Bacteria

All experiments in this study were performed using two P. aeruginosa strains coded as PA57 and PA43 and two S. aureus strains (MSSA60, MRSA75). Bacteria were isolated from the sputum of patients during an exacerbation of the advanced stage of CF. Criteria for selecting the strains were strong or weak ability to produce biofilm in comparison to other strains of the same species. As for the PA57 strain, it was isolated from a patient with a severe form of the disease (FEV1 = 13%) [11], had a strong biofilm production capacity, and in addition a very large amount of mucus observed on the agar around the colony. PA43 was originated from a patient with mild disease, produced a weak biofilm, and had no mucus around the colony observed on the agar plates. Out of S. aureus strains, we chose MSSA60 having strong biofilm production capacity, which was isolated from a patient with a severe form of the disease (the same from which PA57 was isolated), and MRSA75 originated from a patient with mild disease, with relatively weak ability to produce biofilm. Isolated bacteria were cultured in tryptic soy broth (TSB, Oxoid/Thermo Fisher Scientific, Fremont, CA, USA) for 72 h at 37 °C under aerobic conditions. After cultivation, bacteria were centrifuged for 10 min at 500 g and washed with 10 ml of phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich, Steinheim, Germany), killed, and in such form used for in vitro tests with immune cells. In some cases, bacteria cultures were used for isolations of biofilm matrix components (see below).

Killing P. aeruginosa and S. aureus bacterial cells

Bacteria pellets originating from 72 h cultures were treated thrice with high temperature (121 °C) at 0.3 bars in the ASVE-ELMI ESS-207 SMS steam sterilizer and in that form used to stimulate immune cells (see below). The follow-up bacteria culture was verified to be sterile.

Isolation of biofilm matrix componentsDNA extraction

The 72 h bacterial culture was centrifuged (8000 rpm, 10 °C, 15 min). The pellet was washed twice by PBS and then incubated in 0.25 mL of 10 mM Tris–HCl (pH 8) and 2.5 mg/mL of lysozyme at 37 °C for 2 h. Then, 0.5 mL of lysis buffer (50 mM Tris, 100 mM EDTA, 1% SDS, pH 8) and 1 mg/mL of proteinase-K were added and incubated at 50 °C for 2 h in a water bath. The digestion with proteinase-K was followed by the addition of 0.5 mL of phenol:chloroform (1:1). The samples were mixed gently for 3 min and centrifuged (14 000 rpm, 4 °C, 15 min). The upper layer was transferred to a fresh tube and extracted with chloroform:isoamyl alcohol (24:1) by centrifugation at (14 000 rpm, 4 °C, 15 min). This step was repeated. The supernatant was precipitated with double volume of ethanol and left till the precipitate was formed. DNA was collected by centrifuging at 14 000 rpm, 4 °C, for 15 min, and dried. The pellet was suspended in 10 mM Tris–HCl (pH 8) and 1 mM EDTA (pH 8) buffer and incubated at 45 °C in a water bath for 3 h. The quality of DNA was checked using DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA).

Lipopolysaccharide (LPS) isolation

LPS was isolated using hot phenol/water method and purified as described by Westphal et al. [15]. The quantity of LPS was measured after the sample lyophilization.

Exopolysacchairde (EPS) isolation

EPS was isolated and purified as described by Górska et al. [16]. Briefly, polysaccharide was obtained by trichloroacetic acid extraction of bacterial mass, precipitated with ethanol, and purified by DNAse, RNAse, and protease. EPS was purified by ion-exchange chromatography. The fractions containing neutral EPS were pooled, desalted by dialysis against water at 4 °C for 24 h, and lyophilized. Total saccharide concentration was measured by phenol sulfuric acid method according to Dubois’s method [17].

Peptidoglycan (PG) isolation

The isolation protocol was conducted according to the modified method of Schaub and Dillard [18]. Briefly, the bacterial mass was suspended in 25 mM phosphate buffer (pH 6) and added drop by drop to a boiling 8% SDS. Then, the suspension was incubated at 100 °C (30 min), cooled, and ultracentrifuged (45 000 rpm; 15 °C; 30 min). The obtained pellet was extracted one more time, washed (4–6 times) with phosphate buffer, and freeze-dried. To obtain pure PG, the freeze-dried mass underwent digestion by DNase, RNase, and protease.

