Animals
Mlc1 TG mice (B6; CBB6(129)-Tg(Mlc1-tTA)2Rhn) were a gift from K.F. Tanaka (Keio University). SNX25 constitutive KO (Snx25 +/-) mice (B6/N-Snx25tm1a/Nju, Nanjing BioMedical Research Institute of Nanjing University; strain name, B6/N-Snx25tm1a/Nju, strain number, T001400) were obtained from Nanjing BioMedical Research Institute of Nanjing University (NBRI). Snx25 cKO mice were generated by first crossing our Snx25 LacZ/+ mice with CAG-Flpo mice (B6.Cg-Tg(CAG-FLPo)/1Osb), which were a gift from M. Ikawa (Osaka University), in order to excise the LacZ cassette framed by Frt sites and obtain an allele with floxed exon 4 (Snx25loxP/loxP mice)54. We crossed Advillin-cre mice (B6.Cg-Tg(Avil-Cre/ERT2)AJwo/J) (Jackson Laboratory, Stock No: 032027) with Snx25loxP/loxP mice to obtain AvilCreERT2/WT; Snx25loxP/loxP mice. We crossed Cx3cr1CreERT2 mice (B6.129P2(C)-Cx3cr1tm2.1(Cre/ERT2)Jung/J) (Jackson Laboratory, Stock No: 020940) with Snx25loxP/loxP mice to obtain Cx3cr1CreERT2/WT; Snx25loxP/loxP mice. We crossed Cx3cr1CreERT2/WT; Snx25loxP/loxP mice with reporter mice RCL-eNpHR3.0-EYFP (Ai39 mice, Jackson Laboratory, Stock No: 014539) to obtain Cx3cr1CreERT2/WT; Snx25loxP/loxP; Ai39/+ mice. We crossed Cx3cr1CreERT2/WT; Snx25loxP/loxP mice with reporter mice RCL-ChR2(H134R)/EYFP (Ai32 mice, Jackson Laboratory, Stock No: 024109) to obtain Cx3cr1CreERT2/WT; Snx25loxP/loxP; Ai32/+ mice. C57BL/6-Tg (CAG-EGFP) mice were purchased from Japan SLC (Hamamatsu, Japan). They were housed in standard cages under a 12 h light/dark cycle and temperature-controlled conditions. All the protocols for the animal experiments were approved by the Animal Care Committee of Nara Medical University in accordance with the policies established in the NIH Guide for the Care and Use of Laboratory Animals. This study was also carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org/).
Behavioral test
Paw mechanical sensitivity was assessed using von Frey’s filaments based on the up-down method developed by Chaplan55. The von Frey’s filaments used were: 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2, 4 g. Animals were acclimatized for at least 15 min in individual clear acrylic cubicles (10 × 10 × 10 cm) placed on top of an elevated wire mesh. Quick withdrawal or licking of the paw after the 3 s stimulus was considered a positive response. Threshold values were derived according to the method described by Chaplan55. For the formalin test, 10 µl of 5% formalin was injected subcutaneously into the plantar surface of the right hind paw. PBS (10 µL) was injected into the plantar surface of the left hind paw. We calculated the durations of lifting, shaking, and licking of the formalin-injected paw. For hot plate test, mice were acclimatized for at least 2 h (1h/day x 2days) in individual clear acrylic cubicles placed on the preheated plate. The withdrawal latency in response to the stimulus was determined manually. To assess sensitivity to weak tactile stimulus, mice were acclimatized for at least 15 min in individual clear acrylic cubicles (10 × 10 × 10 cm) placed on top of an elevated wire mesh. Calibrated von Frey filaments (0.008-1g) were used to stimulate the plantar surface of the hind paws43, 44. Withdrawal following or immediately after the 1 s stimulus were considered as a positive response. Plantar surface was stimulated 10 times with each filament and the number of positive responses was measured43, 44. In all the behavioral tests, examiners were always blind to the genotypes of mice, the kinds of treatments, and the sides of hind paws that received injections. After the evaluation was done, the behavioral data were analyzed by a different researcher.
