Proteomic and Bioinformatic Characterization of Extracellular Vesicles

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Proteomic and bioinformatic characterization of extracellular vesicles released from human macrophages upon influenza A virus infection Wojciech Cypryk, Martina B Lorey, Anne Puustinen, Tuula A Nyman, and Sampsa Matikainen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00596 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 11, 2016

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Journal of Proteome Research

Proteomic and bioinformatic characterization of extracellular vesicles released from human macrophages upon influenza A virus infection

Wojciech Cypryk1,*, Martina Lorey2,*, Anne Puustinen3, Tuula A. Nyman1,4 and Sampsa Matikainen2

*

Equal Contribution

1

Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 University of Helsinki,

Finland 2

University of Helsinki and Helsinki University Hospital, Rheumatology, Helsinki, Finland

3

Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250 Helsinki, Finland

4

Institute of Clinical Medicine, Sognsvannsveien 20, Rikshospitalet, 0372 Oslo, Norway

Address correspondence and reprint requests to Dr. Sampsa Matikainen, 2University of Helsinki and Helsinki University Hospital, Rheumatology, Helsinki, Finland. Phone: +358 50 3258757, e-mail: [email protected]

KEYWORDS: influenza A virus, innate immunity, macrophages, proteomics, extracellular vesicles

Abbreviations used in this paper:

IAV = Influenza A Virus, EV = extracellular vesicle, MV = Microvesicle, ILV = Intraluminal vesicle, MVB = Multivesicular body, HIV-1 = Human immunodeficiency 1 virus, MS = Mass spectrometry, LC-MS/MS = Liquid chromatography-tandem mass spectrometry, FABP = Fatty acid-binding protein, COMMD = Copper metabolism MURR-1 domain, EM = Electron microscopy, siRNA = small interfering RNA, GO = Gene ontology, KEGG = Kyoto Encyclopedia of Genes and Genomes, Panther = Protein ANalysis THrough Evolutionary Relationships, IPA = Ingenuity Pathway Analysis, GTP = 1

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Guanosine-5’-triphosphate, GAPR-1 = Golgi-associated plant pathogenesis-related protein, ACBP = Acyl-CoA-binding protein

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Abstract

Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics and functional studies. We demonstrate that at 9 h post-infection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAVinfected macrophages compared to 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggests that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.

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Introduction

Influenza A viruses (IAV) are negative-stranded RNA viruses capable of infecting variety of avian and mammalian species. These viruses are responsible for the annual epidemics that cause severe illnesses in millions of people worldwide, leading to 500,000 deaths annually.1 Host defense against IAV infection is initiated by the innate immune system. The innate immune system detects viral genomes and replication intermediates with their pattern recognition receptors. These responses are essential for the development of later adaptive immune responses, which provide specific cell-mediated and humoral protection, and are often necessary for a complete clearance of infection. In humans, IAV infection is initiated in the respiratory tract, where the virus is capable of overcoming the mucus barriers and rapidly infects epithelial cells of nasal, oral and lower respiratory tract cavities, which are the primary host cells for the virus. Once establishing initial infection, IAV subsequently spreads to local immune cells, including macrophages. Macrophages are the principal effector cells of the innate immune system that produce antiviral and pro-inflammatory cytokines in response to IAV infection.2 In addition to cytokines, activated macrophages secrete many other biomolecules (proteins, nucleic acids, lipids) that play important roles in intercellular signaling.3,4 However, the details of IAV-activated macrophage responses involved in intercellular communication are incompletely understood. Protein secretion is mediated via classical, ER-Golgi mechanisms, as well as through unconventional, vesicle-mediated protein release. Proteins that do not contain the N-terminal signal sequence exit the cells via several distinct mechanisms inside membrane-enclosed, nano-sized entities, collectively called “extracellular vesicles” (EV).5 There are two different types of EVs: microvesicles (MVs) bud directly from the plasma membrane through a mechanism called shedding, whereas exosomes are formed when intraluminal vesicles (ILVs) accumulate in multivesicular bodies (MVBs), and are released by fusion of MVBs with the plasma membrane. A third mechanism has been proposed, where MVBs fuse with secretory autophagosomes to generate amphisomes, which

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subsequently release the exosomes into the extracellular space by fusing with the plasma membrane in a process called secretory autophagy.6 EV secretion has attracted significant attention among scientists throughout recent years but their role in viral infections is only beginning to be elucidated.7 Numerous recent studies highlighted the importance of EV secretion in the course of severe fever with thrombocytopenia virus,8 human immunodeficiency 1 (HIV-1) virus,9-11 Epstein-Barr virus,12,13 gamma herpesvirus14 and influenza.15 Many studies have shown that viral components can be found on the EV, suggesting that EV secretion can provide a novel method for virus dissemination in the body. In addition, EVs from HIV-1-infected dendritic cells have been proposed to mediate productive virus trans-infection to T cells.16 Mass spectrometry (MS)-based proteomics has immensely developed during the last decade, and the current methods and high resolution MS instruments make it possible to identify thousands of proteins with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Today, MS-based proteomics is a core component in making fundamental new discoveries in immunology and virology.17,18 Here, we have used high-throughput proteomics combined with bioinformatics to characterize EVs released from human macrophages upon IAV infection. We first demonstrate that IAV infection of human macrophages triggers robust vesicle-mediated protein secretion within 9 h post-infection. We identified more than two thousand human proteins in the EVs of IAV-infected macrophages, and provide evidence that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins (FABPs), antiviral cytokines, copper metabolism Murr-1 domain (COMMD) proteins, and autophagy-related proteins.

