Mineral Fibers
Short fiber amosite (SFA) and long fiber amosite (LFA), kindly provided by F. Turci (Turin, Italy) originated from South Africa. The length range of LFA was 2-100 µm with more than 30% of fibers longer than 5 µm. SFA were obtained by grinding of LFA and its length range was 1-10 µm, with less than 1% fibers longer than 5 µm. The diameter distribution ranged between 0.2-1.5 µm and was similar for both fibers (Davis et al. 1986; Tomatis et al. 2010). All samples were thermally treated at 200°C for 3 h to remove any possible trace of endotoxin and suspensions were prepared by sonication and manual vortexing in sterile Phosphate-buffered saline (PBS) or water for in vivo and in vitro experiments, respectively.
Chemicals and reagents
Human recombinant cytokines IFNɣ, IL-4 and IL-13 were purchased from Peprotech (Neuilly sur Seine, France) and human recombinant M−CSF was obtained from Miltenyi Biotec SAS (Paris, France). Lipopolysaccharide (LPS) from E.coli (serotype: 055:B5) and z-VAD-FMK were purchased from Sigma-Aldrich (St-Quentin Fallavier, France) and MedChemExpress (Clinisciences, Nanterre, France), respectively. Stock solutions human recombinant cytokines were prepared in sterile distilled water containing 0.1% bovine serum albumin. Z-VAD-FMK was dissolved in DMSO and control culture received the same dose of DMSO than their treated counterparts, without exceeding 0.2% (vol/vol).
Animals
Female C57BL/6 mice weighing 20 gr, used at 8 weeks of age, were purchased from Janvier SAS (St Berthevin, France). Animals were kept with sterile rodent feed and acidified water, and housed in positive-pressure air-conditioned units (25 °C, 50% relative humidity) on a 12 h light/dark cycle. After acclimatization, the animals were exposed by intra-tracheal instillation to short or long amosite fibers (0.1 mg) suspended in a volume of 0.5 ml of PBS-BSA. Vehicle controls were injected with an equal volume of PBS-BSA. Mice were then euthanized 4 months (120 days) after particle administration with an intraperitoneal injection of 12 mg sodium pentobarbital (Certa, Braine-l’Alleud, Belgium).
Broncho-alveolar lavage and lung sampling
The broncho-alveolar lavage (BAL) was performed by cannulating the trachea and infusing the lungs with 4x with 1 ml 0.9% NaCl. The BAL fluid was centrifuged 280 g, 4 °C, 10 min using a 5804R centrifuge (Eppendorf, Hamburg, Germany), and the cell-free supernatant was used for biochemical measurements. After resuspension of the pellet in PBS, total BAL fluid cells were counted (at least 200 cells) in Turch (crystal violet 1%, acetic acid 3%), and then pelleted onto glass slides by cyto-centrifugation for differentiation by light optical microscopy, after Diff-Quick staining (Baxter, Lessines, Belgium). Total proteins and lactate dehydrogenase (LDH) activity were assessed on BAL fluid as previously described (Arras et al. 2001). BAL fluid content of double-stranded DNA (dsDNA) was measured using Quant-iT PicoGreen dsDNA reagent (Invitrogen, Carlsbad, USA), according to the manufacturer's protocol.
Quantification of total lung collagen and BAL cytokines
Collagen deposition was estimated by measuring hydroxyproline (OH-proline) content in lung homogenates and tissue inhibitor of metalloproteinase (TIMP-1) content in BAL fluids. Hydroxyproline was assessed by high-pressure liquid chromatography analysis on hydrolyzed lung homogenates (6 N HCl at 108° C during 24 h) as previously described (Biondi et al. 1997). TIMP-1 levels were assessed by ELISA (R&D Systems, Minneapolis, USA). The following ELISA were performed on BAL fluids according to manufacturers’ instructions: CCL2 were investigated using a DuoSet ELISA kit (R&D Systems, Minneapolis, USA); total Ig-G, Ig-A and Ig-M were measured using an Invitrogen Uncoated ELISA kit (ThermoFischer Scientific, Courtaboeuf, France).