Mice

Inbred C57BL/6 mice (8–12 weeks of age, 18–22 g) were maintained at the Animal Breeding Unit of the Department of Immunology of Jagiellonian University Medical College. All mice were held in standard caging conditions with water and standard diet ad libitum.

Isolation of peritoneal exudate cells

Peritoneal mouse exudate cells were induced by an intraperitoneal injection of 1.5 mL of 3% thioglycollate (Sigma-Aldrich). After 18 h (neutrophils) or 96 h (macrophages), mice were euthanized by overdosing of isoflurane vapors (Abbott Laboratories, Chicago, IL, USA) and cervical dislocation was performed. Cells were then collected by washing out the peritoneal cavity with 5 mL of PBS (Lonza, Verviers, Belgium) containing 5 U heparin/mL (Polfa, Warsaw, Poland). Cells were centrifuged, and red blood cells were lysed. Osmolarity was restored by the addition of PBS. At least three mice were used as donors of peritoneal exudate cells for each experiment.

Cell viability

Cell viability was monitored by means of LDH activity (lactate dehydrogenase) using LDH assay kit (Thermo Fisher Scientific, Rockford, IL, USA) according to manufacturer’s instruction. The viability of phagocytes was controlled in all experimental systems to avoid cytotoxic effect of the tested agents.

Stimulation of neutrophils and macrophages with bacterial products

Neutrophils and macrophages were cultured in 24-well flat-bottom cell culture plates at 5 × 105/well in IMDM medium (Lonza) supplemented with 5% fetal bovine serum (FBS; Lonza), 2 mM stable L-glutamine (Lonza), and 50 mg/mL gentamicin (KRKA, Warsaw Poland) at 37 °C in an atmosphere of 5% CO2. To determine the influence of selected bacteria and their components on innate immune cell activity, neutrophils and macrophages were stimulated with heat-killed whole bacterial cells (20:1 bacteria per cell), or with their antigens (EPS, LPS, DNA, and PG at selected concentrations, see Figs. 3 and 4 captions). As a reference stimulus, we used 0.1 µg/mL LPS from Escherichia coli strain 0111:B4 (LPS, Sigma-Aldrich). After 24 h of stimulation, culture supernatants were collected and frozen at − 80 °C until use. Cell lysates were used for western blot analysis.

Cytokine measurement

Cytokine levels in cell culture supernatants were measured by sandwich ELISA. Microtiter plates (Costar EIA/RIA plates, Corning) were coated with a cytokine-specific antibody. The expression levels of IL-6, IL-10, and IL-12p40 were measured according to the manufacturer’s instructions (OptEIA Sets, BD Biosciences). TNF-α level was measured according to the manufacturer’s instructions (ELISA uncoated kits, Invitrogen, Waltham, MA, USA). In all cases, 10% FBS in PBS was used as a blocking solution. TMB substrate solution (Invitrogen) was used to develop a colorimetric reaction, which was stopped with 2 M sulfuric acid. Optical density was measured at 450 (570) nm using a microtiter plate reader (PowerWaveX, Bio-Tek Instruments, Winooski, VT, USA).

Nitric oxide (NO) determination

NO levels in culture supernatants of macrophages were quantified by the accumulation of nitrite as a stable end product, according to a modified Griess method [19]. Cell culture supernatant (100 µL) was mixed with 14 mM 4,4′-diamino-diphenylsulfone (Dapsone, Sigma-Aldrich) in 2 M HCl (50 µL) and 0.1% N-1-naphthylenediamine dihydrochloride (50 µL) in deionized water. The absorbance of the tested culture supernatants at 550 nm was compared with a sodium nitrate standard (NaNO2) curve.

