Cell lines.
HL-60 cells were purchased from American Type Culture Collection (Manassas, VA, USA). HL-60 cells were cultured in RPMI 1640 medium supplemented with L-glutamine (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (Gibco, Billings, MT, USA). Cells were passaged once every two days. For differentiation into neutrophil-like cells, the cells were cultured in a medium supplemented with 1.25% dimethyl sulfoxide (DMSO; Merck, Rahway, NJ, USA) for 4 days.
Animal models.
C57BL/6-Lrrk2tm1.1Miff/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All the mice were housed under standard laboratory conditions in a controlled pathogen-free room. Food and water were provided ad libitum. The protocols used for all animal experiments in this study were approved by Hokkaido University, Japan.
Antibodies and chemicals.
The following antibodies were used in this study: mouse monoclonal antibodies against b-actin (Merck) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal antibodies against MFN2 (Abcam, Tokyo, Japan), LRRK2 (Abcam), LRRK2 pS935 (Abcam), RAB10 (Abcam), RAB10 pT73 (Abcam), and TOMM20 (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit donkey IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Alexa Fluor 488-conjugated anti-rabbit goat IgG and Alexa Fluor 555-conjugated anti-mouse goat IgG antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2) was purchased from Cayman Chemical Co (Ann Arbor, MI, USA).
Bone marrow neutrophils.
Bone marrow neutrophils were prepared by centrifugation using Percoll gradients, as previously described [18]. Purified neutrophils were suspended in ice-cold Hank’s balanced salt solution (HBSS) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.2) and 1% bovine serum albumin (BSA, Rockland Immunochemicals Pottstown, PA, USA) and stored on ice until further use.
Gene silencing.
LRRK2 was silenced by infecting HL-60 cells with lentiviruses constructed by inserting LRRK2 shRNA sequence (sh1: TRCN0000021462; sh2: TRCN0000021460, Merck) into Tet-pLKO-puro (Addgene, Watertown, MA, USA). Tet-pLKO-puro was a gift from Dr. Dmitri Wiederschain [19]. The targeting sequence of the irrelevant (Irr) shRNA has been previously reported [20]. The infected HL-60 cells were selected with 0.8 µg/ml puromycin (Merck). For gene silencing in dHL-60 cells, HL-60 cells expressing tetracycline-inducible LRRK2 shRNA or Irr shRNA were cultured in the presence of 1.25% DMSO for 1 day, followed by 3 days in a medium supplemented with 1.25% DMSO and 0.5 µg/ml doxycycline (Dox; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
Chemotaxis assays.
Transwell chemotaxis assays were performed as previously described [16, 21]. For neutrophils, transwell chemotaxis assays were performed using 24-well transwell chambers (pore size, 3.0 mm; Corning, Corning, NY, USA). For dHL-60 cells, transwell chemotaxis assays were performed using 24-well transwell chambers (pore size, 5.0 mm; Corning). Cells were placed on the upper side of the top chambers, and cells that migrated to the lower side of the chamber membrane were stained with Diff-Quick (Sysmex, Kobe, Japan) and counted. Five microscope fields of view were analyzed for each experiment.
For two-dimensional chemotaxis assays, a Dunn chamber (Hawksley, Sussex, UK) was used. After washing cells, the cells were attached to a coverslip and incubated at 37°C for 10 min in HBSS containing 20 mM HEPES (pH 7.2) and 1% BSA. For neutrophils, coverslips were placed on a Dunn chamber, in which the outer well of the chamber was filled with the same solution supplemented with 10 µM fMLP (MP Biomedicals, Irvine, CA, USA) and a 1:10 dilution of 10% gelatin (Nacalai Tesque) in H2O. Cell migration was recorded by capturing images every 15 s for 20 min. For dHL-60 cells, the outer wells of the Dunn chambers were filled with the same solution, supplemented with 10 nM fMLP. Cell migration was recorded by capturing images every 15 s for 30 min. Image of each cell migration were recorded using an inverted microscope (Axiovert135, Carl Zeiss, Oberkochen, Germany) equipped with a digital camera (AxioCam and Axiovison software; Carl Zeiss). Stacks of images were then analyzed by ImageJ Fiji software (National Institutes of Health, Bethesda, MA, USA), with Chemotaxis and Migration Tool plug-in (Ibidi) for analysis of the cell tracking, velocity, and directional index of each moving cell. Cells that migrated more than 20 µm in their tracks during a 20 min incubation period were used for analysis.
Air pouch analysis.
Air pouch analysis of local inflammation was performed as described previously [22]. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium and injected with 5 ml of air to create a subcutaneous dorsal pouch. After three days, the pouches were reinjected with 3 ml of air. Six days after the first injection, the pouches were injected with 1 ml of 0.5 mg/ml human interleukin-8 (IL-8) (Shenandoah Biotechnology, Warwick, PA, USA) and 0.5% carboxymethylcellulose (FUJIFILM Wako) in PBS. After 4 h, the mice were anesthetized, and the pouches were washed with 2 ml of PBS. The lavage fluid was immediately chilled on ice, the volume was recorded, and the neutrophils were counted.
