Strange IndiaStrange India

Ethical statement for animal experiments

Animal experiments were approved by the Monash Animal Research Platform ethics committee and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University (approval numbers 13294, 13335, 17075, 14013 and 23006).


Wild-type C57BL/6 J mice were from the Monash Animal Research Platform. Sperm from Nav1.8cre+/+ mice (B6.129-Scn10atm2(cre)Jnw/H B6, stock ID EM:04582, European Mouse Mutant Archive) were used for in vitro fertilization to generate Nav1.8cre+/− mice on a C57BL/6 J background. Rosa26DTA+/+ mice (B6.129-Gt(ROSA)26Sortm1(DTA)Mrc/J), strain 010527, Jackson Laboratory) were maintained on a C57BL/6 J background. To delete sensory neurons expressing NaV1.8, Nav1.8cre+/− mice were bred with Rosa26DTA+/+ mice to generate Nav1.8cre+/−/Rosa26DTA+/− mice. Nav1.8cre−/−/Rosa26DTA+/− littermates were used as controls. For visualizing NaV1.8+ neurons, Rosa26tdT reporter mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, strain 007914, Jackson Laboratory) were bred with Nav1.8cre+/+ mice to generate Nav1.8cre+/−/Rosa26tdT+/−. Leprdb/db mice (BKS.Cg-Dock7m + /+Leprdb/J, strain 000642) were obtained from Jackson Laboratory. Mice were bred as heterozygotes to generate Leprdb/db and Leprdb/+ littermates. B6.129S2-Ramp1 mouse sperm was kindly provided K. Tsujikawa and used for in vitro fertilization to generate Ramp1fl/+ mice. Ramp1−/− mice were generated by crossing Ramp1fl/fl mice with CAGcre mice (C57BL/6-Tg(CAG-cre)13Miya, RIKEN BioResource Research Center, strain 09807). Specific deletion of Ramp1 in myeloid cells (LysMcre+/−/Ramp1fl/fl mouse) was done by crossing Ramp1fl/fl mice with LysMcre+/+ mice (B6.129P2-Lyzs, RIKEN BioResource Research Center, strain 02302). LysMcre+/− littermates were used as controls. To obtain mice constitutively expressing tdTomato, Rosa26tdT mice were crossed with B6.C-Tg(CMV-cre)1Cgn/J mice from Jackson Laboratory (strain 006054).

Full-thickness skin wound model

Male 10- to 12-week-old mice were used for most experiments, except for experiment with CGRP variant delivery in which female (10- to 12-week-old non-diabetic or 12- to 14-week-old diabetic) mice were used. Full-thickness punch-biopsy wounds (5 mm in diameter) were created while mice were under isoflurane anaesthesia as described25,26. For analgesia, mice received subcutaneous administration of 0.1 mg kg−1 buprenorphine. In non-diabetic mice, wounds were covered with a round seal spot plaster (22.5 mm, Livingstone International, Australia) secured with 3M surgical tape. In experiments involving CGRP delivery, a nylon ring (Zenith 5/16 inch and M8 Nylon Washer) was attached with superglue (UHU) to prevent wound contraction. Wounds received topical treatment with either 10 μl of saline (PBS) or a CGRP variant in PBS. Solutions were applied in two different dosages: 250 ng of CGRP or equimolar eCGRP on day 1 post-injury for the low dose, and 500 ng of CGRP or equimolar eCGRP on day 1 and day 4 post-injury for the high dose. For diabetic mice, 4 wounds were created and treated with PBS or CGRP variant in PBS (500 ng CGRP or equimolar eCGRP) on day 1 and day 3.

Volumetric muscle loss model

Non-diabetic (10- to 12-week-old) and diabetic (12- to 14-week-old) male mice underwent isoflurane anaesthesia. For analgesia, mice received subcutaneous administration of 0.1 mg kg−1 buprenorphine. A 1-cm unilateral incision was made, exposing the fascia. Muscle injuries were created either with a 3-mm biopsy punch or by excising a 3 mm × 5 mm segment of the quadriceps, including the rectus femoris muscle. In experiments involving CGRP delivery, muscle defects were covered with a fibrin matrix (40 μl total, 8 mg ml−1 fibrinogen (Enzyme Research Laboratories), 12 U ml−1 bovine thrombin (Sigma), 5 mM CaCl2, and 17 μg ml−1 aprotinin (Roche, Sigma)) containing CGRP (250 ng or 1 μg for non-diabetic mice and 1 μg for diabetic mice) or equimolar eCGRP. The incision site was sutured with non-absorbable sutures.

