Quantitative whole-cell MALDI-TOF MS fingerprints distinguishes human monocyte sub-populations activated by distinct microbial ligands
© Portevin et al.; licensee BioMed Central. 2015
Received: 30 October 2014
Accepted: 30 March 2015
Published: 11 April 2015
Conventionally, human monocyte sub-populations are classified according to surface marker expression into classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) lineages. The involvement of non-classical monocytes, also referred to as proinflammatory monocytes, in the pathophysiology of diseases including diabetes mellitus, atherosclerosis or Alzheimer’s disease is well recognized. The development of novel high-throughput methods to capture functional states within the different monocyte lineages at the whole cell proteomic level will enable real time monitoring of disease states.
We isolated and characterized (pan-) monocytes, mostly composed of classical CD16− monocytes, versus autologous CD16+ subpopulations from the blood of healthy human donors (n = 8) and compared their inflammatory properties in response to lipopolysaccharides and M.tuberculosis antigens by multiplex cytokine profiling. Following resting and in vitro antigenic stimulation, cells were recovered and subjected to whole-cell mass spectrometry analysis. This approach identified the specific presence/absence of m/z peaks and therefore potential biomarkers that can discriminate pan-monocytes from their CD16 counterparts. Furthermore, we found that semi-quantitative data analysis could capture the subtle proteome changes occurring upon microbial stimulation that differentiate resting, from lipopolysaccharides or M. tuberculosis stimulated monocytic samples.
Whole-cell mass spectrometry fingerprinting could efficiently distinguish monocytic sub-populations that arose from a same hematopoietic lineage. We also demonstrate for the first time that mass spectrometry signatures can monitor semi-quantitatively specific activation status in response to exogenous stimulation. As such, this approach stands as a fast and efficient method for the applied immunology field to assess the reactivity of potentially any immune cell types that may sustain health or promote related inflammatory diseases.
Five to 15% of human blood mononuclear cells are monocytes. Upon extravasation into inflamed tissue, their ability to differentiate into monocyte-derived macrophages and dendritic cells make them key components of the innate immune system to initiate adaptive immune responses and contribute to tissue homeostasis upon resolution of inflammation . Based on the most recent nomenclature, monocytes can be divided into classical (CD14++CD16−), intermediate CD14++CD16+ and non-classical (CD14+CD16++) short-lived cells which are constantly replenished from a common myeloid progenitor [2-4]. Their phenotypic characterization is usually performed by flow cytometry using a gating strategy in combination with HLA-DR labelling to exclude the measurement of NK cells and granulocytes expressing the CD16 marker as well .
Based on their CD16 expression levels, monocyte subpopulations have different chemotactic, phagocytic and inflammatory abilities  and distinct propensities to differentiate into professional antigen presenting cells . In fact, blood monocyte composition is heterogeneous between human individuals and gender  and within individuals it can be substantially altered by infections such as septicaemia, HIV and M. tuberculosis infection [9-11] during which the CD16+ compartment can increase dramatically. In the case of M. tuberculosis infection, a functional deficiency in the CD16+ subset to recall specific immune responses due to an impaired dendritic cell differentiation has been notably described . In contrast, a human genetic trait associated to an absent CD16+ compartment in the blood was not necessarily associated with disease . Nevertheless, these inherent and induced discrepancies between humans have led to a series of reports describing association of the blood monocyte composition and recruitment with a particular emphasis on CD16+ monocytes in the pathogenesis of non-communicable inflammatory disorders such as atherosclerosis , stroke , rheumatoid arthritis  and diabetes complications  but also Alzheimer’s disease , and experimental models of human disorder multiple sclerosis , tumour genesis and metastasis . In that context, we aimed to develop a method that could assess CD16+ monocyte functionalities for the diagnosis/prognosis but also severity or resolution monitoring of those diseases which may be particularly meaningful in the context of immune-based therapeutics targeting the recruitment of monocytes .
Untargeted mass spectrometry analysis of complex samples such as entire prokaryotic or eukaryotic cell lysates that are directly spotted in the MALDI ionisation/desorption matrix offers a fast method for rapid identification of bacteria  but also the discrimination/authentication of mammalian and insect cell lines [23-26]. This approach notably prevents any potential bias or reproducibility issues due to purification or fractionation steps prior mass spectrometry analysis. Whole-cell MALDI-TOF “fingerprints” or “signatures” performed on human primary blood cells were shown to reproducibly discriminate lymphocytes from monocytes or granulocytes or between macrophage subtypes with relatively low starting material ranging between 25 × 102 to 5 × 104 cells [27,28]. In that context, we aimed to assess whether whole-cell MALDI-TOF fingerprinting would have enough discriminatory power to i) distinguish human monocyte subpopulations and ii) monitor activation profiles of monocytes exposed to distinct microbial ligand. We purified monocytes from healthy individuals (n = 8) and isolated autologous CD16+ subpopulations. Cell preparations were immunologically characterized and following stimulation with LPS and M. tuberculosis derived antigens subjected to MALDI-TOF analysis. We extracted semi-quantitative mass spectrometry data to highlight monocytic subset discriminatory biomarkers as well as specific microbial activation profiles.
Fresh blood packs (buffy coat) were purchased anonymously from the Blutspendezentrum SRK beider Basel, Switzerland. In compliance with the Helsinki Declaration, signed informed consents stating specifically that “the donation or certain components thereof be used for medical research after definitive anonymization” was obtained prior blood donation. Consent form was accessed on October 31st 2014 and can be found here: http://blutspende-basel.ch/fileadmin/BSZ/docs/blutspende/2015_Blutspendeaufgebot_en.pdf. An ethics board approval for this study was consequently not required.
