment and are implicated in the therapeutic mechanism of action of T cell adoptive transfer therapy.5 Treatment with lipopolysaccharide (LPS) is one way of inducing a pro-inflammatory macrophage state. LPS is recognised by the pattern recognition receptor toll-like receptor 4 (TLR4) in complex with CD14 and MD2 proteins and triggers several intracellular pathways that include the IκB kinase (IKK)–NF-κB pathway.6–9 This activation results in expression and subsequent release of a variety of pro-inflammatory cytokines including TNF-α, IL-6 and IL1β.10 Within a given cellular population the response to LPS stimulation is heterogeneous, and both adherent and non-adherent cells are observed following in vitro activation of non-adherent monocytes.11 The aim of our study was to systematically investigate the proteome difference between adherent and non-adherent LPS-treated monocyte subpopulations and to identify which of these are more appropriate for tumor immune therapy. Even though the proteome of LPS-treated monocytes has been previously studied,12–16 as far as we know these subpopulations have never been investigated separately.


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The basic question about the role of the attachment process in differentiation of monocytes into macrophages was first raised in an early review.17 More recent research has revealed that adhesion can lead to early stages of monocyte differentiation through activation of STAT1 protein.18 Adhesion can also lead to up-regulation of genes involved in the phagocytic functions of macrophages, as well as in metabolism of lipids, fatty acids, carbohydrates, amino acids and steroids, in endocytosis and migration.19,20 The processes of activation, attachment and differentiation appear to be so intrinsically interlinked that they are difficult to untangle and characterize separately. Single-molecule biochemical assays are certainly not well suited to such a task, while an unbiased global analysis could be a more appropriate method. In the present study we applied a proteomics approach to address this issue. By measuring abundance changes in thousands of proteins simultaneously we attempted to identify the difference between adherent and non-adherent LPS-treated cells and to distinguish between the activation, attachment and differentiation processes. A common model of monocyte differentiation, the human monocytic THP-1 cell line, was employed.12,21–23 The results of the study were used to predict which cell subpopulation after LPS treatment would be more appropriate for tumor immunotherapy.


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MATERIALS AND METHODS Cell Culture, Treatment and Counting The human monocytic cell line THP-1 was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biochrome), 0.05 mM mercaptoethanol, 2 mM LGlutamine and 100 U/mL penicillin-streptomicin mixture. Details of the experiments performed with the cells are summarized in Table S1, which lists the times of incubation, types of stimulants and their concentrations, culture dish format and the number of replicates. Nonadherent THP-1 cells (0.4 x106 cells/mL) were used at the initial step in all experiments. LPS from E. coli (Sigma, L2654-1MG) was diluted with phosphate buffered saline (PBS). Phorbol 12-myristate 13-acetate (PMA) (Sigma) was diluted with dimethyl sulphide (DMSO); the final concentration of DMSO in culture was 0.005%. Tissue culture flasks and plates used in the study were with traditional Tissue Culture surface (Sarstedt). After collecting cells in suspension cultures were washed with PBS, adherent cells then being incubated with 0.05% trypsin with EDTA and phenol red (Gibco) for collection. Cells were counted using a TC10 cell counter (BioRad) with trypan blue staining. Cell diameter was measured using a NucleoCounter NC-200 Cell Counter (Chemometec). After collecting for proteomics analysis (centrifugation: 6 min; 1,000 x g) cell pellets were washed with PBS and lysed in a lysis buffer (8 M urea in 100 mM ammonium bicarbonate) by sonication on ice (21 sec; in three repeats with breaks in between). Cytokine Measurement with Luminex Concentrations of soluble cytokines were measured in media using a Bio-Plex Pro™ Human Cytokine 10-plex Assay kit (Luminex), following the manufacturers’ instructions.


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FACS Analysis Stimulated THP-1 cells were analyzed by flow cytometry. Cells were stained with antibodies specific for CD86, CD80, PD-L1 or isotype controls (all from BD Pharmingen). Samples were run in a Gallios flow cytometer (Beckman Coulter, Brea, USA) and analyzed using Kaluza v1.1 software (Beckman Coulter). Expression of the marker of interest was quantified using Median Fluorescence Intensity. Protein Isolation and Digestion Cell lysates were centrifuged (20 min; 20,800 x g; 8oC), with supernatants collected. The protein concentration was determined using the BCA kit (Pierce). Following reduction (5 mM dithiotreol, 37oC, 30 min) and alkylation (14 mM iodoacetomide, RT, 30 min), 10 µg of proteins were digested by trypsin (Promega) at a 1:30 trypsin:protein concentration ratio (37oC, overnight) in the presence or absence of 1% ProteseMAX (Promega). Digestion was stopped with formic acid (FA). Digests were cleaned with Stage Tips (Thermo Scientific), dried and resuspended in 0.1% FA prior to analysis. Mass Spectrometry LC-MS/MS analyses were performed using an Easy-nLC chromatography system (Thermo Scientific) directly coupled on-line to a Q Exactive24 mass spectrometer (Thermo Scientific). For each sample, 1 or 2 µg were injected from a cooled autosampler onto an 8 cm long fused silica tip column (SilicaTips™, New Objective Inc., USA) packed in-house with 3 µm C18-AQ ReproSil-Pur® (Dr. Maisch GmbH, Germany) or EASY-ColumnTM. The chromatographic separation was achieved using an acetonitrile (ACN)/water solvent system containing 0.1% FA and a gradient of 90 or 110 min from 5% to 35% of ACN. Mass spectra were acquired with a