Adoptive transfer of bone marrow cells

Bone marrow cells (1 × 107) from 6-week-old Ramp1−/− or wild-type C57BL/6 J mice were intravenously injected into lethally irradiated 6-week-old recipient wild-type or Ramp1−/− mice that received 100 mg l−1 neomycin sulfate for 2 weeks post-irradiation. Skin or muscle defect surgeries were performed 6 weeks after transplantation.

Histological analysis

Skin wounds were collected using an 8-mm biopsy punch, fixed in 10% formalin at room temperature for 24 h, cut at the edge of the wounds, embedded in paraffin and sectioned at 4 μm until the centre of the wound was passed. Re-epithelialization was measured by histomorphometric analysis. Slides were stained with haematoxylin and eosin, and the centre of the wound was determined by measuring the distance between the panniculus carnosus muscle gap using Aperio ImageScope Viewer (Leica Biosystems). Closure was calculated as the ratio of epidermis closure to the length of the panniculus carnosus gap. Muscle injury sites, including the proximal and distal quadriceps segments, were collected, fixed in 10% formalin solution for 24 h, embedded in paraffin, and sectioned at 4 μm thickness for 5 depths, starting from the edge of the patella, passing the centre of the wound, up to the proximal end of the defect site. Cross-sections were stained with Masson’s Trichrome. Muscle regeneration was determined by averaging the percentage of blue-stained fibrotic area (normalized to the total area) and the remaining non-fibrotic muscle area across five tissue section depths, using Aperio ImageScope.

Immunohistochemistry for neuropeptides, TSP-1 and myeloid cells

Tissues and DRGs (L1–L6 vertebrae) were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in OCT compound for 10 μm cryosections. Sections were stored at −20 °C, thawed, permeabilized and blocked with 1% bovine serum albumin (BSA), 10% normal goat serum (NGS) or normal donkey serum in PBS for 1 h. Sudan Black B solution (0.1% in 70% ethanol) was applied for 10 min. For neuropeptide detection, primary antibodies were added in staining buffer (0.5% BSA, 5% NGS or 0.5% BSA, 5% normal donkey serum in PBS) overnight at 4 °C. The primary antibodies included rabbit anti-CGRP (66.7 μg ml−1, Sigma, C8198), rabbit anti-substance P (1:500, Thermo Fisher Scientific, 20064), rabbit anti-VIP (1:500, Thermo Fisher Scientific, 20077), and goat anti-galanin (1 μg ml−1, Abcam, 99452). For TSP-1 detection, sections were incubated with AffiniPure Fab Fragment goat anti-mouse IgG (H + L) at 100 μg ml−1 (Jackson ImmunoResearch Labs, 115-007-003) in PBS for 2 h at room temperature, followed by mouse anti-thrombospondin-1 (5 μg ml−1, Thermo Fisher Scientific, 14-9756-82). For myeloid cell detection, slides were incubated with rat anti-mouse CD11b (5 μg ml−1, Thermo Fisher Scientific, 14-0112-82). Sections were washed and incubated with respective secondary antibodies for 1 h at room temperature. The secondary antibodies included F(ab′)2-Goat anti-Rabbit IgG Alexa Fluor 488 (2.6 μg ml−1, Thermo Fisher Scientific, A-11070), donkey anti-goat IgG Alexa Fluor 488 (2.6 μg ml−1, Thermo Fisher Scientific, A-11055), goat anti-mouse IgG Alexa Fluor Plus 488 (2.6 μg ml−1, Thermo Fisher Scientific, A48286TR), and goat anti-rat IgG Alexa Fluor Plus 594 (2.6 μg ml−1, Thermo Fisher Scientific, A48264). Counterstaining with DAPI for 10 min and mounting with Fluoroshield followed. Imaging was done using Leica DMi8 fluorescent microscope and Leica SP8 inverted confocal microscope.

Evaluation of neuropeptide expression

Skin and muscle samples from male Nav1.8cre+/−/Rosa26tdT+/− mice (10- to 12-week-old) were immunostained as detailed above, imaged on a Leica DMi8 fluorescent microscope and processed using Fiji59. Binary images were created with an optimal threshold, and overlapping areas were determined by combining region of interest binary images. Area fraction values, indicating neuropeptide expression in NaV1.8+ nerves, were calculated based on pixel ratios and converted using a built-in scale bar60,61.