Blood processing and monocyte isolation
Peripheral Blood Mononuclear Cells (PBMCs) were immediately isolated by density centrifugation using pre-filled Greiner Bio-One Leucosep® tubes according to the manufacturer’s recommendations. PBMCs rings were collected and washed twice in PBS before final suspension at 20 × 106 cells/ml in ice-cold freezing medium (50% RPMI-1640, 40% fetal bovine serum, 10% DMSO) and transferred at −80°C in Nalgene® Mr. Frosty for short-term storage (<1 month). For monocyte isolation, 40 × 106 to 60 × 106 cells of cryopreserved PBMCs were thawed and quickly washed with 13 ml ice-cold RPMI-1640. Median cell viability upon recovery was assessed by trypan blue exclusion (88.37%, IQR: 82.1-97). Pan- and CD16+ monocyte purification were performed using Pan Monocyte Isolation Kit and CD16+ Monocyte Isolation Kit respectively according to manufacturer’s instructions (Miltenyi Biotec GmbH).
Flow cytometry analysis
Prior final washing steps, 100 μl suspension of PBMCs, pan-monocytes and CD16 monocytes were spared and washed with PBS 1% FCS before antibody staining for 15 min on ice with anti-CD14 FITC (MΦP9, Becton Dickinson), anti-CD16-PE (Leu11c, Becton Dickinson) and anti-Slan (MDC8, Miltenyi Biotec). After washing, antibody labelled cell preparation were immediately analysed on a BD FACSCalibur apparatus (BD Biosciences) and data analyzed using FlowJo 9.5.2 (Treestar).
Cell culture conditions
Monocyte suspensions were cultivated in 96-well flat-bottom tissue culture treated plates at a density of 2 × 105 cells per well in complete medium (RPMI-1640 (Gibco Life Technologies™) complemented with 5% heat-inactivated fetal bovine serum (PAA Laboratories GmbH), 100 mM Na-pyruvate (Gibco), Penicillin/Streptomycin (50 U & 50 μg/ml respectively, Gibco) for 24 hours at 37°C in a humidified atmosphere with 5% CO2 in the presence or absence (resting) of PPD (RT50, Statens Serum Institute, 5 μg/ml final) or LPS (O111:B4, 10 ng/ml final, Sigma-Aldrich). After 24 hours of resting/incubation period, tissue culture plates were placed on ice for 15 minutes and the cells re-suspended by vigorous pipetting, pelleted by centrifugation (250 RCF) and cell pellets transferred at −80°C.
Cytokine concentrations present in monocyte culture supernatants were measured using Bio-Plex Pro™ Human Cytokine Group I kit according to manufacturer’s instructions and data acquired on a Luminex® 200™ System.
Whole-cell mass spectrometry
Cryopreserved cell pellets were washed with 70% ethanol and briefly vortexed before centrifugation (10 min, 16000 RCF). Supernatants were discarded, cell pellets dried for 1 minute at room temperature and finally solubilized with 10 μl formic acid 10%, mixed with 2 volumes of a saturated sinapinic acid solution (40 mg for 1 ml of acetonitrile 60%/H20 37%/TFA 3%) and spotted in quadruplicates on a MALDI-TOF chip. External calibration was performed using ribosomal corresponding m/z signals from whole-cell E. Coli (DH5α). Mass spectra (m/z mass range: 3000 to 30000) were acquired on a Shimadzu Biotech Axima Confidence.
Quantitative analysis of mass spectrometry data using MALDIquant R package
Mass spectra were pre-aligned to conserved m/z peaks at 4078.8 and 14019.1, exported as mzXML files using MALDI-MS Shimadzu Biotech Launchpad 2.8.1 (Kratos Analytical Ltd) and imported using R Studio version 0.98.945 and MALDIquant Foreign R package. Using MALDIquant package , mass spectra were square-root transformed, smoothed using Savitz-Golay-Filter, baseline corrected using TopHat method and intensity values median-normalized so as to set to one the median of peak intensities of each mass spectrum individually before alignment using the Lowess warping algorithm. Peak detection was performed using a signal-to-noise ratio of 3. Peak list and their respective intensities were retrieved and peak intensities obtained from each quadruplicates averaged using OpenOfficeCalc before further statistical analysis.
Wilcoxon signed rank tests, two-way ANOVA with Bonferroni post-test corrections were performed and scatter-plots generated using GraphPad prism (version 4.03, San Diego, CA, USA). Principal component analyses using correlation matrix were conducted using Paleontological Statistics Software package for education and data analysis, version 2.17c .
Results and discussion
Phenotypic and functional characterization of Pan- versus autologous CD16+ monocytes
Quantitative whole-cell MALDI-TOF MS distinguishes major monocyte subpopulations and captures immune activation events
Whole-cell MALDI-TOF MS captures molecular patterns of cellular activation specific to monocyte subpopulations.
To our knowledge, this is the first report describing the use of quantitative whole-cell MALDI-TOF MS analysis to identify mass spectra that discriminate resting and activated human monocyte subsets. As a next step, we are now aiming to identify the most relevant protein candidates highlighted in this study. This proof of concept could easily be translated to clinical studies to monitor the functional status of monocytes from patients suffering from systemic and chronic inflammatory disorders. Diabetes mellitus has been associated with various immune dysfunctions that could be assessed and monitored by whole-cell mass spectrometry approaches. The identification of disease specific markers could provide relevant biological insights in the pathogenesis of diabetes mellitus and the well described enhanced susceptibility of patients to bacterial infection . High throughput monitoring of functional status of cell subsets in peripheral blood based on whole cell MALDI-TOF MS could provide unique opportunities to monitor disease progression and resolution in clinical settings.
We would like to thank Sebastien Gibb for efficient and swift remote support in handling MALDIquant R package . Damien Portevin’s salary was funded by the Basel University Research Fund (Förderung exzellenter junger Forschender).
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