Ethics statement
No living animal was used to collect blood samples. The procedures for the blood collection were carried out during routine slaughtering procedures, during the exsanguinating phase. Milk for isolating exosomes was collected at the Teaching and Research Farm Frankenforst of the University of Bonn. This farm holds a permit according to the statutory provisions of the European and German animal welfare law (Art 11, Para 1, Clause 1aTierschG) for breeding and keeping farm animals (cattle, sheep, pigs, chicken, and quail). The milk samples were non-invasively obtained during the naturally occurring milk-let down reflex when the piglets were suckled, without using oxytocin injections. For these reasons, following both German and Italian current legislations, which reflect the EU current legislation, the experimental protocols were not required to be submitted to the named institutional Ethical committee.
Purification and characterization of porcine MEx
Purification of MEx from sows. Exosomes were purified through differential ultracentrifugation and size exclusion chromatography (SEC), as previously reported for porcine MEx with some minor modifications 24. Milk from multiparous healthy sows (Teaching and Research Farm Frankenforst, University of Bonn) was collected during natural milk ejection. Briefly, sows’ skimmed milk (7.5 mL) was centrifuged at 10,000 g for 30 min at 4 °C to remove the remaining fat, cellular debris and microvesicles. The supernatant was diluted with double-filtered (0.22 µM) sterile PBS to reach a final volume of 12.5 mL and then it was transferred to Ultra-clear quick seal ultracentrifuge tubes (Beckman Coulter, Indianapolis, CA, USA) and ultracentrifuged at 100,000 g for 1 h at 4 °C, using a fixed rotor (Beckman Coulter TY65 fixed angle rotor, Pasadena, CA, USA). The collected exosomes (2 mL) were thoroughly mixed with the pipette and further purified through SEC, using the qEVOriginal columns from Izon (Izon Science, Oxford, UK), following the manufacturer’s instructions. After the void volume (3 mL), 4 fractions of 500 μL each were collected. The fractions expected to contain the exosomes were pooled and depleted from lipopolysaccharides (LPS) for in vitro studies, using the ToxinEraser Endotoxin Removal Kit (GenScript, Piscataway, NJ, USA) and following the manufacturer’s instructions. The quality of the purification was assessed by Nanoparticle tracking analysis (NTA), Transmission electron microscopy (TEM) and identification of exosome marker protein by western blotting. The purified LPS-depleted exosomes were stored at -80 °C until use.
Nanoparticle tracking analysis (NTA). The Nanoparticle Tracking Analysis (NTA) was conducted using a Nanosight NTA 3.3 (Amesbury, United Kingdom) instrument as per the manufacturer’s instructions. The MEx were diluted in double-filtered PBS (1:50), loaded into the chip and the particles were visualized and analyzed with the NTA 3.3 Dev Build 3.3.301 software. For the analysis, the instrument was set up to operate at 22 °C, with a syringe pump speed of 30 arbitrary units (AU) and for each sample, 5 videos of 60 sec each were recorded. Results (mean of 5 measurements) are expressed as exosome size (nm) and concentration (particles/mL).
Transmission electron microscopy (TEM). MEx (2.5 µL) were applied to glow‐discharged carbon‐coated formvar copper grids, negatively stained with 2% uranyl acetate, air-dried for 10 min and observed in an FEI Talos 120 kV transmission electron microscope (FEI Company, Netherlands). Images of exosomes were acquired by a 4 k × 4 K Ceta CMOS camera.
Identification of exosome marker protein by western blotting. The milk exosome protein concentration was first determined with the Pierce Bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA), following the manufacturer’s instructions. Exosomal proteins (2 g) of fractions before and after removal of LPS from exosomes were loaded on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and Western blotted on nitrocellulose membrane, using Trans-Blot Turbo Midi 0.2 µm Nitrocellulose Transfer Packs (Bio-Rad Laboratories, Hercules, CA, USA), and the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membranes were blocked for 1 h with ROTI®Block 1X (Carl Roth, GmbH Co.KG, Schoemperlen, Germany) and incubated with the primary antibody rabbit anti-human TSG-101 (1:2000) (ab225877, Abcam, Cambridge, UK) - a known exosome marker 24,27,28 - for 2 h at room temperature, and then with the secondary antibody polyclonal anti-rabbit peroxidase (1:3000) (Vector Laboratory, Inc.30, Burlingame, CA, USA) for 1 h at room temperature. The immunodetection of the reactive bands was performed using the Immobilion Western chemiluminescent HRP substrate (Millipore Corporation, Billerica MA, WA, USA).