Immunofluorescence for Ki-67 and KRT14

Paraffin sections underwent 20-minute antigen retrieval in 10 mM sodium citrate buffer (pH 6.0), followed by PBS washes and 5-minute permeabilization (0.2% Triton X-100 in PBS). Blocking with 10% NGS in 1% BSA/PBS occurred for 2 h, and endogenous IgG was blocked with unconjugated affinity-purified F(ab) fragment anti-mouse IgG (H + L) (10 μg ml−1, Jackson ImmunoResearch, AB_2338476) for 1 h at room temperature. Staining overnight at 4 °C utilized rat anti-mouse Ki-67 (5 μg ml−1, Thermo Fisher Scientific, 5698-82) and mouse anti-mouse cytokeratin 14 (4 μg ml−1, Thermo Fisher Scientific, MA5-11599) in 1% NGS in PBS with 0.1% BSA. After PBS-T washes, incubation with secondary antibodies occurred: goat anti-mouse Alexa Fluor 647 (2 μg ml−1, Thermo Fisher Scientific, A-21235) and goat anti-rat Alexa Fluor 488 (2.67 μg ml−1, Thermo Fisher Scientific, A48262TR) for 1 h at room temperature, followed by PBS-T wash. Counterstaining with DAPI (1 μg ml−1) for 10 min at room temperature preceded mounting with Fluoroshield.

TUNEL assay

The In Situ Cell Death Detection Kit, TMR red (Roche, 12156792910) was used, following the manufacturer’s instructions on muscle and skin tissue cryosections. To detect CD11b+ cells, sections were incubated overnight at 4 °C with rat anti-mouse CD11b (5 μg ml−1, M1/70, Thermo Fisher Scientific, 14-0112-82) in staining buffer. After PBS-T washes, sections were incubated with Alexa Fluor 488 goat anti-rat antibody (2.67 µg ml−1, Thermo Fisher Scientific, A48262TR), washed with PBS-T, and counterstained with DAPI (1 μg ml−1) before mounting with Fluoroshield. Two tissue section levels were evaluated per sample to determine the percentage of TUNEL+ apoptotic cells over total CD11b+ cells, examining three fields per section within the injury site.

Fibroblast, keratinocytes, myoblasts and endothelial cell maintenance

Human umbilical vein endothelial cells (HUVECs; Sigma, 200P-05N) cultured in EGM-2 medium (Lonza, CC-4176) up to 3 passages, and primary mouse fibroblasts from C57BL/6 J mouse tails26 (passages 2–3) were used. MCDB-131 medium (Thermo Fisher Scientific) with 100 mg ml−1 penicillin/streptomycin and 2 mM glutamine was employed for proliferation assays. C2C12 mouse myoblasts (CellBank Australia) were cultured in a 1:1 ratio of DMEM to F10 medium (2 mM glutamine, 10% FBS, 100 units ml−1 penicillin/streptomycin). HaCaT keratinocytes (a gift from R. Boyd) were cultured in DMEM without Ca2+ and Mg2+ (2 nM glutamine, 10% Chelex-treated FBS, 0.03 nM calcium chloride, 100 units ml−1 penicillin/streptomycin) for at least 3 passages. Cells obtained from vendors were authenticated and certified negative for Mycoplasma contamination. For proliferation assays, FBS was reduced to 2% or kept at 10% for 24 h. Detached with TrypLE, cells were seeded (2,000 cells per well for HUVECs, fibroblasts, HaCaTs; 1,000 cells per well for C2C12) and treated with CGRP (1 or 20 nM) or 10–20% FBS. Incubation for 48 h (fibroblasts, C2C12) or 72 h (HUVECs, HaCaTs) at 37 °C with 5% CO2 followed. Proliferation was determined using the CyQUANT Cell Proliferation Assay (Invitrogen), presented as fold change over basal proliferation (medium only). PerkinElmer EnVision multi-mode plate reader with EnSpire Manager software was used.

