BLM-induced lung injury in mice
Eight-week-old male C57BL/6J mice were purchased from Charles River Japan (Yokohama, Japan). Mice were anesthetized by an intraperitoneal injection of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg), and then intratracheally injected with 6 U/kg BLM sulfate (Sigma-Aldrich, St. Louis, MO). Lungs were harvested on day 7 or 14 after BLM instillation. All animal experiments were performed in accordance with the Japanese Physiological Society’s guidelines for animal care and were approved by the University of Miyazaki Ethics Committee on Animal Experimentation.
Cell Cultures, Cell Transfection, And TGF-β1 Treatment
Mlg 2908 (CCL-206™), a murine lung fibroblast cell line, was purchased from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco’s Modified Eagle’s Medium/F12 (Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) and 1% penicillin/streptomycin. Cultured Mlg 2908 were grown in 5% CO2 humidified atmosphere at 37 °C. Passage was performed every 3 days. For LOXL2 knockdown experiments, the cells were seeded onto 6-well cell culture plates (2.0 × 105/well, Thermo Fisher Scientific, Waltham, MA) or Lab-Tek II chamber slides (0.4 × 105/well, Sigma-Aldrich). LOXL2 small interfering RNA (siRNA; 10 nM) was transfected into Mlg 2908 using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) 72 hours after seeding. The LOXL2 siRNA target sequences were as follows: first, 5’-GAACAGAAGUUCGAACACUTT-3’; second, 5’-GGAGUGAAGUGCUCAGGAATT-3’ (Life Technologies Japan, Tokyo, Japan). A scrambled siRNA was used as a control. At 72 hours after siRNA treatment, human recombinant TGF-β1 (5 ng/ml; R&D, Minneapolis, MN) or dimethyl sulfoxide (DMSO, control) was added to the medium. The cells were harvested for quantitative real-time PCR (qRT-PCR) and western blotting 24 hours after TGF-β1 treatment. Immunostaining and actin filament staining of cells on chamber slides were also performed 24 hours after TGF-β1 treatment, as described below.
Western Blotting
Whole lung tissues and cultured Mlg 2908 were homogenized and centrifuged at 10,000 × g to remove cell debris. Protein content of lysates was measured by Bradford protein assay. Equal amount of proteins was fractionated by 10% SDS-PAGE, and transferred to Immobilon-P transfer membrane (Merck Millipore, Burlington, MA). Expression levels of each molecule were analyzed by western blotting using antibodies recognizing the following proteins: LOX (Santa Cruz Biotechnology, Dallas, TX), LOXL2, Snail (Abcam, Cambridge, MA), pSmad2, Smad2 (Cell Signaling Technology, Danvers, MA), and β-actin (Sigma-Aldrich). Protein expression was quantified by densitometry using the Fusion FX software (Vilber Lourmat, Collégien, France).
qRT-PCR
Total RNA was extracted from lungs and Mlg 2908 cells using the Trizol reagent (Invitrogen, Carlsbad, CA). First-strand complementary DNA was synthesized using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, CA). qRT-PCR was performed with the following TaqMan/Applied Biosciences primers (Thermo Fisher Scientific): Lox (Mm00495386_m1), Loxl2 (Mm00804740_m1), Col1a1 (Mm00801660_g1), and Postn (Mm01284919_m1). The transcription levels of target genes were standardized against those of the Hprt1 (Mm03024075_m1) gene and calculated by the ΔΔCt method.
