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The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury

The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells... Citation: Yagura K, Ohtaki H, Tsumuraya T, Sato A, Miyamoto K, Kawada N, et al. (2020) The Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) have shown enhancement of CCL2 and CCL5 by human bone potential in facilitating recovery from spinal cord injury (SCI) through communicating with marrow-derived mesenchymal stem/stromal cells microglia/macrophages (MG/MΦ). We here focused on chemokines as a candidate for the might contribute to inflammatory suppression and axonal extension after spinal cord injury. PLoS ONE communication. Selected MG/MΦ-related chemokines were determined gene expression 15(3): e0230080. https://doi.org/10.1371/journal. after SCI and further focused CCL2/CCR2 and CCL5/CCR5 to estimate role of the chemo- pone.0230080 kines by hMSCs. Male C57/BL6 mice were subjected to spinal cord transection. Gene Editor: Xing-Ming Shi, Augusta University, UNITED expression was assayed in the spinal cords following SCI for selected MG/MΦ-related che- STATES mokines and their receptors. hMSCs (5×10 cells) were then transplanted into parenchyma Received: October 31, 2019 of the spinal cord, and the expressions of the Ccl2/Ccr2 and Ccl5/Ccr5 axes, inflammation, Accepted: February 20, 2020 MG/MΦ-polarization, and axonal regeneration were evaluated to measure the influence of the hMSCs. Finally, mouse CCL5 was injected into the spinal cords. Acute increases in Published: March 10, 2020 gene expression after SCI were observed for most chemokines, including Ccl2; chronic Peer Review History: PLOS recognizes the + + + increases were observed for Ccl5. CCL2 -cells merged with NeuN -neurons. CCR2 immu- benefits of transparency in the peer review + + process; therefore, we enable the publication of noreactivity was principally observed in Ly-6G /iNOS -granulocytes on postoperative day + + + all of the content of peer review and author (pod) 1, and CCL5 and CCR5 immunoreactivity overlapped with NeuN -neurons and F4/ responses alongside final, published articles. The 80 -MG/MΦ on pod 14. The hMSC transplantation enhanced Ccl2 and Ccl5 and improved editorial history of this article is available here: + + locomotor activity. The hMSC implantation did not alter the number of Ly-6G /CCR2 but https://doi.org/10.1371/journal.pone.0230080 decreased Il1, Elane, and Mpo on pod 3. Conversely, hMSC transplantation increased Copyright:© 2020 Yagura et al. This is an open expression of Zc3h12a (encodes MCP-1-induced protein) on pod 14. Moreover, hMSC access article distributed under the terms of the Creative Commons Attribution License, which increased the Aif1, and two alternatively activated macrophage (AAM)-related genes, Arg1 permits unrestricted use, distribution, and and Chil3 (Ym1), as well as axonal regenerative markers, Dpysl2 and Gap43. Gene expres- reproduction in any medium, provided the original sion indicative of AAM polarization and axonal regeneration were partially recovered by author and source are credited. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 1 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Data Availability Statement: All relevant data are CCL5 injection. These results suggest that hMSC implantation increases Ccl2 and Ccl5, within the paper and its Supporting Information improves locomotor activity, enhances MG/MΦ polarization to AAM, and increases the files. gene expression of axonal regenerative markers. These functions of hMSCs might be par- Funding: YES. This work was supported by tially mediated by the CCL2/CCR2 and CCL5/CCR5 axes. KAKENHI, as follows: 16H05443 (Hirokazu Ohtaki), 17K10943 (Yutaka Hiraizumi), and 26861214 (Atsushi Sato). Competing interests: The authors declare that they have no competing interests. Introduction Every year, more than 10,000 people in the United States experience spinal cord injuries (SCI) due to trauma-related accidents. While medication regimens during the acute period of injury involve the extensive administration of steroids and other anti-inflammatory drugs, the recov- ery of neurologic function relies on the neural plasticity to each patient. The considerable number of patients that become permanently paralyzed despite medical intervention reflects the inadequacy of inherent mechanisms in repairing the nervous system [1, 2]. Bone marrow-derived mesenchymal stem/stromal cells (MSCs) are multipotent tissue stem cells that have shown promise in facilitating regeneration and/or recovery after neuronal injury [3–6]. We have previously demonstrated that xenografting human MSCs (hMSCs) into immunocompetent mice subjected to SCI [7] or brain ischemia [8] protected against short- term neurological deficits and damage; however, the levels of the grafted hMSCs decreased sig- nificantly within the first week. Further experiments indicated that hMSCs might communi- cate with recipient tissues and thereby convert the phenotypic polarization of host microglia/ macrophages (MG/MF) to an alternatively activated phenotype (AAM), which is known to promote tissue repair and regeneration after injury [9]. Several immune- and nonimmune- related disease models and isogenic graft experiments have provided evidence for the immu- nomodulatory functions of hMSCs [10, 11]. However, precisely how hMSCs communicate with or regulate MG/MF remains unclear. Chemokines guide the migration of immune cells, such as leukocytes and macrophages, and stimulate inflammatory responses, and are divided into the CC, CXC, C, and CX3C fami- lies from the structural feature [12]. Levels of many chemokines are increased in patients with central nervous system (CNS) disorders, such as SCI [13] and cerebral infarction [14], increase the levels of many chemokines. Indeed, enhanced levels of chemokines were observed in iso- genic grafted bone marrow-derived MSCs of a hypoxia-ischemia rat model, but not in adipose tissue-derived MSCs [15]. Cultured hMSCs secrete several chemokines and express several chemokine receptors on the cell surface [16]. An additional study further reported that MSCs elevate the secretion of several chemokines by stimulating lipopolysaccharide (LPS) in vitro [17]. Hence, the communication between hMSCs and MG/MF may be partially mediated by chemokines. Research has indicated that some chemokines play other roles in addition to chemotaxis response. Chemokines such as CCL2 (monocyte chemoattractant protein-1; MCP-1), CX3CL1 (fractalkine), CXCL12 (Stromal cell-derived factor 1; SDF-1), and CCL5 (regulated on activa- tion, normal T cell expressed and secreted; RANTES) are reportedly involved in neuroprotec- tion [18–21], axonal regeneration [22, 23], and the differentiation of MG/MF to AAM [24]. In present study, we firstly examined the effect and fate of transplanted hMSCs after SCI. Following the study, we determined gene expression for selected some MG/MF-related che- mokines and their receptors on SCI, and then focused on two representative major MG/MF- related chemokine axes, CCL2/CCR2 and CCL5/CCR5, whose corresponding increases in the gene expression had been characterized as acute or chronic. The influences of hMSC PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 2 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury implantation following SCI on the CCL2/CCR2 and CCL5/CCR5 axes, inflammation, MG/ MF polarization, and/or axonal regeneration were examined to gain insight into the actions of hMSCs. Materials and methods Animals Wild-type, male C57BL/6 mice (8 to 12 weeks old, weighing 17 to 25 g) were purchased from Sankyo Lab Service Corporation (Tokyo, Japan) and used for the experiments (total of 139 ani- mals including 7 euthanized animals). Mice were maintained on a 12-h light/dark cycle at 24 ± 2˚ C with constant humidity (55 ± 10%) and free access to food and water. All experimen- tal procedures involving animals were approved and followed by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Showa University (#06105 and 07079). SCI model The SCI mouse model was generated in accordance with previously described methods [7, 25]. Anesthesia was induced via inhalation of 4.0% sevoflurane and maintained with 3.0% sevoflur- ane in N O/O (2:1). Under aseptic conditions, an incision was made along the midline of the 2 2 skin on the back, and the muscles, soft tissues, and yellow ligaments overlying the spinal col- umn between T9 and T10 were removed. The intervertebral spinal cord between T9 and T10 was then transected with a thin-bladed razor (FEATHER, Osaka, Japan). After bleeding had stopped and the coagulated blood had been removed, the incision was closed, and the animals were administered 1.0 mL of lactated Ringer’s solution (s.c.; Otsuka, Tokyo, Japan) to prevent dehydration of which were given once a day until they gain the body weight. To support their eating, animal food was placed on the cage floor, and the water bottle was lowered to allow easy access. Moreover, to support urination, the region of the lower abdomen in all mice was gently stimulated a few times every day. Although we did not administer any pain relievers or anesthesia during the experiments because they may influence SCI and locomotor activity, we made all efforts to minimize animal numbers and animal discomfort. However, total of 7 ani- mals were excluded from the study by humane endpoints. The 3-animals scored the Basso Mouse Scale (BMS) [26] more than 3 immediately after SCI were excluded from the experi- ments for incomplete hindlimb paralysis by a surgical error. Moreover, the 4-mice were excluded from the study because the mice exhibited peritoneal infection, severe body weight loss more than 25% before surgery, hindlimb wounds, and/or tail or foot autophagia. These animals were euthanized by cervical dislocation or CO -gas exposure. Preparation of implanted hMSCs Frozen vials of hMSCs from the bone marrow were obtained from Dr. Prockop (The Center for the Preparation and Distribution of Adult Stem Cells [http://medicine.tamhsc.edu/irm/ msc-distribution.html]) under the auspices of a National Institutes of Health (NIH)/National Center for Research Resources grant (P40 RR 17 447–06). The experiments were performed with hMSCs from donor 281L [7, 8, 27]. To expand hMSCs, a frozen vial of 1.0×10 passage 3 cells was thawed and plated at 100 cells/cm in multiple 150-mm plates (Nunclon, Thermo Fisher Scientific, Waltham, MA) with a 20-mL complete culture medium (CCM) that con- sisted ofα-minimal essential medium (α-MEM; Invitrogen, Carlsbad, CA), 20% heat-inacti- vated fetal bovine serum (FBS; Hyclone; Thermo Fisher Scientific), 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). The cultures were PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 3 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury incubated, and the medium was replaced every 3 days for approximately 8 days until the cells were 70–80% confluent. The medium was then discarded, and the culture plates were washed with phosphate-buffered saline (PBS). Adherent cells were harvested using 0.25% trypsin and 1 mM EDTA (Invitrogen) for 5 min at 37˚ C and resuspended at 5 × 10 cells in 0.5 μL of ster- ile Hank’s balanced salt solution (HBSS; Invitrogen) for injection. Injection of hMSCs into the spinal cord The hMSCs were injected into the spinal cord in accordance with previously described meth- ods [7]. Briefly, on the day following surgery to induce SCI, mice were re-anesthetized via inhalation of sevoflurane. The animals were placed face-down, and a truncated 29G needle with a 5.0-μL glass syringe (Hamilton, Reno, NV) was inserted directly into the intervertebral spinal cord (D/V -1.0 mm) between T10 and T11. We trained investigators to insert the needle to the depth of the anterior horn of the spinal cord by the injection of dye. Then, the depth of needle insertion was standardized at -1.0 mm from surface of which did not leak the dye from the needle tract during infusion or from the ventral spinal cord. The hMSCs (5 × 10 cells/μL, n = 40 mice) or HBSS (n = 26 mice) was infused at a rate of 0.5 μL/min with an Ultra Micro Pump (World Precision Instruments, Sarasota, FL). Following infusion, the needle was left in place for 