Flow cytometry with tissue samples

Skin wounds were collected using an 8-mm biopsy punch, and muscle defects were dissected to isolate the quadriceps. Samples were minced with scissors and subjected to two serial digestions with collagenase XI (1 mg ml−1) at 37 °C (two times 20 min for skin, two times 15 min for muscle). After the first digestion, the supernatant was collected and mixed with neutralization buffer (DMEM/F12 with 10% FBS and 5 mM EDTA). The first collection was kept on ice and fresh collagenase XI was added to the undigested tissue for the second digestion. Digestion mixtures were passed through a 70-μm cell strainer and stained with LIVE/DEAD Fixable Aqua dye (Thermo Fisher Scientific, 1:400 dilution in PBS) for 20 min on ice. Cells were incubated with TruStain FcX anti-CD16/32 (10 μg ml−1; clone 93, BioLegend) diluted in staining buffer (5% FBS and 2 mM EDTA in PBS) for 20 min and subsequently incubated with primary antibodies in staining buffer for a further 30 min on ice. The following anti-mouse antibodies from BioLegend were used: FITC anti-CD11b (clone M1/70, 6.6 μg ml−1) or BV711 anti-CD11b (clone M1/70, 2 μg ml−1); PE anti-F4/80 (clone BM8, 4 μg ml−1); BV421 anti-Ly6G (clone 1A8, 2 μg ml−1); BV711 anti-Ly6C (clone HK1.4, 1 μg ml−1) or FITC anti-Ly6C (clone HK1.4, 5 μg ml−1); PE-Cyanine7 anti-CD206 (clone C068C2, 2.6 μg ml−1); APC anti-CD206 (clone C068C2, 2 μg ml1); PE-Cyanine7 anti-CD3 (clone 17A2, 4 μg ml−1); BV711 anti-CD3 (clone 17A2, 4 μg ml−1);APC anti-CD4 (clone GK1.5, 2 μg ml−1); BV421 anti-CD8 (clone 53-6.7, 2 μg ml−1); PE anti-TCR β (clone H57-597, 2 μg ml−1); APC/Fire 750 anti-TCR γ/δ (clone GL3, 2 μg ml−1); PE-Cyanine anti-CD11c (clone N418, 2 μg ml−1); APC/Fire 750 anti-MHC Class II (clone M5/114.15.2, 2 μg ml−1). Cells were washed once with a large volume of staining buffer before analysis with BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

Mouse bone marrow neutrophil and monocyte isolation

Bone marrow cells were flushed from femora and tibiae of C57BL/6 J mice (8- to 12-week-old) with HBSS without Ca2+ and Mg2+ containing 2% FBS and 1 mM EDTA. Cell suspension was passed through a 70-μm strainer. Next, EasySep Mouse Neutrophil Enrichment Kit or EasySep Mouse Monocyte Isolation Kit (STEMCELL Technologies) was used to isolate neutrophils or monocytes according to the manufacturer’s instructions. Neutrophils were resuspended in RPMI containing 100 units ml−1 penicillin/streptomycin and 10% FBS for cell migration assay and cell death assay or 2% FBS for efferocytosis. Monocytes were cultured in DMEM/F12 (Thermo Fisher Scientific) containing 10% FBS, 2–10 ng ml−1 M-CSF (PeproTech) and 100 units ml−1 penicillin/streptomycin for subsequent experiments. RAMP1 and CALCRL were detected on neutrophils and monocytes using rabbit anti-RAMP1 (8.5 μg ml−1, Alomone Lab, ARR-021) and rabbit anti-calcitonin receptor-like receptor (5 μg ml−1, Biorbyt, orb526584).

Neutrophil cell death

Bone marrow-isolated neutrophils were cultured in RPMI 1640 medium (10% FBS). Cells were incubated with CGRP (1-20 nM, Tocris Bioscience, 83651-90-5) for 10 min, followed by treatment with IL-1 (5 ng ml−1) and TNF (50 ng ml−1) for 12 h at 37 °C with 5% CO2 to induce cell death. After 12 h, cells were washed with PBS and incubated with LIVE/DEAD Fixable Aqua dye (Thermo Fisher Scientific, 1:400 dilution) in PBS on ice for 20 min. Cell death was assessed using BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