Chemiluminescence/ROS production

The effect of killed bacteria (PA57, PA43 MSSA and MRSA) on the production of ROS by neutrophils was evaluated in vitro using luminol-dependent chemiluminescence. Chemiluminescence was counted at 37 °C in a temperature-stabilized luminometer (Luminoscan, Thermo Fisher Scientific). Briefly, 18 h peritoneal cells induced by thioglycolate (5 × 105/well) were mixed with luminol (0.8 mg/mL) in 1:1 volume ratio (both Sigma-Aldrich) on a 96-well flat-bottom black plate and incubated at 37 °C for 30 min (Thermo Fisher Scientific). After incubation, the cells were quickly stimulated with bacteria in 1:20 ratio (neutrophil:bacteria) or yeast zymosan (200 µg/mL, Sigma-Aldrich) and photon emission was measured for 75 min with 3 min intervals. Results are expressed as relative light units (RLU) where photons were counted every 5 s.

Western blot

The levels of iNOS and arginase-1 protein were determined using western blot technique. Macrophages were lysed in lysis buffer containing a mixture of protease inhibitors (PBS, Triton X-100, 10% SDS, Sigma-Aldrich). The total protein concentration in the lysates was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). Samples containing equal amounts of protein (14 mg) were suspended in loading buffer in a 2:1 ratio and denatured for 4 min at 100 °C. Samples were applied to polyacrylamide gel with 10% SDS and separated electrophoretically in the Laemmli system using Mighty Small II apparatus (Amersham Biosciences, UK). Separated proteins were transferred to the nitrocellulose membrane (Bio-Rad, Hercules, CA USA) using Hoefer TE22 transfer equipment (Amersham Biosciences). After overnight incubation with protein blocking solution at 4 °C (3% skimmed milk), membranes were incubated for 2 h at RT with rabbit polyclonal anti-iNOS (Enzo Lifesciences, Farmingdale, NY, USA) or anti-arginase-1 antibodies (Invitrogen) and mouse monoclonal anti-β-actin antibodies (Sigma-Aldrich). Then, membranes were incubated for 2 h with secondary antibodies, anti-rabbit IgG (Sigma-Aldrich), and goat IgG conjugated with phosphatase alkaline (Sigma-Aldrich,) at RT. The bands were detected with alkaline phosphatase substrate BCIP/NBT (Sigma, St. Louis, MO, USA). Protein bands were scanned and analyzed using freeware Scion Image for Windows (Scion, Frederick, MD, USA). Results are presented as a ratio of optical density of protein to β-actin, which is constitutively expressed in cells.

ProteomicsSample preparation for LC–MS/MS analysis

Macrophages were lysed in 150 µL of lysis buffer (2% SDS, 50 mM DTT in 0.1 M Tris–HCl pH 7.6), vortexed, incubated in 95 °C for 5 min, and clarified by centrifugation at 14 000 g for 30 min. Before protein digestion, the total protein concentration in collected lysates was determined by WF assay [20, 21]. Next, a volume containing 70 µg of total protein was transferred to Microcon-30 kDa centrifugal filter units (Merck, Darmstadt, Germany), denatured with 8 M urea in 0.1 M Tris–HCl, pH 8.5, and digested to peptides with a use of filter-aided sample preparation (FASP) protocol [20]. Briefly, proteins were alkylated with iodoacetamide and cleaved with LysC–trypsin mix (Thermo Scientific, Waltham, MA, USA) with the enzyme to protein ratio 1:50. Digestions were carried out overnight in 50 mM Tris–HCl, pH 8.5, at 37 °C. After digestion, the peptide yields were determined by WF assay and the aliquots containing equal amount of total peptides were desalted on 96-well MiniSpin C18 columns (Harvard Apparatus, Holliston, MA, USA). Samples were then concentrated to a volume of ~ 5 µL and stored at − 80 °C. For project-specific spectral libraries preparation, equal amount of peptides from 40 samples distributed across all experimental conditions were combined and subjected to the fractionation protocol. HpH fractionation on C18 Micro SpinColumns (Harvard Apparatus) was performed in 50 mM ammonium formate buffer (pH 10) with 13 consecutive injections of the eluent buffer, comprising 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 35, 50, and 80% acetonitrile in 50 mM ammonium formate buffer (pH 10), collected by centrifugation (300 × g, 2 min), and dried in a speedvac concentrator (Eppendorf, Hamburg, Germany). In this way, peptides were distributed across 13 HpH fractions and analyzed by LC–MS/MS in DDA acquisition mode for library generation. Prior to the analysis, all samples and library peptide fractions were solubilized in 0.1% formic acid in a concentration of 0.5 µg/µL and spiked with the iRT peptide mix (Biognosys, Schlieren, Switzerland) for normalization of the retention time.

