N formyl methionyl leucyl phenylalanine

Alloxan, Glucagon, Des-his1-[Glu9]-Glucagon amide, Rolipram, Percoll, RPMI medium, Hank’s balanced salt solution without Ca⁺² or Mg⁺² (HBSS), mouse CXCL1/KC, human CXCL8/IL-8, N-formyl methionyl-leucyl-phenylalanine (fMLP), H-89, SQ 22536, DMSO, LPS, Penicillin, Zymosan A and Streptomycin were purchased from Sigma Chemical Co (St. Louis, MO, USA). Ficoll-Paque PLUS (density 1.077 g/mL) was purchased from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA), platelet-activating factor (PAF) was purchased from Calbiochem (San Diego, CA, USA), and fetal bovine serum (FBS) was purchased from Gibco (Albuquerque, NM, USA). Meropenem was purchased from ABL – Antibióticos do Brasil (São Paulo, SP, Brazil) and Luminol was donated by Prof. Margareth Queiroz from Oswaldo Cruz Institute/Fiocruz (Rio de Janeiro, RJ, Brazil). All the solutions were freshly prepared immediately before use.

Neutrophils (5 x 10⁶/ml in phosphate buffered saline with cations (PBS+, Sigma-Aldrich)) were stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP, 100 nM; Sigma-Aldrich), interleukin-8 (IL-8), TNF-α (100 ng/ml and 10 ng/ml respectively; R&D Systems) or buffer for 10 or 30 min. Cells were incubated with 100 μl phosphate buffered saline without cations (PBS-, Sigma-Aldrich) containing 5 μl mouse anti-human CD13 fluorescein isothiocyanate (FITC)-conjugated antibody (Clone WM-47; Millipore UK Ltd, FCMAB180F) or an isotype control for 30 min and analysed by flow cytometry (FACSCalibur^™, BD Biosciences). Mean fluorescence values were calculated from 10,000 events per sample using FLOWJO 7.2.5 software (Tree Star, Inc. Ashland, USA).

Chemotaxis was evaluated by the Cell Invasion Assay (Millipore Sigma, Cat. ECM 555). Blood neutrophils (n=10) and oral neutrophils (n=6) from HCs (n=10) were isolated and 0.5 million cells were used per well. Cells were pre-incubated in a 96 well plate with the anti-CD44 antibody (TermoFisher, Cat. 16-0441-85) at 37°C, for 30 min. Then cells were transported to the upper chamber of the migration assay and incubated in the presence or absence of soluble Galectin-9 (1.5 ng/ml) for 3 hours at 37°C. N-Formylmethionyl-leucyl-phenylalanine fMLP (50 μm, Sigma Aldrich, Cat. F3506-10MG) was used as a chemoattractant in the lower chamber. Culture media was used as the negative control. The migrated cells were lyzed and quantified by measuring the relative fluorescence units per manufacturing protocol using the plate reader a5 480/520 nm.

CBG, with purity greater than 99%, was purchased from the Chinese National Institute for Food and Drug Control. It was dissolved in dimethyl sulfoxide (DMSO) from Sigma and stored in stock concentration of 20 mg/ml at -20°C. The Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit was purchased from Thermo Scientific. As a reference for mass spectrometry analysis showed in S1 Fig, CBG (C1272) was bought from Sigma-Aldrich, Sweden, dissolved to 1 mg/ml in MeOH. LPS from Escherichia coli O111:B4, adenosine 5′-triphosphate (ATP), staurosporine solution, cytochalasin B (CytB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were purchased from Sigma. The caspase-1 inhibitor Z-YVAD-FMK was from MBL. The lactate dehydrogenase (LDH) Cytotoxicity Detection Kit ^PLUS was from Roche Applied Science.

To induce and observe cell migration, 3D-µ-Slide migration-chambers (Ibidi GmbH, Graefelfing, Germany) were used. One slide contains three separated systems or chambers, each chamber consists of a central channel and a left and a right reservoir (Figure 3).
The three chambers were filled as described in Table 1 and Figure 3b. In this setup, the PMNs—placed in the right reservoir—migrated along the gradient of the chemoattractant n-formylmethionyl-leucyl-phenylalanine (fMLP; Sigma Aldrich, St. Louis, USA), which was brought into the left reservoir. During migration, cells had to pass through the collagen matrix inside the central channel and could thereby be examined via live cell imaging (Figure 3c). Based on preliminary tests (data not shown), where we had already proven that only granulocytes were able to pass the collagen matrix, we were assured that solely this cell type (CD11b⁺; CD62L⁺; CD66⁺) is observed.
Cell observation was performed with a Leica DMi8 microscope and recorded with Leica DFC9000 GT camera (both Leica Microsystems GmbH, Wetzlar, Germany) for 22 h. Both camera and microscope were controlled by Leica Application Suite X software platform, version 3.4.2.18368 (Leica Microsystems GmbH). A stage top incubator consisting of Ibidi Blue Line gas mixer and Ibidi heating chamber (both Ibidi GmbH) was used to keep conditions constant at 37 °C and 5% CO₂.