Lung Histology
Lungs were instilled and perfused with 0.9% NaCl and superior-left lung lobe was fixed in 3.6% formaldehyde solution (Sigma-Aldrich, Saint Louis, USA) for one night. Sections embedded in paraffine were stained with hematoxylin and eosin (H&E, nucleus and cytoplasm staining). Images were acquired with a slide scanner SCN400 (Leica, Diegem, Belgium) and processed with Tissue Image Analysis 2.0 (Leica).
Preparation of Human Monocyte-Derived Macrophages (MDMs)
As alveolar macrophages, and especially those derived from monocytes, seem play a key role in asbestos-induced lung fibrosis development (Joshi et al. 2020), the model of human monocyte-derived macrophages is well adapted to study and compare the cellular effects of short and long asbestosis fibers.
Patients exposed to silicate particles and healthy donors (HD): Peripheral blood mononuclear cells were obtained from HD or patients exposed to silica particles through Ficoll (Eurobio, Les Ulis, France) gradient centrifugation. Blood buffy coat from HD were provided by the Etablissement Français du sang (Rennes, France) after their consent. Patients exposed to silica from the Occupational Health and Occupational Pathology Service of Rennes University hospital were consecutively included after written informed consent. This study was approved by the local ethics committees (Committees for protection of persons approval Ile de France 5, N°2020-A01990-39).
Differentiation of monocytes in MDMs and treatment: Monocytes, selected after a 1-h adhesion step, were differentiated into MDM for 6 days using M−CSF (50 ng/ml) in RPMI 1640 medium GlutaMAX (Gibco, Life technologies SAS, Courtaboeuf, France) supplemented with antibiotics (20 IU/ml penicillin and 20 μg/ml streptomycin (ThermoFisher Scientific) and 10% heat inactivated fetal bovine serum (FBS, Lonza, Levallois-Perret, France), as previously described (Lescoat et al. 2020b). After 6 days, the supernatant of cells was removed and replaced with medium containing 5% of heat-inactivated FBS and M−CSF (10 ng/ml). These cells corresponding to M0-MDMs were then exposed to SFA or LFA from 0 to 50 ng/cm2. In some case, M0-MDMs were pre-incubated to 10 ng/ml of LPS in the presence or not of 10 µM of z-VAD-FMK before amosite fibers. According to experiments, conditioned media were removed and stocked at −20 °C for ELISA analysis whereas the cells were washed and harvested for RNA extraction or flow cytometry analysis.
Evaluation of cell viability, cytokine/chemokine levels and Caspase-1 activity
Cell viability was assessed with CyQUANT assay (in vitrogen, Thermofisher). MDMs plated at 2x105 cells/cm2 were exposed at different concentrations of fibers from 0 to 50 µg/cm2) for 4 h or 24 h. After particle exposure, CyQUANT was added to MDMs in complete medium, incubated for 1 h at 37°C and read on using appropriate wavelengths (485/540 nm) a spectrofluorimeter SPECTROStar OMEGA (BMG Labtech, Champigny s/Marne, France).
Levels of human IL-1β, IL-6, IL-8, and TNFα secreted in MDM culture media were quantified by ELISA using specific Duoset ELISA development system kits (R&D Systems, Minneapolis, MN, USA).
Caspase-1 activity was quantified by luminescence using the Caspase-Glo 1 inflammasome assay according to the recommendations of providers (Promega, Charbonnières-les-Bains, France) on a Enspire 2300 luminometer (Perkin-Elmer, Waltham, MA, USA).
Reverse transcription-quantitative polymerase chain reaction (RTqPCR) experiments
Total RNAs were extracted from cells with Nucleospin RNA extraction Kit (Macherey-Nagel) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher Scientific). qPCR assays were next performed using the fluorescent dye SYBR Green methodology and a CFX384 Real-Time PCR detector (Bio-Rad Laboratories, Marnes-la-Coquette, France). The KiCqStart® SYBR® Green primers for human and mouse cDNA were provided by Sigma-Aldrich. The specificity of amplified genes was evaluated using the comparative cycle threshold method (CFX Manager Software). These mean Cq values were used to normalize the target mRNA concentrations to those of the 18S ribosomal protein by the 2(−ΔΔCq) method.