Surgery of spared nerve injury (SNI) model
Surgical procedures were performed under 2% isoflurane anesthesia. SNI was made by a 6-0 polypropylene thread with tight ligation of the two branches of the right sciatic nerve, the common peroneal and the tibial nerves, followed by transection and removal of a 2-mm nerve portion. The sural nerve remained intact and any contact with or stretching of this nerve was carefully avoided. Muscle and skin were closed in two distinct layers.
Reagents
For tamoxifen (TAM) treatment, we employed oral administration. TAM (Sigma-Aldrich, St. Louis, MO, USA) was mixed with powdered chow (0.5 mg/g normal chow). This oral administration method is convenient for continuous administration and results in efficient induction of recombination while minimizing stress on the mice20. For Snx25 deletion in BMDMs, we treated cells with Snx25-specific siRNA (Sigma Aldrich) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher). For Snx25 deletion in BMDMs derived from Cx3cr1CreERT2/WT; Snx25loxP/loxP mice, we treated cells with 1 mM 4-OH-tamoxifen (4-OHT, Sigma Aldrich) for 24 h. For inhibition of proteasomes, we used 5 mM MG132 (Sigma Aldrich) for 4 h. For depletion of macrophages in hind paw skin, we used clodronate liposome (MKV300, Cosmo Bio, Tokyo, Japan).
Clodronate liposome treatment
Twenty-microlitter of 10 mg/ml clodronate liposomes or control liposomes were subcutaneously injected into the right side of the hind paw skin on days 0 and 3. Skin sections were stained with anti-CD206 or anti-MHCII at day 6.
4-OHT treatment
For depletion of SNX25 in dermal macrophages, we administered 4-OHT (40 ng/mL, 10mL) by intradermal injection daily for seven days into Cx3cr1CreERT2/WT; Snx25loxP/loxP mice or Cx3cr1CreERT2/WT; Snx25loxP/loxP; Ai32/+ mice. Vehicle was injected into the contralateral side of the same animal. At 8 days after the last injection, von Frey test was performed. Sections were stained with anti-GFP and anti-MHCII at day 11. For depletion of SNX25 in DRG, we administered 4-OHT (200 ng/mL, 20mL) into exposed DRG (L4) of Cx3cr1CreERT2/WT; Snx25loxP/loxP; Ai32/+ mice. At 5 days after administration, von Frey test was performed. DRG sections were stained with anti-GFP and anti-MHCII at day 5.
Immunohistochemistry
Mice were anesthetized and perfused transcardially with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) (PB). Skin, DRG, sciatice nerve and spinal cord were removed, postfixed overnight in the same fixative, and then immersed in 30% sucrose in PB overnight. Next, the tissues were frozen in powdered dry ice, embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), and stored at -80°C prior to sectioning. Eighteen-micrometer-thick sections were immersed in PBS containing 5% bovine serum albumin and 0.3% Triton X-100 for 1 h. Antibodies against mouse anti-CGRP (1:500, ab1887, abcam, Cambridge, UK), rabbit anti-c-Fos (1:10000, 226003, Synaptic Systems, Gottingen, Germany), rabbit anti-TRPV1 (1:100, KM018, Trans Genic, Fukuoka, Japan), rabbit anti-TrkA (1:150, ab76291, abcam), mouse anti-NF200 (1:1000, N0142, Sigma-Aldrich), mouse anti-PGP9.5 (1:500, ab8189, abcam), rat anti-MHCII (1:100, NBP1-43312, Novus Biologicals, Centennial, CO, USA), goat anti-CD206 (1:500, AF2535, R&D Systems, Minneapolis, MN, USA), rabbit anti-Iba1 (1:500, 019-19741, Wako, Osaka, Japan), rabbit anti-NGF (1:1000, sc-548, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-GFP (1:5000, 04404-84, nacalai tesque, Kyoto, Japan), rabbit anti-GFP (1:5000, A6455, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-SNX25 (1:500, 13294-1-AP, Proteintech, Rosemont, IL, USA), biotin mouse anti-CD4 (1:200, 100403, BioLegend, San Diego, CA), biotin mouse anti-CD8a (1:200, 100703, BioLegend), biotin mouse anti-Gr1 (1:200, 108403, BioLegend), biotin mouse anti-NK1.1 (1:200, 108703, BioLegend), biotin mouse anti-CD19 (1:200, 13-0193-81, eBioscience San Diego, CA), were applied overnight at 4°C. Alexa Fluor 488- and 594- (1:1000, Life Technologies, Grand Island, NY, USA) conjugated IgG were used as secondary antibodies. Sections were subjected to fluorescent Nissl staining (Neurotrace, Molecular Probes, Eugene, OR, USA). Images were captured using a confocal laser scanning microscope (C2, Nikon, Tokyo, Japan). For 3,3’-diaminozidine (DAB) staining, 8 mm-thick sections were immersed in PBS containing 5% bovine serum albumin and 0.3% Triton X-100 for 1 h. Antibodies against mouse anti-PGP9.5 (1:500, ab8189, abcam) were applied overnight at 4°C. After immunoreaction with DAB containing 0.03% H2O2 solution, sections were enclosed with mounting medium.
