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Combined proteome and eicosanoid profiling reveals novel implications of human fibroblasts in chronic inflammation Ammar Tahir, Andrea Bileck, Besnik Muqaku, Laura Niederstaetter, Dominique Kreutz, Rupert Laurenz Mayer, Denise Wolrab, Samuel M. Meier, Astrid Slany, and Christopher Gerner Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04433 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017
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Analytical Chemistry
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Combined proteome and eicosanoid profiling reveals novel
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implications of human fibroblasts in chronic inflammation
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Ammar Tahir1, Andrea Bileck1, Besnik Muqaku1, Laura Niederstaetter1, Dominique Kreutz1,
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Rupert L. Mayer1, Denise Wolrab1, Samuel M. Meier1, Astrid Slany1, Christopher Gerner1*
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1
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Austria
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, 1090 Vienna,
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* Corresponding author
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Christopher Gerner
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Department of Analytical Chemistry
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University of Vienna
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Waehringerstr. 38
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1090 Vienna
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Austria
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[email protected] 19
+43-1-4277-52302
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Short Title: Multi-omics of inflammation mediators
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Abstract
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During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses.
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Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid
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formation by human fibroblasts. Aim of this study was to analyze the formation of the most relevant
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inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation
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and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification
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was applied for proteome profiling, while an in-house established data-dependent analysis method based
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on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites
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was determined by targeted analysis. The secretion of fourty proteins including cytokines, proteases and
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other inflammation agonists as well as fourteen pro-inflammatory and nine anti-inflammatory eicosanoids
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was found significantly induced, while several acylcarnithins and sphingomyelins were found
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significantly
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downregulated most cytokines and proteases, abrogated the formation of pro-, but also anti-inflammatory
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eicosanoids and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the
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chemokines CXCL1, CXCL5, CXCL6 and complement C3, known to contribute to chronic inflammation,
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were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal
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stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring.
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Comparative proteome profiling regarding other cells demonstrated cell type specific synthesis of,
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amongst others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better
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understand cell type-specific regulation of inflammation mediators and shedding new light on the role of
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fibroblasts in chronic inflammation.
downregulated
upon
inflammatory
stimulation.
Treatment
with
dexamethasone
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Keywords: eicosadomics, lipidomics, proteomics, metabolomics, mass spectrometry 2 ACS Paragon Plus Environment
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Analytical Chemistry
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Introduction
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Throughout decades of intense research, eicosanoids have been proven to play important roles in
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physiological and pathophysiological processes related to inflammation, such as diabetes, atherosclerosis
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and cancers.1-3 Neutrophils, macrophages and platelets are known as major producers of eicosanoids that
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regulate important processes such as antibody and cytokine release, as well as cell proliferation,
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differentiation and migration.
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Based on the pro-inflammatory effects of eicosanoids,4,5 most anti-inflammatory drugs are designed to
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target enzymes responsible for the synthesis of eicosanoids, either by inhibiting the enzymatic functions,
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or by repressing the transcription of the genes encoding for the enzymes.6,7 However, these drugs may also
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interrupt the formation of anti-inflammatory eicosanoids,8,9 thus potentially interfering with the resolution
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of inflammation. This is a scenario with some potential relevance in case of chronic inflammation and
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related diseases. Actually, while classical antiphlogistic drugs have been successfully implemented in the
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treatment of acute inflammation, chronic inflammation responds in an unsatisfying fashion to sustained
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treatments with these drugs and still represents a great challenge in clinical practice.10,11
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Fibroblasts are important players of inflammation, displaying strong chemokine secretion activities.12,13
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Moreover, fibroblasts have been identified as important players in chronic inflammation over the last few
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years.14,15 One reason therefore may be that, after they have been activated upon inflammation, fibroblasts
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hardly undergo cell death as typically observed in case of leukocytes,16 so that inflammation-associated
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activities may be maintained for long time periods in the body. Furthermore, these cells may not be
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affected by classical antiphlogistic drugs in a same way as leukocytes. For an improved understanding of
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the molecular processes occurring in these cells, a reliable determination of inflammation mediators,
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which are eicosanoids and cytokines, is mandatory. Referring to a previous study about proteome
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alterations induced in inflammatory stimulated primary human dermal fibroblasts,12 we here analyzed
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these cells with respect to their responsiveness to one of the most important anti-inflammatory drugs,
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dexamethasone. Thereby, focus was put on eicosanoids and chemokines, as well as other lipids, proteins
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and metabolites, applying mass spectrometry-based shotgun and targeted analyses.