1 min to facilitate the diffusion of the solution into the tissue. Previously, we demon- strated that the fate of hMSCs does not differ between immunocompetent and immunode- ficient animals after ischemia [8]. Therefore, in the present study, we refrained from using immunosuppressant following cell implantation [10]. Assessment of motor function Motor function after SCI was evaluated using an open-field behavior test that focused on hin- dlimb locomotor function according to the BMS. The BMS consists of an open-field locomotor rating scale that ranges from 0 (complete paralysis) to 9 (normal mobility). Briefly, individual mice were placed in the center of an open field (50 × 50 cm ) with a smooth, non-slip floor and monitored for 4 min. Hindlimb movements, trunk/tail stability, and forelimb-hindlimb coordination were assessed and graded. Mice were tested daily for 14 days after the operation. Mice were randomly numbered to ensure that the investigators remained unaware of the treat- ment groups during scoring. Isolation of RNA and production of cDNA Under sodium-pentobarbital (100 mg/kg, i.p.) anesthesia, spinal cord samples from the T7 to T12 vertebrae were dissected on postoperative days (pod) 0 (sham-operated non-SCI control), 1, 3, 7, and 14 with/without hMSC or vehicle injection. The tissue was snap-frozen in liquid nitrogen and stored at −80˚ C until use. Total RNA was isolated using the TRIZOL Reagent (Invitrogen) in accordance with the manufacturer’s instructions and dissolved in RNase-free water. The purity and concentration of the extracted RNA were determined spectrophotomet- rically (NanoDrop, Wilmington, DE). A High-Capacity RNA-to-cDNA Kit (Applied Biosys- tems, Foster City, CA) was used to synthesize cDNA using 2 μg of total RNA in accordance with the manufacturer’s instructions. To confirm the species specificity of the primers, we also extracted total RNA from cultured hMSCs and synthesized the cDNA. Semi-quantification of the number of hMSCs in the spinal cord On days 1, 3, or 14 after SCI (0, 2, or 13 days after cell injection; n = 4 mice at each time- point), mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and the spinal PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 4 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury cord was collected and subjected to cDNA preparation as described above. Semi-quantifica- tion of the number of hMSCs was determined based on the gene expression level of human GAPDH in mouse Gapdh via qPCR. Standard curves were generated by adding serial dilutions 2 6 of hMSCs (1 × 10 to 1 × 10 cells) to spinal cord samples (T7 to T12 vertebrae) of uninjured mice prior to homogenization according to the methods published by previous studies [7, 8, 28–30]. Finally, the estimated number of resident hMSCs was calculated from the standard curve. The human-specific primers cross-hybridized less than 0.07% with mouse cDNA obtained from spinal cord samples that had not been injected with hMSCs. Polymerase chain reaction (PCR) PCR analyses were performed using TaKaRa Ex Taq (TaKaRa, Shiga, Japan) to confirm the species specificity of primers. The reaction mixture was created with an appropriate volume of the cDNA mixture: 0.25 μL of forward and reverse primers (50 nmol/mL), 2.0 μL of dNTP mixture (0.25 mM each), 0.1 μL of TaKaRa Ex Taq (5 units/μL), and 2.0 μL of 10 × Ex Taq Buffer in a total volume of 20 μL. Thermal-cycling parameters were set as follows: 95˚ C for 1 min for initial denaturation, followed by a cycling regime of 40 cycles at 95˚ C for 45 s, 60˚ C for 30 s, and 72˚ C for 45 s. At the end of the final cycle, an additional 7-min extension step was conducted at 72˚ C. Ten microliters of each reaction mixture was electrophoresed onto 2.0% agarose gel, and the bands were visualized using ethidium bromide. Quantitative PCR (qPCR) analyses were performed with SYBR Premix Ex Taq II reagent (TaKaRa) and an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosys- tems, Lincoln, CA). Relative gene expression was calculated using the absolute quantification method. The mouse glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh) was used as a housekeeping gene to normalize the cDNA levels. All primers were designed as described in Table 1. All data are expressed the relative level (fold) to compare with pod 0 (basal level) after normalization against mouse Gapdh as a housekeeping gene (n = 6 mouse at each time-point). Immunostaining Under sodium-pentobarbital (100 mg/kg, i.p.) anesthesia, the animals were transcardially per- fused with 0.9% saline followed by 2% paraformaldehyde (PFA) in 50 mM phosphate buffer (PB) (pH 7.2), and the spinal cord (T7 to T12 vertebrae) was carefully removed. The spinal cords were then post-fixed overnight, immersed in 20% sucrose in 0.1 M PB for two nights, and embedded in liquid nitrogen-cooled isopentane using an embedding solution (20% sucrose in 0.1 M PB:O.C.T. compound [Sakura Finetech, Tokyo, Japan]: 2:1]. Sagittal spinal cord sections (thickness, 5 μm) were obtained using a cryostat (Hyrax50, Carl Zeiss, Inc.; Oberkochen, Germany) and stored at –80˚ C until use. For single staining, the sections were washed with PBS and incubated in 0.3% H O to 2 2 quench internal peroxidase reactions. The sections were then immersed in 2.5% normal horse serum (NHS; Vector, Burlingame, CA) in PBS containing 0.05% Tween 20 (PBST) to block non-specific reactions for 1 h. Subsequently, the sections were incubated overnight with poly- clonal rabbit anti-GFAP (1:10; DAKO, Glostrup, Denmark) or polyclonal rabbit anti-Iba1 antibodies (1:500; WAKO, Tokyo, Japan). After washing with PBST, the sections were incu- bated with biotinylated goat anti-rabbit immunoglobulin G (IgG) (1:200, Santa Cruz, Santa Cruz, CA) for 2 h. They were then incubated in an avidin-biotin complex solution (Vector) followed by diaminobenzidine (DAB; Sigma, St. Louis, MO) as a chromogen. Immunopositive cells were observed via light microscopy (AX70) using DP2-BSW image analysis software (Olympus, Tokyo, Japan). PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 5 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Table 1. Primers used for real-time polymerase chain reaction. 0 0 0 0 Name Symbol Species Forward (5 to 3 ) Reverse (5 to 3 ) Size (bp) AIF1 Aif1 mouse atcaacaagcaattcctcgatga cagcattcgcttcaaggacata 144 Arg1 Arg1 mouse ctccaagccaaagtccttagag aggagctgtcattagggacatc 185 CCL2 Ccl2 mouse ttaaaaacctggatcggaaccaa gcattagcttcagatttacgggt 121 CCL3 Ccl3 mouse ttctctgtaccatgacactctgc cgtggaatcttccggctgtag 100 CCL4 Ccl4 mouse ttcctgctgtttctcttacacct ctgtctgcctcttttggtcag 121 CCL5 Ccl5 mouse gctccaatcttgcagtcgtg gagcagctgagatgcccatt 242 CCL7 Ccl7 mouse gctgctttcagcatccaagtg ccagggacaccgactactg 135 CCR1 Ccr1 mouse tggtgggcaatgtcctagtg aagtaacagttcgggccctc 300 CCR2 Ccr2 mouse atccacggcatactatcaacatc caaggctcaccatcatcgtag 104 CCR3 Ccr3 mouse accccgtacaacctggttct ccaacaaaggcgtagattactgg 158 CCR5 Ccr5 mouse ttttcaagggtcagttccgac ggaagaccatcatgttacccac 158 CRMP2 Dpysl2 mouse tcaaaggtggcaagattgtgaa ggaatcaccattctggagtgg 145 CXCL1 Cxcl1 mouse ctgggattcacctcaagaacatc cagggtcaaggcaagcctc 117 CXCL2 Cxcl2 mouse tccagagcttgagtgtgacg tggttcttccgttgagggac 201 CXCR2 Cxcr2 mouse gggtggggagttcgtgtaga aggtgctaggatttgagcct 200 ELANE Elane mouse caggaacttcgtcatgtcagc agcagttgtgatgggtcaaag 162 ENO2 Eno2 mouse tgagaataaatccttggagctggt ggtcatcgcccactatctgg 302 GAP43 Gap43 mouse tctgctactaccgatgcagc tggaggacggggagttatcag 181 GAPDH Gapdh mouse gctacactgaggaccaggttgt ctcctgttattatgggggtctg 306 GAPDH Gapdh human ggagcgagatccctccaaaat ggctgttgtcatacttctcatgg 197 GAPDH Gapdh mouse aggtcggtgtgaacggatttg tgtagaccatgtagttgaggtca 123 IL-1β Il1b mouse gcaactgttcctgaactcaact atcttttggggtccgtcaact 89 IL-4 Il4 mouse tctcgaatgtaccaggagccatatc agcaccttggaagccctacaga 183 MCPIP Zc3h12a mouse acgaagcctgtccaagaatcc taggggcctctttagccaca 115 MPO Mpo mouse agggccgctgattatctacat ctcacgtcctgataggcaca 152 Ym-1 Chil3 mouse ggtggacacagaatggttcttc ccaggagcgttagtgacagc 128 AIF: allograft inflammatory factor, Arg: arginase, CCL: chemokine (C-C motif) ligand, CCR: chemokine (C-C motif) receptor, Chil: chitinase-like, CRMP: collapsin response mediator protein, CXCL: chemokine (C-X-C motif) ligand, CXCR: chemokine (C-X-C motif) receptor, ELANE: neutrophil elastase, Eno: neuron-specific enolase, GAP: growth associated protein, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, GFAP: glial fibrillary acidic protein, IL: interleukin, MCPIP: monocyte chemotactic protein-1 induced protein, MPO: myeloperoxidase, = not species specific. https://doi.org/10.1371/journal.pone.0230080.t001 For multiple staining, sections that had been immersed in 0.3% H O were incubated with 2 2 an M.O.M. Immunodetection Kit (Vector), followed by 2.5% NHS/PBST. The sections were then incubated overnight with primary antibodies. The sections were rinsed with PBST and immersed with appropriate fluorescently labeled secondary antibodies for 2 h. Subsequently, cell nuclei were stained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI, 1:10,000; Roche, Manheim, Germany) and incubated in 1.0 mM CuSO in 50 mM ammonium acetate buffer (pH 5.0) to diminish autofluorescence [31]. Fluorescence was detected using an Axio Imager optical sectioning microscope with ApoTome (Carl Zeiss, Inc.). All primary and sec- ondary antibodies used for immunostaining are listed in Tables 2 and 3. For control staining, the same steps were performed with the exception of incubation with each primary antibody. Cell counting Five sagittal sections (thickness: 5 μm) were collected from each mouse: at the midline (3), which included the central canal adjacent to the core injury site; and bilaterally at 150 μm (2 and 4) and 300 μm (1 and 5) lateral to the midline. The numbers of immunopositive cells PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 6 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Table 2. Primary antibodies used for immunohistochemistry. Primary antibodies (Clone #) Host Companies Catalog # Dilution CCL2 Rabbit Bioss (Boston, MA) bs-1101R 1,500 CCL5 (25H14L17) Rabbit Thermo Fisher Scientific (Waltham, MA) 701030 100 CCL5 Goat R&D systems (Minneapolis, MN) AF478 300 CCR2 Goat Novus Biologicals (Littleton, Co) NB100-701 300 CCR2 Goat GeneTex (Irvine, CA) GTX45788 300 CCR5 Rabbit Bioss (Boston, MA) bs-2514R 500 NeuN (A60) Mouse Merc Millipore (Burlington, MA) MAB377 1,000 GFAP Rabbit DAKO (Glostrup, Denmark) N1506 10 GFAP (G-A-5) Mouse SIGMA (St. Louis, MO) G3893 1,000 Iba1 Rabbit Wako (Osaka, Japan) 019–19741 500 Alexa635labeled-Iba1 Rabbit Wako (Osaka, Japan) 013–26471 500 F4/80 (Cl:A3-1) Rat BMA Biomedicals (Augst, Switzerland) T-2008 500 Ly-6G (RB6-8C5) Rat BD Biosciences (Franklin Lakes, NJ) 557445 400 iNOS Rabbit Merc Millipore (Burlington, MA) 06–573 250 Ym-1 Rabbit StemCell Technologies (Vancouver, BC Canada) 1404 200 CRMP2 Rabbit LifeSpan BioSciences (Seattle, WA) LS-B5657 3,000 GAP43 (EP890Y) Rabbit Abcam (Cambridge, UK) ab75810 500 CCL: chemokine (C-C motif) ligand, CCR: chemokine (C-C motif) receptor, CRMP: collapsin response mediator protein, GAP: growth associated protein, GFAP: glial fibrillary acidic protein, Iba1: ionized calcium-binding adapter molecule, iNOS: inducible nitric oxide synthase, NeuN: neuronal nuclei. https://doi.org/10.1371/journal.pone.0230080.t002 against CCR2, Iba1, Ly-6G, and inducible nitric oxide (iNOS) antibodies were counted in four random fields at the injury site in each animal at 500× magnification and expressed as the aver- age total number in a square millimeter (mm ). Injection of recombinant mouse CCL5 (RANTES) into the spinal cord To investigate the role of CCL5 in the spinal cord after injury, recombinant mouse CCL5 was directly injected into the spinal cord between T10 and T11 according to the cell-injection pro- cedure described above. Recombinant mouse CCL5 (RANTES; Miltenyi Biotec, Bergisch Gladbach, Germany) was dissolved in saline containing 0.1% bovine serum albumin (BSA; Wako) under aseptic conditions to obtain a concentration of 20 ng/μL. Seven days after SCI, mouse spinal cords were injected with a bolus of CCL5 (20 ng, n = 6 mice) or vehicle (n = 6 mice) at a rate of 0.5 μL/min. Mice were anesthetized with sodium pentobarbital (100 mg/kg, i. p.) at 24 h following injection, and spinal cord tissue was dissected from T7 to T12 for qPCR. Table 3. Secondary antibodies used for immunohistochemistry. Secondary antibodies Host Companies Catalog # Dilution Rabbit IgG (biotin) Goat Santa Cruz Biotechnology (Santa Cruz, CA) SC-2040 200 Goat IgG (biotin) Horse Vector Laboratory (Burlingame, CA) BA-9500 200 Mouse IgG (Alexa 546) Goat ThermoFisher Scientific (Waltham, MA) A11030 800 Rabbit IgG (Alexa 488) Goat ThermoFisher Scientific (Waltham, MA) A11034 400 Rabbit IgG (Alexa 546) Goat ThermoFisher Scientific (Waltham, MA) A11035 400 Rabbit IgG (Alexa 647) Goat ThermoFisher Scientific (Waltham, MA) A21245 400 Rat IgG (FITC) Goat Jackson ImmunoResearch (West Grove, PA) 112-096-071 200 Goat IgG (Alexa FITC) Donkey ThermoFisher Scientific (Waltham, MA) A11055 400 Goat IgG (Alexa 546) Donkey ThermoFisher Scientific (Waltham, MA) A1056 400 https://doi.org/10.1371/journal.pone.0230080.