Characterization of porcine milk exosomes’ immunomodulatory effects on porcine monocytes
Purification of porcine monocytes. Peripheral blood (100 mL) from twenty 60-100 kg healthy pigs (TOPIGS) was collected during routine slaughtering procedures in sterile flasks containing 0.2% of EDTA (Sigma-Aldrich, St. Louis, MO, USA) as an anticoagulant. PBMC were isolated first through Ficoll density gradient centrifugation, as previously described for bovine blood 46. Briefly, blood was first centrifuged at 1260 g for 30 min at 18 °C to obtain the buffy coat. The buffy coat was diluted 1:5 in cold sterile Dulbecco’s PBS without Ca2+ and Mg2+ + 2 mM EDTA (Sigma-Aldrich) and carefully layered onto 3 mL of Ficoll-Paque Plus (1.077g/mL) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells were then centrifuged at 1700 g (without brakes) for 30 min at 4 °C to obtain the PBMC ring. PBMC were collected at the interface, washed twice with cold sterile PBS without Ca2+ and Mg2+ + 2 mM EDTA, centrifuged at 500 g for 7 min at 4 °C and incubated with Red Blood Cell Lysis (Roche Diagnostics GmbH, Mannheim, Germany) buffer for 3 min at room temperature to remove the red blood cells. Three consequent washes with cold sterile PBS+ 2 mM EDTA were performed to remove contaminant platelets. CD14+ monocytes were further purified from PBMC through magnetic-activated cell sorting technique (MACS), using CD14 MicroBeads, LS (large size) columns and 30 mm pre-separation filters (Miltenyi-Biotech, Bergisch Gladbach, Germany), as previously described for bovine samples 47, and following the manufacturer’s instructions. Monocytes were counted using an automatic cell counter (TC20TM, BioRad), and cells were resuspended in complete medium Roswell Park Memorial Institute 1640 medium (RPMI) with L-glutamine+ 25 mM HEPES + 1% P/S + 1% non-essential amino acids and 10% exosome-depleted Fetal Bovine Serum (FBS), purchased from Sigma-Aldrich.
Exosome uptake assay. To evaluate if porcine monocytes could internalize porcine MEx, monocytes of healthy animals (four biological replicates) were treated with LPS-depleted MEx and visualized using fluorescence microscopy. First, 108 LPS-depleted exosomes (ratio of 200 exosomes/cell) were labelled with the PKH26 Red Fluorescent Cell Linker Mini (Sigma-Aldrich), following the manufacturer’s instructions with minor modifications. Briefly, LPS-depleted MEx or PBS (negative control) were mixed with 250 μL of Diluent C and then rapidly added to a PKH26 dye solution in diluent C (0.04 x 10-6 final concentration), which was prepared immediately before staining. The exosomes were incubated with periodic mixing for 5 minutes in dark, and then 10% of exosome depleted serum was added to stop the staining and allow the binding of excess dye. The excess of unincorporated dye was further removed with the Exosome Spin Columns (MW3000) (Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions. Before cell seeding, the sterile 4-well Nunc Lab-Tek II Chamber Slides w/Cover RS Glass Slide (Thermo Fisher Scientific, Waltham, MA, USA) were treated with 100 μL of Poly-D-lysine (50 μg/mL) (Sigma-Aldrich) for 2 h, to enhance the cells’ adherence, and washed with pyrogen-free water. Then, 5 x 105 monocytes, purified from 4 animals, were seeded and co-incubated with 108 PKH26-labeled exosomes (150 μL) or PBS as a negative control for 22 h at 39 °C in a humidified atmosphere and 5% CO2. After the incubation, cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and the nuclei were stained with Hoechst 33342 (Sigma-Aldrich) (1 μg/mL) for 15 min. Finally, a drop of Invitrogen™ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific) was added to the slides and the cells were visualized using a fluorescence microscope (Eclipse E600; Nikon). The images were then analyzed with the ImageJ/Fiji software. The percentage of PKH26-exosome positive cells was determined by calculating the ratio between the cells positive for the red fluorescent dye and the total number of cells observed in each field, multiplied by 100. Results are expressed as the mean percentage of three different fields.