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Based on our practical experience with in-depth proteome profiling of inflammatory processes using a
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QExactive orbitrap,12,17,18 we developed a data-dependent profiling strategy for eicosanoids similar to
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shotgun analysis of peptides. Only few research groups have yet employed orbitrap mass spectrometry for
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eicosanoid profiling,19 and, to the best of our knowledge, this is the first analytical study screening for
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eicosanoids released by human fibroblasts. The present study demonstrates that the combined application
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of eicosadomics, proteomics and metabolomics uncovers previously unrecognized features of fibroblasts,
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highlighting their relevance for chronic inflammation and potentially offering new molecular targets for
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improved therapy.
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Analytical Chemistry
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Material and Methods
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Cell culture
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Normal human dermal fibroblasts (NHDF), kindly provided by Verena Paulitschke, were cultured as
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previously described.12 Experiments were performed with cells at same passages (20 to 22) for three
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biological replicates each of control and treated cells, using 1.5x106 cells per 25cm2-culture flask. Control
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cells were incubated for 24h at 37°C and 5% CO2. In parallel, inflammatory stimulated NHDF were
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obtained by treating cells with 10ng/mL of IL-1β (Sigma-Aldrich, Vienna, Austria) for 24h, based on
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experience from previous studies.20-22 One aliquot of stimulated cells was additionally treated with
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100ng/ml of dexamethasone (Sigma-Aldrich) one hour after stimulation and cultured like the other for
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additional 23 hours. Thereafter, all cells were cultured for additional 6h in 3ml of serum-free medium to
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obtain the fraction of secreted proteins.23 Similar experiments were performed with NHDF derived from
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another donor at passage 6 to 7 (Lonza, France, Amboise) and human mesenchymal stem cells (hMSC,
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passage 4 to 5; Lonza). hMSC were cultured in mesenchymal stem cell growth medium (Lonza)
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supplemented with the associated Bulletkit and 100U/ml penicillin/streptomycin (ATCC/LGC Standards,
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London, UK). All further processing steps and shotgun proteomics were done in the same way as for
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NHDF.
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Extraction of eicosanoids
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Supernatants from cell culture (3mL each) were collected each in an individual 15mL falcon tube. 100nM
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of each of the internal standards (PGE2-d4, 15S-HETE-d8 and PGF2a-d4; from Cayman Europe, Tallinn,
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Estonia) were added. After protein precipitation, EtOH was evaporated in a speedvac at 35°C for 25min
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until the original sample volume - before protein precipitation - was restored. Samples were then diluted
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1:3 with MS-grade water and extracted using 30mg/mL StrataX solid phase extraction (SPE) columns
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(Phenomenex, Torrance, CA, USA). Columns were conditioned and equilibrated with HiPerSolv grade
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methanol (MeOH; VWR International, Vienna, Austria) and LC-MS grade water (Sigma-Aldrich),
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respectively. After sample loading, the columns were washed with 15% MeOH, and eicosanoids were 5 ACS Paragon Plus Environment
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eluted with 1mL of acetonitrile (ACN, VWR International), MeOH and formic acid (FA; Sigma-Aldrich)
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(ACN:MeOH:FA, 49:49:2). Solvents were evaporated by vacuum centrifugation and lipids were dissolved
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in 200µL reconstitution buffer (ACN:H2O:FA, 30:70:0.02).