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 7 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Statistical analysis As each mouse was assigned a random number, all data were collected and analyzed without the investigator being aware of the group identities. Data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were conducted with one-way analyses of variance (ANOVAs) following non-parametric multiple comparisons, as indicated in each fig- ure legend. A value of P < 0.05 was considered to indicate statistical significance. Analyses were performed using Statcel4 (OMS Ltd., Tokyo, Japan). Results Injection of hMSCs suppressed SCI symptoms We have previously reported that transplant viable hMSCs, but not inviable hMSCs into injured spinal cord significantly improved locomotor activity and decreased lesion size which is surrounded by GFAP immunoreactions within a week [7]. These effects were likely medi- ated through MG/MF-hMSC communication. To confirm the reproducibility of these find- ings, we determined the BMS score, the damaged site by performing GFAP immunostaining and assessing the gene expression of Eno2 (neuron-specific enolase) as well as the retention of hMSCs in the spinal cord (Fig 1). On day 1 after SCI, hMSCs (5 × 10 cells/μL) or HBSSs were injected into the intervertebral spinal cord. To validate the injection and retention of the hMSCs, human GAPDH was semi- quantified via qPCR, and the estimated hMSC number was calculated from the standard curve (Fig 1A). Immediately after hMSC injection (pod 1), approximately 260,000 hMSCs were Fig 1. Injection of human mesenchymal stem/stromal cells (hMSCs) suppresses spinal cord injury (SCI) symptoms. (A) Retention of hMSCs after injection into the � � spinal cord 1 day after SCI (n = 4 mouse at each time-point). : P < 0.01 (Dunnett’s post hoc test, vs day 1). (B) Locomotor activity was determined based on BMS scores after SCI with/without hMSC injection. We injected hMSCs (filled circles, n = 40 mouse) or vehicle (HBSS; open circles, n = 26 mouse) into the spinal cord on postoperative day (pod) 1. Locomotor activity was then tracked for 14 days. Control animals (filled triangle, n = 45 mouse) received no injection into the spinal cord � � � after SCI. : P < 0.05, : P < 0.01 (Tukey’s post hoc test). (C) Representative images of lesion site in the spinal cord 3 and 14 days after SCI (n = 3 mouse in each group). The lesion site was identified as the area surrounded by enhanced GFAP expression. Vehicle-treated control animals (upper) exhibited a larger lesion area than hMSC- treated animals (bottom). R: rostral, C: caudal, D: dorsal, V: ventral. (D) Expression of Eno2 was quantified via real time-PCR after SCI with/without hMSCs in a time- dependent fashion. Eno2 expression was temporarily decreased on pod 3 in the HBSS-treated group (n = 5 mouse). Significant suppression of Eno2 expression was � � observed in the hMSC-treated group (n = 10 mouse) on pod 3. : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. The gene expression levels are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. All data are expressed as the mean ± SEM. https://doi.org/10.1371/journal.pone.0230080.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 8 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury detected–representing 50% of the injected cells–at the five vertebral sites (Th 7–12), including the site of injection. The numbers of hMSCs decreased to approximately 40,000 cells on pod 3, and have few-to-no hMSCs were detected on pod 14. SCI induced severe locomotor disability, with BMS scores reaching approximately 1.5 points within a few hours; they gradually increased through pod 14. Animals treated with hMSCs were associated with significantly higher BMS scores than were the vehicle-treated ani- mals from pod 3 through pod 14 (Fig 1B). Typical images of the lesion sites surrounded by GFAP immunoreactions in the hMSC-treated and HBSS-treated groups on pod 3 and 14 are shown in Fig 1C. The hMSC-treated mice exhibited fewer lesions (i.e., glial scarring) than the HBSS-treated mice at pod 14. Eno2 is a neuron-specific glycolytic enzyme which is considered to indicate neuronal viabil- ity. Expression of Eno2 was significantly higher in hMSC-treated animals than in HBSS-treated animals (Fig 1D). All of these results support the findings of our previous study [7]: hMSCs suppress SCI-induced adverse events. Expression of chemokines and their receptors after SCI We next quantified the levels of MG/MF-related chemokines and the expression of their receptor genes in mice during the experimental periods without hMSC injection after the SCI (Fig 2). The expression of selected chemokine genes, including Ccl2, Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl2, significantly increased by 48-, 41-, 228-, 52-, 17-, and 56-fold, respectively, on pod 1. With the exceptions of Ccl3, Ccl4, and Cxcl1, decreases in gene expressions were mostly observed on pod 7. Levels of Ccl3, Ccl4, and Cxcl1 expression remained 41-, 112-, and 7-fold Fig 2. Gene expression of chemokines and their receptors after spinal cord injury (SCI). Expression of Ccl2 (A), Ccl3 (B), Ccl4 (C), Ccl7 (E), Cxcl1 (F), and Cxcl2 (G) significantly increased on post-operative day (pod) 1. In contrast, increases in Ccl5 (D) expression were observed on pod 14. Increases in gene expression were observed for all chemokine receptors (H—L) on pod 1. While Ccr1 (H), Ccr3 (J), and Cxcr2 (L) expression decreased by pod 7, Ccr2 (I) and Ccr5 (K) levels remained significantly elevated on pod 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level) after normalization against Gapdh as a housekeeping gene (n = 6 � � � mouse at each time-point). Data are expressed as mean ± SEM. : P<0.05, : P<0.01 (Dunnett’s post hoc test, vs day 0). https://doi.org/10.1371/journal.pone.0230080.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 9 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury greater than pre-injury levels on pod 14. Although no increases in Ccl5 expression were observed during the acute period, levels of Ccl5 expression had increased by 82-fold on pod 14. By contrast, the expression of all chemokine receptors, including Ccr1, Ccr2, Ccr3, Ccr5, and Cxcr2 significantly increased by 12-, 44-, 6-, 4-, and 144-fold, respectively, on pod 1. Although Ccr1, Ccr3, and Cxcr2 expression decreased on pod 7, Ccr2 and Ccr5 levels remained 18-fold and 5-fold greater, respectively, at pod 14. We divided these chemokines into two groups: those exhibiting acute increases following SCI (CCL2, CCL3, CCL4, CCL7, CXCL1, and CXCL2) and those exhibiting increases in the chronic phase after SCI (CCL5). Then, we fur- ther analyzed the cellular localization of CCL2 and CCL5 and their receptors in the spinal cord via immunohistochemistry because they have been reported well for MG/MF migration as a representative gene. Distribution of CCL2 and CCR2 expression in the spinal cord on day 1 We examined the cellular localization of CCL2 and its receptor CCR2 via immunohistochem- istry before inducing SCI and at pods 1 and 14 after SCI. Few-to-no CCL2 immunoreactions were observed in the spinal cord of pre-SCI mice. Immunopositive reactions for CCL2 were observed around the site of SCI at pod 1. CCL2 immunoreactions at pod 1 merged with NeuN (a neuronal marker) but not with GFAP (an astrocytic marker) or F4/80 (an MG/MF marker) (Fig 3A and 3B). Similar to the findings observed for Ccl2 expression (Fig 2A), CCL2 immuno- reactivity had diminished by pod 14 (Fig 3C). Few-to-no CCR2 immunoreactions were observed in the spinal cord pre-SCI. However, immunoreactivity was obviously recognized at the epicenter of injury on pod 1 but diminished Fig 3. Localization of CCL2 and CCR2 after spinal cord injury (SCI). (A) CCL2 cells (green) were observed around the site of SCI on day 1 and were merged with the neuronal marker NeuN (red) (arrows). (B) CCL2 immunoreactions (green with arrows) did not overlap with the astrocyte marker GFAP (upper in red) or the MG/ + + MF marker F4/80 (bottom in red). (C) The intensity of CCL2 immunoreactivity decreased by postoperative day (pod) 14. (D) CCR2 immunoreactivity (green) was + + + + mainly expressed in Ly-6G cells (pink) around the site of the spinal cord lesion (arrows), but not in Iba1 cells (red) (arrowhead). (E) Numbers of CCR2 , Ly-6G , + � � + Iba1 , and merged cells were counted at the site of injury (n = 5 mouse). Data are expressed as the mean ± SEM. : P<0.01 (Dunnett’s post hoc test). (F) Many CCR2 + + + (green)/Ly-6G+ (pink) cells were also merged with iNOS (red) (arrows). (G) Numbers of CCR2 , Ly-6G , iNOS , and merged cells were counted (n = 5 mouse). Most CCR2 cells were co-stained with Ly-6G and iNOS. Data are expressed as the mean ± SEM. Blue indicates nuclear staining with DAPI. https://doi.org/10.1371/journal.pone.0230080.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 10 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury once again by pod 14. These results were consistent with those observed regarding Ccr2 expression (Fig 2I). Most CCR2-positive (CCR2 ) cells exhibited a round shape and a seg- mented nucleus. Multiple-staining experiments revealed that CCR2 cells had merged, sug- gesting that these cells were granulocytes (Fig 3D). Few-to-no CCR2 reactions were observed + + + in cells stained with Iba1, NeuN, and GFAP. The numbers of CCR2 , Ly-6G , and Iba1 cells at the site of injury were 292 ± 32, 394 ± 42, and 27 ± 6 cells/mm , respectively. Moreover, we 2 + + observed 245 ± 26 cells/mm CCR2 /Ly-6G cells at the site of injury: a significantly greater + + 2 number than that of CCR2 /Iba1 cells (4 ± 2 cells/mm ) (Fig 3E). Our results also indicated + + + 2 that CCR2 /Ly-6G cells had merged with iNOS cells (236 ± 21 cells/mm ) (Fig 3F and 3G). Distribution of CCL5 and CCR5 expression in the spinal cord on day 14 We aimed to determine the localization of CCL5 and its receptor. Previous studies have reported that CCL5 binds to three receptors: CCR1, CCR3, and CCR5 [32]. Because Ccr1 and Ccr3 expression temporarily increased in the acute phase and decreased by pod 7, we focused only on the levels of CCR5, whose underlying gene expression exhibited sustained increases by pod 14 (Fig 2H, 2J and 2K). Few-to-no CCL5 immunoreactions were observed in the spinal cord immediately following SCI or pod 1. However, CCL5 immunoreactions were observed + + on pod 14 around the SCI site. The CCL5 immunoreactions merged with NeuN and F4/80 cells but not with GFAP cells (Fig 4A). Although the immunoreactions were prominent at the epicenter of the injury on pod 1, CCR5 immunoreactivity was rarely observed post-SCI. CCR5 cells exhibited a round shape with lobed nuclei; moreover, similar to the findings observed for CCR2 on pod 1, CCR5 cells were merged with Ly-6G (Fig 4B). However, unlike the findings for CCR2, CCR5 + + Fig 4. Localization of CCL5 and CCR5 after spinal cord injury (SCI). (A) CCL5 cells were observed at the peri-injury site on pod 14 and were merged with NeuN + + + + (red) and F4/80 (red) cells, respectively (arrows). However, CCL5 cells were not merged with GAFP cells (red). (B) CCR5 immunoreactivity (green) was observed in + + the epicenter on pod 1, and merged with Ly-6G cells (red) (arrows). (C) CCR5 cells (green) were observed at the peri-injury site on pod 14, and were merged with + + + + NeuN (red with arrows) and F4/80 (arrows with dashed lines) cells. (D) F4/80 cells (red) on pod 14 co-stained with Ym1 immunoreactions (green), a marker for M2-type alternatively activated MG/MF (arrows). Nuclei were detected using DAPI. https://doi.org/10.1371/journal.pone.0230080.