The thrombin-derived peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), IVE25 (IVEGSDAEIGMSPWQVMLFRKSPQE), and HVF18 (HVFRLKKWIQKVIDQFGE) were synthesized by Biopeptide Co., Inc. (USA). The purity (>95%) of these peptides was confirmed by mass spectral analysis (MALDI-TOF Voyager).

Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PAO1 were purchased from American Type Culture Collection (ATCC, USA). Bacteria from overnight cultures were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium, centrifuged at 2,000 × g for 10 min and washed in 10 mM Tris buffer (pH 7.4). Next, E. coli was heat-inactivated for 20 min and P. aeruginosa for 30 min at 65°C. Killing was confirmed by plating the heat-inactivated suspensions on TH-agar plates.

RAW 264.7 cells (ATCC) and the derived RAW-Blue NF-κB/AP-1 reporter cells (InvivoGen, USA) were cultured in DMEM (HyClone, GE Healthcare Life Science, USA) supplemented with 10% (v/v) heat-inactivated FBS (FBSi; Invitrogen, USA) and 1% (v/v) antibiotic–antimycotic solution (AA; Invitrogen). In addition, 200 µg/ml of the selective antibiotic Zeocin (InvivoGen) was added to the RAW-Blue cells.

Human peripheral blood mononuclear cells were isolated from buffy coats (obtained from Skåne University Hospital, Lund, Sweden) by Lymphoprep (ρ = 1.077 g/ml; Axis-Shield, Norway) density centrifugation at 700 × g for 20 min. Cells from the interphase were washed three times with phosphate-buffered saline (PBS) and monocytes were purified using anti-CD14-coated microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s instruction (monocyte purity was >96%). To obtain M1 or M2 macrophages, cells were cultured for 6 days in RPMI-1640-GlutaMAX-I medium (Life Technologies, UK), 10% (v/v) FBSi, 1% (v/v) AA, and 5 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, UK) or 12.5 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems) as described previously (18).

RAW-Blue reporter cells (4 × 10⁵ cells/ml) in DMEM supplemented with 10% FBSi were seeded in 96-wells plates and incubated overnight to allow adherence. Next, the cells were incubated with 4 × 10⁵ CFU/ml of heat-inactivated E. coli or 4 × 10⁶ CFU/ml of heat-inactivated P. aeruginosa in the presence or absence of peptides using various conditions. After 20 h, NF-κB/AP-1 activation was determined by the release of secreted embryonic alkaline phosphatase (SEAP) into the culture supernatants. For this purpose, 20 µl supernatant was mixed with 180 µl SEAP detection reagent (QUANTI-Blue, InvivoGen), incubated for 1.5 h at 37°C, and the absorbance was measured at 600 nm.

RAW 264.7 cells (4 × 10⁵ cells/ml) in DMEM with 10% FBSi were seeded in 96-wells plates and incubated overnight to allow adherence. Cells were stimulated with heat-inactivated E. coli (4 × 10⁵ CFU/ml) or P. aeruginosa (4 × 10⁶ CFU/ml), as described previously for 20 h in the presence or absence of GKY25 and IVE25. Cytokines in supernatants of RAW 264.7 cell cultures were determined using the CBA Mouse Inflammation Kit (BD Biosciences, USA) according to the manufacturer’s protocol.

To determine cytokine release by human cells, fresh venous blood taken from healthy donors, collected in the presence of the anticoagulant lepirudin (50 µg/ml) was mixed with 3 parts RPMI-1640-GlutaMAX-I medium. Next, 1 ml of this mixture was stimulated in 24-wells plates with E. coli (4 × 10³ CFU/ml) or P. aeruginosa (4 × 10⁴ CFU/ml) in the presence or absence of 10 µM GKY25. After 6 h of incubation at 37°C and 5% CO₂, plates were centrifuged for 5 min at 200 × g without break and plasma was collected for cytokine analysis. The levels of IL-6, IL-8, IL-12p40 and TNF-α were determined using Human CytoSets (Invitrogen), whereas the Human Cytokine Array panel A (R&D Systems) was used to analyze a panel of 36 cytokines and chemokines according to the manufacturer’s instructions.