Microarray
Total RNA was isolated from the bone marrow of C57BL/6 mice and Mlc1 TG mice using the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany). The RNA samples were analyzed with Affymetrix GeneChip mouse genome 430 2.0 Arrays by Takara Bio (Otsu, Shiga, Japan).
Next-generation sequencing (NGS)
Whole-genome DNA was isolated from Mlc1 TG mice using the NucleoBond AXG Column (Macherey-Nagel). Identification of the loci of transgene insertion was performed by Takara Bio, followed by NGS on the Illumina sequencing platform.
qRT-PCR
Total RNA of cells or tissues was extracted using a NucleoSpin RNA kit (Macherey-Nagel). Total RNA extracts were reverse-transcribed using random primers and a QuantiTect Reverse Transcription kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Real-time PCR was performed using a LightCycler Quick System 350S (Roche Diagnostics), with THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan). PCR primers used in this study were as follows: β-actin sense primer, 5¢-AGCCATGTACGTAGCCATCC-3¢; β-actin antisense primer, 5’-CTCTCAGCTGTGGTGGTGAA-3’; Mlc1 sense primer, 5’-CTGACTCAAAGCCCAAGGAC-3’; Mlc1 antisense primer, 5’-AGCGCAAATAATCCATCTCG-3’; Mov10l1 sense primer, 5’-TGCTTCTGAACGTGGGACAGG-3’; Mov10l1 antisense primer, 5’-ACACAGCCAATCAGCACTCTGG-3’; Ngf sense primer, 5’-TCAGCATTCCCTTGACACAG-3’; Ngf antisense primer, 5’-GTCTGAAGAGGTGGGTGGAG-3’; Nrf2 sense primer, 5’-GCAACTCCAGAAGGAACAGG-3’; Nrf2 antisense primer, 5’-GGAATGTCTCTGCCAAAAGC-3’; Scn9a sense primer, 5’-AAGGTCCCAAGCCCAGTAGT-3’; Scn9a antisense primer, 5’-AGGACTGAAGGGAGACAGCA-3’; Scn10a sense primer, 5’-GCCTCAGTTGGACTTGAAGG-3’; Scn10a, antisense primer, 5’-AGGGACTGAAGAGCCACAGA-3’; Trpv1 sense primer, 5’-CCCTCCAGACAGAGACCCTA-3’; Trpv1 antisense primer, 5’-GACAACAGAGCTGACGGTGA-3’.
