Mice and human samples

All procedures in vivo followed the EU-adopted regulations (Directive 2010/63/EU). Ethical and regulatory approvals were consented by Direção Geral de Alimentação e Veterinária (DGAV, 003671) and conducted according to the guidelines sanctioned by the DGAV. Mice were supplied by Professor Massimiliano Mazzone, Center for Cancer Biology, VIB, KU Leuven, Belgium. Eight-week-old gender- and age-matched C57BL/6 mice were bred under specific pathogen-free condition and kept at the Life and Health Sciences Research Institute (ICVS) animal facility. Mice were fed ad libitum and kept under light/dark cycles of 12 h, temperature of 18–25 °C and humidity of 40–60%. Male, 10 to 12 weeks old, wild-type (WT, Tie2:c-Metwt/wt) and conditional knockout (cKO, Tie2:c-Metfl/fl) C57BL/6J mice were used.

This study was reviewed and approved by the Ethics Committee of Hospital de São João (CES 72–12). Transbronchial cryobiopsies and cryopreserved bronchoalveolar lavage (BAL) from ILD patients were obtained. Demographic and clinical features of studied patients are reported in Table S1 and Table S2, respectively.

BLM-induced pulmonary fibrosis mouse model

Mice were sedated with ketamine and medetomidine and intratracheally administered with vehicle or with BLM sulphate (BML-AP302-0010, Enzo Life Sciences) as a single dose of 2.5 mg/kg in 50 µL saline.

BAL collection and preparation of mouse lung single-cell suspensions

Briefly, mice were over anesthetized by intraperitoneal injection, then trachea was exposed by a midline incision and cannulated. Bilateral BAL recovered by a three times lavage with 1 mL of PBS. The supernatant from the BAL was frozen for the LDH and multi‐analyte flow assay. Then, animals were perfused with PBS. After dissection, the lungs were placed in dispase (734-1312, Corning) while minced into 2–3 mm3 pieces for 20 min. Then, 1 mg/mL DNase I (10104159001, Roche) and 5 mg/mL collagenase II (17101-015, Gibco) solution in Dulbecco's Modified Eagle Medium (21969-035, Gibco) was added to the sample. After, an enzymatic digestion period of 10 min at 37 °C, tissue disruption was performed by mechanical titrations with P1000 tip, and then a second incubation period of 10 min at 37 °C was conducted. After incubation, the disrupted tissue suspension was collected and sequentially filtered through 100 µm and 70 µm cell strainers. The supernatant was collected and stored at -80 °C for the LDH assay. BAL cells were added to the lung cell suspension, centrifuged and the pellet was resuspended in ammonium-chloride-potassium lysis buffer (A1049201, Gibco), and incubated for 4 min. Cells were then maintained in cold PBS containing 4% fetal bovine serum (10270-106, Gibco).

Flow cytometric immunophenotyping analysis of mouse and human samples

Single-cell suspension, 1 × 106 cells were preincubated with TruStain FcX anti-mouse CD16/32 antibody (101320, Biolegend) for 10 min at RT. After the blocking of non-specific binding of immunoglobulin to the Fc receptors, cells were stained with the membrane antibodies listed in Table S3, for 30 min on ice in the dark. After, cells were fixed by incubation in 4% paraformaldehyde (PFA) for 20 min on ice in the dark. Defrosted human BAL cells were preincubated with TruStain FcX anti-human CD16/32 antibody (422301, Biolegend) and then stained for 30 min with the membrane antibodies listed in Tables S4. Data were acquired on a BD® LSRII flow cytometer and BD® LSRFortessa™ Cell Analyzer, for mouse and human samples, respectively. FlowJo Software was used to analyze the data. Gating strategy is illustrated in Fig. S1A, B, for the identification of myeloid and lymphoid cells, respectively, in mouse lung and in Fig. S2 for human samples.

Survival analysis

After BLM administration, animals were followed for 21 days. Animals’ weight was monitored every 1–3 days. Mice that reached the humane endpoint were sacrificed, evaluated with a score that accounts for the percentage weight loss, labored breathing, increased respiratory rate, decreased activity, hunched posture and ruffled fur.