RAW 264.7, M1, or M2 macrophages (8 × 10⁵ cells/ml) were seeded in 96-wells plates and incubated overnight to allow adherence. The following day, cell medium was refreshed, and the cells were preincubated with different concentrations of GKY25. After 3 h of incubation, the cell medium was removed and fluorescent pHrodo Green E. coli BioParticles (Life Technologies), suspended in Hank’s balanced salt solution (HBSS) according to the manufacturer’s instructions, were added to the cells. In a separate set of experiments, E. coli BioParticles were preincubated with GKY25 in the presence or absence of AB serum for 1 h prior to addition to naive cells. After 1.5 h incubation of cells and particles at 37°C, the mean fluorescence of phagocytosed particles was measured using a VICTOR3 Multilable Plate counter spectrofluorometer (PerkinElmer, USA) at excitation/emission wavelengths of 485/535 nm. Wells containing only fluorescent E. coli BioParticles were used as a negative control, whereas cells incubated with fluorescent E. coli BioParticles in the absence of GKY25 were used as a positive control. Phagocytosis is expressed as the percentage of fluorescence as compared to the positive (100%) and negative (0%) controls. Each experiment was carried out in quadruplicate, and the results were calculated from three to six independent experiments.

The viability of RAW-Blue and RAW 264.7 cells was examined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) reduction assay. In brief, cells were incubated with 10% (v/v) of a 5 mg/ml MTT solution for 1 h at 37°C, washed with PBS and 100 µl dimethyl sulfoxide was added followed by measuring of the absorbance at a wavelength of 550 nm.

Samples were diluted in reducing Tricine SDS Sample Buffer (Life Technologies) and denatured at 95°C for 7 min. Next, samples were separated by electrophoresis on a 10–20% Tris-Tricine gel (Life Technologies) using 1× Tris–Tricine SDS running buffer (Life Technologies) for 100 min at 100 V. Proteins and peptides were transferred to PVDF membranes at 25 V for 10 min using a Trans Blot Turbo system (BioRad, USA). Subsequently, membranes were blocked with 3% non-fat dry milk in PBS containing 0.05% Tween-20 (PBST) for 30 min at RT. TCPs were detected using polyclonal rabbit antibodies against the thrombin C-terminal epitope VFRLKKWIQKVIDQFGE (VFR17; Innovagen AB, Sweden) for 1 h at RT, followed by three times washing with PBST and incubation with swine anti-rabbit immunoglobulin-HRP conjugated antibodies (1:1,000; Dako, Denmark) for 1 h at RT. Membranes were washed thrice with PBST, and TCPs were visualized after 3 min incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Denmark) using the Chemi Doc MP imaging system (BioRad).

Fibrin slough from a patient with a chronic venous ulcer was fixed with 2.5% glutaraldehyde in cacodylate buffer and processed as previously described (13). For immunostaining of TCPs on bacteria, samples were blocked with 5% (v/v) goat serum in incubation buffer [0.2% bovine serum albumin (BSA) in PBS, pH 7.6] for 15 min at RT followed by overnight incubation with 1 µg/ml of VFR17 at 4°C. Next, samples were washed and incubated with 1 µg/ml EM goat anti-Rabbit IgG 10 nm Au (BBI, UK), diluted in incubation buffer, for 2 h at 4°C. After additional washing and post fixation with 2% glutaraldehyde, samples were examined using a FEI/Philips CM 100 transmission electron microscope (operated at 80 kV accelerating voltage) connected to an Olympus Veleta camera (Japan).

RAW 264.7 cells (2 × 10⁵ cells/ml) were seeded on round glass cover slips (Menzel-Gläser, Thermo Scientific) and incubated overnight to allow adherence. pHrodo Green E. coli BioParticles were suspended as described above and incubated with 2 µM GKY25 or 20 µM HVF18 for 1 h at 37°C. Next, preincubated E. coli BioParticles were added to RAW 264.7 for 1.5 h at 37°C to allow phagocytosis. Afterward, cells were washed with PBS, fixed with 2% formaldehyde for 30 min at 4°C, washed again and permeabilized with 0.5% (v/v) Triton for 2 min at RT. After washing, samples were blocked with 5% (w/v) BSA in PBS, incubated with VFR17 for 30 min at 37°C, washed again with PBS and incubated with secondary goat anti-rabbit Alexa Fluor 568 antibody (1:1,000; Invitrogen) for 30 min at 37°C. Subsequently, samples were washed and mounted with ProLong Gold with DAPI (Life Technologies) to stain the nuclei. Mounted samples were examined using an LSM 700 laser-scanning confocal microscope (Zeiss, Germany) with λ_ex = 405 nm for DAPI, λ_ex = 488 nm for E. coli BioParticles, and λ_ex = 568 nm for GKY25 or HVF18. A C-Apochromat 63×/1.20 W Korr M27 glycerol immersion objective was used. Images were collected and processed with Zen 2012 software and analyzed using the ImageJ software (version 1.49q).