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Materials and Methods

Cell culture and infections Human macrophages were derived from leukocyte-rich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Monocytes (three donors per experiment) were isolated and differentiated into macrophages as described previously.19 In total, 1.4 x 106 monocytes were seeded per well on 6-well plates. The monocytes were cultured in serum free macrophage media (Macrophage-SFM, Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (ImmunoTools, Germany) and 50 U/mL penicillin–streptomycin (Lonza, Basel, Switzerland) at 37 °C and 5 % CO2 for 6 days, polarizing the monocytes into macrophages of the acute pro-inflammatory M1-phenotype. On day six the cells were washed with PBS, supplied with fresh RPMI 1640 medium (Gibco) supplemented with L-glutamate and antibiotics and subsequently infected with human pathogenic influenza A (H3N2) strain Udorn/72 (2.56 hemagglutination unit/mL) for the times indicated.

Sample preparation For EV isolation, 9h cell culture supernatants from untreated and IAV-infected macrophages were collected and centrifuged at 500 g for 10 min to remove suspended cells and further at 3,000 g for 30 min to remove residual cell debris. Cleared cell culture supernatants were concentrated with Amicon 100 kDa MWCO centrifugal filter units (Millipore) and EVs were isolated by ultracentrifugation as previously described.20 For proteomic analysis and Western blotting, washed EV pellets were solubilized in Laemmli sample buffer before SDS-PAGE. For electron microscopy (EM), washed EV pellet was fixed with 2 % PFA in PBS and directly used for EM grids preparation.

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For total secretome analysis by SDS-PAGE and silver staining or Western blotting, the culture medium was centrifuged at 400 g to remove detached cells and cell debris and subsequently concentrated 50-100-fold on 10 kDa MWCO centrifugal filter units (Millipore).

Electron microscopy of vesicles The EM of vesicles was performed as described by Théry et al.21 with minor modifications. Briefly, 5 µL of fixed exosome suspension was transferred to Pioloform-carbon-coated copper grids and allowed to absorb for 20 minutes. The grids were subsequently washed and contrasted with uranyl acetate, embedded in the mixture of uranyl acetate and methyl cellulose and dried. Vesicles were observed at 80 kV with Jeol JEM-1400 transmission electron microscope in the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki.

Proteomic analysis using GeLC-MS/MS Human macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 9 hrs or left untreated. We analyzed two independent biological replicates with cells from three individual donors each. EVs were isolated from equal supernatant volumes (40-60 mL of medium per condition) by ultracentrifugation, then washed in PBS, and pelleted again. The washed EV pellet was solubilized in Laemmli sample buffer, and the proteins were identified using GeLC-MS/MS as previously described.22 Briefly, the proteins were separated by SDS-PAGE, silver stained and the gel lanes were cut into 5-6 pieces. The proteins were reduced with 20 mM dithiotreitol for 30 min in 56 °C, alkylated with 55 mM iodoacetamide for 15 min in the dark, and in-gel digested with trypsin (Promega) overnight in 37 °C. The peptides were eluted twice with 0.1 % TFA/50 % ACN, dried and solubilized in 40 µL 0.1 % TFA for mass spectrometry analysis. Each peptide mixture was analyzed by automated nanoflow capillary LC–MS/MS using an EASY nanoLC 1000 (Proxeon, Thermo Fisher Scientific Inc., USA) coupled to a quadrupole Orbitrap mass spectrometer (QExactive, Thermo Fisher Scientific Inc., USA). Aclaim Pepmap 100 (75 µm x 2 cm, nanoViper, C18, 3µm, 100Å, Proxeon, Thermo Fisher Scientific Inc., USA) was used as the trap 7



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column. Reverse-phase separation of peptides was carried out using a 75 µm × 15 cm Acclaim PepMap100 C18 column (Dionex, Thermo Fisher Scientific Inc., USA) at a flow rate of 300 nL/min. The mobile phases were 5 % acetonitrile, 0.1 % formic acid (A) and 95 % acetonitrile, 0.1 % formic acid (B). Peptides were eluted from the column with a linear gradient of 5–35 % solvent B in 90 minutes. Fractions with very high peptide concentration were re-analyzed with a 3h separation gradient. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 400 to m/z 1800 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 80 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation with an isolation window of 2 and normalized collision energy of 27. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 100 ms, without a lowest fixed mass defined. Dynamic exclusion duration was 30 s. The obtained MS/MS spectra were searched with the Mascot database search engine (version 2.4.0) through the Proteome Discoverer 1.4.0.288 interface (Thermo Fisher Scientific Inc., USA) against the SwissProt database (SwissProt 2015_11, 549,832 sequences, 196,078,138 residues). The data was processed separately for the two biological replicates. The search criteria specified trypsin as the digestion enzyme, fixed modification: carbamidomethyl (C), variable modifications: oxidation (M), deamidation (N, Q). Precursor masses were defined to be between 350 and 5000 kDa, precursor mass tolerance was 10 ppm, and fragment mass tolerance 20 mmu. Maximum missed cleavage sites were set to 2, and preferred taxonomy to “human”. The maximum p-value for identification confidence was 0.05. The false discovery rate (FDR) was determined with a decoy database, target FDR (Strict): 0.01, target FDR (Relaxed): 0.05, based on q-values. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE23 partner repository with the dataset identifier PXD004424.