Liquid chromatography–tandem mass spectrometry

Peptides (1 µg) were injected onto a nanoEase M/Z Peptide BEH C18 75 µm i.d. × 25 cm column (Waters, Milford, MA, USA) via a trap column nanoEase M/Z Symmetry C18 180 µm i.d. × 2 cm column (Waters). For library generation, each peptide fraction was separated using a 98 min 1% to 40% B phase linear gradient (A phase—0.1% FA; B phase—80% ACN and 0.1% FA) operating at a flow rate of 300 nL/min on an UltiMate 3000 HPLC system (Thermo Scientific) and applied to a TripleTOF 6600 + (Sciex, Framingham, MA, USA) mass spectrometer. The main working nano-electrospray ion source (Optiflow, Sciex, Framingham, MA, USA) parameters were as follows: ion spray voltage 3.2 kV, interface heater temperature (IHT) 200 °C, ion source gas 1 (GS1) 10, and curtain gas (CUR) 25. For DDA acquisition, spectra were collected in full scan mode (350–1400 Da), followed by 100 CID MS/MS scans of the 100 most intense precursor ions from the preceding survey full scan exceeding 100 cps intensity under dynamic exclusion criteria. Samples analyzed in SWATH acquisition mode were separated using a 63 min 1–40% B phase linear gradient at a flow rate of 300 nL/min. For SWATH acquisition, spectra were collected in full scan mode (400–1250 Da), followed by 100 SWATH MS/MS scans using a variable precursor isolation window approach, with m/z windows ranging from 6 to 90 Da.

Mass spectrometric raw data analysis, spectral library generation, and SWATH quantitation

DDA data were searched against the murine UniProt database (release 2021_01_04, 17 056 entries) using the Pulsar search engine implemented in Spectronaut 16 software (Biognosys) [22] with default parameters (± 40 ppm mass tolerance on MS1 and MS2 level, mutated decoy generation method, trypsinP enzyme specificity). Deep learning-assisted iRT regression was set as iRT reference strategy for RT to iRT calibration with minimum R2 set to 0.8. Peptide, protein and PSM FDR were set to 1%. Library was generated using 3–6 fragment ions per precursor.

Project-specific library was then used to analyze the SWATH data in Spectronaut 16 (Biognosys). Data were filtered by 1% FDR on peptide and protein level, while quantitation and interference correction were done on the MS2 level. Protein grouping was performed based on the ID picker algorithm [23]. Protein quantities were calculated by averaging the respective peptide intensities, while the latter were obtained as mean precursor quantities. The protein coefficients of variation (CVs) were calculated based on the summed intensities of their respective peptides. Data were normalized by global regression strategy, while statistical testing for differential protein abundance was done using t tests with multiple testing correction after Storey [24]. Statistically significant differences (q value < 0.05) with quantitative cutoff for absolute 1.5-fold change were considered as differentially regulated. The LC–MS data, library and Spectronaut project have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository [25] with the dataset identifier PXD036521.

Statistical analysis

Statistical significance of differences between groups was analyzed using one-way ANOVA, followed, if significant, by a Dunnett’s test or Tukey’s for post hoc comparison. Results are expressed as mean ± SEM values. A P value < 0.05 was considered statistically significant. Analysis was performed using GraphPad Prism v. 5.01 (GraphPad Software, Inc. San Diego, CA, USA). The exact statistical analysis is named in the relevant figure’s caption.