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 11 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury immunoreactivity was still observed on pod 14 at the peri-injury site. Furthermore, CCR5 cells were merged with NeuN+ and F4/80+ immunoreactions (Fig 4C) on pod 14. Moreover, the F4/80 cells were co-stained with Ym1, which is an AAM marker (Fig 4D). Effects of hMSCs on CCL2, CCR2, and the inflammatory response We subsequently investigated the effect of hMSC treatment on the levels of gene expression corresponding to chemokines and their receptors. The mouse specificity of the chemokine primers was examined using mouse spinal cords and naïve hMSCs via RT-PCR (Fig 5A). A house-keeping gene, Gapdh (glyceraldehyde-3-phosphate dehydrogenase), primers for which amplified both human and mouse samples gene expression in our previous study [27], was rec- ognized signals from both mouse and human samples. However, all tested primers for Ccl2, Ccl5, and their receptors were amplified with estimated size in mouse spinal cord samples (Fig 5A). We then compared Ccl2 levels in the spinal cord between mice injected with HBSS or hMSCs after SCI. Ccl2 expression on pod 3 was approximately 16-fold higher in mice treated with hMSCs than in those treated with HBSS; these increases remained significant at pod 14 (Fig 5B). Although levels of Ccr2 expression in the spinal cord were similar in hMSC- and HBSS-treated mice on pod 3, they increased in hMSC-treated mice by pod 14 (Fig 5C). + + No obvious differences were observed in CCL2 or CCR2 staining, which had merged with NeuN staining around the site of injury, between the groups on pod 3. However, the intensity of immunoreactivity tended to be greater in mice treated with hMSCs (Fig 5D). While Ly-6G cells were easily recognized at the sites of injury, their numbers did not differ significantly between the groups. However, there were few CCR2 cells, and they did not over- lap with Ly-6G cells (Fig 5E and 5F). On pod 3, CCR2 immunoreactivity was observed in larger cells at the site of injury, overlapping with NeuN cells (Fig 5G). To determine the effect of hMSC treatment on inflammation involving granulocytes, we examined gene expression for inflammatory markers, such as Il1b, Elane (neutrophil expressed elastase), and Mpo (myeloperoxidase) on pod 3. While HBSS-injected SCI mice exhibited an increase in the expression of all three genes in the spinal cord, this expression was significantly suppressed in mice treated with hMSCs (Fig 5H–5J). Zc3h112a (Zinc finger CCCH-type con- taining 12A, also known as monocyte chemotactic protein-induced protein) is a protein whose expression is induced by CCL2; Zc3h112a is thereby implicated in immunosuppression and M2-type macrophage polarization [33]. Mice treated with hMSCs exhibited significant increases in Zc3h112a expression in the spinal cord on pod 14 (Fig 5K). Effects of hMSCs on CCL5 and CCR5 expression We next investigated the effects of hMSCs on CCL5 and CCR5 at pod 14. We observed no sig- nificant differences in the expressions of Ccl5 or Ccr5 in the spinal cord between the hMSC- and HBSS-treated groups on pod 3. Although no differences in Ccr5 expression were observed, Ccl5 expression in the spinal cord increased by approximately 11-fold in hMSC-treated mice by pod 14 relative to the levels observed in HBSS-treated mice (Fig 6A and 6B). + + Immunostaining experiments confirmed that CCL5 merged with NeuN and F4/80 cells + + (Fig 3). However, the CCL5 /F4/80 cells were more dominant in hMSC-treated mice (Fig + + 6C). In hMSCs-treated mice, CCR5 cells were merged with F4/80 cells around the site of injury (Fig 6D). Influence of hMSCs on MG/MF polarization Injection of hMSCs on pod 1 influenced MG/MF levels on pod 14. Quantification experi- ments revealed that hMSC-treated animals exhibited significantly greater Aif1 (a pan-MG/MF PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 12 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 5. Human mesenchymal stem/stromal cells (hMSCs) increased Ccl2 expression and reduced inflammatory responses. (A) All tested chemokine and receptor primers were detected at the expected rates in mouse samples (Ms). Ccl2 (B) and Ccr2 (C) expressions in the spinal cord were quantified via real time-PCR after spinal cord injury (SCI) and hMSC injection. Ccl2 levels were significantly increased in hMSC-treated mice (n = 19 mouse) on postoperative days (pod) 3 and 14 relative to the levels observed in HBSS-treated mice (n = 13 mouse) on the same pod. Temporary increases in Ccr2 expression were observed in HBSS-treated mice, with levels � � decreasing by pod 14. However, increases in Ccr2 expression remained significant in hMSC-treated mice on pod 14. Data are expressed as mean ± SEM. : P < 0.01 + + (Dunnett’s post hoc test), n.s.: not significant. (D) CCL2 cells (green) were co-labeled with NeuN cells (red) in both groups on pod 3. Blue indicates nuclear staining + + + using DAPI. (E) Slight CCR2 immunoreactivity (green) was observed in Ly-6G (pink with arrows) and Iba1 (arrows with dashed lines) cells. Moreover, some large PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 13 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury cells were labeled with CCR2 antibody (arrowhead). Blue indicates nuclear staining using DAPI. (F) No significant differences in the number of positive cells were + + observed between the hMSCs- and HBSS-treated groups (n = 3 mouse at each group). (G) Large CCR2 cells (red; arrows) co-localized with NeuN reactions (green) but not with GFAP reactions (pink). Blue indicates nuclear staining using DAPI. (H-K) The transplantation of hMSCs influenced inflammation-induced gene expression in the spinal cord on pod 3. Animals treated with hMSCs (n = 10 mouse) exhibited significant decreases in Il1b (H), Elane (I), and Mpo (J) expression in the spinal cord on pod 3 relative to HBSS-treated animals (n = 5 mouse). (K) Conversely, Zc3h12a expression was significantly greater in hMSC-treated mice (n = 9 mouse) than in HBSS-treated mice (n = 8 mouse) on pod 14. The gene expression levels are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as mean ± SEM. : P < 0.05 (Dunnett’s post hoc test), n.s.: not significant. https://doi.org/10.1371/journal.pone.0230080.g005 marker, also known as Iba1) expression than did HBSS-treated animals on pod 14 (Fig 7A). Immunostaining for Iba1 in the spinal cord revealed that Iba1 reactions clustered at both the peri-injury and injection sites in hMSC-treated mice; no such clustering was observed around the site of injection in the HBSS-treated group (Fig 7B). Fluorescent staining revealed that F4/ + + 80 MG/MF-like cells were co-expressed with Ym1 cells around the injection site, suggestive of M2-type AAMs (Fig 7C). Moreover, the number of F4/80 MG/MF-like cells was greater in hMSC-treated mice than in HBSS-treated mice. Expression of the AAM markers Arg1 (Arginase1) and Chil3, which encodes Ym1, was sig- nificantly lower in the hMSCs-treated group than in the HBSS-treated group on pod 3. How- ever, levels of gene expression had increased by 15- and 2-fold, respectively, in the hMSCs- treated group on pod 14 (Fig 7D and 7E). Moreover, the expression of the anti-inflammatory cytokine IL-4 had increased by 3-fold in hMSCs-treated animals by pod 14 (Fig 7F). Influence of hMSCs on axonal regeneration To determine the effect of hMSCs on axonal regeneration, we examined gene expression and protein localization corresponding to CRMP2 (encoded by Dpysl2) and GAP43 in the mouse spinal cord after SCI. Expression of Dpysl2 remained nearly unchanged in HBSS-treated mice Fig 6. Human mesenchymal stem/stromal cells (hMSCs) increased levels of CCL5 expression during the chronic phase. Expression of mouse (A) Ccl5 and (B) Ccr5 in the spinal cord was quantified via real-time PCR in a time-dependent fashion after spinal cord injury (SCI) and hMSC injection. Mice treated with hMSCs (n = 19 mouse) exhibited significant increases in Ccl5 expression relative to HBSS-treated mice (n = 13 mouse) on postoperative day (pod) 14. However, no significant differences in Ccr5 expression were observed between the two groups during the experimental period. Data are expressed the relative level (fold) to compare with pod 0 � � (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. : P < 0.01 (Dunnett’s post hoc test), n.s.: + + not significant. (C) CCL5 immunoreactivity (green) was observed at the peri-injury site on pod 14 and merged with NeuN (red) and F4/80 (pink) cells. Mice treated + + + with hMSCs exhibited greater CCL5 immunoreactivity than did HBSS-treated mice. (D) Similar CCR5 reactions (green) were merged with F4/80 (red) cells in both experimental groups. Blue indicates nuclear staining using DAPI. https://doi.org/10.1371/journal.pone.0230080.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 14 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 7. Human mesenchymal stem/stromal cells (hMSCs) increased levels of alternatively activated macrophages (AAM) and anti-inflammatory cytokines. (A) The transplantation of hMSCs significantly increased the levels of Aif1 expression on postoperative day (pod) 14 (n = 9 mouse) relative to those observed in the HBSS- � � treated animals (n = 8 mouse). Data are expressed as the mean ± SEM. : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. (B) Iba1-positive MG/MF were + + clustered around the site of hMSC injection on pod 14 (n = 3 mouse in each group). (C) F4/80 cells (green) exhibited greater overlap with Ym1 cells (red) around the injection site in the hMSC-treated group than in the HBSS-treated group. Nuclear staining was performed using DAPI. Transplantation of hMSCs significantly increased the expression of the AAM markers Arg1 (D) and Chil3 (E), as well as that of the anti-inflammatory cytokine IL-4 (F) on pod 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. � � � : P<0.05, : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. https://doi.org/10.1371/journal.pone.0230080.g007 during the experimental period. However, Dpysl2 expression increased on pod 14 and was sig- nificantly higher in hMSC-treated mice than in HBSS-treated mice (Fig 8A). Similar tenden- cies were observed for Gap43 expression, which was significantly higher in hMSCs-treated + + animals on pod 14 (Fig 8B). CRMP2 and GAP43 immunoreactions were detected in the peri-injury region and clearly merged with NeuN neurons on pod 14 (Fig 8C and 8D). CCL5 promotes AAM activity and axonal regeneration To confirm whether increases in Ccl5 expression following hMSC injection contribute to MG/ MF polarization and axonal regeneration, mouse recombinant CCL5 was injected into the spi- nal cord after SCI on pod 7. On the following day, we examined the gene expression of AAM and axonal regeneration markers. Aif1 expression remained unchanged following CCL5 injec- tion (Fig 9A). However, CCL5-treated mice tended to increase the expression of Chil3 and Arg1 and exhibited significantly greater Chil3 expression than did vehicle-treated mice (p = 0.004) (Fig 9B and 9C), suggesting that CCL5 contributes to MG/MF polarization. CCL5-injected mice also exhibited increases in Dpysl2 (p = 0.097) and Gap43 (p = 0.033) expression (Fig 9D and 9E), suggesting that CCL5 also contributes to axonal regeneration. