Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein γ-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the pro-inflammatory cytokines TNFα, IL-1β and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.

Isolation of human leukocytes

Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) were isolated using a Ficoll (Biochrom AG, Germany) gradient centrifugation, and contaminating erythrocytes present in the PMN pellet were lysed with an ammonium chloride solution (0.8% (w/v) NH4Cl, 0.1 mM EDTA, buffered with KHCO3 to pH 7.2–7.6) in a ratio 1:9 (v/v) cell suspension/solution. Monocytes and T lymphocytes were independently isolated from the PBMC fraction, by positive selection using MACS magnetic beads (Miltenyi Biotec, Germany) against CD14 and CD3 respectively, following manufacturer’s instructions. Neutrophils were isolated from the PMN fraction by negative selection, using EasySep Human Neutrophil Enrichment Kit (StemCell Technologies, France) according to manufacturer’s instructions. Each leukocyte subset was obtained from 3 different donors (n = 3). The non-circulating leukocytes, monocyte-derived macrophages and dendritic cells, were obtained by culturing monocytes according to established protocols [35].

Flow cytometry

To confirm cell purity, isolated leukocyte subsets were analyzed by flow cytometry with an Attune® Acoustic Focusing Cytometer (Applied Biosystems) by accessing their dispersion pattern or after staining with specific cell surface markers, such as anti-human CD3 antibody (BioLegend, San Diego, CA, USA) to stain T lymphocytes, and anti-human CD16 antibody (BD Biosciences, San Jose, CA, USA) to stain neutrophils.

Cell culture

Freshly purified monocytes were cultured in complete RPMI 1640 media (1% L-glutamine (R&D Systems, Minneapolis, MN, USA), 10% heat-inactivated FBS, 1% penicillin-streptomycin (PS), 1% non-essential amino acids and 1 mM sodium pyruvate (all from Sigma-Aldrich, St. Louis, MO, USA)). For monocyte-derived cultures, cells were plated in 24 wells plate (1×106 cells per well) in complete RPMI 1640 medium with 75 U/ml IL-4 and 1000 U/ml recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (both from R&D Systems) for 6 days, to obtain monocyte-derived dendritic cells (MoDC), or with 1000U/ml GM-CSF (Peprotech, London, UK) for 7 days, to obtain monocyte-derived macrophages. Cultures were incubated at 37°C in a 5% CO2 humidified atmosphere and fresh medium was added every 2/3 days. THP-1 cell line was kindly given by Dr Nuno Santos (CBME, University of Algarve, Faro, Portugal) and was cultured according to ATCC instructions, in RPMI 1640 with L-Glutamine (Lonza, Visp, Switzerland), 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA, USA) and 1% PS (Thermo Scientific, Waltham, MA, USA). Differentiation into macrophagic THP-1 (THP-1 MoM) was achieved by culturing cells in 25 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) in complete RPMI for 48h.

Inflammatory stimulation assays

THP-1 and/or THP-1 MoM were seeded in 12-well plates at 1x106 cells and cultured for 12 h with media supplemented with 100 ng/ml of LPS (Sigma-Aldrich) or 250 μg/ml of synthetic hydroxyapatite nano crystals (HA) (Sigma). At determined time points, cells were harvested for RNA extraction and quantification of gene expression, as described below.

Protein extraction

Leukocytes and cell lines total protein was obtained by extraction with RIPA buffer (50mM Tris HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%SDS) for 1h at 4°C, with agitation, followed by a centrifugation at 16 000 xg for 15 min at 4°C. Protein concentration was assessed using Micro BCA kit (Thermo Scientific), according to the manufacturer’s instructions.