Western blotting
Samples (cells or tissues) were lysed with 10 mM Tris, pH 7.4, containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, and 0.1% sodium dodecyl sulfate (SDS). The homogenate was centrifuged at 20,600 g for 5 min, and the supernatant was stored at -20°C. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce). Equal amounts of protein per lane were electrophoresed on SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane. The blots were probed with rabbit anti-SNX25 (1:1000, 13294-1-AP, Proteintech), rabbit anti-TRPV1 (1:100, KM018, Trans Genic), rabbit anti-TrkA (1:10000, ab76291, abcam), goat anti-CD206 (1:1000, AF2535, R&D Systems), rabbit anti-NGF (1:200, sc-548, Santa Cruz Biotechnology), rabbit anti-Nrf2 (1:500, sc-722, Santa Cruz Biotechnology), rabbit anti-HO-1 (1:500, ADI-SPA-896, Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-TGFbRI (1:200, sc-398, Santa Cruz Biotechnology) and rabbit anti-GAPDH (1:2000, ABS16, Burlington, MA, USA) antibodies. Immunoblot analysis was performed with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG using enhanced chemiluminescence Western blotting detection reagents (Wako). Data were acquired in arbitrary densitometric units using Scion image software.
Co-immunoprecipitation
Cells were lysed with 50 mM Tris, pH 7.4, containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and then incubated 4°C for 20 min with rotation. The lysate was centrifuged at 21,500 g for 15 min and the supernatant was collected. A rabbit IgG against Nrf2 (sc-722, Santa Cruz Biotechnology) was incubated with SureBeads Protein G Magnetic Beads (Bio-Rad, Berkeley, CA) for 10 min. The mixture was added to the supernatant for immunoprecipitation, incubated for 1 h with rotation, and then the immunobound protein was eluted.
Primary DRG neurons
DRGs from Snx25 +/- and WT littermate mice were quickly collected in DMEM/F12 medium and incubated for 90 min at 37°C in a 0.2% collagenase solution. After dissociation, DRGs were transferred to a tube containing DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution. Ganglia were gently triturated using pipettes. After centrifugation, cells were resuspended in DMEM/F12 supplemented as above and plated on poly-L-lysine-coated culture dishes. Neurons were kept at 37°C in 5% CO2 and the medium was changed to DMEM/F12 with B27 supplement 8 h after plating.
Fluo-4 Calcium assay
DRG neurons were seeded in 96-well cell culture plates at a density of 1.5 × 104 cells per well and cultured overnight. Intracellular calcium responses to capsaicin were measured using Calcium kit II-Fluo4 (CS32, Dojindo, Kumamoto, Japan) in accordance with the manufacturer's instructions. The temperature of the platform was controlled to 37°C. Cells were fluorescently imaged at 495-nm excitation every 7 s, and the fluorescence intensities of neurons were quantified at 515 nm. Fluorescence intensities of neurons were quantified simultaneously for the entire well. Capsaicin (10 mM) was added to measure the response.
Bone marrow transplantation (BMT)
BM recipients were male 8-week-old C57BL/6J, Snx25 +/+, Snx25 +/-, or Snx25loxP/loxP mice. Mice were intraperitoneally injected with the chemotherapeutic agent busulfan (30 μg/g body weight; B2635, Sigma-Aldrich) in a 1:4 solution of dimethyl sulfoxide and PBS 7, 5, and 3 days prior to bone marrow transfer. All mice were treated with antibiotics (trimethoprim/sulfamethoxazole) for 14 days after busulfan treatment. Bone marrow-derived cells were obtained from the femur and tibia of 5-week-old C57BL/6-Tg (CAG-EGFP), Snx25 +/+, Snx25 +/-, or Cx3cr1CreERT2/WT; Snx25loxP/loxP mice and resuspended in PBS with 2% FBS. Bone marrow-derived cells (1 × 106) were transferred to 8-week-old male C57BL/6, Snx25 +/+, Snx25 +/-, or Snx25loxP/loxP recipient mice by tail vein injection (100 μL). For quantitative analysis, engraftment was verified by determining the percentage of EGFP-expressing cells in the blood. We counted the numbers of EGFP+ cells in peripheral blood by flow cytometry and confirmed efficient chimerism as demonstrated by the large proportions of circulating blood leukocytes expressing EGFP.
