Subjects and Samples
IPF tissue samples were obtained from explanted lungs removed at the time of lung transplantation. Nonfibrotic control tissue samples were obtained from lungs declined for organ donation.
TBXA2R floxed mice17 were obtained from Jackson Labs (#21985) . These mice have loxp sites surrounding TBXA2R exon 2, which under the influence of Cre recombinase is excised, eliminating protein production. They had been backcrossed onto a B6 strain. These were crossed to mice with tamoxifen-inducible cre expression under the control of the universally expressed Rosa26 locus (Rosa26-CreER, Jackson Labs #4847).58 In combination, these Rosa26-CreER+TBXA2Rf/f mice are referred to as TBXA2RiKO, and have global deletion of TBXA2R when induced by Tamoxifen. Hermansky-Pudlak Syndrome mice included “pale ear” mice (Jackson Laboratories #525) which carry a constitutive mutation in Hps1,59 and “pearl” mice (Jackson Laboratories #3215), which carry a mutation in Hps2, also called Ap3b1.60
Bleomycin-induced fibrosis models
Bleomycin (Hospira Inc.) was purchased from Vanderbilt University Medical Center pharmacy. Bleomycin (0.04 units) in 100 μl saline was delivered by direct i.t. instillation under anesthesia as described previously.25,26,61 Tamoxifen-inducible transgenic mice were treated with tamoxifen (400 mg tamoxifen citrate /kg of chow ad libitum) for 2 weeks prior to bleomycin instillation (4 weeks prior to bleomycin instillation). Lungs were harvested following euthanasia by pentobarbital at designated time points. Right lungs were tied off and snap frozen for estimation of collagen and extraction of RNA and protein, and left lungs were inflated to 25 cm H20 with 10% neutral buffered formalin for histology.
Mice were randomized to receive either 25 mg/kg/day CPI211 (ifetroban; Cumberland Pharmaceuticals, Nashville, TN) in drinking water or normal drinking water (vehicle). In a follow-up experiment, 50 mg/kg/day ozagrel HCl hydrate (CombiBlocks, San Diego, CA) was given to mice, and several mice were alternatively treated with ethanol vehicle as negative controls. Final concentration of ethanol in drinking water was approximately 0.04%. All drugs were pretested for palatability to ensure normal consumption of drinking water. Mice were weighed and water was changed once a week.
Radiation-induced fibrosis model
10-12 week-old male/female mice were randomly assigned to vehicle- or Ifetroban-treated groups. Isoflurane anesthetized mice were placed on a 37°C recirculating water heating pad and the thorax was administered 17 Gy (300 kVp/10 mA X-rays) at 1.64 Gy per min as we previously described.62 With the exception of the thorax the entire animal was shielded by a custom lead block 2.5 cm thick.
Measurement of collagen (in vivo)
Total collagen in right upper lobes or whole right lungs of the lung was measured using the hydroxyproline assay (Biovision, kit #K555) or Sircol assay (Biocolor S1000 kit) as per the manufacturer’s instructions and as previously published61.
Treatment with F2-IsoPs or U46619 (in vitro)
Cells were seeded at a density of 8 × 104 cells/ml in DMEM supplemented with 20% FBS and allowed to grow to conﬂuence. Twelve hours before the treatment by F2-IsoPs or U46619, the medium was changed to DMEM with 2.5% FBS and 50 μg/ml ascorbic acid. Three hours before the treatment by F2-IsoPs or U46619, Ifetroban (300nM) was added to the culture media. Cells were then treated with either 15-F2-IsoPs (100nM) (Cayman Chemical, USA) or with TBXA2R-agonist, U46619 (Cayman Chemical, USA) in the same concentration. A stock solution of 15-F2t-IsoPs and U46619 (both 1 mg/ml in ethanol) was diluted to a concentration of 10−5 M and then further diluted to ﬁnal concentrations with DMEM.
Measurement of collagen (in vitro)
Cells were grown to confluence in 60–mm dishes and the medium was replaced with DMEM containing 2.5% FBS. The cells were incubated with either F2-IsoPs or TxB2 (Cayman Chemical, Ann Arbor, MI) for 48 h at 37°C. In some experiments, Ifetroban or Z-LLY-FMK (Abcam) was added 3h before stimulation, respectively. The amount of total soluble collagen in culture supernatants was measured by the Sircol dye binding assay kit (BioColor Ltd.) according to the manufacturer’s protocol.
Mouse lung fibroblast culture
At the time of harvest, the lungs were perfused blood free with 30 ml PBS containing 10 U/ml heparin from the right ventricle, then minced and digested in an enzyme cocktail of DMEM containing 1% of BSA , 2 mg/ml of collagenase type IV, 100 ug/ml of DNase, and 2.5 mg of Dispase II (Roche Diagnostics, Mannheim, Germany) at 37°C for 30 min, then strained through a 100-um nylon cell strainer. Mouse lung cells were pretreated with anti-CD16/32 antibody (BioLegend) to block Fc receptors and then incubated with speciﬁc antibodies at 4°C in the dark. Lung fibroblasts were isolated by FACS Aria, defined as CD140a+/CD31-/CD45-/CD326-. Cells were isolated from 3-5 mice per group for each experiment.
Isolation and culture of primary human lung fibroblasts
Primary lung fibroblasts were isolated from IPF lungs removed at the time of lung transplant surgery as previously described63,64.