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 15 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 8. Human mesenchymal stem/stromal cells (hMSCs) promoted axonal regeneration. The transplantation of hMSCs increased the expression of the axonal regeneration markers Dpysl2 (A) and Gap43 (B). Mice treated with hMSCs (n = 9 mouse) exhibited significantly greater gene expression than did the HBSS-treated mice (n = 8 mouse) on postoperative day (pod) 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. : P < 0.05 (Student’s t-test), n.s.: not significant. (C) CRMP2 immunoreactivity (green) + + was observed in NeuN neurons (red) at the peri-injury site on pod 14. (D) GAP43 immunoreactivity (green) was merged with NeuN neurons (red) at the peri-injury site on pod 14 (n = 3 mouse in each group). Blue indicates nuclear staining using DAPI. https://doi.org/10.1371/journal.pone.0230080.g008 Discussion Human-MSC therapy has shown promise in supporting regeneration and/or recovery from diverse tissue injuries, including neuronal injuries [3–6]. Although we have reported that implanted hMSCs suppress neural injuries in mice by modulating MG/MF after brain ische- mia and SCI [7, 8], the mechanism and form of communication between hMSCs and MG/MF remain unclear. In the present study, we examined the influences of hMSCs on chemokine expressions because chemokines guide the migration of immune cells, including MF. Using our SCI model, we especially focused on hMSC-induced changes in two MF-related chemo- kine axes: CCL2/CCR2 and CCL5/CCR5. We here xenografted hMSCs into mouse spinal cord PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 16 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 9. Microglia/macrophage (MG/MF) polarization and axonal regeneration were enhanced by CCL5. To determine whether increases in CCL5 following hMSC transplantation contribute to MG/MF polarization and axonal regeneration, recombinant mouse CCL5 (rmCCL5, n = 6 mouse) or vehicle (n = 6 mouse) was injected into the spinal cord on postoperative day (pod) 7. We examined levels of gene expression for MG/MF polarization (A-C) and axonal regeneration (D and E) 24 hours after injection. There were no obvious differences in the levels of the pan-macrophage marker Aif1 (A). Expression of the M2-type AAM marker Chil3 (B) significantly increased following the injection of rmCCL5 (p<0.01). Although Arg1 expression (C) also tended to increase, these increases were nonsignificant. Expression of the axonal regeneration marker Gap43 (E) significantly increased following the injection of rmCCL5 (p<0.05), while Dpysl2 (D) expression tended to increase (p = 0.097). All data are expressed the relative level (fold) to compare with vehicle treated animals (n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are � � � expressed as the mean ± SEM. : P < 0.01, : P < 0.05 (Dunnett’s post hoc test). https://doi.org/10.1371/journal.pone.0230080.g009 without administration of immunosuppressants to not influence an immune response. Many studies were given immunosuppressants for decrease of the rejection by xenografting. How- ever, we have previously reported that injection of the hMSCs into immunocompetent mouse brain did not change the fate of cells to compare with immunodeficient mice [8]. These are supported and reproduced by many studies [10]. The Ccl2 and Ccr2 expressions drastically increased on pod1, and CCL2 immunoreactions were recognized in neurons in the acute phase following SCI. While previous studies have reported that CCR2 is mainly expressed in monocytes and infiltrated MF [34], CCR2 re- + + actions had barely merged with F4/80 MG/MF; however, CCR2 reactions overlapped greatly with Ly-6G granulocytes, which were likely neutrophils, at the epicenter of the injury on pod 1. Aberrations in the CCL2/CCR2 axis are reportedly associated with neuroinflamma- tory disease. [35–39]. CCR2 gene-deficient (KO) animals exhibit decreases in lesion size and recruitment of monocytes into the lesion following traumatic brain injury (TBI) [18] and SCI, [40]. Moreover, CCR2 neutrophils are activated under inflammatory conditions, thereby exacerbating the inflammatory response [41, 42]. According to these studies, most CCR2 / Ly-6G granulocytes on pod 1 exhibited co-localization of iNOS, suggesting an increase of inflammation. In the present study, the hMSC implantation resulted in improvements in locomotor activ- ity and tended to decrease lesion size; these findings were consistent with our previous report [7]. While we initially expected that the implanted hMSCs would diminish Ccl2 expression, PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 17 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury the levels of Ccl2, and not those of Ccr2, increased following hMSC transplantation. However, + + + the numbers of Ly-6G and CCR2 /Ly-6G cells did not change significantly with hMSC transplantation. Interestingly, CCR2 immunoreactions after hMSC transplantation were + + observed in NeuN -neurons as well as Ly-6G cells. Some recent studies have speculated that, in addition to its role in chemotaxis, CCL2 exerts neuroprotective and immunomodulatory effects [18, 19]. CCL2 aids in the migration of neural precursor cells and promotes neuroregeneration in patients with TBI [39] and stroke [43]. Kwon et al. suggested that the overexpression of CCL2 derived from neurons promotes axonal regeneration via AAM activation after CNS injury [44]. Investigating the condition of inflam- mation and neutrophil activation, we found that the transplantation of hMSCs decreased the gene expression levels of Il1b, Elane and Mpo; these observations indicated reduced inflamma- tion and neutrophil activation. Recent findings are consistent with our results: neutrophils polarized after infiltration can exert both beneficial and deleterious effects following tumori- genesis [45–47], stroke [48] and TBI [49]. Further, we found that mice treated with hMSCs exhibited persistent Ccl2 expression for 2 weeks as well as increases the expression of Zc3h12a, which encodes MCPIP1 and is known to induce by CCL2 and IL-1β, on pod14. MCPIP1 plays a role in anti-inflammation via the down-regulation of NF-κB signaling [50], and MCPIP1-KO mice reportedly exhibit significant increases in infarct volume after brain ischemia [51]. Although further studies are required to determine the precise roles of chronic increases in CCL2, the enhancement of Ccl2 expression via hMSC transplantation may contribute to anti- inflammation. We also examined the expression of Ccl5 after inducing SCI: it drastically increased by pod 14. While CCL5 binds to three receptors, CCR1, CCR3, and CCR5, we focused on CCR5 because Ccr5 expression remained significantly elevated through pod 14. Our findings vary in their consistency with previous research, as the role of the CCL5/CCR5 axis in CNS injuries is controversial in the literature. CCL5 deletion reduces infarct volume in cerebral ischemia [52], and CCR5 blockade improves locomotor function in a murine model of SCI [53]. These obser- vations suggest that CCL5 acts as a deleterious chemokine. Conversely, CCR5-KO mice exhibit increases in infarct volume in models of ischemia [54] and accelerated motor neuronal death in models of hypoglossal nerve injury [55]. Application of CCL5 to cultured neural cells signif- icantly suppressed cell death and increased the expression of some growth factors [21]. More- over, CCL5 decreased IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), and iNOS gene expression in lipopolysaccharide-treated primary MG cultures [55]. The present study found that the hMSC implantation significantly increased Ccl5 expres- sion by pod 14 but observed no changes in Ccr5 expression. Previous investigations have reported that CCL5 expression in neurons, MF, and astrocytes of the spinal cord of models of neuropathic pain and ischemia increased from pod 3 [21, 56]. We observed that CCL5 and CCR5 localized to neurons and MG/MF; this was not affected by hMSC implantation. How- ever, the intervention increased the expressions of pan-MG/MF (Aif1) and M2-type AAM (Arg1 and Chil3) markers. Moreover, the animals that received the hMSC implantation exhib- ited increases in Il4 expression by pod 14. IL-4 is a major activator of AAM polarization [57]. Interestingly, CCL2, CCL5, and insulin-like growth factor 1 (IGF-1) are also reportedly involved in the differentiation of MG/MF toward AAMs in osteoblasts [24], suggesting that greater hMSC-induced Il4 and Ccl5 expression by might stimulate MG/MF migration and polarization. Axonal extension and reconstruction of neuronal networks are important events in the recovery of spinal function. Research has suggested that AAMs promote axonal growth and overcome the effects of inhibitory substrates [58]. Several studies have also indicated that the implantation of AAMs into the injured spinal cord induces axonal regrowth and functional PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 18 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury improvement [59, 60]. In the present study, implantation of hMSCs increased the expression of the genes encoding axonal regenerative markers, including Dpysl2 and Gap43, on pod 14, and corresponding immunoreactions were observed in the neurons at the peri-injury site. To obtain direct evidence for the contribution of the increased Ccl5 expression to axonal exten- sion, we injected rmCCL5 into the spinal cord on pod 7 and determined the gene expression of M2-type AAM as well as the levels of axonal regenerative markers. We observed signifi- cantly greater Chil3 and Gap43 expression in rmCCL5-treated mice, which also tended to exhibit increases in Arg1 and Dpysl2 expression; however, no obvious changes in Aif1 expres- sion were observed following CCL5 injection. These results suggest that the CCL5 may par- tially participate in AAM polarization and axonal extension. In the present study, we examined the role of hMSCs after SCI by focusing on two represen- tative MF-related chemokine axes, CCL2/CCR2 and CCL5/CCR5, both of which exhibited acute or chronic increases following SCI. hMSC implantation upregulated the levels of these chemokines. We also confirmed improvements in the clinical symptoms of SCI following hMSC injection. While the CCL2/CCR2 axis contributes to the enhancement of inflammation, hMSC-mediated increases in CCL2 did not alter the number of granulocytes. Moreover, hMSCs might induce MG/MF polarization to the M2-type AAM and drive axonal generation. While chemokines could participate in these functions, the direct mechanisms by which the communication between hMSCs and the recipient tissues yield these phenomena remain to be elucidated. Future studies should examine mice subjected to chemokine KO or gene silencing to determine whether hMSCs influence CCL2/CCR2 and CCL5/CCR5 axes in post-SCI neuroregeneration. Conclusions In conclusion, the post-SCI implantation of hMSCs enhances the expression of Ccl2 and Ccl5 and improves locomotor activity. Moreover, the implantation of hMSCs enhances MG/MF polarization to AAM and the gene expression of axonal regenerative markers. The function of hMSCs might be partially mediated by the chemokine axes. Supporting information S1 Checklist. (DOCX) Acknowledgments We would like to thank Editage (www.editage.jp) for English language editing. Author Contributions Conceptualization: Kazumichi Yagura, Tomomi Tsumuraya, Atsushi Sato. Data curation: Kazumichi Yagura, Hirokazu Ohtaki, Naoto Kawada, Keisuke Suzuki, Motoyasu Nakamura. Formal analysis: Kazumichi Yagura, Hirokazu Ohtaki, Kazuyuki Miyamoto. Funding acquisition: Hirokazu Ohtaki, Atsushi Sato, Yutaka Hiraizumi. Investigation: Kazumichi Yagura, Hirokazu Ohtaki, Tomomi Tsumuraya, Atsushi Sato. Methodology: Kazumichi Yagura, Hirokazu Ohtaki. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 19 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Project administration: Kazumichi Yagura, Hirokazu Ohtaki, Masahiko Izumizaki, Yutaka Hiraizumi, Kazuho Honda. Resources: Hirokazu Ohtaki, Atsushi Sato, Yutaka Hiraizumi. Software: Kazumichi Yagura, Hirokazu Ohtaki. Supervision: Hirokazu Ohtaki, Koji Kanzaki, Kenji Dohi, Masahiko Izumizaki, Yutaka Hirai- zumi, Kazuho Honda. 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Nat Med. 1998; 4 (7):814–21. https://doi.org/10.1038/nm0798-814 PMID: 9662373. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 23 / 23 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Public Library of Science (PLoS) Journal

The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury

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Copyright: © 2020 Yagura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: YES. This work was supported by KAKENHI, as follows: 16H05443 (Hirokazu Ohtaki), 17K10943 (Yutaka Hiraizumi), and 26861214 (Atsushi Sato). Competing interests: The authors declare that they have no competing interests.