Bone marrow-derived macrophage (BMDM) culture and application of mechanical stretch
Bone marrow cells were obtained from femur and tibia of 8-week-old male C57BL/6, Snx25 +/+, Snx25 +/-, or Cx3cr1CreERT2/WT; Snx25loxP/loxP mice and cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, and 0.01% macrophage colony stimulating factor (M-CSF). After 6 days, the BMDMs were transferred to 3.5-mm dishes in RPMI-1640 containing 10% FBS and 1% penicillin/streptomycin. After overnight incubation, qPCR or Western blot analysis of BMDMs was performed. For mechanical stretch experiment, BMDMs were seeded to stretch chamber (STB-CH-04, STREX, Osaka, Japan) coated with 0.05 mg/ ml fibronectin (Millipore, #FC010) and exposed to stretch up to 12.5 %. Un-stretch control BMDMs were treated equally without application of mechanical stretch.
PCR array
The mouse inflammatory response and autoimmunity RT2 Profiler PCR Array kit (PAMM-077Z, Qiagen) in a 96-well format was used. This kit profiles the expression of 84 genes that encode inflammatory response, autoimmunity, and other genes related to inflammation. Hind paw skins were quickly dissected 3 d after formalin injection, frozen rapidly, and stored at −80°C until use. Total RNA was purified using the NucleoSpin RNA kit (Macherey-Nagel) in accordance with the manufacturer's instructions. cDNA was obtained from purified RNA using the RT2 First Strand Kit (Qiagen) provided with the PCR Array kit. cDNA template mixed with PCR master mix was dispensed into each well and real-time PCR was performed. Three independent arrays (three animals) were performed.
Fluorescent in situ hybridization (FISH)
FISH was performed with a probe targeting Cx3cr1 mRNA using the RNAscope Fluorescent multiplex reagent kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions.
Nerve ligation assay
To assess NGF/TrkA complex trafficking from the periphery toward the DRG cell bodies, we carefully exposed the left sciatic nerve and tightly ligated the nerve with one 6.0 suture in WT and Snx25 +/- mice. Eight hours after the surgery, mice were terminally anesthetized and quickly perfused with 4% PFA. After perfusion, the left sciatic nerve was excised, post-fixed for 24 h in the same perfusion fixative, cryoprotected in 30% sucrose for 48 h at 4°C, and then frozen in tissue freezing medium (O.C.T.). Longitudinal sections (18 μm) of the left sciatic nerve were cut on a cryostat and then stored at -30°C before staining. Sciatic nerve sections were stained with the primary antibody against rabbit anti-TrkA (1:150, ab76291, abcam). Alexa Fluor 594- (Life Technologies) conjugated IgG was used as the secondary antibody.
Generation of constructs and transient transfection of 293T cells
PCR cloning was performed to amplify Snx25 and Nrf2 cDNA with a primer having an optimal Kozak consensus sequence just before the in-frame first ATG of the mouse Snx25 and Nrf2 genes. Fragments were inserted into the pcDNA3.1/Myc-His vector (Invitrogen). Using the LipofectAMINE reagent (Invitrogen), 293T cells were transfected with a Snx25 and Nrf2 construct according to the manufacturer’s instructions.
Fluorescence activated cell sorting (FACS)
Hind paw skin from Cx3cr1CreERT2/WT; Snx25loxP/loxP mice (±TAM) were collected and dissociated using Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, Germany). After dissociation, the cells were stained with various combinations of mAbs. The mAbs used in this study were Alexa Fluor 488–anti-CD11b (1:100, Biolegend, San Diego, CA), Alexa Fluor 647-anti-F4/80 (1:100, Biolegend), PE/Cyanine7 anti-CD45 (1:100, Biolegend). After washing, the cells were stained with 7-AAD (BD Biosciences) to exclude the dead cells from analysis. Macrophages (CD45+ CD11bhigh F4/80high) from the hind paw skin were isolated by the FACSAria (> 96% purity).
Quantification and statistical analysis
Quantifications were performed from at least three independent experimental groups. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test or Welch’s t-test for two groups or one-way ANOVA for multiple groups, and significant differences between group means were identified with the Tukey–Kramer test. Statistical significance is indicated as asterisks. *p < 0.05, **p < 0.01. All n are indicated in Figure legends.