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resolution of R = 70,000, followed by up to 10 consecutive data-dependent MS/MS spectra taken using higher-energy dissociation (HCD) with the energy set at 25 units. Samples were analyzed in a randomized order. Protein Identification and Quantification MS/MS data were extracted and processed according to a previously described protocol25 and searched against the concatenated version of UniProtKB/Swiss-Prot database (release 2012_06 (20,257 human sequences) or release 2014_10 (20,194 human sequences)) using the Mascot search engine v. 2.3 (Matrix Science Ltd., UK). The following parameters were used: trypsin digestion with a maximum of two missed cleavages; carbamidomethylation (C) as a fixed modification; pyroglutamate (Q) and oxidation (M) as variable modifications; a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.1 Da. The list of identified proteins was filtered using 1% false discovery rate (FDR) and at least two peptides per protein as limiting parameters. Quantification was performed using the program Quanti that employs extracted-ionchromatogram-based quantification.25 Further details and results of analyses are provided in Supporting Information (Tables S2, S3 and S4). In total from 1595 to 3161 proteins (depending on the experiment) were quantified with at least two unique peptides per protein. Bioinformatics and Statistical Analysis Protein abundances were normalized with the assumption that equal amounts of protein digests were injected for each sample. Proteins that likely originated from the media (serum albumin and hemoglobin) or sample handling (keratins) were excluded from the results. Mean values of technical duplicates were calculated, log transformed and used in further analyses. Principal component analysis (PCA) and Orthogonal Projections to Latent Structures (OPLS) were


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performed using Simca software version 13.0.3 (Umetrics). Unpaired Student’s T-test with equal or unequal variance (depending on the result of Excel F-test) was applied to calculate the pvalues. FDR (Benjamini-Hochberg)26 adjusted p-values (q-values) below 0.05 were used to identify significantly up/down-regulated proteins and pathways. Pathway analysis was conducted using String version 10 (http://string-db.org/) and q-values for Gene Ontology (GO) terms were calculated against the dataset with all identified proteins. All the error bars in the figures represent sample standard errors of the mean. Fold change was calculated as a ratio of the average values of biological replicates. Quality control of proteomics data was performed in several ways. (1) The retention times of peptides were compared for consistency among different samples within the same experiment. As a result, the first seven samples were excluded from the last experiment due to problems with the LC column. (2) Whether protein abundances correlated with the order of injection was assessed. No protein with a correlation better than R2>0.80 was identified. (3) PCA plots were analysed for the presence of outliers. No outliers were identified in any experiment except the last one, where two outliers were found and removed from further analysis.


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RESULTS Cell Responses to LPS and Cell Subpopulations following LPS Stimulation THP-1 cells incubated with LPS for 48 h released cytokines (IL-1β, TGFα, and IL-6) into media (Fig. 1A) as expected.10 LPS treatment also induced growth inhibition (Fig. 1B) and a higher percentage of adherent cells (Fig. 1C) compared to unstimulated cells. Cells in suspension were collected separately for proteome analysis from those attached to the culture dish (Fig. 2, inset). Proteomes of four cell subpopulations were analyzed and compared (Table S2 and Fig. 2): 1) non-adherent LPS-treated cells; 2) adherent LPS-treated cells; 3) untreated non-adherent cells (used as a control); and 4) untreated adherent cells. In total 1,595 proteins were quantified with at least two peptides per protein. PCA of the proteomics data revealed that both technical and biological replicates were close to each other, while cell subpopulations were well separated.


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Figure 1. Effect of lipopolysaccharide (LPS) on human monocytic THP-1 cells. Incubation for 48 h of THP-1 cells with LPS (100 ng/mL) induced: (A) release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6); (B) growth inhibition leading to reduction in total cell count compared to control (p=0.04); (C) higher percentage of adherent cells compared to control (p=0.03).


