Animals
MiR-155−/−C57BL/6 knock-out (miR-155 KO) mice, Csf1r+155Tg/Tg knock-in (miR-155 KI) mice, and Csf1r−155Tg/Tg wild-type (WT) mice were kindly provided by the CBR Institute for Biomedical Research, Harvard Medical School, Boston, USA. C57BL/6 as WT mice were purchased from the Jackson Laboratory. Colony-stimulating factor 1 receptor (Csf1r), also known as macrophage colony-stimulating factor (M-CSF) receptor, controls the production, differentiation and function of macrophages. As we know, macrophages play crucial roles (such as phagocytosis and trigging the inflammatory response) in acute gout including in the initial phase, in development, and in remission. The mice were housed at 24 ± 2 °C under 12-h light/12-h dark cycles in a pathogen-free facility; 8- to 10-week-old males were used to perform the experiments. The handling of mice and experimental procedures in this study were performed in accordance with the requirements of the Institutional Animal Care and Use Committee of Henry Ford Health System.
Preparation of MSU crystals
Briefly, 1.0 g uric acid (Sigma-Aldrich) was dissolved in 200 ml boiling distilled water containing 6.0 ml 1 M NaOH. After adjusting the pH of the solution to 7.2 with HCl, crystals that formed were sterilized by heating at 180 °C for 2 h [13]. The solution was gradually cooled by stirring at room temperature and stored overnight at 4 °C. The precipitate was filtered from the solution, dried under low heat, and suspended in phosphate-buffered saline (PBS) at a concentration of 50 mg/ml. All reagents were prepared under pyrogen-free conditions.
BMDM culture and MSU stimulation
Bone marrow cells obtained from the femoral bones of C57BL/6(WT), miR-155 KO, Csf1r+155Tg/Tg (miR-155 KI), and Csf1r−155Tg/Tg (WT) mice, respectively, were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml). To induce the proliferation and differentiation of myeloid progenitors to macrophages, the medium was supplemented with 30 ng/ml M-CSF (#0914245, Peprotech). The cells were washed and received fresh medium with M-CSF every 2–3 days. After 7 days the cells were harvested with 0.25% trypsin. Dead cells were first gated out by propidium iodide (PI) staining. The phenotypic validation of BMDMs was performed by flow cytometry with staining using fluorescein isothiocyanate (FITC)-conjugated anti-CD11b and phycoerythrin (PE)-conjugated anti-F4/80 antibodies (both diluted 1:200). The BMDMs are double-positive for CD11b and F4/80. According to the experimental protocol, the MSU crystal suspension (MSU 2.5 mg/ml concentration) was added to the incubated BMDMs (MSU 100 μg/ml final concentration) for 4 or 8 h and the ratio of TNF-α production from BMDMs treated with MSU for 2 or 4 h were measured.
Gout model
Mice were placed under anesthesia (150:10 mg/kg ketamine:xylazine injected intraperitoneally) and were injected with MSU crystals into the right foot pad (1 mg in 40 μl PBS) or ankle joint (0.5 mg in 20 μl PBS). The same volume of sterile saline was injected into the left foot pad or ankle joint at the same time to serve as the control. Inflammation parameters were evaluated following MSU crystal injection at different time points (6, 24, and 48 h). Paw swelling and the size of ankle joints were measured with an electronic caliper at the indicated time points [14,15,16]. For an air pouch model, 5 ml sterile air was first injected subcutaneously into the back of mice to form an air pouch, and then 3 ml sterile air was injected to the air pouch at day 3 and day 5. At day 7, MSU suspension (3 mg in 1 ml PBS) was injected into the air pouch [17]. For MSU-induced peritonitis, MSU crystals (3 mg in 0.5 ml PBS) were injected into the peritoneal cavity as part of the intraperitoneal gouty model [7]. The total number of air pouch and peritoneal cavity exudate cells were harvested after 4 or 8 h and counted by a hemocytometer.
Real-time quantitative polymerase chain reaction
The cultured BMDMs were harvested after 7 days. Total RNA of BMDMs was extracted using Trizol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using reverse transcription reagents (Invitrogen, USA) according to the manufacturer’s protocols. Real-time quantitative polymerase chain reaction (qPCR) was performed using the ABI Prism 7900HT Detection System (Applied Biosystems, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The gene primers sequences were synthesized by eurofins genomics (Louisville, USA) as follows. TNF-α: forward 5’-ACAAAGGTGCCGCTAACCACATGT-3′, reverse 5’-ATGCTGCTGTTTCAGTCGAAGGCA-3′; IL-1β: forward 5’-GGGCCTCAAAGGAAAGAATC-3′, reverse 5’-CTCTGCTTGTGAGGTGCTGA-3′; β-actin: forward 5’-CAACGAGCGGTTCCGATG-3′, reverse 5’-GCCACAGGATTCCATACCCA-3′. The β-actin as an internal control was used to normalize the gene expression. Gene expression was analyzed using the 2–ΔΔCT method.
The expression of miR-155 in BMDMs was measured using Taqman MicroRNA Assays (Applied biosystems, Foster City, CA, USA) according to the manufacturer’s protocols. The Taqman MicroRNA Assay for U6 snRNA was used to normalize the relative abundance of miRNAs.
Enzyme-linked immunosorbent assay (ELISA)
IL-1β protein levels in lavage fluids of the air pouch and peritoneal cavity models were measured by ELISA using kits from eBioscience (cat. no. 88–7013-88; San Diego, CA, USA) following the manufacturer’s instructions. The 96-well microplates were read using a VICTOR X3 plate reader.
Statistical analysis
Data were analyzed with GraphPad Prism 5. Differences between experimental groups were tested using the unpaired t test. Data are expressed as mean ± SEM. P < 0.05 was considered to denote statistical significance.
