Mice

C57BL/6J male mice were purchased from the Charles River Laboratories (Sulzfeld, Germany; via AnimaLab). Mice were used in experiments at the age of 8–12 weeks. Mice were housed in standard environmental conditions and were fed a commercial diet and had access to water (both ad libitum). All experimental animal protocols were approved by the Local Ethical Committee No. II in Krakow (294/2017 and 244/2019).

Endotoxemic model

Mice were intraperitoneally (i.p.) injected with 1 mg/kg body weight (b.w.) of LPS (Escherichia coli, serotype 0111:B4; Sigma-Aldrich, Saint Louis, MO, USA) in saline to induce endotoxemia [36]. At selected time points, mice were subjected to intravital imaging, or their blood and peritoneal exudate were collected as detailed below.

Sample collection for microparticle isolation

Mice were anesthetized with a mixture of ketamine hydrochloride (200 mg/kg b.w.; Biowet Pulawy, Pulawy, Poland) and xylazine hydrochloride (10 mg/kg b.w.; aniMedica, Südfeld, Germany) and subjected to blood and peritoneal exudate collection. Blood was collected by cardiac puncture in a heparinized syringe, and peritoneal fluid/exudate was collected by lavage of the peritoneal cavity with 1 mL of saline, and after 30 s of gentle manual massage, peritoneal fluid was retrieved. Samples were centrifuged at 400 × g and 1200 × g for 10 min at room temperature (RT) and then at 1500 × g for 15 min at RT for the retrieval of plasma and debris-free peritoneal fluid. Both blood and peritoneal exudate were collected at selected time-points (as indicated below and in figure legends; for in vitro RAW 264.7 cell stimulation MPs were collected at 8 h post-LPS administration) and were immediately subjected to the MP isolation procedure. Mice were euthanized by overdosing of ketamine/xylazine mixture.

Microparticle isolation

MPs were isolated by sequential centrifugation. The first step was a negative isolation of leukocytes (400 × g and 1200 × g, 10 min at RT) and then of platelets (1500 × g, 15 min at RT) to remove cell debris but also apoptotic bodies. In the following step, the collected supernatant was pelleted in a centrifuge twice at 14,000 × g for 35 min at 4 °C (Sorvall WX+ Ultracentrifuge Series) [37].

Nanoparticle tracking analysis

Before the second centrifugation as in Microparticle Isolation, the MP pellet was washed with sterile 1x phosphate-buffered saline (PBS) and was analyzed with Nanoparticle Tracking Analysis (NTA) using the NanoSight system–LM10HS microscope equipped with the LM14 488 nm laser module (Malvern Instruments Ltd., Malvern UK). The size, distribution, and concentration of blood plasma- and peritoneal exudate-derived MPs were determined by NTA which track in real time each particle individually in liquid suspension, using both Brownian motion and light scattering. For this purpose, samples with MPs were diluted 500 times in prefiltered (0.2 μm) sterile PBS to the total volume of 1 mL. Subsequently, the solution was placed into an insulin syringe and loaded into the sample chamber. The MP size and concentration measurements were taken from 60-s video duration, captured by the sCMOS camera and calculated using the NanoSight NTA 2.3 analytical software. The samples were analyzed with the same settings.

Flow cytometry analysis

Employing flow cytometry (FACSCanto, BD Biosciences Immunocytometry Systems, San Jose, CA, USA), MPs isolated from blood, and peritoneal fluid were gated as neutrophil-derived Ly6G+ MPs (PE anti-mouse Ly6G, 1A8, BioLegend, San Diego, CA, USA), and monocyte/macrophage-derived F4/80+ MPs (PE anti-mouse F4/80, BM8; eBioscience, San Diego, CA, USA). Microparticles were analyzed using FACSDiva v8.0.1 software (BD Biosciences). The corresponding isotype control rat IgG2a, κ (PE IgG2a antibody, κ; RTK2758; BioLegend, San Diego, CA, USA), was analyzed in parallel.

Preparation of cremaster muscle for intravital microscopy (IVM)

Prior to IVM, healthy and endotoxemic mice (8 h post LPS-administration) were anesthetized with a mixture of ketamine and xylazine hydrochloride as described above. Subsequently, cannulation of the right jugular vein was performed to administrate anesthetics/antibodies. Preparation of the cremaster muscle for IVM was performed as previously described [8, 38]. Briefly, mice were placed on the Plexiglas board dedicated for cremaster muscle visualization. A small piece of the scrotum was cut to expose the left testis from which cremaster muscle was carefully extracted. Then, the muscle was cleaned of connective tissue and separated from the testis by thermal cutting (with a cautery) of the thin ligaments connecting the muscle with the testis. Visualized cremaster muscle was placed on a previously prepared optically clear board. The testis and epididymis were put back inside, into the abdominal cavity.