Electrophoresis and Western blot

Aliquots of total protein extracts, ranging from 5 μg to 30 μg, were size separated in a 4–12% (w/v) gradient polyacrylamide precast gel containing 0.1% (w/v) SDS (NuPage, Invitrogen, Carlsbad, CA, USA)) and transferred onto a nitrocellulose membrane (Biorad, Richmond, CA, USA). Western blot detection of tarp, cGRP, tMGP, cMGP and GAPDH was performed using specific primary antibodies CTerm-GRP (5 μg/ml), cGRP (1:1000 v/v) (GenoGla Diagnostics, Faro, Portugal), tMGP and cMGP (1:1000 v/v, both from VitaK BV, Maastricht, The Netherlands) previously validated as described [16, 30, 31, 33, 34, 36], and anti-GAPDH (1:500 v/v) (Santa Cruz Biotechnology). Detection was achieved using species-specific secondary horseradish peroxidase-conjugated antibodies and Western Lightning Plus-ECL (PerkinElmer Inc., Waltham, MA, USA). Image acquisition was obtained using an IQ LAS 4000 mini biomolecular imager (GE Healthcare, Lisbon, Portugal) and enhancement was performed with Adobe Photoshop. Semi-quantification was performed using ImageJ software, and relative protein levels normalized to GAPDH are presented as arbitrary units.

RNA extraction

Total RNA was extracted from purified and cultured leukocytes (3 donors) and cell lines as described by Chomczynski and Sacchi. The concentration of total RNA was determined by spectrophotometry at 260 nm and quality evaluated by agarose-denaturating gel electrophoresis.

PCR analysis

One microgram of total RNA obtained from isolated leukocytes, THP-1 cell line, and 25 nanograms of total RNA isolated from exosomes were first treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) according to manufacturer’s instructions and reverse transcribed using Moloney-murine leukemia virus reverse transcriptase, RNase Out (both from Invitrogen, Carlsbad, CA, USA), and an oligo(dT) adapter (5’-ACGCGTCGACCTCGAGATCGATG (T)13–3’), according to manufacturer’s recommendations. PCR reactions and set up of technical replicates, were as follows: 5 μl of cDNA (from 1:10 or 1:5 cDNA synthesis), 0.6 μl of forward and reverse primers (0.3 μM final concentration) (Table 1) and 10 μl of SsoFast™ EvaGreen® Supermix (Bio-Rad) in reactions of 45 cycles. Fluorescence was measured at the end of each extension cycle in the FAM-490 channel and the melting profiles of each reaction were performed for monitoring of unspecific amplifications. Levels of gene expression were calculated using the comparative method (ΔΔCt) and normalized using gene expression levels of 18S or GAPDH housekeeping genes.

Anti-inflammatory assays

Purified and characterized fully γ-carboxylated GRP from sturgeon (A. Naccarii) and uncarboxylated recombinant human GRP produced in E. Coli [33], further referred as cGRP and ucGRP, respectively, were used in these assays as previously described [16, 31]. For the anti-inflammatory assays using LPS as the inflammatory stimulating agent, THP-1 cells were seeded in 96 well plates at a density of 2.5 x105cells/well and differentiated as described above. After differentiation, media was removed and cells were treated for 24 h with 0.5, 0.75 and 1.5 μg/ml of cGRP and ucGRP, followed by addition of 50 ng/ml of LPS for additional 24 h. Dexamethasone treated cells (2 μM) were used as positive control. Conditioned media were collected and anti-inflammatory activity was analyzed using commercially available ELISA assays to measure TNFα (PreproTech) and PGE2 (Thermo Scientific) levels in cell supernatants, according to the manufacturer’s protocols. ELISA results were analyzed using a four parameter logistic curve fitting model in GraphPad Prism software.

For the anti-inflammatory assay using BCP crystals and protein mineral complexes (PMCs), BCP crystals were produced as previously described [16, 33] and sterilized overnight at 180°C to minimize endotoxins contamination. PMCs were prepared by incubating BCP crystals, resuspended in RPMI media at 100 μg/ml, with approximately 750 ng/ml of cGRP/ucGRP for 30 min at 37°C [16]. 100 μg/ml of BCP crystals and PMCs (PMC-cGRP and PMC-ucGRP), as well as 1.5 μg/ml of cGRP/ucGRP proteins alone, were added to differentiated THP-1 as described above, for a period of 24 h (controls consist of non-treated THP-1 MoM culture media). Conditioned media were collected and TNFα levels were measured using a commercially available ELISA (Thermo Scientific) according to the manufacturer’s protocol.

Cell viability of THP-1-MoM supplemented with 50 ng/ml LPS, 1.5 μg/ml of cGRP and ucGRP, 100 μg/ml of BCP crystals and PMC-cGRP/ucGRP, was determined at appropriate times using the CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA), following manufacturer’s instructions.

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