Histological examination

Lungs were perfused with PBS, fixed in 4% PFA for 24 h, subsequently paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. Inflammatory scoring system for mouse lung was performed according to Gori et al. [41], consisting of a scale from 0 to 4 (corresponding to no inflammation to severe inflammation, respectively, depending on the density of inflammatory infiltrates and on the alveolar septum thickness). The fibrotic score was determined according to the modified Aschcroft score [42]. Histological scoring system for lesions extension quantification in the lung was randomly performed by blindly score lung lobes, using a × 10 magnification. In both scores, the slides were independently evaluated by two researchers in a double-blind fashion. The average score from all fields was used to reach the inflammatory/fibrotic score of each animal. Representative images from the H&E and Masson’s trichrome stained tissue sections were acquired using the Olympus BX61 light microscope equipped with an Olympus DP-70 digital camera in a × 10 or × 20 magnification and processed by cellSens™ software.

Measurement of lactate dehydrogenase (LDH) activity

The LDH activity in BAL and dissociated tissue supernatants was assayed using the LDH kit (K726, BioVision). Briefly, 2 µL of NADH standards and supernatant samples were transferred to a 96-well plate and 100 µL of LDH substrate mixture were added, the absorbance was measured at the wavelength of 450 nm in a microplate reader Varioskan Flash (Thermo Scientific). Then the plate was incubated for 30 min at 37 °C. LDH activity in samples was calculated based on the standard curve, according to the manufacturer instructions, and LDH activity in the samples was expressed in nmol/min (mU) per mL.

RNA isolation and RT-qPCR

On day 7 after BLM administration, single-cell suspensions were frozen at -80 °C and RNA was isolated using ExtractMe total RNA kit (EM09, Blirt) according to the manufacturer instructions. On day 14 after BLM administration, the lungs were flash frozen in liquid nitrogen. For the RNA extraction, the tissue was firstly homogenized in TRIzol reagent (15596-026, Invitrogen), through mechanical disruption using microcentrifuge pestle and then needles with crescent gauge values.

First-strand cDNA was synthesized using the commercial Maxima First Strand cDNA synthesis kit (K1671, Thermo Fisher Scientific), following the manufacturer instructions. Real-time PCR amplifications were performed using commercial 2 × Maxima probe/ROX master mix (K0231, Thermo Fisher Scientific), containing a Hot Start Taq DNA Polymerase and TaqMan® gene expression premade assays from Applied Biosystem.

Real-time PCR amplifications were performed using commercial 2 × Maxima probe/ROX master mix (K0231, Thermo Fisher Scientific), containing a Hot Start Taq DNA Polymerase and TaqMan® gene expression premade assays from Applied Biosystem. The PCR probes analyzed at 7 days were glyceraldehyde-3-phosphate dehydrogenase (Gapdh, Mm99999915_g1), insulin-like growth factor (Igf)-1 (Mm00439560), transforming growth factor (Tgf)-β1 (Mm01178820_m1), and connective tissue growth factor (Ctgf, Mm01192933_g1). The PCR probes assessed at 14 days were Gapdh (Mm99999915_g1), Collagen (Col) 1a1 (Mm00801666_g1), Col1a2 (Mm00483888_m1), Col3a1 (Mm00802300_m1), Actin-α 2 (acta-2, Mm00725412_s1), S100a4 (Mm00803372_g1), Tgf-β1 (Mm01178820_m1), Ctgf (Mm01192933_g1), Elastin (Eln, Mm0051470_m1) and Igf-1 (Mm00439560_m1). Gene expression levels were normalized to endogenous control Gapdh and performed accordingly to the 2−ΔΔCT method [43]. Gene expression data was presented in relative expression fold-change based on 2−ΔΔCT (cKO value) / average (2−ΔΔCT (WT group)).

LEGENDplex™ (multi‐analyte flow assay kit)

Cell suspensions from dissociated lung tissue were homogenized in 1 mL of PBS with protease inhibitors (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, 04693159001, Roche Diagnostic) and 0.05% Tween 20. The respective supernatants were analyzed with the following Mouse LEGENDplex™ panels. Mouse chemokines were analyzed using a mouse 13-plex multiplex bead-based assay panel, including CXCL1 (KC), Eotaxin (CCL11), MCP-1 (CCL2), IP-10 (CXCL10), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), TARC (CCL17), MIG (CXCL9), MIP-3α (CCL20), LIX (CXCL5), BLC (CXCL13) and MDC (CCL22), according to the manufacturer’s instructions (740451, Biolegend). Mouse proinflammatory cytokines, were evaluated using a mouse 8-plex multiplex bead-based assay panel, including IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, IL-1β and IL-6, according to the manufacturer’s instructions (740848, Biolegend). Additional mouse cytokines, were quantified using a mouse 12-plex multiplex bead-based assay panel, including, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ and TNF-α, according to the manufacturer’s instructions (741044, Biolegend).