Visualization of microparticles and leukocytes in the cremaster vasculature with IVM

Monocytes/monocyte-derived MPs were visualized with PE anti-F4/80 (1.2 μg/mouse, clone BM8; eBioscience, San Diego, CA, USA), and neutrophils and neutrophil-derived MPs were stained with anti-mouse Ly6G antibodies (1.2 μg/mouse; PE or Brilliant Violet 421 anti-Ly6G, clone 1A8, BioLegend, San Diego, CA, USA). Endothelial cells were stained with anti-CD31 antibodies (5 μg/mouse; Alexa Fluor 647 anti-CD31, clone 390, BioLegend, San Diego, CA, USA). Cells and MPs were visualized per 20× field of view (FOV).

Spinning disk confocal intravital microscopy (SD-IVM)

The cremaster muscle was imaged with a ZEISS Axio Examiner.Z1 upright microscope equipped with a metal halide light source (AMH-200-F6S; Andor, Oxford Instruments, Abingdon, UK) with motorized 6 position excitation filter wheel and laser-free confocal spinning disk device (DSD2; Andor, Oxford Instruments, Abingdon, UK) with ZEISS EC Plan-NEOFLUAR 10×/0.3 and/or ZEISS EC Plan-NEOFLUAR 20×/0.5 air objective. The following filters were used, four excitation filters (DAPI: 390/40 nm; GFP: 482/18 nm; RFP: 561/14 nm; Cy5: 640/14 nm), and appropriate emission filters (DAPI: 452/45 nm; GFP: 525/45 nm; RFP: 609/54 nm; Cy5: 676/29 nm). For fluorescence detection, the 5.5-megapixel sCMOS camera (Zyla 5.5; Andor, Oxford Instruments, Abingdon, UK) was used and the iQ 3.6.1 acquisition software (Andor, Oxford Instruments, Abingdon, UK) to drive the microscope.

Cell culture of RAW 264.7 macrophages

RAW 264.7 mouse macrophage cell line (American Type Culture Collection; Manassas, VA, USA) was used in the study. Cells were cultured in RPMI 1640 medium (Lonza, Switzerland), supplemented with 10% fetal bovine serum (FBS; Biowest, USA), 2% penicillin/streptomycin and 1% L-glutamine (Sigma-Aldrich, Germany), at 37 °C in a 5% CO2 incubator (Thermo electron, USA). Cells were subcultivated in T-75 cell culture flasks (Nunc, Denmark). Cells used in the experiments were collected between 4 and 15 passages. Cells from passages 4–8 were defined as originating from “low passages” and cells from > 10 passages as “high passages.” The majority of studies (if not clearly stated otherwise) was performed on cells from “low passages”. All cell culturing was performed in a laminar chamber (ESCO, Singapore). Once the cells were 80–85% confluent, they were detached using mechanical scrapers (Biologix, USA), gently re-suspended, and then placed in a sterile 15-mL centrifuge tube (NEST, China). Next, they were centrifuged at 1500 rpm for 10 min (MPW, Poland). The supernatant was removed, and the cells were re-suspended in 1 mL of fresh complete medium. Cell counts were performed using a Bürker hemocytometer. Additionally, cell viability was determined by Trypan blue assay (Sigma-Aldrich, Germany). The cells were then subcultured to 96-well tissue culture plates (NEST, China), adding 1 × 105 cells per well and then incubated at 37 °C in 5% CO2 for 30 min to let them adhere.

Macrophage stimulation

RAW 264.7 macrophages were stimulated with LPS and/or MPs. LPS-treated cultures served as a positive control [39] and some cells were left unstimulated (negative control). Experimental groups consisted of cells stimulated with MPs isolated from blood plasma or peritoneal exudate that were added in two concentrations: 8 × 106 and 16 × 106 per well. Numbers of MPs were established by NTA as described above. Some of the cells were additionally stimulated by LPS at a concentration of 10 μg/mL. The cells were then incubated overnight at 37 °C in 5% CO2 until further analysis. In some experiments, cells were incubated for 1, 3, 6, and 9 hours instead of 18 hours (overnight) wherever indicated. In some studies, prior (30 min) or after (30 min) LPS and MP stimulation following inhibitors were added: NG-nitro-L-arginine methyl ester (L-NAME; NOS inhibitor) and N-(3-aminomethyl)-benzylacetamidine (1400W; specific iNOS inhibitor) in concentration of 2 mM and 50 μM, respectively. After overnight stimulation, NO was measured in supernatants and fixed cells were subjected to immunocytochemistry to detect iNOS as described below.