Viability assay. The viability assay was performed to assess the potential cytotoxicity of LPS-depleted MEx on porcine monocytes by using the Cell proliferation kit I (MTT) (Roche Diagnostics) as already reported for bovine monocytes 47. A total amount of 1 x 105 cells (25 µL) per well, was seeded in duplicate in sterile 96-well plates (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The plates were incubated for 12 h and 22 h at 39 °C in a humidified atmosphere and 5% CO2 with increasing numbers (103, 105, 107, and 108) of LPS-depleted MEx (25 µL) or with the medium as control (no exosomes). The study was carried out on an average of five biological replicates. To measure the cells’ viability, 10 µL of the MTT labeling reagent were added to each well and incubated at 39 °C for 4 h, following the manufacturer’s instructions. The formazan crystals were solubilized by adding 100 µL of solubilizing buffer and incubating the plates overnight at 39 °C. The absorbance was read at 550 nm with a LabSystem Multiskan plate reader Spectrophotometer (LabX, Midland, Canada).
Apoptosis assay. To evaluate whether MEx could affect porcine monocytes’ apoptosis, the enzymatic activity of Caspase-3/7 was measured. Briefly, 5 x 104 cells (12.5 µL) were seeded in duplicate in sterile 384-well black plates (Corning Inc., Kennebunk, ME, USA), as previously described for bovine samples 46. The study was carried out on an average of five biological replicates. The cells were incubated for 12 h and 22 h at 39 °C in a humidified atmosphere and 5% CO2 with increasing numbers (103, 105,107, and 108) of LPS-depleted porcine MEx (12.5 µL) or the medium as control (no exosomes). The apoptosis assay was carried out using the Apo-ONE® reagent Homogeneous Caspase-3/7 kit (Promega, Madison, WI, USA), following the manufacturer’s instructions. The fluorescence of 485/538 nm (absorbance/emission) was measured with a fluorescence plate reader Fluoroscan Ascent (Thermo Fisher Scientific).
Chemotaxis assay. The monocytes’ chemotaxis towards zymosan activated serum (ZAS) – a known chemoattractant - was measured as previously reported, with some minor changes 47. The study was carried out on an average of five biological replicates. Briefly, ZAS was prepared as previously described 48, and 50 microliters of purified monocytes (1 x 105) were seeded in duplicate in the upper chamber of a sterile 24-well Transwell migration plates (Corning Inc.), equipped with an 8 µm pore size membrane. Cells were then pretreated with 2 x 107 LPS-depleted MEx (a ratio of 200 exosomes/cell), in the absence of a chemoattractant, for 22 h at 39 °C and 5% CO2. After the incubation, ZAS (3 mg/mL) was added as chemoattractant in the lower chamber, and the cells were incubated either in the presence of MEx or only medium (RMPI-1640 with 1% of exosome-depleted FBS) as positive control (no exosomes) again for 2 h at 39 °C and 5% CO2. Finally, non-migrated cells were gently removed with a swab moistened with PBS, and migrated cells were stained with Diff-Quick (Sigma-Aldrich) and counted in 10 different fields, using light microscopy (inverted microscope).
