Animals
Nine-week-old male C57BL/6 mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). We chose this strain because it is well characterized and is susceptible to BLM-induced pulmonary fibrosis. Interleukin (IL)-10 knock-out mice with C57BL genetic background (B6.129P2-Il10tm1Cgn/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The experimental protocols in this study were approved by the animal care and use committee of Nippon Medical School (Tokyo, Japan, Approval Number; 27–098).
BLM treatment and adoptive transfer of splenocytes
Osmotic pumps (ALZET model 2001; DURECT Corporation, Cupertino, CA, USA) containing 200 μL saline, with or without BLM (100 mg/kg of mouse body weight; Nippon Kayaku Co., Tokyo, Japan), were implanted in recipient C57BL/6 mice on day 0 (Fig. 1a) [13]. Incision wounds were sealed using a surgical suture. BLM was continuously infused via the pumps over 7 days, according to the manufacturer’s instructions.
Spleens were removed from donor C57BL/6 mice without BLM treatment and minced to obtain single-cell suspensions. Recipient C57BL/6 mice treated with BLM received 1 × 105 donor C57BL/6 splenocytes on day 7 or 14 via caudal vein injection. On day 28, mice were sacrificed and the lungs were removed for examination.
Histological examination
Lung samples were fixed in 10% formalin buffer (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for histological examination. Paraffin sections (3.5 μm-thick) were cut from fixed lungs, stained with hematoxylin and eosin (HE) to assess gross morphology and Masson’s trichrome stain to visualize collagen deposition [14], and examined by microscopy. Lung fibrosis was measured using quantitative histology following Ashcroft’s method [15].
Antibody-mediated Treg depletion
To deplete Tregs, we incubated single-cell suspensions of splenocytes (1 × 105) with 1 μL of an anti-CD25 monoclonal antibody (mAb) (clone: PC61; BioLegend, San Diego, CA, USA) dissolved in sterile phosphate-buffered saline (PBS) for 20 min on ice. Then, these cells were adoptively transferred to BLM-treated mice on day 14 via the caudal vein. On day 28, the mice were sacrificed, and the lungs were removed and subjected to HE and Masson’s trichrome staining. The extent of lung fibrosis was measured by quantitative histology according to Ashcroft’s method to determine the effect of CD25 neutralization with the anti-CD25 mAb.
Fluorescence-activated cell sorting (FACS) analysis of splenocytes
Splenocyte subpopulations were analyzed by evaluating the relative proportions of Tregs, macrophages, and B cells. Single-cell suspensions of splenocytes were stained with anti-mouse CD4 (BioLegend) and CD25 (eBioscience, Waltham, MA, USA) antibodies to analyze Tregs. To analyze macrophages and their polarization, splenocytes were stained with anti-mouse F4/80 (BioLegend), CD80 (eBioscience), and CD206 (BioLegend) antibodies. Splenocytes were stained with an antibody against CD45R (B220; BioLegend) for B cell detection. To eliminate nonspecific staining, isotype- control antibodies, matched to the surface marker antibody’s host species and class, were used. FACS analysis was performed using a BD FACSCanto II and BD FACSVerse flow cytometers (BD Biosciences, San Diego, California, USA). Data collected were analyzed with FlowJo software (Tree Star, Inc., Ashland, Oregon, USA).
Isolation of Tregs from the spleen and adoptive transfer to BLM-treated mice
After single-cell suspensions were obtained from the spleens of C57BL/6 mice, Tregs were purified using a MiniMacs CD4+CD25+ Regulatory T-cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. To achieve the highest purity, we separated positive and negative cell fractions by passing the cells through a second column. BLM-treated recipient C57BL/6 mice received an intravenous tail vein injection of 1 × 106 isolated donor cells on day 14 after initiating BLM treatment. The dose of Tregs was determined according to a study performed by D’Alessio FR and colleagues [16]. Mice were sacrificed on day 28, and the lungs were removed and subjected to histological and biochemical analyses. Blood collected by cardiac punctures and lung homogenates were subjected to cytokine or chemokine analyses.
Hydroxyproline measurement
The total collagen content of the right lung was determined by hydroxyproline assay [17]. After acid hydrolysis of the right lung with 12 N HCl at 100 °C for 20 h in a sealed glass tube (Iwaki, Tokyo, Japan), the hydroxyproline content was determined by high-performance liquid chromatography.