Macrophage cell death and polarization marker expression

Bone marrow cells from 8- to 12-week-old C57BL/6 J mice were flushed, filtered and cultured in conditioned medium (DMEM/F12 with 10% heat-inactivated FBS, 100 units ml−1 penicillin/streptomycin, and 20% L929 fibroblasts-conditioned medium) at 37 °C with 5% CO2. After 7–9 days, differentiated macrophages were collected and seeded in 12-well or 6-well plates. The next day, cells were treated with CGRP (1 or 20 nM, Tocris Bioscience, 83651-90-5) for 20 min before exposure to mouse IL-1 (5 ng ml−1) and TNF (50 ng ml−1), IL-4 (2 ng ml−1) and IL-13 (2 ng ml−1), or IL-10 (2 ng ml−1) (PeproTech Inc) for 24 or 72 h. Macrophages were detached with TrypLE (Gibco) containing 3 mM EDTA, stained with LIVE/DEAD Aqua dye for 20 min on ice, and incubated with blocking solution (10 μg ml−1 TruStain FcX anti-CD16/32 (clone 93, BioLegend)) for 20 min before staining with antibodies for 30 min on ice. Antibodies from BioLegend included PE anti-CD11b (clone M1/70, 1 μg ml−1), BV711 anti-F4/80 (clone BM8, 2 μg ml−1), APC anti-CD80 (clone 16-10A1, 0.5 μg ml−1) and PE-Cyanine7 anti-CD206 (clone C068C2, 1 μg ml−1). For intracellular staining, cells were fixed and permeabilized using FluoroFix Buffer and Intracellular Staining Permeabilization Wash Buffer (Perm buffer, BioLegend). APC anti-mouse arginase-1 (Thermo Fisher, Clone AlexF5, 1 μg ml−1) was added to the Perm buffer and incubated with the cells for 30 min on ice. After washing with Perm buffer and staining buffer, cells were analysed using BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

Neutrophil and macrophage migration

Assays were conducted using 6.5-mm-diameter culture plate inserts (Corning) with 5-μm and 3-μm pore sizes for macrophages and neutrophils, respectively. Macrophages (1 × 105) or neutrophils (3 × 105) in migration media (DMEM/F12 with 0.25% BSA) were added to the inserts. The lower chambers contained migration buffer alone or chemoattractant (mouse CCL2 10 ng ml−1 for macrophages or mouse CXCL1/KC 150 ng ml−1 for neutrophils, PeproTech) with or without CGRP. Cells were allowed to migrate through the insert membrane for 3-4 h at 37 °C with 5% CO2. For macrophages, the inserts were then fixed with 4% paraformaldehyde, and cells on the upper side were removed. DAPI (1 μg ml−1) was used to stain cells on the bottom side, and they were counted using a fluorescent microscope. For neutrophils, cells that migrated into the lower chamber were collected and counted using a haemocytometer. The data are presented as the fold change, calculated by dividing the number of cells that migrated in response to treatments by the number of cells that migrated spontaneously (migration media only).


An efferocytosis assay kit (Cayman, 601770) was used following the manufacturer’s instructions. Neutrophils were labelled with CFSE and cultured in RPMI with 2% serum for 12 h to induce cell death. Bone marrow-derived macrophages cultured for 7 days were seeded at a density of 4 × 105 cells per well in a 6-well plate with DMEM/F12 containing 10% FBS and 100 units ml−1 penicillin/streptomycin. Prior to the assay, macrophages were pre-treated with CGRP (1 or 20 nM) for 24 h. Macrophages were collected, labelled with CytoTell Blue, and then incubated with CFSE-labelled dead/dying neutrophils at different ratios (1:1, 1:2, and 1:4) at 37 °C for 15 min. The reaction was stopped by washing cells with ice-cold PBS containing 5% FBS and 1 mM EDTA. Cells were analysed with BD LSR Fortessa X-20 and FlowJo software (BD Biosciences). Macrophages were identified by CytoTell Blue-positive staining, and the efferocytosis index was calculated as the percentage of CFSE-positive cells in CytoTell Blue-labelled macrophages.

Adoptive transfer of tdTomato+ cells for in vivo migration and efferocytosis

tdTomato+ bone marrow cells from CMV-cre/Rosa26tdTomato male mice (8- to 12-week-old) were adoptively transferred into Nav1.8cre/Rosa26DTA and Rosa26DTA mice either directly after red blood cell lysis (migration assay) or following neutrophil isolation (efferocytosis assay). In the migration assay, 1 × 107 cells were intravenously delivered on day 2 after skin or muscle injury. On day 3, collected tissues were analysed via flow cytometry to detect tdTomato+ cells. For the efferocytosis assay, neutrophils were cultured in low serum (2%) for 24 h to induce cell death, and 2 × 106 dead or dying neutrophils were intradermally injected at the skin wound border on day 3 post-injury. After 30 min, collected tissues were assessed via flow cytometry to quantify efferocytosis as the number of monocytes or macrophages that had taken up tdTomato+ apoptotic neutrophils. Results were presented as fold change relative to Rosa26DTA control mice.