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Figure 2. Principal component analysis (PCA) of proteomics data and a schematic overview of the experimental design (inset in the upper right corner). Human monocytic THP-1 cells were incubated with and without LPS (100 ng/mL) for 48 h in 3 biological replicates. Adherent and non-adherent cell subpopulations were collected separately and analyzed using label-free, massspectrometry-based proteomics. PCA (R2=0.3; Q2=0.2; 4 principal components) revealed good reproducibility of biological and technical replicates. Untreated Non-adherent (UN) cells were used as a control population because they remained in the same morphological state after incubation. The two vectors UN and to Untreated Adherent (UA) and to LPS-treated Nonadherent (LN) are almost orthogonal, suggesting that the activation and attachment to surface are two largely independent processes in terms of the proteome change. The LPS-treated Adherent (LA) cells are well separated from other cell populations along the main t1 axis, which indicates a large difference between them, likely due to LA cells being closer to macrophages.


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Non-adherent Cells following LPS Stimulation are Activated by Cytokines The proteome changes in non-adherent cells following LPS stimulation (LN in Fig. 2) were identified in comparison to control cells. OPLS analysis was performed on these two groups (mean values of technical replicates were used). The up-regulated proteins with more than twofold change (20 proteins; Table S5) were mapped using a pathway database to identify the most up-regulated pathways (Table S6). Half of these proteins were linked to response to interferons (IFN) type I (q(GO:0060337)=7.7x10-5). This pathway was second in the list sorted by q-value, following the general pathway ‘innate immune response’. IL-1β (IL1B_HUMAN) among the upregulated proteins was likely the precursor of IL-1β protein because the dataset contained peptides belonging to its cleavable part (Fig. 3A). Since 70% of the up-regulated proteins represented a response to cytokines, with the related pathways being significantly over-represented (q(GO:0019221)=4.7x10-3), we hypothesized that the non-adherent cells were activated not by LPS itself but by the factors released from the LPSactivated cells. This hypothesis was supported by the fact that IFN-γ, which is also produced by macrophages in response to LPS,27 prevented cellular attachment (0 % attached cells upon IFN-γ treatment versus 12.4±0.3 % in control). When non-adherent LPS-treated cells were transferred into a new culture dish and incubated for 48 h with fresh media, the fraction of adherent cells was five times smaller compared to control (p=0.02; Fig. S1). The cells remaining in suspension following LPS-treatment therefore have a reduced propensity towards attachment compared to the original cell population.


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Figure 3. Relative abundances of selected proteins and peptides (in logarithmic scale). (A) Average relative peptide abundance of two peptides from the cleavable part of the IL-1β precursor protein (‘Peptide 1’: QAASVVVAMDK; ‘Peptide 2’: CSFQDLDLCPLDGGIQLR) and one peptide from the chain (‘Peptide 3’: NLYLSCVLKDDKPTLQLESVDPK) in all studied cell subpopulations (Fig. 2, insert). The presence of these peptides makes it likely that the identified protein is a precursor form of the IL-1β protein. (B) Average protein abundances of Caspase-1 (CASP_HUMAN) and IL-1β (IL1B_HUMAN) in cell subpopulations from the same experiment correlate.


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Adherent Cells Differentiate into Macrophages following LPS Stimulation The proteomes of the LPS-treated adherent cells were most distant along the main t1 axis of the PCA plot from control compared to other cell subpopulations (Fig. 2). We hypothesized that these cells began their differentiation into macrophages. To test this, the proteins with abundances significantly changed in LPS-treated adherent cells (LA in Fig. 2) compared to in control cells were identified using a T-test (Table S7) and mapped onto pathways (Table S8). A total of 100 proteins were determined to be up-regulated (Table S7). Half of the proteins were found to be involved in localization, interaction with environment and migration (q(GO:0016477; GO:0007154)=4.1x10-2 and q(GO:0048870; GO:0051674)=4.5x10-2). The cell adhesion was mediated by integrin (M) beta 2, ICAM-1 (ICAM1_HUMAN), CD44 (CD44_HUMAN) and Protein Tyrosine Kinase 2 Beta (PTK2B_HUMAN). Integrin beta 2 (ITGB2_HUMAN) was found by proteomics to be up-regulated in adherent LPS-treated cells, while integrin M (CD11b) was confirmed to be up-regulated by FACS analysis (Fig. S2). Among

up-regulated

proteins,

27%

were

involved

in

cell

differentiation

(p(GO:0030154)=6.8x10-4). This list included proteins playing important roles in macrophage differentiation, such as NF-κB28,29 (fold change 3.7), IFI1630 (fold change 3.1), and MMP931 (fold change 43.1). Neither NF-κB nor IFI16 were significantly up-regulated in the non-adherent LPS-treated cells. The second in the list of up-regulated pathways sorted by q-values (following the general pathway ‘innate immune response’) was ‘immune effector processes’ (q(GO:0002252) =3.4x10-6). Cell division pathways were down-regulated in the cells (p(GO:0006270)=1.5x10-5, p(GO:0006271)=3.6 x10-5, and p(GO:0006268)=4.4x10-4).