BAL supernatants were analyzed with LEGENDplex™ mouse macrophage 13-plex multiplex bead-based assay panel, including CXCL1 (KC), TGF-β1, IL-18, IL-23, MDC (CCL22), IL-10, IL-12p70, IL-6, TNF-α, G-CSF, TARC (CCL17), IL-12p40, and IL-1β, according to the manufacturer’s instructions (740852, Biolegend).

The assays were performed in a V‐bottom plate according to the manufacturer's protocol. Data acquisition was done using the BD® LSRII flow cytometer. BioLegend's LEGENDplex™ data analysis software was applied for analysis.

Measurement of lung hydroxyproline

Hydroxyproline concentration of the lungs was measured to determine the total collagen content in the lung using a colorimetric hydroxyproline assay kit (MAK008, Sigma), according to the manufacturer’s instructions. In brief, the lungs were homogenized and then hydrolyzed in 6 N HCl at 120 °C for 3 h in a dry block heater. Hydrolyzed samples were transferred to a 96-well plate and evaporated to dryness at 70 °C. Then, 100 µL of chloramine T reagent was added to each sample and incubated at room temperature (RT) for 5 min, followed by the addition of 100 µL of 4-(Dimethylamine) benzaldehyde (DMAB) reagent to each sample and standard well and incubated at 60 °C for 90 min. Absorbance of oxidized hydroxyproline was read at 560 nm in a microplate reader Varioskan Flash (Thermo Scientific). The amount of hydroxyproline was calculated from the standard curve and its concentration was expressed in micrograms per milligram of lung.

Immunofluorescence

Human lung cryobiopsy slides were incubated for 1 h at 60 °C, deparaffinized in xylene and dehydrated in 100% ethanol. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) heated on microwaved (500 W) during 15 min. Non-specific antibody binding sites in lung tissue sections were blocked for 1 h at RT with blocking solution (BS), 1:20 horse serum in PBS. Slides were incubated at RT overnight with the primary antibody 1:200 CD3 (ab16669, Abcam), 1:50 CD68 (orb197999, Biorbyt) or 1:300 myeloperoxidase (14569, Cell Signaling Technology) diluted in BS. Slides were then stained with the secondary antibody 1:150 horse anti-rabbit IgG (BA-1100, Vector Labs) diluted in BS, for 2 h at RT. Tissue sections were further incubated with 1:100 Alexa Fluor™ 568 streptavidin (S11226, invitrogen) diluted in BS for 1 h at RT. For sequential double staining, non-specific antibody binding sites in lung tissue sections were blocked for 1 h at RT in BS, PBS with 1:5 donkey serum. Slides were incubated at 4ºC overnight with the primary antibody 1:100 c-MET (ab51067, Abcam) diluted in BS. Slides were then stained with the secondary antibody 1:150 DyLight™ 488 donkey anti-rabbit IgG (406404, Biolegend) diluted in BS for 2 h at RT. Nuclei were counterstained with 0.1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) for 5 min at RT. After each mentioned incubation, slides were rinsed with PBS containing 0.02% (v/v) Tween-20. Slides were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher) and images acquired using an Olympus BX61 light microscope equipped with an Olympus DP-70 digital camera in a × 20 magnification, processed by cellSens software.

Single cell RNA-sequencing analysis

Data from a single cell RNA-sequencing analysis of lungs from eight donor and nine patients with various forms of pulmonary fibrosis, including IPF, HP and CTD-ILD, were used [44]. Raw data were retrieved from GEO with the identifier GSE122960, imported into the R environment (version 3.4), and analyzed with the Seurat package (http://satijalab.org/seurat/, version 2.2). R code used for data analysis is available at https://github.com/NUPulmonary/Reyfman2018.

Statistical analysis

Data were analyzed and plotted using the GraphPad Prism 6.00 (GraphPad Software, San Diego, CA). All results were expressed as group mean ± standard error of the mean (SEM). For all comparisons, normality was assessed with Shapiro-Wilk test, if normality is not verified non-parametric tests were used. Comparisons between two groups were performed either with parametric unpaired two-tailed Student’s t-test or non-parametric two-tailed Mann–Whitney test. Comparisons between three groups were performed either with parametric one-way ANOVA or non-parametric Kruskall-Wallis test. Survival curves (Kaplan–Meier plots) were compared using the log-rank test. A p value of less than 0.05 was considered a statistically significant difference.