Immunohistochemistry for fibroblast growth factor (FGF) 9
Sections of paraffin-embedded lung lobes were deparaffinized and rehydrated. Antigen retrieval was achieved by boiling at 105 °C for 7 min in 10 mM citrate buffer (pH 6.0), followed by gradual cooling to room temperature. Then, the sections were treated with 3% hydrogen peroxide in methanol for 20 min and blocked with 10% normal goat serum (NICHIREI BIOSCIENCES, INC., Tokyo, Japan) at room temperature for 10 min. Sections were incubated with an anti-FGF9 polyclonal antibody (Abcam, Cambridge, MA, USA) for 30 min at room temperature. For FGF9 staining, tissue sections were incubated with a secondary anti-rabbit antibody (NICHIREI BIOSCIENCES, INC.) for 30 min at room temperature. FGF9 expression was visualized using Histofine Simple Stain Mouse MAX-PO (R) and Histofine Simple Stain AEC Solution (NICHIREI BIOSCIENCES, INC.).
Enzyme-linked immunosorbent assay (ELISA) experiments
Mouse plasma FGF9 and IL-10 levels were measured using ELISA kits purchased from Cloud-Clone Corp. (Houston, TX, USA) and R&D Systems Inc. (Minneapolis, MN, USA), respectively. Approximately 0.5–1.0 mL of blood was collected from each mouse on day 28 by cardiac puncture, placed in a tube containing EDTA and aprotinin, incubated at 4–8 °C for 15 min, and then centrifuged at 5000 rpm for 2 min at 4 °C to separate the plasma. Plasma samples were stored at − 20 °C until analysis. For chemoattractant chemokine (CC motif) ligand 2 (CCL2) measurement in the lungs, the right lung of each mouse was added to 1.0 mL ice-cold lysis buffer (100 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail [Complete mini; Roche Diagnostics, Basel, Switzerland]), and then homogenates were prepared with a 2-mL tissue grinder (Wheaton Industries, Millville, New Jersey, USA). After centrifuging the homogenate at 10,000×g for 5 min at 4 °C, supernatants were prepared from the lung homogenates, and CCL2 concentration was measured using ELISA kits from Cloud-Clone Corp.
ELISA was performed to determine the concentrations of FGF9, IL-10, and CCL2 following the manufacturer’s protocols. Briefly, supernatants of each sample were diluted in PBS, and 100 μL of the diluted samples was assayed with the kits. The optical density was measured at a wavelength of 450 nm using a microtiter plate reader. All samples were measured in duplicate.
Immunohistochemistry for col-I and CD45
Sections of paraffin-embedded lung lobes were deparaffinized and rehydrated. Antigen retrieval was achieved by boiling at 105 °C for 7 min in 10 mM citrate buffer (pH 6.0) (LSI Medience Corporation, Tokyo, Japan), followed by gradual cooling to room temperature. Then, the sections were blocked with 1% bovine serum albumin (BSA) (Sigma–Aldrich Corporation, St. Louis, MO, USA) for 20 min at room temperature. Tissue sections were incubated with anti-CD45 and anti-Col-I antibodies (Abcam) overnight at 4 °C followed by Alexa Fluor 488-conjugated and Alexa Fluor 568-conjugated antibodies (Abcam) for 1 h at room temperature. VECTASHIELD mounting medium dispersed over the entire section to counterstain the nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc. Burlingame, CA, USA).
Immunohistochemistry for FGF9 and E-cadherin
Sections of paraffin-embedded lung lobes were deparaffinized and rehydrated. Antigen retrieval was achieved by boiling at 105 °C for 7 min in 10 mM citrate buffer (pH 6.0), followed by gradual cooling to room temperature. Then, the sections were blocked with 1% BSA for 20 min at room temperature. For FGF9 staining, tissue sections were incubated with an anti-FGF9 antibody (Abcam) for 1 h at room temperature followed by an Alexa Fluor 568-conjugated antibody (Abcam) for 30 min at room temperature. Subsequently, they were blocked with 1% BSA for 20 min at room temperature and incubated with an anti-E-cadherin antibody (Merck KGaA, Darmstadt, Germany) at 4 °C overnight followed by an Alexa Fluor 488 antibody for 30 min at room temperature. VECTASHIELD mounting medium dispersed over the entire section to counterstain the nuclei with DAPI.
Splenectomy
Either on day 0 or 14 after BLM challenge, the abdominal cavity of mice was opened above the left kidney during isoflurane anesthesia. Then, the spleen vessels were carefully cauterized and the spleen was removed. On day 28, mice were sacrificed and lungs were subjected to HE and Masson’s trichrome staining to quantify the fibrotic score using Ashcroft method.
Statistical analysis
Animal experiments involved at least 6 mice in each treatment group, unless otherwise stated. Comparisons among multiple groups were analyzed by 1-way analysis of variance with the Tukey–Kramer post-hoc correction to adjust for multiple comparisons. An unpaired 2-tailed Student’s t test was used for single comparisons. Data were analyzed using JMP 9 software, version 9.0.3 (SAS Institute Inc., Cary, NC, USA). Differences were considered statistically significant when P values were less than 0.05. Data are expressed as the mean ± standard deviation.