Crystal violet (CV) assay

Macrophage numbers were estimated by the crystal violet (CV) assay that indirectly verifies cell numbers and viability by evaluation of cell adherence to culture plates [40]. After overnight incubation with LPS and/or MPs, culture medium was carefully removed from the wells. Next cells were fixed in absolute methanol (Chempur, Poland) for 10 minutes at RT. Methanol was then removed and crystal violet solution (Sigma-Aldrich, Germany) was added to each well (25 mg CV was dissolved in 5 mL 20% methanol). Plates were incubated for 5 min, washed twice in tap water to remove the unbound dye and then dried. To extract CV from the cells, 100% methanol was added to wells. Plates were agitated for 10 min, and absorbance was measured at 570 nm in a microplate reader (Tecan, Infinity F200 Pro, Männedorf, Switzerland).

Presto Blue viability assay

After overnight stimulation with LPS and/or MPs, proliferation/viability of RAW 264.7 macrophages was evaluated by the PrestoBlue assay (BioVision, USA) [41]. Briefly, at the end of the incubation time, PrestoBlue reagent was added to each well and then plates were incubated for another 30 min at 37 °C in 5% CO2 in darkness. The fluorescence was measured at 610 nm (excitation 535 nm, emission 595 nm) in a microplate reader.

MTT assay

To determine mitochondrial activity of RAW 264.7 macrophages, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT; Sigma-Aldrich, Germany) was added to each well in a final concentration of 5 mg/mL after overnight incubation with LPS and/or MPs. The cells were incubated for 3 h at 37 °C and 5% CO2. The medium was then removed, and the formazan precipitate was solubilized in acidic isopropanol (Chempur, Poland). The absorbance was measured at 570 nm in a microplate reader.

Nitric oxide (NO) production assay

The generation of nitric oxide was measured with the Griess assay [42]. The RAW 264.7 cells were incubated in 96-well plates overnight in a presence of LPS and/or MPs as described above. After this time, cell culture supernatants were collected and frozen prior to further analyses. To run the assay, the samples were thawed and 50 μL was mixed with equal volume (25 μL each) of Griess reagents A (5% phosphoric acid containing 1% sulfanilamide) and B (0.1% solution of N-(1-naphthyl)ethylenediamine in distilled water (dH2O)); both from Sigma-Aldrich (Saint Louis, MO, USA). After 10 min at RT, the absorbance was measured at the wavelength 570 nm using a microplate reader.

NBT assay: respiratory burst measurement

The intracellular reactive oxygen species (ROS) production by RAW 264.7 macrophages was determined using 3,3′-(3,3′-dimethoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (NBT) reduction assay [43]. Phorbol myristate acetate (PMA) was used as a positive control as it induces ROS synthesis [44]. One hour before the end of the overnight incubation with LPS and/or MPs, 50 nM PMA (Sigma-Aldrich, Germany) was added into some wells (RPMI 1640 was added to PMA-non-stimulated wells). Plates were incubated for 1 h and then NBT solution (10 mg/mL; Sigma Aldrich, Germany) was added, and the plates were further incubated for 1 h at 37 °C and 5% CO2. The supernatants were removed, and the cells were fixed for 15 min in absolute methanol, washed twice with 70% methanol, then dried. The formazan deposits were solubilized in 120 mL of 2 M KOH and 140 mL dimethyl sulfoxide (DMSO; both from Sigma-Aldrich, Germany). After homogenization of the well content, the absorbance was read at 590 nm using a microplate reader.