Bioinformatic analysis 8


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For gene ontology (GO) classification and KEGG pathway analysis the proteomics data of EVs from IAV-infected and control cells from two biological replicates were combined into one IAV and one Control file. These two datasets were then submitted to EnrichR.24 The output files of the enrichment analysis were tables which included p-values, Benjamini-Hochberg adjusted p-values, the z score of the deviation from the expected rank, as well as the “combined score” which is the combination of the p-value with the z-score by multiplying these two numbers for as follows: c=ln(p)⋅z. The adjusted pvalues instead of regular p-values were used in all analyses for additional level of confidence. To compare the most differentially enriched GO terms between the two conditions we used R (R Core Team (2013). R: A language and environment for statistical computing. R (Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/)) to define the absolute difference between the two adjusted p-values in the two conditions. Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Mountain View, CA, www.ingenuity.com) was used for mapping the identified proteins into “Subcellular location and molecular type”, “Canonical Pathways” and “Diseases and biological functions”.

Western blotting The protein samples were denatured at 95°C for 5 min and separated on SDS-PAGE, transferred to Immobilon-P (PVDF) transfer membranes (Millipore), blocked with 5 % non-fat milk solution in TBS-Tween (TBS-T) and incubated overnight at 4 °C with primary antibodies. The membranes were washed with TBS-T and incubated with appropriate HRP-conjugated secondary antibody for 1 h at room temperature and after that washed with TBS-T and visualized with Western Lightning ECL (Perkin-Elmer, Waltham, MA). The antibodies against annexin 1 (sc-12740), alix (sc-53540), GAPR-1 (sc-398783), E-FABP (sc-365166) and MDGI (sc-58275) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against CD11c (ITGAX, ab52632) was purchased from Abcam PLC (Cambridge, UK). Secondary antibodies were purchased from Dako (Dako Denmark A/S).

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GAPR-1 silencing On day 5 of cell culture in 12-well plates, macrophages were transfected with 100 nM non-targeting control small interfering RNA (siRNA, AllStars Negative Control siRNA, Qiagen, Hilden, Germany) and with 50 nM each of two GAPR-1 siRNAs (FlexiTube siRNA 5nmol, Qiagen: Hs_C9orf19_8, Hs_C9orf19_6; final concentration = 100 nM) using HiPerFect Transfection Reagent (Qiagen), according to the manufacturer’s instruction. After 3 h of siRNA treatment, cells were supplemented with fresh macrophage-SFM. On the following day, the siRNA treatment was repeated and cells were left unstimulated or infected with IAV for 9 h, after which the cell-culture supernatants were collected, concentrated with Amicon 10 kDa MWCO filter units (Millipore), and proteins were separated by SDS-PAGE and visualized with silver staining and Western blotting.

Results and Discussion

IAV infection activates extracellular vesicle-mediated protein secretion in human macrophages We have previously shown that IAV infection of human monocyte-derived macrophages causes major alterations to subcellular protein trafficking inside the cell and triggers robust protein secretion.25 Majority of the proteins identified from the total secretomes upon IAV infection were found in ExoCarta26 and did not contain signal sequence required for classical, ER-Golgi secretion pathway, suggesting that they are secreted in EVs.27 Here we have characterized the EV-mediated protein release in global manner during IAV infection of human macrophages. First we characterized the kinetics of protein secretion during IAV infection of human macrophages. Macrophages were left untreated or infected with IAV for different time periods. After this the cell culture supernatants were collected, concentrated, and proteins were separated by SDSPAGE and subsequently visualized with silver staining (Fig. S-1A). IAV infection of human macrophages resulted in marked protein secretion already at 3 h post-infection steadily increasing up to 9 h after IAV infection. Protein secretion dramatically increased at 18 h after IAV infection, probably due to cell death. This is in agreement with our earlier observations that IAV infection of 10


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human macrophages leads to cell death at 12 h post-infection.25 In accordance with silver staining results, secretion of Alix, a well-defined marker of EV secretion, was clearly enhanced 9 h post-IAV infection (Fig. S-1A). Based on kinetic experiments, we isolated EVs from untreated and IAV-infected macrophages at 9 h post-infection for subsequent analysis. Electron microscopy analysis of isolated vesicles revealed large amount of EVs in samples isolated from IAV-infected macrophage cell culture supernatants (Fig. 1A, left). The observed vesicles were predominantly small size (

Proteomic and Bioinformatic Characterization of Extracellular Vesicles

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