Histology, Immunostaining, And Confocal Microscopy
Lungs were removed, fixed in 4% buffered formalin, and embedded in paraffin. Lung sections (4 µm) were mounted on slides for staining with hematoxylin–eosin and Masson’s trichrome (American Master Tech Scientific, Lodi, CA). For immunostaining, lung sections were permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 30 minutes, and then pre-treated with 3% hydrogen peroxide to remove endogenous peroxidase. For immunostaining to detect surfactant protein C (SP-C), antigen activation was performed by heating the sections in an autoclave for 20 minutes at 105 °C in a HistoVT One (Nacalai Tesque, Kyoto, Japan), followed by cooling at room temperature. All sections were blocked with 2.5% goat serum / 2.5% donkey serum and 0.1% bovine serum antigen (BSA) in PBS-T for 60 minutes at room temperature. Each section was incubated overnight at 4 °C with primary antibodies against the following proteins: LOXL2 (1:400; Abcam, Cambridge, MA), Podoplanin (1:100; MBL, Nagoya, Japan), SP-C (1:200; Sigma-Aldrich), CD11b (1:200; Cell Signaling Technology), fibroblast surface protein (FSP, 1:800; Cell Signaling Technology), Snail (1:1,000; Abcam), and α-SMA (1:400; Sigma-Aldrich). After washing with PBS, the following secondary antibodies were used: Alexa Fluor 488 anti–rabbit IgG, Alexa Fluor 594 anti–rabbit IgG, Alexa Fluor 594 anti–goat IgG (Thermo Fisher Scientific). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). Images of immunofluorescence staining of cells and tissues were acquired using a Nikon C2 + confocal laser scanning microscope (Nikon, Tokyo, Japan). For quantification of co-localized cells from double-immunostained slides, one investigator took photos of 10 random fields at 200-fold magnification in a blinded fashion (i.e., without knowledge of the experimental or control groups from which the sections were taken), and the cells in each field were counted independently by two other investigators in a blinded fashion.
Immunocytochemistry And Actin Filament Staining
Individual siRNA-transfected and TGF-β1-treated Mlg 2908 cells grown on chamber slides were rinsed in PBS, fixed with 4% buffered formalin for 20 minutes, and blocked in 5% donkey serum and 0.1% BSA in PBS-T for 30 minutes at room temperature. Immunostaining on chamber slides with anti–α-SMA antibody (1:400; Sigma-Aldrich) was performed overnight at 4 °C. Nuclei were counterstained with DAPI. The α-SMA–positive and DAPI-positive cells were counted in 10 random fields per chamber at 200-fold magnification. The percentages of α-SMA–positive cells were calculated by dividing the number of α-SMA–positive cells by the number of DAPI-positive cells. For actin filament staining, cells were stained with Acti-stain 488 phalloidin (1:150; Cytoskeleton, Denver, CO).
Quantification Of Intracellular Actin Filament
Intracellular actin filaments were quantified as follows. Twenty equally distributed horizontal lines were overlaid on the actin-stained images. For each line, the number of the intersection points with actin filaments was manually counted. Intracellular actin filaments were quantified in 50 cells in each sample. The index of intracellular actin filaments was defined as the number of intersections per cell.
Human BALF Preparation
We retrospectively collected clinical data and BALF samples of patients with ARDS or pulmonary sarcoidosis (as disease control) admitted to the University of Miyazaki Hospital between 2009 and 2015. Subjects included 33 patients with ARDS (20 males and 13 females; mean age 67.6 [28–90] years), and 7 patients with pulmonary sarcoidosis (2 males and 5 females; mean age 58.6 [34–73] years). Patients with cancer in any organ were excluded from the study. The study protocol was approved by the University of Miyazaki Research Ethics Committee (O-0531). Informed consent was obtained in the form of opt-out on our hospital website. BALF samples were obtained from individual patients when they underwent diagnostic bronchoalveolar lavage. BALF was centrifuged at 1,000 rpm for 5 minutes, and supernatants were stored at − 80 °C prior to use.
Enzyme-linked Immunosorbent Assay (ELISA)
LOXL2 concentration in BALF was measured using the human LOXL2 ELISA kit (Abcam).
Statistical analysis
Data are expressed as means ± SEM. Statistical analyses were performed using GraphPad PRISM 7 (GraphPad Software, La Jolla, CA). Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The Mann–Whitney U test was used for single-parameter comparisons. P < 0.05 was considered statistically significant.