RT–PCR, qPCR and RNA-seq

Isolated neutrophils were treated with CGRP (1 nM) in RPMI with 10% FBS and 100 units ml−1 penicillin/streptomycin for 4 h at 37 °C with 5% CO2. Isolated monocytes cultured in DMEM/F12 with 10% FBS, 100 units ml−1 penicillin/streptomycin, and M-CSF (10 ng ml−1) for 3 days, had their medium replaced with CGRP (1 nM) for 4 h at 37 °C with 5% CO2. After collection, RNA extraction used the RNeasy Plus Micro Kit (Qiagen). For PCR with reverse transcription (RT–PCR) and quantitative PCR (qPCR), reverse transcription used ReverTra Ace (Toyobo). RT–PCR primers were: Human_Calcrl 5′-CATGCACATCCTTATGCAC-3′ and 5′-CCATCACTGATTGTTGACAC-3′; Human_Ramp1 5′-GCCAGGAGGCTAACTACG-3′ and 5′-GAAGAACCTGTCCACCTCTG-3′; Mouse_Calcrl 5′-GGTACCACTACTTGGCATTG-3′ and 5′-GTCACTGATTGTTGACACTG-3′; Mouse_Ramp1 5′-GACGCTATGGTGTGACT-3′ and 5′-GAGTGCAGTCATGAGCAG-3′. Human or mouse GAPDH primers were from Integrated DNA Technologies (51-01-07-12 and 51-01-07-13, respectively). PCR products were analysed by gel electrophoresis. qPCR was performed using LightCycle96 with software LightCycle96 (Roche Diagnostics) and TaqMan Assay primers from Thermo Fisher Scientific (Thbs1, Mm00449032_g1; Gapdh, Mm99999915_g1). For RNA-seq, RNA quantity and quality assessment, library preparation and sequencing were performed at the Medical Genomics Facility, Monash Health Translation Precinct (MHTP). RNA quantity was assessed using Qubit. RNA samples (20 ng) with RNA integrity number (RIN) value ≥ 7 were used for library preparation. First strand synthesis was performed using a dT primer which adds the Illumina P7 (5′-CAAGCAGAAGACGGCATACGAGAT-3′), 8-bp i7 index for each sample and a 10-bp unique molecular identifier. The modified reverse transcriptase reaction also adds a template switching sequence at the 5′ end of the RNA during the generation of indexed cDNA. These first stand indexed cDNA were pooled and amplified using primers to P7 and the template switch sequence. Illumina P5 was added by tagmentation by Nextera transposase during amplification. Standard Illumina R1 primer was used (main cDNA read), followed by standard i7 primer for index or unique molecular identifier. R2 primer was present but not used as it will read into poly-A tail. Sequencing was performed on the NextSeq2000 (Illumina), using NextSeq 1000/2000 P2 Reagents (100 cycles) v3 (Illumina) in accordance with the Illumina Protocol 1000000109376 v3 Nov2020.

Demultiplexing and mapping

Fastq files were processed using the nfCore/RNAseq (v3.2) pipeline using the umi function62. Reads were aligned to the Mus musculus GRCm38 reference using STAR aligner63. Reads were quantified using featureCounts producing the raw genes count matrix and various quality control metrics which were summarized in a multiQC report64,65. Raw counts were analysed with Degust66, a web tool which performs normalization using trimmed mean of M values (TMM)67. Differential gene expression analysis was performed using limma/voom68 in Degust and genes with a FDR-adjusted P value < 0.05 were considered significantly upregulated or downregulated. Volcano plots were made using the web tool, VolcaNoseR69. Gene ontology enrichment analysis for biological processes was performed with the web tool, ShinyGO 0.77, by providing all upregulated or downregulated DEGs separately as the input for each experimental group70.

siRNA-mediated knockdown

Macrophages (4 × 105 cells per well in a 6-well plate) were transfected with 10 nM scrambled siRNA (Silencer Select Negative Control No. 1 siRNA, Thermo Fisher, 4390843) or Silencer Select Pre-Designed siRNA against mouse TSP-1 (Thermo Fisher, s124596) using Reduced-Serum Medium (Opti-MEM, Gibco) and Lipofectamine RNAiMAX (Invitrogen, 51985034) for 6 h. The medium was then replaced with fresh culture medium (DMEM/F12 with 10% FBS). After 24 h, cells were collected for the migration assay. For the efferocytosis assay, cells were cultured with 1 nM CGRP immediately after transfection. After 24 h, cells were collected and co-cultured with dead or dying neutrophils. The evaluation of cell death and polarization used the same methods as those for assessing macrophage death and polarization marker expression.