RAW 264.7 cells (2 × 10⁵ cells/ml) were cultured in glass bottom cell culture dishes (VWR, Sweden) overnight to allow adherence. The next day, cell medium was changed to DMEM supplemented with 10% (v/v) AA (no FBSi), and E. coli BioParticles (50 µg/ml), suspended in HBSS medium, were added together with 2 µM TAMRA-labeled (T-) GKY25 to the RAW 264.7 cells. The culture dishes were placed in an environmental chamber (37°C and 5% CO₂), and images were immediately recorded every third minute for 2 h, from three different areas, with the Nikon A1+ confocal on a TiE inverted microscope (Nikon, Japan) with GaAsO detectors. Images were collected using a 60×/1.4 plan Apo λs lens. The λ_ex = 488 nm was selected for green-labeled E. coli BioParticles and λ_ex = 561 nm for T-GKY25. Images were captured and processed using the NIS-Elements software (Nikon).

Values are shown as means ± SEM of “n” independent experiments. Evaluation of the differences between control and test samples was done in GraphPad Prism version 6.0 using a one-way ANOVA with a Dunnett’s multiple comparisons test or a paired t-test. A p value of <0.05 was considered significant. The heat map was created in R (19) with the R package ggplot2 (20).

As chronic, non-healing ulcers are commonly colonized or infected by bacteria such as S. aureus and P. aeruginosa (21, 22), we explored whether TCPs are associated with bacteria in these wounds. For this purpose, fibrin slough was collected from a wound containing P. aeruginosa (rod shaped) and S. aureus (cocci) and analyzed using electron microscopy. Figure 1A illustrates the presence of both rods and cocci in a phagocytic cell located in the fibrin slough; TCPs were found to be present on both types of bacteria (Figures 1B,C). Furthermore, TCPs also interacted extracellularly, in the fibrin slough, with these bacteria (Figures 1D,E). The presence of TCPs in acute wound fluids and chronic wound fluids was confirmed by western blot analysis (Figure 1F). TCPs of approximately 4 kDa are comparable with both the TCP HVF18, a peptide cleaved by neutrophil elastase (13), and the three amino acid larger TCP FYT21, cleaved by Pseudomonas elastase (16), as well as the prototypic TCP GKY25.

To investigate if TCPs are interacting with bacteria intracellularly or extracellularly, E. coli BioParticles were used and added together with TAMRA-labeled (T)-GKY25 to RAW 264.7, and the uptake was recorded for 2 h by using live cell imaging. The videos (Videos S1 and S2 in Supplementary Material) demonstrate direct binding of T-GKY25 to E. coli BioParticles extracellularly. Phagocytosis by RAW 264.7 cells of E. coli BioParticles with bound T-GKY25 was observed 10 min after the start of the experiments. In addition, RAW 264.7 cells internalized small aggregates of E. coli BioParticles with bound T-GKY25 as well as peptide alone. Control samples without peptides show uptake of E. coli BioParticles in RAW 264.7 cells, whereas control samples without E. coli particles show the presence of GKY25 and HVF18 in the cytoplasma of the RAW 264.7 cells (Figure S1 in Supplementary Material). Of note, the control peptide IVE25 did not bind to E. coli BioParticles or RAW 264.7 cells (data not shown).

Furthermore, to confirm the interaction of TCPs with E. coli BioParticles before entering the cells, E. coli BioParticles were preincubated with T-GKY25 or T-HVF18 for 1 h followed by the addition to RAW 264.7 cells. The results show internalization of E. coli BioParticles colocalized with GKY25 or HVF18 in RAW 264.7 cells (Figure 2).

