All chemical reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Arhalofenate acid (MBX-102 acid), the active form of arhalofenate, was used for in-vitro studies. Arhalofenate (MBX-102) was used for in-vivo studies. MSU crystals were prepared as described previously , suspended at 25 mg/mL in sterile, endotoxin-free phosphate-buffered saline (PBS), and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, MD). A-769662 was from LC laboratories (Woburn, MA). Antibodies to phospho-AMPKα (Thr172) and total AMPKα (recognizing both AMPKα isoforms), SIRT1, TFAM, TRX1, TRX2, and TXNIP were from Cell Signaling Technology (Danvers, MA). Antibodies to pro-caspase-1 and cleaved caspase-1 (p10) were from Biovision (Milpitas, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.
Subcutaneous air pouch model and flow cytometry analysis
C57BL/6 mice (n = 8–10/group) were subcutaneously injected under the skin adjacent to the back of the neck with sterile air (day 1, 5 mL; day 4, 3 mL) to form air pouches as described previously . On day 4, mice were dosed daily with vehicle (1% carboxymethylcellulose/2% Tween-80), arhalofenate (at a loading dose of 250 mg/kg per oral), or dexamethasone (20 mg/kg intraperitoneally) for 3 days. On day 7, 30 min after the last dose, MSU crystals (20 mg in 5 mL saline) were injected into the air pouch to elicit an acute immune response. After 4 h, the mice were sacrificed and 5 mL of heparinized saline was injected into the air pouch to collect the exudates.
Exudates were centrifuged and resuspended in PBS. The cell suspension was incubated with phycoerythrin (PE)-conjugated rat anti-mouse CD45+ antibody (diluted 1:100; MCA1031PE; AbD Serotech) for all leucocyte staining, and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse Ly-6B.2 alloantigen (diluted 1:100; #MCA771FB; AbD Serotech) for neutrophil staining according to the manufacturer’s protocol. Propidium iodide staining solution (BD Pharmingen™; #556463) was added to the cell suspension to exclude non-viable cells. All live cells were further gated based on CD45 expression for leukocytes. BD™ Compbeads (BD Biosciences; #552845) were used for nonspecific binding of antibodies to optimize fluorescence compensation settings. Counting beads (Spherotech Accuount fluorescent particles; #ACFP-100-3) were added to the stained cells to obtain absolute cell number. FACS analysis was performed on a BD LSR II flow cytometer using Diva software (v6.1.2, Becton Dickinson) and analyzed using FlowJo software (v9.5.3, Tree Star Inc.).
Bone marrow-derived macrophages (BMDMs) were generated as described previously . Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA). After 5–7 days, the M-CSF-derived macrophages were re-plated onto 24-well (5 × 105/well) or six-well (2 × 106/well) plates and primed with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Gemini Bio-products, West Sacramento, CA) for 24 h in complete RPMI medium before treatment with the indicated reagents in fresh RPMI containing only 1% FBS.
Cells were lysed in RIPA buffer with 2 mM sodium vanadate and protease inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates (10–15 μg) were separated by gradient 4–20% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA), probed with antibodies, exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA), and visualized by radiography.
Mouse IL-1β and CXCL1 (KC) were measured using DuoSet enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN).
BMDMs were incubated with MitoSOX Red reagent (Thermo Scientific) reagent (1 μM) to examine mitochondrial ROS generation which was visualized by fluorescence microscopy. Cells were also incubated with 10 nM MitoTracker Green (which is insensitive to ROS) to confirm the localization of MitoSOX Red to mitochondria. Immunofluorescence microscopy was carried out to visually identify p62 puncta and lysosomes and to determine co-localization of p62 and lysosomal-associated membrane protein 1 (LAMP1), which indicates autophagosome and lysosome fusion (i.e., activated autophagy). In brief, cells were fixed and permeabilized with cold methanol. Immunocytochemical staining of cells used rabbit anti-p62 monoclonal antibody (Cell Signaling, #23214) or rabbit anti-LAMP1 antibody (Abcam, #ab24210). Alexa Fluor 488 goat anti-rabbit IgG (Thermo Scientific) and Alexa Flour 555 goat anti-rabbit IgG (Thermo Scientific) secondary antibodies were used to detect p62 and LAMP1, respectively. Imaging was acquired via a confocal microscope (Zeiss LSM 880 Confocal with FAST Airyscan).
Transmission electron microscopy (TEM)
Cells were fixed with 2.5% glutaraldehyde in 0.15 M cacodylate buffer, and postfixed in 1% OsO4 in 0.1 M cacodylate buffer for 1 h on ice, followed by staining en bloc with 2–3% uranyl acetate for 1 h on ice. The cells were dehydrated in a graded series of washes with ethanol (20–100%) on ice followed by one wash with 100% ethanol and two washes with acetone (15 min each) and embedded with Durcupan. Ultrathin (50–60 nm) sections were cut on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 min and Sato’s lead stain for 1 min. Grids were viewed using a JEOL JEM1400-plus TEM (JEOL, Peabody, MA). TEM images were taken using a Gatan OneView digital camera with 4 k × 4 k resolution (Gatan, Pleasanton, CA). Mitochondrial area was determined using the free-hand tool in ImageJ and manually tracing around the mitochondrial outer membrane. The area of each crista membrane was also calculated in the same manner. The sum of the areas of the total complement of cristae was then divided by the sum of the mitochondrial area to obtain the cristae volume density as described previously .
Data are presented as either mean values ± standard deviation (SD) or mean ± standard error of the mean (SEM) as indicated. Statistical analyses were performed by one-way or two-way analysis of variance with Bonferroni post-hoc testing using GraphPad Prism software, version 6. p values less than 0.05 were considered significant.