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Citation: Yagura K, Ohtaki H, Tsumuraya T, Sato A, Miyamoto K, Kawada N, et al. (2020) The Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) have shown enhancement of CCL2 and CCL5 by human bone potential in facilitating recovery from spinal cord injury (SCI) through communicating with marrow-derived mesenchymal stem/stromal cells microglia/macrophages (MG/MΦ). We here focused on chemokines as a candidate for the might contribute to inflammatory suppression and axonal extension after spinal cord injury. PLoS ONE communication. Selected MG/MΦ-related chemokines were determined gene expression 15(3): e0230080. https://doi.org/10.1371/journal. after SCI and further focused CCL2/CCR2 and CCL5/CCR5 to estimate role of the chemo- pone.0230080 kines by hMSCs. Male C57/BL6 mice were subjected to spinal cord transection. Gene Editor: Xing-Ming Shi, Augusta University, UNITED expression was assayed in the spinal cords following SCI for selected MG/MΦ-related che- STATES mokines and their receptors. hMSCs (5×10 cells) were then transplanted into parenchyma Received: October 31, 2019 of the spinal cord, and the expressions of the Ccl2/Ccr2 and Ccl5/Ccr5 axes, inflammation, Accepted: February 20, 2020 MG/MΦ-polarization, and axonal regeneration were evaluated to measure the influence of the hMSCs. Finally, mouse CCL5 was injected into the spinal cords. Acute increases in Published: March 10, 2020 gene expression after SCI were observed for most chemokines, including Ccl2; chronic Peer Review History: PLOS recognizes the + + + increases were observed for Ccl5. CCL2 -cells merged with NeuN -neurons. CCR2 immu- benefits of transparency in the peer review + + process; therefore, we enable the publication of noreactivity was principally observed in Ly-6G /iNOS -granulocytes on postoperative day + + + all of the content of peer review and author (pod) 1, and CCL5 and CCR5 immunoreactivity overlapped with NeuN -neurons and F4/ responses alongside final, published articles. The 80 -MG/MΦ on pod 14. The hMSC transplantation enhanced Ccl2 and Ccl5 and improved editorial history of this article is available here: + + locomotor activity. The hMSC implantation did not alter the number of Ly-6G /CCR2 but https://doi.org/10.1371/journal.pone.0230080 decreased Il1, Elane, and Mpo on pod 3. Conversely, hMSC transplantation increased Copyright:© 2020 Yagura et al. This is an open expression of Zc3h12a (encodes MCP-1-induced protein) on pod 14. Moreover, hMSC access article distributed under the terms of the Creative Commons Attribution License, which increased the Aif1, and two alternatively activated macrophage (AAM)-related genes, Arg1 permits unrestricted use, distribution, and and Chil3 (Ym1), as well as axonal regenerative markers, Dpysl2 and Gap43. Gene expres- reproduction in any medium, provided the original sion indicative of AAM polarization and axonal regeneration were partially recovered by author and source are credited. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 1 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Data Availability Statement: All relevant data are CCL5 injection. These results suggest that hMSC implantation increases Ccl2 and Ccl5, within the paper and its Supporting Information improves locomotor activity, enhances MG/MΦ polarization to AAM, and increases the files. gene expression of axonal regenerative markers. These functions of hMSCs might be par- Funding: YES. This work was supported by tially mediated by the CCL2/CCR2 and CCL5/CCR5 axes. KAKENHI, as follows: 16H05443 (Hirokazu Ohtaki), 17K10943 (Yutaka Hiraizumi), and 26861214 (Atsushi Sato). Competing interests: The authors declare that they have no competing interests. Introduction Every year, more than 10,000 people in the United States experience spinal cord injuries (SCI) due to trauma-related accidents. While medication regimens during the acute period of injury involve the extensive administration of steroids and other anti-inflammatory drugs, the recov- ery of neurologic function relies on the neural plasticity to each patient. The considerable number of patients that become permanently paralyzed despite medical intervention reflects the inadequacy of inherent mechanisms in repairing the nervous system [1, 2]. Bone marrow-derived mesenchymal stem/stromal cells (MSCs) are multipotent tissue stem cells that have shown promise in facilitating regeneration and/or recovery after neuronal injury [3–6]. We have previously demonstrated that xenografting human MSCs (hMSCs) into immunocompetent mice subjected to SCI [7] or brain ischemia [8] protected against short- term neurological deficits and damage; however, the levels of the grafted hMSCs decreased sig- nificantly within the first week. Further experiments indicated that hMSCs might communi- cate with recipient tissues and thereby convert the phenotypic polarization of host microglia/ macrophages (MG/MF) to an alternatively activated phenotype (AAM), which is known to promote tissue repair and regeneration after injury [9]. Several immune- and nonimmune- related disease models and isogenic graft experiments have provided evidence for the immu- nomodulatory functions of hMSCs [10, 11]. However, precisely how hMSCs communicate with or regulate MG/MF remains unclear. Chemokines guide the migration of immune cells, such as leukocytes and macrophages, and stimulate inflammatory responses, and are divided into the CC, CXC, C, and CX3C fami- lies from the structural feature [12]. Levels of many chemokines are increased in patients with central nervous system (CNS) disorders, such as SCI [13] and cerebral infarction [14], increase the levels of many chemokines. Indeed, enhanced levels of chemokines were observed in iso- genic grafted bone marrow-derived MSCs of a hypoxia-ischemia rat model, but not in adipose tissue-derived MSCs [15]. Cultured hMSCs secrete several chemokines and express several chemokine receptors on the cell surface [16]. An additional study further reported that MSCs elevate the secretion of several chemokines by stimulating lipopolysaccharide (LPS) in vitro [17]. Hence, the communication between hMSCs and MG/MF may be partially mediated by chemokines. Research has indicated that some chemokines play other roles in addition to chemotaxis response. Chemokines such as CCL2 (monocyte chemoattractant protein-1; MCP-1), CX3CL1 (fractalkine), CXCL12 (Stromal cell-derived factor 1; SDF-1), and CCL5 (regulated on activa- tion, normal T cell expressed and secreted; RANTES) are reportedly involved in neuroprotec- tion [18–21], axonal regeneration [22, 23], and the differentiation of MG/MF to AAM [24]. In present study, we firstly examined the effect and fate of transplanted hMSCs after SCI. Following the study, we determined gene expression for selected some MG/MF-related che- mokines and their receptors on SCI, and then focused on two representative major MG/MF- related chemokine axes, CCL2/CCR2 and CCL5/CCR5, whose corresponding increases in the gene expression had been characterized as acute or chronic. The influences of hMSC PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 2 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury implantation following SCI on the CCL2/CCR2 and CCL5/CCR5 axes, inflammation, MG/ MF polarization, and/or axonal regeneration were examined to gain insight into the actions of hMSCs. Materials and methods Animals Wild-type, male C57BL/6 mice (8 to 12 weeks old, weighing 17 to 25 g) were purchased from Sankyo Lab Service Corporation (Tokyo, Japan) and used for the experiments (total of 139 ani- mals including 7 euthanized animals). Mice were maintained on a 12-h light/dark cycle at 24 ± 2˚ C with constant humidity (55 ± 10%) and free access to food and water. All experimen- tal procedures involving animals were approved and followed by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Showa University (#06105 and 07079). SCI model The SCI mouse model was generated in accordance with previously described methods [7, 25]. Anesthesia was induced via inhalation of 4.0% sevoflurane and maintained with 3.0% sevoflur- ane in N O/O (2:1). Under aseptic conditions, an incision was made along the midline of the 2 2 skin on the back, and the muscles, soft tissues, and yellow ligaments overlying the spinal col- umn between T9 and T10 were removed. The intervertebral spinal cord between T9 and T10 was then transected with a thin-bladed razor (FEATHER, Osaka, Japan). After bleeding had stopped and the coagulated blood had been removed, the incision was closed, and the animals were administered 1.0 mL of lactated Ringer’s solution (s.c.; Otsuka, Tokyo, Japan) to prevent dehydration of which were given once a day until they gain the body weight. To support their eating, animal food was placed on the cage floor, and the water bottle was lowered to allow easy access. Moreover, to support urination, the region of the lower abdomen in all mice was gently stimulated a few times every day. Although we did not administer any pain relievers or anesthesia during the experiments because they may influence SCI and locomotor activity, we made all efforts to minimize animal numbers and animal discomfort. However, total of 7 ani- mals were excluded from the study by humane endpoints. The 3-animals scored the Basso Mouse Scale (BMS) [26] more than 3 immediately after SCI were excluded from the experi- ments for incomplete hindlimb paralysis by a surgical error. Moreover, the 4-mice were excluded from the study because the mice exhibited peritoneal infection, severe body weight loss more than 25% before surgery, hindlimb wounds, and/or tail or foot autophagia. These animals were euthanized by cervical dislocation or CO -gas exposure. Preparation of implanted hMSCs Frozen vials of hMSCs from the bone marrow were obtained from Dr. Prockop (The Center for the Preparation and Distribution of Adult Stem Cells [http://medicine.tamhsc.edu/irm/ msc-distribution.html]) under the auspices of a National Institutes of Health (NIH)/National Center for Research Resources grant (P40 RR 17 447–06). The experiments were performed with hMSCs from donor 281L [7, 8, 27]. To expand hMSCs, a frozen vial of 1.0×10 passage 3 cells was thawed and plated at 100 cells/cm in multiple 150-mm plates (Nunclon, Thermo Fisher Scientific, Waltham, MA) with a 20-mL complete culture medium (CCM) that con- sisted ofα-minimal essential medium (α-MEM; Invitrogen, Carlsbad, CA), 20% heat-inacti- vated fetal bovine serum (FBS; Hyclone; Thermo Fisher Scientific), 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). The cultures were PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 3 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury incubated, and the medium was replaced every 3 days for approximately 8 days until the cells were 70–80% confluent. The medium was then discarded, and the culture plates were washed with phosphate-buffered saline (PBS). Adherent cells were harvested using 0.25% trypsin and 1 mM EDTA (Invitrogen) for 5 min at 37˚ C and resuspended at 5 × 10 cells in 0.5 μL of ster- ile Hank’s balanced salt solution (HBSS; Invitrogen) for injection. Injection of hMSCs into the spinal cord The hMSCs were injected into the spinal cord in accordance with previously described meth- ods [7]. Briefly, on the day following surgery to induce SCI, mice were re-anesthetized via inhalation of sevoflurane. The animals were placed face-down, and a truncated 29G needle with a 5.0-μL glass syringe (Hamilton, Reno, NV) was inserted directly into the intervertebral spinal cord (D/V -1.0 mm) between T10 and T11. We trained investigators to insert the needle to the depth of the anterior horn of the spinal cord by the injection of dye. Then, the depth of needle insertion was standardized at -1.0 mm from surface of which did not leak the dye from the needle tract during infusion or from the ventral spinal cord. The hMSCs (5 × 10 cells/μL, n = 40 mice) or HBSS (n = 26 mice) was infused at a rate of 0.5 μL/min with an Ultra Micro Pump (World Precision Instruments, Sarasota, FL). Following infusion, the needle was left in place for 1 min to facilitate the diffusion of the solution into the tissue. Previously, we demon- strated that the fate of hMSCs does not differ between immunocompetent and immunode- ficient animals after ischemia [8]. Therefore, in the present study, we refrained from using immunosuppressant following cell implantation [10]. Assessment of motor function Motor function after SCI was evaluated using an open-field behavior test that focused on hin- dlimb locomotor function according to the BMS. The BMS consists of an open-field locomotor rating scale that ranges from 0 (complete paralysis) to 9 (normal mobility). Briefly, individual mice were placed in the center of an open field (50 × 50 cm ) with a smooth, non-slip floor and monitored for 4 min. Hindlimb movements, trunk/tail stability, and forelimb-hindlimb coordination were assessed and graded. Mice were tested daily for 14 days after the operation. Mice were randomly numbered to ensure that the investigators remained unaware of the treat- ment groups during scoring. Isolation of RNA and production of cDNA Under sodium-pentobarbital (100 mg/kg, i.p.) anesthesia, spinal cord samples from the T7 to T12 vertebrae were dissected on postoperative days (pod) 0 (sham-operated non-SCI control), 1, 3, 7, and 14 with/without hMSC or vehicle injection. The tissue was snap-frozen in liquid nitrogen and stored at −80˚ C until