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Proteins up-regulated in adherent and non-adherent cells following LPS treatment (LA and LN in Fig. 2, respectively) contra the control were compared to up-regulated proteins in PMA-treated cells (Table S9) that are known to differentiate into adherent macrophages21. LPS-treated adherent cells had, as would be expected for differentiating cells, more up-regulated proteins in common with PMA-treated cells than did non-adherent cells (30% versus 20%; Fig. S3). Since the protein isolation procedure for the proteomics study was likely biased towards soluble proteins, additional FACS analysis was performed to identify surface membrane-bound markers. The B7 complex (its subunit CD80), which is responsible for T-cell activation, was up-regulated in response to LPS. Significantly higher amounts of the complex were evident in adherent compared to non-adherent LPS-treated cells (Fig. 4). The subunit CD86 did not exhibit such a clear difference (data not included). At the same time PD-L1, which is responsible for T-cell inhibition, was also up-regulated. The data supports the idea that adherent LPS-treated cells might be more efficient in signal transduction to T-cells than are non-adherent LPS-treated cells.


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Figure 4. FACS analysis of CD80 and PD-L1 on the surface of the studied cell subpopulations (Fig. 2). The B7 complex (CD80 subunit), which is responsible for T-cell activation, was upregulated in response to LPS, more-so in LPS-treated adherent cells compared to in non-adherent cells. At the same time, PD-L1, which is responsible for T-cell inhibition, is also up-regulated, indicating the well-tuned system of T-cell activation. The data supports the idea that the LPStreated adherent cells might conduct more efficient signal transduction to T-cells. Attachment Prepares Cells for Differentiation The untreated adherent cells (UA in Fig. 2), whose proteome groups on the PCA plot were separated from both adherent LPS-treated and control cells are likely the result of the spontaneous attachment process. The proteomes of untreated adherent cells were compared with control using OPLS. The intercellular adhesion molecule 1 (ICAM1_HUMAN) was evident among the up-regulated proteins with more than two-fold change (Table S10, Fig. S4). Other proteins on the list included CD14, MARCKS-related protein (MRP_HUMAN), cathepsins (CATB_HUMAN and CATL1_HUMAN), and neutrophil cytosolic factor 1 (NCF1_HUMAN).


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Based on the nature of these up-regulated proteins we hypothesized that the attachment process prepares untreated cells for responses to stimuli and differentiation. Consistent with this suggestion the diameter of adherent cells was determined to be larger than that of non-adherent cells (13.0 ± 0.1 µm versus 12.8 ± 0.2 µm, 20 replicates, p=0.0004). This hypothesis was tested in an additional proteomics experiment in which THP-1 cells were incubated for 48 h in 4 replicates without any stimulus to permit spontaneous cell adherence (Fig. S5). The adherent cells were separated from non-adherent cells by transferring the latter to another dish with fresh media and adding new media to the first dish. Both cell subpopulations were then treated with LPS at four concentrations (35, 100, 500, and 800 ng/mL) for 24 h, followed by extraction and analysis of the proteomes. The OPLS analysis separated the samples with different LPS concentrations along the main principle axis (Fig. S6). Almost all (95%, 53/56) up-regulated proteins in LA cells with more than two-fold change were among the upregulated proteins in LPS-treated cells which significantly influenced separation along the t1 axis (Fig. S7). It was concluded that the LPS treatment results in our experiments were reproducible, and that the t1 axis corresponds to differentiation and pro-inflammatory polarization. The effect of attachment was apparent as a shift along the t1 axis even for the lowest concentration of LPS employed (Fig. 5, only two lowest concentrations are depicted), which supports the priming effect of attachment. The priming was not reflected in proteomes at higher LPS concentrations, most likely because most of the cells were already close to being macrophages.


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Figure 5. Distances in an OPLS model separating proteomes of adherent and non-adherent THP1 cells treated with LPS at different concentrations from the proteomes of untreated cells (Fig. S6, right). THP-1 monocytes were primed with attachment (‘Adherent before treatment with LPS’) or not (‘Non-adherent before treatment with LPS’), as depicted in Fig. S5, then treated with different concentrations of LPS for 24 h, collected and analyzed using LC-MS/MS. Distances were calculated between the mean projections on the main t1 axis. Priming through attachment is apparent (p

Proteomics Reveals a Role for Attachment in Monocyte Differentiation

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