Phagocytosis assay. The phagocytosis assay was carried out by measuring the fluorescence of fluorescein-labelled Escherichia coli (E. coli) K-12 strain bioparticles (Invitrogen) as previously performed 49. The study was carried out on an average of seven biological replicates. Opsonisation of fluorescein-labelled E. coli bioparticles (K-12 strain) was performed by incubating 80 µL of bacteria suspension (5 x 106 E.coli/µL) with 20% of exosome-depleted FBS (20 µL) for 30 min at 39 °C. The suspension was centrifuged at 800 x g for 15 min and suspended in PBS. A total of 3 x 105 monocytes (100 µL) were seeded in duplicate in 96-well plates and treated with 100 µL of LPS-depleted MEx (ratio of 200 exosomes/cell) or with the medium as control (no exosomes). Cells were then incubated at 39 °C and 5% CO2 for 22 h. The cells were washed with PBS and fluorescein-labeled E. coli bioparticles with a ratio of 45 particles/cell were added and co-incubated for 2 h at 39 °C. Cells were washed with PBS to remove non-internalized bioparticles and incubated with 50 µL trypan blue 0.4% for 1 min at room temperature to remove and quench the fluorescence of the non-internalized bacteria, respectively. After removal of the trypan blue, the fluorescence (485/538 nm) was measured using the microplate reader Fluoroscan Ascent (Thermo Fisher Scientific).
Killing capability assay. The intracellular bacterial killing capacity was determined as reported previously 50. The E. coli American Type Culture Collection (ATCC) 25922 (strain Seattle 1946; LCG Standards Ltd., Teddington, UK) were opsonized with 20% exosome depleted FBS (20 µL), incubated at 37 °C for 30 min. The bacteria were washed twice by centrifugation at 1500 g for 10 min at 4 °C and suspended with PBS. A total of 3 x 105 monocytes (100 µL) was seeded in duplicate in cryogenic vials. The study was carried out on an average of five biological replicates. Cells were then treated with 6 x 107 LPS-depleted MEx (100 µL), in a ratio of 200 exosomes/cell, or with the medium as control (no exosomes) and were incubated for 22 h at 39 °C and 5% CO2. After the incubation, cells were incubated for 1 h at 39 °C and 5% CO2 with 1 x 107 opsonized live E. coli. The unbound bacteria were removed by centrifugation and by further treating the cells with 100 µg/mL of gentamicin for 1 h. Gentamicin was eliminated by washing the cells with PBS and centrifuging at 110 x g for 5 min. Finally, cells were lysed with 0.5% Triton x-100 (Sigma-Aldrich) on ice for 10 min, and after overnight incubation at 37 °C, the colonies forming units (CFU) of surviving bacteria were counted on MacConkey agar plates. Results were then expressed as CFU/mL.
Reactive oxygen species (ROS) production assay. As previously described, the production of extracellular superoxide anions (O2-) was determined with the cytochrome C reduction assay 49. The study was carried out on an average of seven biological replicates. A total of 1 x 105 monocytes (50 μL) was seeded in complete medium without phenol red in triplicate in 96-well sterile plates and co-incubated for 22 h at 39 °C + 5% CO2 with 2 x 107 exosomes (50 μL) in a ratio of 200 exosomes/cell. After the incubation period, the production of O2- was measured under non-inflammatory and proinflammatory conditions when challenged with phorbol myristate acetate (PMA). In the non-inflammatory conditions, 10 μL of Cytochrome C from the equine heart (1 mM), (Sigma-Aldrich) were added; while to mimic a proinflammatory challenge 10 μL of Cytochrome C and 2 μL PMA (2.5 μg/mL final concentration; Sigma-Aldrich) were added. Medium without phenol red was added to all wells to have a final volume of 200 μL. The absorbance was measured every 30 min for 4 h at 550 nm with a LabSystem Multiskan plate reader spectrophotometer (LabX).
Statistical analyses
Statistical analyses were performed in GraphPad Prism 8.0.2. Data normality was assessed with the Shapiro Wilk test. Repeated measures one-way ANOVA and Tukey’s multiple comparison tests were used for normally distributed samples from viability and apoptosis at 22 h assays. In contrast, a Friedman test and Dunn’s multiple comparisons tests were applied for apoptosis at 12 h. Paired t-tests were used for phagocytosis, killing capacity, chemotaxis, ROS production and exosome uptake assays. Statistical differences were accepted at P ≤ 0.05.