use. Total RNA was isolated using the TRIZOL Reagent (Invitrogen) in accordance with the manufacturer’s instructions and dissolved in RNase-free water. The purity and concentration of the extracted RNA were determined spectrophotomet- rically (NanoDrop, Wilmington, DE). A High-Capacity RNA-to-cDNA Kit (Applied Biosys- tems, Foster City, CA) was used to synthesize cDNA using 2 μg of total RNA in accordance with the manufacturer’s instructions. To confirm the species specificity of the primers, we also extracted total RNA from cultured hMSCs and synthesized the cDNA. Semi-quantification of the number of hMSCs in the spinal cord On days 1, 3, or 14 after SCI (0, 2, or 13 days after cell injection; n = 4 mice at each time- point), mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and the spinal PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 4 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury cord was collected and subjected to cDNA preparation as described above. Semi-quantifica- tion of the number of hMSCs was determined based on the gene expression level of human GAPDH in mouse Gapdh via qPCR. Standard curves were generated by adding serial dilutions 2 6 of hMSCs (1 × 10 to 1 × 10 cells) to spinal cord samples (T7 to T12 vertebrae) of uninjured mice prior to homogenization according to the methods published by previous studies [7, 8, 28–30]. Finally, the estimated number of resident hMSCs was calculated from the standard curve. The human-specific primers cross-hybridized less than 0.07% with mouse cDNA obtained from spinal cord samples that had not been injected with hMSCs. Polymerase chain reaction (PCR) PCR analyses were performed using TaKaRa Ex Taq (TaKaRa, Shiga, Japan) to confirm the species specificity of primers. The reaction mixture was created with an appropriate volume of the cDNA mixture: 0.25 μL of forward and reverse primers (50 nmol/mL), 2.0 μL of dNTP mixture (0.25 mM each), 0.1 μL of TaKaRa Ex Taq (5 units/μL), and 2.0 μL of 10 × Ex Taq Buffer in a total volume of 20 μL. Thermal-cycling parameters were set as follows: 95˚ C for 1 min for initial denaturation, followed by a cycling regime of 40 cycles at 95˚ C for 45 s, 60˚ C for 30 s, and 72˚ C for 45 s. At the end of the final cycle, an additional 7-min extension step was conducted at 72˚ C. Ten microliters of each reaction mixture was electrophoresed onto 2.0% agarose gel, and the bands were visualized using ethidium bromide. Quantitative PCR (qPCR) analyses were performed with SYBR Premix Ex Taq II reagent (TaKaRa) and an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosys- tems, Lincoln, CA). Relative gene expression was calculated using the absolute quantification method. The mouse glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh) was used as a housekeeping gene to normalize the cDNA levels. All primers were designed as described in Table 1. All data are expressed the relative level (fold) to compare with pod 0 (basal level) after normalization against mouse Gapdh as a housekeeping gene (n = 6 mouse at each time-point). Immunostaining Under sodium-pentobarbital (100 mg/kg, i.p.) anesthesia, the animals were transcardially per- fused with 0.9% saline followed by 2% paraformaldehyde (PFA) in 50 mM phosphate buffer (PB) (pH 7.2), and the spinal cord (T7 to T12 vertebrae) was carefully removed. The spinal cords were then post-fixed overnight, immersed in 20% sucrose in 0.1 M PB for two nights, and embedded in liquid nitrogen-cooled isopentane using an embedding solution (20% sucrose in 0.1 M PB:O.C.T. compound [Sakura Finetech, Tokyo, Japan]: 2:1]. Sagittal spinal cord sections (thickness, 5 μm) were obtained using a cryostat (Hyrax50, Carl Zeiss, Inc.; Oberkochen, Germany) and stored at –80˚ C until use. For single staining, the sections were washed with PBS and incubated in 0.3% H O to 2 2 quench internal peroxidase reactions. The sections were then immersed in 2.5% normal horse serum (NHS; Vector, Burlingame, CA) in PBS containing 0.05% Tween 20 (PBST) to block non-specific reactions for 1 h. Subsequently, the sections were incubated overnight with poly- clonal rabbit anti-GFAP (1:10; DAKO, Glostrup, Denmark) or polyclonal rabbit anti-Iba1 antibodies (1:500; WAKO, Tokyo, Japan). After washing with PBST, the sections were incu- bated with biotinylated goat anti-rabbit immunoglobulin G (IgG) (1:200, Santa Cruz, Santa Cruz, CA) for 2 h. They were then incubated in an avidin-biotin complex solution (Vector) followed by diaminobenzidine (DAB; Sigma, St. Louis, MO) as a chromogen. Immunopositive cells were observed via light microscopy (AX70) using DP2-BSW image analysis software (Olympus, Tokyo, Japan). PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 5 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Table 1. Primers used for real-time polymerase chain reaction. 0 0 0 0 Name Symbol Species Forward (5 to 3 ) Reverse (5 to 3 ) Size (bp) AIF1 Aif1 mouse atcaacaagcaattcctcgatga cagcattcgcttcaaggacata 144 Arg1 Arg1 mouse ctccaagccaaagtccttagag aggagctgtcattagggacatc 185 CCL2 Ccl2 mouse ttaaaaacctggatcggaaccaa gcattagcttcagatttacgggt 121 CCL3 Ccl3 mouse ttctctgtaccatgacactctgc cgtggaatcttccggctgtag 100 CCL4 Ccl4 mouse ttcctgctgtttctcttacacct ctgtctgcctcttttggtcag 121 CCL5 Ccl5 mouse gctccaatcttgcagtcgtg gagcagctgagatgcccatt 242 CCL7 Ccl7 mouse gctgctttcagcatccaagtg ccagggacaccgactactg 135 CCR1 Ccr1 mouse tggtgggcaatgtcctagtg aagtaacagttcgggccctc 300 CCR2 Ccr2 mouse atccacggcatactatcaacatc caaggctcaccatcatcgtag 104 CCR3 Ccr3 mouse accccgtacaacctggttct ccaacaaaggcgtagattactgg 158 CCR5 Ccr5 mouse ttttcaagggtcagttccgac ggaagaccatcatgttacccac 158 CRMP2 Dpysl2 mouse tcaaaggtggcaagattgtgaa ggaatcaccattctggagtgg 145 CXCL1 Cxcl1 mouse ctgggattcacctcaagaacatc cagggtcaaggcaagcctc 117 CXCL2 Cxcl2 mouse tccagagcttgagtgtgacg tggttcttccgttgagggac 201 CXCR2 Cxcr2 mouse gggtggggagttcgtgtaga aggtgctaggatttgagcct 200 ELANE Elane mouse caggaacttcgtcatgtcagc agcagttgtgatgggtcaaag 162 ENO2 Eno2 mouse tgagaataaatccttggagctggt ggtcatcgcccactatctgg 302 GAP43 Gap43 mouse tctgctactaccgatgcagc tggaggacggggagttatcag 181 GAPDH Gapdh mouse gctacactgaggaccaggttgt ctcctgttattatgggggtctg 306 GAPDH Gapdh human ggagcgagatccctccaaaat ggctgttgtcatacttctcatgg 197 GAPDH Gapdh mouse aggtcggtgtgaacggatttg tgtagaccatgtagttgaggtca 123 IL-1β Il1b mouse gcaactgttcctgaactcaact atcttttggggtccgtcaact 89 IL-4 Il4 mouse tctcgaatgtaccaggagccatatc agcaccttggaagccctacaga 183 MCPIP Zc3h12a mouse acgaagcctgtccaagaatcc taggggcctctttagccaca 115 MPO Mpo mouse agggccgctgattatctacat ctcacgtcctgataggcaca 152 Ym-1 Chil3 mouse ggtggacacagaatggttcttc ccaggagcgttagtgacagc 128 AIF: allograft inflammatory factor, Arg: arginase, CCL: chemokine (C-C motif) ligand, CCR: chemokine (C-C motif) receptor, Chil: chitinase-like, CRMP: collapsin response mediator protein, CXCL: chemokine (C-X-C motif) ligand, CXCR: chemokine (C-X-C motif) receptor, ELANE: neutrophil elastase, Eno: neuron-specific enolase, GAP: growth associated protein, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, GFAP: glial fibrillary acidic protein, IL: interleukin, MCPIP: monocyte chemotactic protein-1 induced protein, MPO: myeloperoxidase, = not species specific. https://doi.org/10.1371/journal.pone.0230080.t001 For multiple staining, sections that had been immersed in 0.3% H O were incubated with 2 2 an M.O.M. Immunodetection Kit (Vector), followed by 2.5% NHS/PBST. The sections were then incubated overnight with primary antibodies. The sections were rinsed with PBST and immersed with appropriate fluorescently labeled secondary antibodies for 2 h. Subsequently, cell nuclei were stained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI, 1:10,000; Roche, Manheim, Germany) and incubated in 1.0 mM CuSO in 50 mM ammonium acetate buffer (pH 5.0) to diminish autofluorescence [31]. Fluorescence was detected using an Axio Imager optical sectioning microscope with ApoTome (Carl Zeiss, Inc.). All primary and sec- ondary antibodies used for immunostaining are listed in Tables 2 and 3. For control staining, the same steps were performed with the exception of incubation with each primary antibody. Cell counting Five sagittal sections (thickness: 5 μm) were collected from each mouse: at the midline (3), which included the central canal adjacent to the core injury site; and bilaterally at 150 μm (2 and 4) and 300 μm (1 and 5) lateral to the midline. The numbers of immunopositive cells PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 6 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Table 2. Primary antibodies used for immunohistochemistry. Primary antibodies (Clone #) Host Companies Catalog # Dilution CCL2 Rabbit Bioss (Boston, MA) bs-1101R 1,500 CCL5 (25H14L17) Rabbit Thermo Fisher Scientific (Waltham, MA) 701030 100 CCL5 Goat R&D systems (Minneapolis, MN) AF478 300 CCR2 Goat Novus Biologicals (Littleton, Co) NB100-701 300 CCR2 Goat GeneTex (Irvine, CA) GTX45788 300 CCR5 Rabbit Bioss (Boston, MA) bs-2514R 500 NeuN (A60) Mouse Merc Millipore (Burlington, MA) MAB377 1,000 GFAP Rabbit DAKO (Glostrup, Denmark) N1506 10 GFAP (G-A-5) Mouse SIGMA (St. Louis, MO) G3893 1,000 Iba1 Rabbit Wako (Osaka, Japan) 019–19741 500 Alexa635labeled-Iba1 Rabbit Wako (Osaka, Japan) 013–26471 500 F4/80 (Cl:A3-1) Rat BMA Biomedicals (Augst, Switzerland) T-2008 500 Ly-6G (RB6-8C5) Rat BD Biosciences (Franklin Lakes, NJ) 557445 400 iNOS Rabbit Merc Millipore (Burlington, MA) 06–573 250 Ym-1 Rabbit StemCell Technologies (Vancouver, BC Canada) 1404 200 CRMP2 Rabbit LifeSpan BioSciences (Seattle, WA) LS-B5657 3,000 GAP43 (EP890Y) Rabbit Abcam (Cambridge, UK) ab75810 500 CCL: chemokine (C-C motif) ligand, CCR: chemokine (C-C motif) receptor, CRMP: collapsin response mediator protein, GAP: growth associated protein, GFAP: glial fibrillary acidic protein, Iba1: ionized calcium-binding adapter molecule, iNOS: inducible nitric oxide synthase, NeuN: neuronal nuclei. https://doi.org/10.1371/journal.pone.0230080.t002 against CCR2, Iba1, Ly-6G, and inducible nitric oxide (iNOS) antibodies were counted in four random fields at the injury site in each animal at 500× magnification and expressed as the aver- age total number in a square millimeter (mm ). Injection of recombinant mouse CCL5 (RANTES) into the spinal cord To investigate the role of CCL5 in the spinal cord after injury, recombinant mouse CCL5 was directly injected into the spinal cord between T10 and T11 according to the cell-injection pro- cedure described above. Recombinant mouse CCL5 (RANTES; Miltenyi Biotec, Bergisch Gladbach, Germany) was dissolved in saline containing 0.1% bovine serum albumin (BSA; Wako) under aseptic conditions to obtain a concentration of 20 ng/μL. Seven days after SCI, mouse spinal cords were injected with a bolus of CCL5 (20 ng, n = 6 mice) or vehicle (n = 6 mice) at a rate of 0.5 μL/min. Mice were anesthetized with sodium pentobarbital (100 mg/kg, i. p.) at 24 h following injection, and spinal cord tissue was dissected from T7 to T12 for qPCR. Table 3. Secondary antibodies used for immunohistochemistry. Secondary antibodies Host Companies Catalog # Dilution Rabbit IgG (biotin) Goat Santa Cruz Biotechnology (Santa Cruz, CA) SC-2040 200 Goat IgG (biotin) Horse Vector Laboratory (Burlingame, CA) BA-9500 200 Mouse IgG (Alexa 546) Goat ThermoFisher Scientific (Waltham, MA) A11030 800 Rabbit IgG (Alexa 488) Goat ThermoFisher Scientific (Waltham, MA) A11034 400 Rabbit IgG (Alexa 546) Goat ThermoFisher Scientific (Waltham, MA) A11035 400 Rabbit IgG (Alexa 647) Goat ThermoFisher Scientific (Waltham, MA) A21245 400 Rat IgG (FITC) Goat Jackson ImmunoResearch (West Grove, PA) 112-096-071 200 Goat IgG (Alexa FITC) Donkey ThermoFisher Scientific (Waltham, MA) A11055 400 Goat IgG (Alexa 546) Donkey ThermoFisher Scientific (Waltham, MA) A1056 400 https://doi.org/10.1371/journal.pone.0230080.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 7 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Statistical analysis As each mouse was assigned a random number, all data were collected and analyzed without the investigator being aware of the group identities. Data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were conducted with one-way analyses of variance (ANOVAs) following non-parametric multiple comparisons, as indicated in each fig- ure legend. A value of P < 0.05 was considered to indicate statistical significance. Analyses were performed using Statcel4 (OMS Ltd., Tokyo, Japan). Results Injection of hMSCs suppressed SCI symptoms We have previously reported that transplant viable hMSCs, but not inviable hMSCs into injured spinal cord significantly improved locomotor activity and decreased lesion size which is surrounded by GFAP immunoreactions within a week [7]. These effects were likely medi- ated through MG/MF-hMSC communication. To confirm the reproducibility of these find- ings, we determined the BMS score, the damaged site by performing GFAP immunostaining and assessing the gene expression of Eno2 (neuron-specific enolase) as well as the retention of hMSCs in the spinal cord (Fig 1). On day 1 after SCI, hMSCs (5 × 10 cells/μL) or HBSSs were injected into the intervertebral spinal cord. To validate the injection and retention of the hMSCs, human GAPDH was semi- quantified via qPCR, and the estimated hMSC number was calculated from the standard curve (Fig 1A). Immediately after hMSC injection (pod 1), approximately 260,000 hMSCs were Fig 1. Injection of human mesenchymal stem/stromal cells (hMSCs) suppresses spinal cord injury (SCI) symptoms. (A) Retention of hMSCs after injection into the � � spinal cord 1 day after SCI (n = 4 mouse at each time-point). : P < 0.01 (Dunnett’s post hoc test, vs day 1). (B) Locomotor activity was determined based on BMS scores after SCI with/without hMSC injection. We injected hMSCs (filled circles, n = 40 mouse) or vehicle (HBSS; open circles, n = 26 mouse) into the spinal cord on postoperative day (pod) 1. Locomotor activity was then tracked for 14 days. Control animals (filled triangle, n = 45 mouse) received no injection into the spinal cord � � � after SCI. : P < 0.05, : P < 0.01 (Tukey’s post hoc test). (C) Representative images of lesion site in the spinal cord 3 and 14 days after SCI (n = 3 mouse in each group). The lesion site was identified as the area surrounded by enhanced GFAP expression. Vehicle-treated control animals (upper) exhibited a larger lesion area than hMSC- treated animals (bottom). R: rostral, C: caudal, D: dorsal, V: ventral. (D) Expression of Eno2 was quantified via real time-PCR after SCI with/without hMSCs in a time- dependent fashion. Eno2 expression was temporarily decreased on pod 3 in the HBSS-treated group (n = 5 mouse). Significant suppression of Eno2 expression was � � observed in the hMSC-treated group (n = 10 mouse) on pod 3. : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. The gene expression levels are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. All data are expressed as the mean ± SEM. https://doi.org/10.1371/journal.pone.0230080.