Ex-vivo TBXA2R deletion
Cre recombinase activity was induced by 4-hydroxytamoxifen (4-OH TAM) (Sigma) at a dose of 0.2 µM, which was added into cell culture medium for 8 days. After 8 days of treatment the dose of 4-OH TAM was decreased to 0.05 µM and kept at this level until the end of experiments. Fresh 4-OH TAM was added every 2 days for MLFs. Excision of TBXA2R exon 2 was confirmed by PCR analysis on days 1–8 of treatment in MLFs.
Transient transfection and dual-luciferase assay of murine lung fibroblasts (MLF), human lung fibroblasts (HLF)
MLFs, and HLFs were seeded at a density of 60,000 cells/well into 24-well plates. Cells were transfected with Smad Binding Element (SBE) reporter containing a mixture of 40∶1 SBE luciferase and CMV Renilla (Qiagen, Valencia, CA), using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Transfection solutions were replaced by phenol-free medium with DMEM with 10%FBS 4 h after transfection. Forty-eight hours after transfection, cells were treated with and without several concentration of F2-IsoPs and U46619. Cells were treated for the indicated time (8h) prior to cell lysis and measurement of firefly and Renilla luciferase luminescence using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Twenty microliters of extracts was used for dual-luciferase assay, with 100 μL of substrate. Activity was determined using a luminometer (BioTek Instruments, Winooski, VT) for 15 seconds for each sample. Firefly luciferase luminescence was quenched by Stop and Glo buffer (Promega) for 30 seconds before measuring Renilla activity. Luciferase activities of each transfection were normalized by Renilla activity. A value of 1.0 indicates the basal normalized activity without any added treatment, and values >1 represent the fold of induction for each reporter construct. Each experiment was performed three times to obtain means and standard errors of the mean.
Measurement of F2-IsoPs and TxB2
The levels of F2-IsoPs and TxB2 in lung tissues were determined by gas chromatography- mass spectrometry as previously described65,66.
Western blotting was performed as previously described67. PVDF membranes were used for transfer, and antibodies used include anti-phospho-SMAD2/3 (1∶1,000; Cell Signaling Technology, Danvers, MA), anti-phospho-Akt (1:3000; Cell Signaling Technology), anti-phospho-p44/42 (1:1000; Cell Signaling Technology), anti-Timp1 (1:3000; Thermo Fisher Scientific, Waltham, MA), anti-alpha smooth muscle Actin (1:5000; Abcam, Cambridge, UK), anti-TBXA2R (1:3000; Proteintech, Rosemont, IL), anti-HSP-70 (1:3000; Abcam), and anti-beta Actin (1:5000; Abcam). All antibodies to phosphorylated proteins were diluted in 5% BSA/TBST, and the rest were diluted in 2% milk/TBST.
Flow Cytometry of Lung Cells
Flow Cytometry of mouse lung cells were performed as previously described68. In brief, mouse lung cells were pretreated with anti-CD16/32 antibody (BioLegend, San Diego, CA) to block Fc receptors and then incubated with speciﬁc antibodies at 4°C in the dark. The following antibodies were used for cell surface staining: anti-CD31-FITC (BioLegend), anti-CD45-APC (BioLegend), anti-CD45-PE (BioLegend), anti-CD326-APC/Cy7 (BioLegend), anti-CD140a-PE (BioLegend), anti-CD11b-Pacific Blue (BioLegend), anti-CD103-PE (BioLegend), anti-F4/80-APC (BioLegend), anti-CD11c-PE/Cy7 (BioLegend), anti-Ly6G-APC/Cy7 (BioLegend), and 7AAD (BioLegend). To assess DNA degradation (apoptosis), the cells were incubated with 10 μg/ml of Hoechst 34580 (Life Technologies, GrandIsland, NY) for 30min at 37°C in the dark. Cell ﬂuorescence was measured with the FACS Canto II instrument (Becton Dickinson, San Jose, CA) and analyzed by employing FlowJo software (Tree Star, San Carlos, CA). We defined MLFs as CD31-/CD45-/CD326-/CD140a+ lung cells, and murine lung epithelial cells as CD31-/CD45-/CD326+ lung cells as previously reported. For immune/inflammatory cells, we defined IMs as CD45+/CD11bhigh/F4/80high/Ly6G-/CD11c+ lung cells, AMs as CD45+/CD11bmoderate/CD11c+/CD103- lung cells, DCs as F4/80high/Ly6G-/CD11c+/CD103+ lung cells, monocytes as CD45+/CD11bhigh/F4/80high/Ly6G-/CD11c- lung cells, and neutrophils as CD45+/CD11bhigh/Ly6G+ lung cells using our previously published protocol68 (Extended Fig. 4b).
Single-cell RNA-sequencing analysis
Single-cell RNA-sequencing (scRNA-seq) data generated from single-cell suspensions generated from pulmonary fibrosis and nonfibrotic control lungs underwent quality control filtering and unbiased clustering followed by cell-type annotation as previously reported6. Raw and processed 10X genomics data can be found on GEO using accession number: GSE135893. The code used to analyze the data can be found at https://github.com/tgen/banovichlab/.
Values are expressed as mean ± SEM, and sample size is given for each ﬁgure. Two-way analysis of variance (ANOVA) or 1-way ANOVA followed by the Sidak post-test was performed on Prism (GraphPad, San Diego, CA) to determine statistical signiﬁcance.