As TCPs are interacting with bacteria extracellularly, we explored whether TCPs affect phagocytosis. To investigate if GKY25 has opsonizing effects, E. coli BioParticles were preincubated for 1 h with a range of GKY25, before addition to the cells. We found that the uptake of the particles by RAW 264.7 cells was slightly affected with 2 µM of GKY25 (Figure 3A) but not with any of the other tested concentrations or when using monocyte-derived M1 or M2 macrophages. Interestingly, 3 h preincubation of RAW 264.7 cells with GKY25 significantly increased phagocytosis of E. coli BioParticles (Figure 3B) in a dose-dependent fashion. Incubation times up to 24 h of cells with peptide did not further affect the phagocytic activity of RAW 264.7 cells (data not shown). Of note, phagocytosis by monocyte-derived M1 and M2 macrophages was not significantly affected by the peptide. Furthermore, no difference in phagocytosis of BioParticles was observed when serum-opsonized in the presence of GKY25 (Figure 3C). Finally, preincubation of E. coli BioParticles with 20 µM HVF18 did not affect phagocytosis by RAW 264.7 cells (data not shown).

As GKY25 is preserving the bacterial uptake by RAW macrophages, we investigated whether the peptide would modulate bacteria-induced cell activation. The results showed that GKY25 dose-dependently inhibited NF-κB/AP-1 activation by RAW-Blue cells in response to heat-killed E. coli and P. aeruginosa (Figures 4A,B).

Preincubation of heat-killed bacteria with peptide for 2 h, followed by a washing step before addition to RAW-Blue cells, resulted in inhibited NF-κB/AP-1 activation as well, indicating that binding of GKY25 to heat-killed bacteria adequately inhibits cell activation (Figures 4C,D). The endogenous HVF18 showed similar effects as GKY25. However, to get a significant reduction in NF-κB/AP-1 activation, 20 µM of peptide was needed (Figures 4E,F).

In line with these results, the presence of GKY25 reduced the release of the cytokines IL-6, IL-10, MCP-1, and TNF by RAW 264.7 cells stimulated for 20 h with heat-killed E. coli and P. aeruginosa (Figure 4G); the release of IFN-γ and IL-12p70 was below detection limit (2 pg/ml). Interestingly, stimulation of the cells was much weaker in the presence of P. aeruginosa as compared to E. coli, except for the release of TNF, even though 10 times less CFU were used for the latter species. IVE25, derived from the N-terminal part of thrombin, was used as a control peptide and did not reduce NF-κB/AP-1 activation (Figures 4A–D) or the release of cytokines (data not shown). Of note, the used concentrations of bacteria and peptides did not significantly influence the viability of the cells (Figure S2 in Supplementary Material).

To investigate whether GKY25 can inhibit pro-inflammatory responses in the presence of live bacteria, whole blood was stimulated for 6 h with E. coli or P. aeruginosa together with 10 µM GKY25, followed by cytokine analysis of the cell-free supernatants. The results showed significant decreases in the release of TNF-α from 6.6 (range 3.1–10.9) to 2.9 (0–5.7) ng/ml, IL-6 from 17.1 (9.1–24.8) to 5.0 (0–10.8) ng/ml, IL-8 from 6.1 (3.8–9.3) to 2.8 (0.4–4.4) ng/ml, and IL-12p40 from 1.6 (0.7–2.9) to 0.2 (0.06–0.5) ng/ml by GKY25 in response to E. coli stimulation (Figure 5A). Furthermore, GKY25 significantly decreased the release of IL-6 from 2 (0.4–3.7) to 0.6 (0.3–1.2) ng/ml and IL-12 from 0.2 (0.04–0.6) to 0.1 (0.3–0.4) ng/ml after P. aeruginosa stimulation, whereas TNF-α (0.18 ng/ml) and IL-8 (1.2 ng/ml) were not significantly affected. More extensive cytokine and chemokine analysis of whole blood stimulated with E. coli using profiler arrays showed that GKY25 (10 µM) also reduced the cytokines IFN-γ, IL-1α, and IL-1β, and the chemokines IP-10, I-309, MCP-1, and MIP-1α/MIP-1β, as well as serpin E1, ICAM-1, and CD154 (Figure 5B). GKY25 (10 µM) itself did not induce increased cytokine or chemokine release as compared to unstimulated cells (data not shown).