CGRP variants

CGRP and eCGRP were synthesized by ProteoGenix. eCGRP was designed to contain PlGF residues 123–141 at the N terminus followed by a plasmin-sensitive sequence from vitronectin (KGYR)71. For both variants, a disulfide bond was formed between the two cysteine residues and the C-terminal phenylalanine was amidated. Peptide purity, determined by high performance liquid chromatography, was 89.63% for CGRP and 87.33% for eCGRP.

Cleavage of eCGRP by plasmin

CGRP (4 μg) and equimolar eCGRP in 20 μl of PBS (pH 7.2) were incubated with plasmin (0.0005 U μg−1, Sigma) at 37 °C for 60 min. Aprotinin (25 μg ml−1, Sigma) was added for 5 min at 37 °C to stop plasmin activity. Samples were analysed by SDS–PAGE.

Retention of CGRP and eCGRP into skin and muscle

CGRP (1 μg) or an equal molar amount of eCGRP was intradermally administered to the shaved dorsal skin of male 10- to 12-week-old Nav1.8cre+/−/Rosa26tdT+/− mice, with injection sites marked using a marker. For muscle, CGRP variants were injected into the quadriceps. After 24 h, collected injection sites underwent cryosectioning and immunostaining. Fiji59 was used for analysis, excluding the co-localization area of CGRP with tdTomato fluorescence, indicating endogenous CGRP expression.

cAMP quantification

Freshly isolated neutrophils or bone marrow-derived macrophages (1 million cells) were treated with CGRP (1 nM) in RPMI with 10% FBS for 30 min at 37 °C with 5% CO2. cAMP levels were quantified using a cAMP ELISA kit from Cayman Chemical (581001) according to the manufacturer’s instructions.

Spontaneous pain behaviour assessment

Eight mice per group (4 males, 4 females, C57BL/6 J, 10- to 12-week-old) were acclimatized for 1 h in empty cages. The right hind paw received an intraplantar injection 1 μg of wild-type CGRP, equimolar amount of eCGRP, 0.05% capsaicin (Sigma, M2028), or 20 μl saline. Mice were immediately placed in the cage, and their behaviour was recorded. The number of episodes and the time spent licking, shaking, flinching and lifting the paw were recorded for first 5 min and for 5 min after 1, 6, 24 and 48 h.

Hot plate test

Eight mice per group (4 males, 4 females, C57BL/6 J, 10- to 12-week-old) received an intraplantar injection of 1 μg of wild-type CGRP, equimolar amount of eCGRP, or 20 μl saline in the right hind paw. After 30 min, mice were individually placed on a metal hot plate set to 52 °C. The latency, from mouse placement on the surface to the first behavioural sign of nociception (for example, lifting, shaking, licking the hind paw or jumping), was measured. Mice were immediately removed from the hot plate after responding or after a 30 s cut-off. The test was repeated after 1, 6, 24 and 48 h.

ELISAs for cytokines and MMPs

Homogenized skin wound and muscle tissues were incubated for 30 min on ice in T-PER Tissue Protein Extraction Reagent (10 ml per g of tissue, Thermo Fisher Scientific) containing 1 tablet of protease inhibitor for 7 ml (Roche). Samples were then centrifuged at 10,000g for 5 min and supernatants were stored at −80 °C. Total protein concentration was measured with a Bradford assay (Millipore). Cytokines and MMPs were detected by ELISA from R&D Systems; Mouse IL-1 beta/IL-1F2 DuoSet ELISA; Mouse CCL2/JE/MCP-1 DuoSet ELISA, Mouse CXCL2/MIP-2 DuoSet ELISA; Total MMP-2 Quantikine ELISA Kit; Mouse Total MMP-9 DuoSet ELISA.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad). Significant differences were calculated with Student’s t-test, one-sample t-test, and by ANOVA when performing multiple comparisons between groups. P < 0.05 was considered as a statistically significant difference.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Source link


Leave a Reply

Your email address will not be published. Required fields are marked *