Cell surface receptors and markers analyses by flow cytometry
Phenotypic analysis of MDMs was performed using flow cytometric direct immunofluorescence. After washing and plastic detachment using AccutaseTM (BioLegends, Paris, France), cells were stained with Fixable Viability Stain 780 (BD Biosciences, Le Pont de Claix, France) for 10 min at room temperature to measure viability. MDM were first blocked in phosphate-buffered saline (PBS) supplemented with 2 % FBS solution and with FcR blocking reagent (Miltenyi Biotec SAS, Paris, France) for 10 min at room temperature to avoid nonspecific binding, and then re-suspended and incubated with specific antibodies or appropriate isotype controls for 30 min at 4°C. Cells were washed with PBS, collected by centrifugation (2500 rpm for 5 min) and then analyzed on a LSR II cytometer (BD Biosciences, San Jose, CA, USA) and FlowLogicTM software (Miltenyi Biotec SAS, Paris, France). The phenotypic characterization of MDMs was performed using the following antibodies: BUV395 anti-MHCII, BV421 anti-CD200R, BV605 anti-CD80, BB515 anti-CD206, PE anti-CD163 and APC anti-CD86 their respective isotype control, as recommended by BD Biosciences (Le Pont de Claix, France). Results are expressed as the mean ratio of median fluorescence intensity (MFI) calculated as follows: MFI (mAb of interest)/MFI (isotype control mAb).
Phagocytosis assays by flux cytometry
MDMs plated in 12-well tissue culture plates were exposed to fluorescents YG beads (Fluoresbrite™ Plain YG 1.0 Micron Microsphere, Polysciences, Warrington, USA, in a 10:1 ratio (fluorescent beads/MDM) for 45 min at 37°C or 4°C in a 5% CO2 humidified incubator. Some M0-MDMs were pretreated by 5 µM cytochalasin D (Sigma-Aldrich) for 1 hour, thus acting as a negative control as cytochalasin D inhibits actin polymerization required for engulfment. After phagocytosis, MDMs were washed, detached and stained for viability as already detailed above before YG fluorescence quantification on the cytometer. Results are expressed as % of phagocytosis calculated as follows: % fluorescent MDMs (37°C) - % fluorescent MDMs (4°C).
Efferocytosis Assays by real-time fluorescence microscopy
Human Jurkat CD4 T-lymphocyte cells (1.106 cells/ml), cultured in RPMI 1640 Glutamax culture medium with 10 % FBS, 20 IU/ml penicillin and 20 μg/ml streptomycin, were exposed for 4 h to 10 μM camptothecin (Sigma-Aldrich) to induce apoptosis as previously described (Ballerie et al. 2019). Apoptotic Jurkat cells were then stained for 15 min with 250 ng/ml pHrodo (IncuCyte® pHrodo® Red Cell Labeling Kit, Sartorius, Ann Arbor, USA), washed and added to MDMs plated in 96-well-tissue culture plates, in 10:1 ratio (apoptotic cells/MDM) for 180 min at 37◦C in a 5% CO2 humidified incubator. Engulfment efficiency of MDMs was quantified by real-time fluorescence microscopy (IncuCyte® live-cells Analysis system, Sartorius), measuring total integrated red intensity (ex:560nm/em:585nm) of labeled Jurkat cells when entering the acidic phagosome every 15 min. Mean fluorescence intensity of phagocytosed Jurkat cells after 90 min is used to compare the efferocytosis level.
Antinuclear antibodies detection
The anti-nuclear antibodies detection was performed by indirect immunofluorescence technique using HEp-2 (human epithelial cell line) substrate slides and FITC anti-human IgG conjugate (Nova Lite™ Inova Diagnostics, San Diego, USA). Serums were tittered from 1/80 to endpoint. Titrations were expressed as the last positive dilution and aspect were described in accordance to the recommendations from the International Consensus on ANA Patterns (ICAP) group (von Mühlen et al. 2021).
Statistical Analysis
Data are presented as means ± standard error on the mean (SEM). Comparison between more than 2 groups were performed by repeated measure analysis of variance for paired or one-way analysis of variance followed by Dunnett’s or Newman–Keuls multiple comparison post-hoc test. Depending on conditions and Gaussian distribution, Student’s t test, paired-t-test or Mann–Whitney test were used to compare 2 groups. A P < 0.05 was considered significant. Data analyzes were performed with GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).