Immunofluorescence microscopy.
Briefly, dHL-60 cells were attached to coverslips with HBSS containing 20 mM HEPES (pH 7.2) and 1% BSA for 10 min at 37°C. After treatment with or without 10 nM fMLP for 5 min, the cells were fixed with 2% paraformaldehyde at 37°C for 10 min, washed with PBS, and fixed with methanol at -20°C for 5 min. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed with PBS, and then incubated with 1% BSA in PBS for 30 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Nacalai Tesque). High-resolution structured illumination microscopy (SIM) images were acquired using an N-SIM microscope (Nikon, Tokyo, Japan) and NIS-elements software (Nikon), as previously described [16]. The percentage of cells with tubular mitochondria was calculated by counting cells with tubular mitochondria (length > 3 µm) and dividing this number by the total number of cells observed within each microscopic field, as described previously [16].
Mitochondrial OXPHOS activity.
The mitochondrial respiratory capacity was measured at 37°C by using a high-resolution respirometer (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) as described previously [16]. Briefly, 2×106 dHL-60 cells were added to the respirometer chamber filled with 2 ml of MiR05 medium (110 mM sucrose, 60 mM potassium lactobionate, 0.5 mM ethylene glycol tetraacetic acid, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES [pH 7.1], and 1% BSA). The cells were then stimulated with 1 µM fMLP for 5 min and permeabilized with 5 µM digitonin (Merck). Substrates of OXPHOS and ADP were added to the respirometer chamber in the following order: (1) 2 mM malate, 10 mM glutamate, and 5 mM pyruvate (complex I-linked substrates); (2) 5 mM ADP + 3 mM MgCl2; and (3) 10 mM succinate (complex II-linked substrates). The O2 consumption rates (OCR) were expressed as O2 flux normalized to 1×106 cells. Data acquisition and analysis of data were performed using DatLab software (Oroboros Instruments), as described previously [16].
Mitochondrial fractionation.
Mitochondrial fractionation was performed as previously described with some modifications [23]. dHL-60 cells were pre-incubated in HBSS containing 20 mM HEPES (pH 7.2) for 5 min at 37°C and then stimulated with or without 1 µM fMLP at 37°C for 5 min. The cells were lysed in hypotonic buffer (10 mM Tris-MOPS [pH7.4], 1 mM EDTA-Tris [pH7.4]) with rotation at 4°C for 20 min. The lysed cells were homogenized by 15 repeated passes through a 30-gauge needle attached to a 1 ml syringe. The homogenate was centrifuged at 700 × g and 4°C for 10 min, and the supernatant was centrifuged again at 700 × g and 4°C for 10 min. The supernatant was then centrifuged at 7,000 × g and 4°C for 10 min. The pellet was suspended with isotonic buffer (10 mM Tris-MOPS [pH7.4], 1 mM EDTA-Tris [pH7.4], 200 mM sucrose) and was centrifuged at 7,000 × g and 4°C for 10 min. The pellets were again suspended with isotonic buffer and was centrifuged at 7,000 × g and 4°C for 10 min. The pellets were resuspended in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 20 mM NaF, 10 mM sodium diphosphate, and a protease inhibitor cocktail for Use with Mammalian Cell and Tissue Extracts (Nacalai Tesque)) and incubated on ice for 10 min. The lysates were sonicated and centrifuged at 21,900 × g and 4°C for 30 min. This supernatant was used as the mitochondrial fraction.
GTP-binding assay.
The GTP-binding assay for MFN2 was performed as previously described with some modifications [24]. dHL-60 cells were pre-incubated in HBSS containing 20 mM HEPES (pH 7.2) for 5 min at 37°C, and then stimulated with or without 1 µM fMLP at 37°C for 5 min. After stopping the reaction by adding ice-cold HBSS containing 20 mM HEPES (pH 7.2), the cells were centrifuged at 200 × g and 4°C for 3 min. The cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 5 mM MgOAc, 1 mM DTT, 1 mM Na3VO4, 20 mM NaF, 10 mM sodium diphosphate, and a protease inhibitor cocktail) on ice for 10 min. Cell lysates were sonicated and centrifuged at 21,900 ×g and 4°C for 10 min. After preclear, 250 µg cell lysates were incubated with GTP-agarose beads (Abcam) for 2 h at room temperature. The beads were then washed four times with lysis buffer, and proteins were eluted from the beads using the SDS sample buffer. The GTP-bound form of MFN2 was detected by western blotting with an anti-MFN2 antibody.
Statistical analysis.
All statistical analyses were performed using GraphPad Prism version.8 (GraphPad Software, Inc., Boston, MA, USA). Differences between multiple groups were calculated using one-way ANOVA followed by the Tukey-Kramer test. Differences between the two groups were calculated using a two-tailed Student’s t-test.