Immunocytochemistry: iNOS expression

In some experiments, prior to seeding RAW 264.7 macrophages in 96-well plates, coverglasses were inserted into the wells. Then the cells were added, and they were let to adhere for 30 min. Subsequently they were stimulated with LPS and/or MPs as described above. After the overnight incubation, supernatants were carefully removed and the cells were fixed in a sequence of 1%, 2%, and 3% paraformaldehyde in PBS for 2, 10, and 20 min, respectively, and then washed in PBS for 5 min [45]. Prior to immunocytochemistry, for cell membrane permeabilization, the cells were washed for 5 min in TBS solution which contained Triton X-100 (Sigma-Aldrich, Germany), Na2HPO4 × 12H2O, Na2HPO4 × 1H2O (both from Avantor Performance Materials Poland), bovine serum albumin (BSA; Sigma-Aldrich, Germany), NaCl (Polfa–Lublin S.A., Poland), and dH2O. After washing, the coverglasses were incubated in blocking solution, namely 3% BSA in PBS, for 45 min at RT. Next the cells were labelled with rabbit monoclonal anti-iNOS antibodies (EPR16635 clone; Abcam, Cambridge, UK) diluted 1:200 in 1% BSA/PBS and incubated overnight at 4 °C in a humid chamber. The coverglasses were then washed twice in PBS and further incubated with secondary Cy3-conjugated goat anti-rabbit antibodies (Jackson Immunoresearch Laboratories, Ely, UK) diluted 1:300 in 1% BSA/PBS for 1 h at RT. Following the incubation, the slides were washed in PBS for 5 min and counterstained with 5 μM Sytox green (Invitrogen, Carlsbad, CA, USA) to visualize nuclei. After 5 min of staining, the coverglasses were washed in PBS and mounted with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Fluorescent signal was detected with a ZEISS Axio Examiner.Z1 upright microscope equipped with confocal spinning disk device DSD2 (Andor, Oxford Instruments, Abingdon, UK; the microscope details as above). The percentage of iNOS positive cells was estimated with ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Cytokine measurement

The cell culture supernatant content of mouse IL-1β and IL-6 was measured by Mouse IL-1β ELISA Ready-SET-Go (Affymetrix/ThermoFisher Scientific, Waltham, MA, USA) and Mouse IL-6 ELISA Ready-SET-Go (eBioscience/ThermoFisher Scientific, Waltham, MA, USA). The assays were carried out as indicated by the manufacturers.

Western blot analysis

Freshly isolated MPs were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) in the presence of the protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The prepared samples were subjected to thermal denaturation (5 min, 95 °C) by placing them on a thermoblock (OHAUS, Poland) and then mechanical disruption using a sonicator (Bandelin, Germany). Protein concentration in samples was determined using the Bradford method. For this purpose, concentrated Bradford reagent (Bio-Rad Protein Assay Dye Reagent Concentrate; Bio-Rad; USA) was used, which, after appropriate (5x) dilution in dH2O, was added to the samples and to the standard curve (BSA solution). Absorbance was measured at the 595 nm wavelength, and the ELx808 ELISA plate reader and the KC Junior software (BioTek Instruments, Winooski, VT, USA) were used. Then the samples (proteins) were normalized for further analysis. Commercially purchased 4-20% Mini-PROTEAN® TGXTM Precast Protein Gel (BioRad Laboratories, Hercules, CA, USA) were used for protein separation in a Mini-PROTEAN TetraSystem vertical electrophoresis apparatus (BioRad Laboratories, Hercules, CA, USA). The transfer of separated proteins was performed using the Trans-Blot Turbo Transfer System (BioRad Laboratories, Hercules, CA, USA) in commercially purchased transfer packets Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs (BioRad Laboratories, Hercules, CA, USA), and the manufacturer’s instructions were followed. After transfer, the blots were placed in blocking buffer (5% BSA [w/v] in 0.02 M Tris-buffered saline containing 0.1% Tween 20 (TBST) [v/v]) for 1.5 h (at RT) to block non-specific antibody binding to the membrane. Then, the membranes were probed overnight at 4 °C with primary recombinant rabbit monoclonal antibody (anti-ceruloplasmin, ARC5018-06-01 clone; Thermo Fisher Scientific, Waltham, MA, USA) 1:1000 at 5% BSA/TBST [w/v]). Next, the membranes were washed with TBST (3 times for 10 min) and then incubated for 1 h (at RT) with a horseradish peroxidase (HRP)-conjugated secondary antibody, anti-goat (Cell Signalling Technology, Massachusetts, DA, USA) 1:1000 at 5% BSA/TBST [w/v]. An anti-β-actin monoclonal antibody (13E5 clone; Cell Signalling Technology, Massachusetts, DA, USA) 1:1000 at 5% BSA/TBST [w/v] was used as a control. After that, the blots were washed in TBST and 0.02 M Tris-buffered saline (TBS; twice and once, respectively, for each 10 min). Clarity Max Western ECL Substrate (BioRad Laboratories, Hercules, CA, USA) was used to detect chemiluminescence signal which was visualized by the GeneGnome Bio Imaging System (Syngene, Cambridge, UK). Densitometric analysis of the visible bands was performed with ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Statistical analyses

All data are presented as mean values ± SD. Data were compared either by unpaired two tailed Student’s t-test (to evaluate the means of two populations) or one-way analysis of variance (ANOVA; to compare the means of more than two groups) with Bonferroni’s multiple comparisons post hoc test. Statistical significance was set at P < 0.05.