Isolation and purification of PMNs

For each participant, the oPMNs and cPMNs were isolated and subsequently analyzed on the same day without delay. The collection procedure was based on previously described protocol [11]. All participants were instructed neither to gargle nor to clear their throat during the sampling procedure. The oPMNs were obtained by 4 serial rinses of the oral cavity with 20 ml of sterile sodium chloride solution (0.9% NaCl) for 30 s, with a 4½ min intermission. The pooled sample was kept on ice in a 50 ml centrifuge tube (Sigma) until the end of the collection procedure. One ml from the first rinse of each participant was pipetted into a 2 ml microtube and stored at − 80 °C for further analysis (see below Protease activity). Venous blood samples were drawn from the antecubital fossa of all subjects into 9 ml sodium heparin blood collection tubes (BD Vacutainer™, Breda, the Netherlands) and maintained at room temperature until the PMN isolation procedure. The isolation procedure of oPMNs and cPMNs was carried out as described before [11]. Cell counts were obtained using a Muse® Cell Analyzer (Merck Millipore, Darmstadt, Germany) and verified with a Bürker-Türk counting chamber and Trypan Blue exclusion for the viability of the PMNs.