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LC/MS method for eicosanoids
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Samples were pipetted into 200µL glass inserts. All eluents such as mobile phase A (H2O:FA, 100:0.02)
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and mobile phase B (ACN:MeOH:FA, 90:10:0.02) were degassed prior to their usage. Using Infinity 1290
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UHPLC (Agilent Technologies Austria GmbH, Vienna, Austria), a 40 minutes gradient flow method was
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applied using a Kinetex™ 2.1mm x 15cm, 2.6 µm, C18, 100 Å reversed phase column (Phenomenex); the
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flow rate was set to 250µL/min and a gradient was applied (0-2min: 5% mobile phase B; 2-28min: 5-85%
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mobile phase B; 28-35min: 95% mobile phase B; re-equilibration with 5% mobile phase B). All samples
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(20µL injected) were analyzed in technical duplicates. Mass spectrometric detection was performed with a
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Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) using the HESI-source to achieve
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negative ion mode ionization. MS scans were performed with an m/z range from 250 to 750 and a
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resolution of 35 000 (at m/z = 300). A lock mass was set; drift was +/- 5ppm over all experiments. MS/MS
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scans of the six most abundant ions were achieved through HCD fragmentation at 30% normalized
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collision energy and analyzed in the orbitrap at a resolution of 17 500 (at m/z = 300). Data have been
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converted into mzXML to be readable independent of vendor-specific software and made freely
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available to download following the link:
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https://anchem.univie.ac.at/ueber-uns/internes/geschuetzt/download9156546/
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Eicosanoid data interpretation
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Raw files generated by the Q-Exactive Orbitrap were analyzed and assessed manually using Thermo
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Xcalibur 2.2 Sp1.48 (Qual broswer). Candidate ions were extracted and further evaluated using an in-
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house developed data processing software (programmed by AT). Libraries from Lipid Maps depository
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were used and implemented as references.24,25 6 ACS Paragon Plus Environment
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Analytical Chemistry
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Extraction and digestion of proteins from secretomes, cytoplasm and nuclear extracts
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Secreted proteins were prepared by overnight precipitation with ethanol at -20°C of three cell supernatants
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each of control, activated and dexamethasone treated NHDF. After centrifugation, proteins were dissolved
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in sample buffer (7.5M urea, 1.5M thiourea, 4% CHAPS, 0.05% SDS, 100mM dithiothreitol). In case of
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dexamethasone treated NHDF, two biological replicates were further processed in order to obtain
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cytoplasmic and nuclear proteins, proceeding as previously described.17 In short, cells were lysed in
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isotonic lysis buffer supplemented with protease inhibitors by applying mechanical shear stress.
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Cytoplasmic and nuclear proteins were extracted and dissolved in sample buffer. Protein concentrations
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were determined by means of a Bradford assay (Bio-Rad-Laboratories, Germany). Thereafter, in-solution
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digestion of proteins was performed with trypsin (Roche Diagnostics, Germany).
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LC/MS method for proteins
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Samples were solubilized in 5µl 30% FA containing 10fmol each of 4 synthetic standard peptides and
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diluted with 40µl mobile phase A (98% H2O, 2% ACN, and 0.1% FA). 10µl were injected into the nano
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HPLC-system (Dionex Ultimate 3000) loading peptides on a 2cm x75µm C18 Pepmap100 pre-column
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(Thermo Fisher Scientific) at a flow rate of 10µl/min using mobile phase A. Peptides were then eluted to a
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50cmx75µm Pepmap100 analytical column (Thermo Fisher Scientific) with a flow rate of 300nl/min,
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using a gradient from 8 to 40% mobile phase B (80% ACN, 20% H2O, 0.1% FA) over 95min for secreted
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proteins, and over 235min for cytoplasmic and nuclear proteins. The nano HPLC system was coupled to a
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QExactive orbitrap with a nanospray ion source (Thermo Fisher Scientific). MS scans were performed in
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the range from m/z 400 to 1400 at a resolution of 70000 (at m/z =200), MS/MS scans at a resolution of
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17500 (at m/z =200), using a top 8 method for secreted proteins, and a top 12 method for cytoplasmic and
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nuclear proteins and applying HCD fragmentation at 30% normalized collision energy.
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Protein data interpretation
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Identification of proteins and label-free quantification (LFQ) were performed using the MaxQuant 1.5.2.8
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software including the Andromeda search engine and the Perseus statistical analysis package version 7 ACS Paragon Plus Environment
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1.5.2.3,26,27 searching against the UniProt database for human proteins (version 102014). A minimum of
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two peptide identifications, at least one of them unique was required for positive protein identification. For
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peptides and proteins, a false discovery rate (FDR) of less than 0.01 was applied. Protein regulation was
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determined by comparing the LFQ values for each individual protein in the different samples using
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Perseus, normalizing to the same initial protein amount of 20µg. Changes in protein abundance values
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(Supplementary Tables S1-S3) were determined by a two-sided t test, considering proteins as significantly
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regulated when the abundance difference was at least twofold with p