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 8 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury detected–representing 50% of the injected cells–at the five vertebral sites (Th 7–12), including the site of injection. The numbers of hMSCs decreased to approximately 40,000 cells on pod 3, and have few-to-no hMSCs were detected on pod 14. SCI induced severe locomotor disability, with BMS scores reaching approximately 1.5 points within a few hours; they gradually increased through pod 14. Animals treated with hMSCs were associated with significantly higher BMS scores than were the vehicle-treated ani- mals from pod 3 through pod 14 (Fig 1B). Typical images of the lesion sites surrounded by GFAP immunoreactions in the hMSC-treated and HBSS-treated groups on pod 3 and 14 are shown in Fig 1C. The hMSC-treated mice exhibited fewer lesions (i.e., glial scarring) than the HBSS-treated mice at pod 14. Eno2 is a neuron-specific glycolytic enzyme which is considered to indicate neuronal viabil- ity. Expression of Eno2 was significantly higher in hMSC-treated animals than in HBSS-treated animals (Fig 1D). All of these results support the findings of our previous study [7]: hMSCs suppress SCI-induced adverse events. Expression of chemokines and their receptors after SCI We next quantified the levels of MG/MF-related chemokines and the expression of their receptor genes in mice during the experimental periods without hMSC injection after the SCI (Fig 2). The expression of selected chemokine genes, including Ccl2, Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl2, significantly increased by 48-, 41-, 228-, 52-, 17-, and 56-fold, respectively, on pod 1. With the exceptions of Ccl3, Ccl4, and Cxcl1, decreases in gene expressions were mostly observed on pod 7. Levels of Ccl3, Ccl4, and Cxcl1 expression remained 41-, 112-, and 7-fold Fig 2. Gene expression of chemokines and their receptors after spinal cord injury (SCI). Expression of Ccl2 (A), Ccl3 (B), Ccl4 (C), Ccl7 (E), Cxcl1 (F), and Cxcl2 (G) significantly increased on post-operative day (pod) 1. In contrast, increases in Ccl5 (D) expression were observed on pod 14. Increases in gene expression were observed for all chemokine receptors (H—L) on pod 1. While Ccr1 (H), Ccr3 (J), and Cxcr2 (L) expression decreased by pod 7, Ccr2 (I) and Ccr5 (K) levels remained significantly elevated on pod 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level) after normalization against Gapdh as a housekeeping gene (n = 6 � � � mouse at each time-point). Data are expressed as mean ± SEM. : P<0.05, : P<0.01 (Dunnett’s post hoc test, vs day 0). https://doi.org/10.1371/journal.pone.0230080.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 9 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury greater than pre-injury levels on pod 14. Although no increases in Ccl5 expression were observed during the acute period, levels of Ccl5 expression had increased by 82-fold on pod 14. By contrast, the expression of all chemokine receptors, including Ccr1, Ccr2, Ccr3, Ccr5, and Cxcr2 significantly increased by 12-, 44-, 6-, 4-, and 144-fold, respectively, on pod 1. Although Ccr1, Ccr3, and Cxcr2 expression decreased on pod 7, Ccr2 and Ccr5 levels remained 18-fold and 5-fold greater, respectively, at pod 14. We divided these chemokines into two groups: those exhibiting acute increases following SCI (CCL2, CCL3, CCL4, CCL7, CXCL1, and CXCL2) and those exhibiting increases in the chronic phase after SCI (CCL5). Then, we fur- ther analyzed the cellular localization of CCL2 and CCL5 and their receptors in the spinal cord via immunohistochemistry because they have been reported well for MG/MF migration as a representative gene. Distribution of CCL2 and CCR2 expression in the spinal cord on day 1 We examined the cellular localization of CCL2 and its receptor CCR2 via immunohistochem- istry before inducing SCI and at pods 1 and 14 after SCI. Few-to-no CCL2 immunoreactions were observed in the spinal cord of pre-SCI mice. Immunopositive reactions for CCL2 were observed around the site of SCI at pod 1. CCL2 immunoreactions at pod 1 merged with NeuN (a neuronal marker) but not with GFAP (an astrocytic marker) or F4/80 (an MG/MF marker) (Fig 3A and 3B). Similar to the findings observed for Ccl2 expression (Fig 2A), CCL2 immuno- reactivity had diminished by pod 14 (Fig 3C). Few-to-no CCR2 immunoreactions were observed in the spinal cord pre-SCI. However, immunoreactivity was obviously recognized at the epicenter of injury on pod 1 but diminished Fig 3. Localization of CCL2 and CCR2 after spinal cord injury (SCI). (A) CCL2 cells (green) were observed around the site of SCI on day 1 and were merged with the neuronal marker NeuN (red) (arrows). (B) CCL2 immunoreactions (green with arrows) did not overlap with the astrocyte marker GFAP (upper in red) or the MG/ + + MF marker F4/80 (bottom in red). (C) The intensity of CCL2 immunoreactivity decreased by postoperative day (pod) 14. (D) CCR2 immunoreactivity (green) was + + + + mainly expressed in Ly-6G cells (pink) around the site of the spinal cord lesion (arrows), but not in Iba1 cells (red) (arrowhead). (E) Numbers of CCR2 , Ly-6G , + � � + Iba1 , and merged cells were counted at the site of injury (n = 5 mouse). Data are expressed as the mean ± SEM. : P<0.01 (Dunnett’s post hoc test). (F) Many CCR2 + + + (green)/Ly-6G+ (pink) cells were also merged with iNOS (red) (arrows). (G) Numbers of CCR2 , Ly-6G , iNOS , and merged cells were counted (n = 5 mouse). Most CCR2 cells were co-stained with Ly-6G and iNOS. Data are expressed as the mean ± SEM. Blue indicates nuclear staining with DAPI. https://doi.org/10.1371/journal.pone.0230080.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 10 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury once again by pod 14. These results were consistent with those observed regarding Ccr2 expression (Fig 2I). Most CCR2-positive (CCR2 ) cells exhibited a round shape and a seg- mented nucleus. Multiple-staining experiments revealed that CCR2 cells had merged, sug- gesting that these cells were granulocytes (Fig 3D). Few-to-no CCR2 reactions were observed + + + in cells stained with Iba1, NeuN, and GFAP. The numbers of CCR2 , Ly-6G , and Iba1 cells at the site of injury were 292 ± 32, 394 ± 42, and 27 ± 6 cells/mm , respectively. Moreover, we 2 + + observed 245 ± 26 cells/mm CCR2 /Ly-6G cells at the site of injury: a significantly greater + + 2 number than that of CCR2 /Iba1 cells (4 ± 2 cells/mm ) (Fig 3E). Our results also indicated + + + 2 that CCR2 /Ly-6G cells had merged with iNOS cells (236 ± 21 cells/mm ) (Fig 3F and 3G). Distribution of CCL5 and CCR5 expression in the spinal cord on day 14 We aimed to determine the localization of CCL5 and its receptor. Previous studies have reported that CCL5 binds to three receptors: CCR1, CCR3, and CCR5 [32]. Because Ccr1 and Ccr3 expression temporarily increased in the acute phase and decreased by pod 7, we focused only on the levels of CCR5, whose underlying gene expression exhibited sustained increases by pod 14 (Fig 2H, 2J and 2K). Few-to-no CCL5 immunoreactions were observed in the spinal cord immediately following SCI or pod 1. However, CCL5 immunoreactions were observed + + on pod 14 around the SCI site. The CCL5 immunoreactions merged with NeuN and F4/80 cells but not with GFAP cells (Fig 4A). Although the immunoreactions were prominent at the epicenter of the injury on pod 1, CCR5 immunoreactivity was rarely observed post-SCI. CCR5 cells exhibited a round shape with lobed nuclei; moreover, similar to the findings observed for CCR2 on pod 1, CCR5 cells were merged with Ly-6G (Fig 4B). However, unlike the findings for CCR2, CCR5 + + Fig 4. Localization of CCL5 and CCR5 after spinal cord injury (SCI). (A) CCL5 cells were observed at the peri-injury site on pod 14 and were merged with NeuN + + + + (red) and F4/80 (red) cells, respectively (arrows). However, CCL5 cells were not merged with GAFP cells (red). (B) CCR5 immunoreactivity (green) was observed in + + the epicenter on pod 1, and merged with Ly-6G cells (red) (arrows). (C) CCR5 cells (green) were observed at the peri-injury site on pod 14, and were merged with + + + + NeuN (red with arrows) and F4/80 (arrows with dashed lines) cells. (D) F4/80 cells (red) on pod 14 co-stained with Ym1 immunoreactions (green), a marker for M2-type alternatively activated MG/MF (arrows). Nuclei were detected using DAPI. https://doi.org/10.1371/journal.pone.0230080.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 11 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury immunoreactivity was still observed on pod 14 at the peri-injury site. Furthermore, CCR5 cells were merged with NeuN+ and F4/80+ immunoreactions (Fig 4C) on pod 14. Moreover, the F4/80 cells were co-stained with Ym1, which is an AAM marker (Fig 4D). Effects of hMSCs on CCL2, CCR2, and the inflammatory response We subsequently investigated the effect of hMSC treatment on the levels of gene expression corresponding to chemokines and their receptors. The mouse specificity of the chemokine primers was examined using mouse spinal cords and naïve hMSCs via RT-PCR (Fig 5A). A house-keeping gene, Gapdh (glyceraldehyde-3-phosphate dehydrogenase), primers for which amplified both human and mouse samples gene expression in our previous study [27], was rec- ognized signals from both mouse and human samples. However, all tested primers for Ccl2, Ccl5, and their receptors were amplified with estimated size in mouse spinal cord samples (Fig 5A). We then compared Ccl2 levels in the spinal cord between mice injected with HBSS or hMSCs after SCI. Ccl2 expression on pod 3 was approximately 16-fold higher in mice treated with hMSCs than in those treated with HBSS; these increases remained significant at pod 14 (Fig 5B). Although levels of Ccr2 expression in the spinal cord were similar in hMSC- and HBSS-treated mice on pod 3, they increased in hMSC-treated mice by pod 14 (Fig 5C). + + No obvious differences were observed in CCL2 or CCR2 staining, which had merged with NeuN staining around the site of injury, between the groups on pod 3. However, the intensity of immunoreactivity tended to be greater in mice treated with hMSCs (Fig 5D). While Ly-6G cells were easily recognized at the sites of injury, their numbers did not differ significantly between the groups. However, there were few CCR2 cells, and they did not over- lap with Ly-6G cells (Fig 5E and 5F). On pod 3, CCR2 immunoreactivity was observed in larger cells at the site of injury, overlapping with NeuN cells (Fig 5G). To determine the effect of hMSC treatment on inflammation involving granulocytes, we examined gene expression for inflammatory markers, such as Il1b, Elane (neutrophil expressed elastase), and Mpo (myeloperoxidase) on pod 3. While HBSS-injected SCI mice exhibited an increase in the expression of all three genes in the spinal cord, this expression was significantly suppressed in mice treated with hMSCs (Fig 5H–5J). Zc3h112a (Zinc finger CCCH-type con- taining 12A, also known as monocyte chemotactic protein-induced protein) is a protein whose expression is induced by CCL2; Zc3h112a is thereby implicated in immunosuppression and M2-type macrophage polarization [33]. Mice treated with hMSCs exhibited significant increases in Zc3h112a expression in the spinal cord on pod 14 (Fig 5K). Effects of hMSCs on CCL5 and CCR5 expression We next investigated the effects of hMSCs on CCL5 and CCR5 at pod 14. We observed no sig- nificant differences in the expressions of Ccl5 or Ccr5 in the spinal cord between the hMSC- and HBSS-treated groups on pod 3. Although no differences in Ccr5 expression were observed, Ccl5 expression in the spinal cord increased by approximately 11-fold in hMSC-treated mice by pod 14 relative to the levels observed in HBSS-treated mice (Fig 6A and 6B). + + Immunostaining experiments confirmed that CCL5 merged with NeuN and F4/80 cells + + (Fig 3). However, the CCL5 /F4/80 cells were more dominant in hMSC-treated mice (Fig + + 6C). In hMSCs-treated mice, CCR5 cells were merged with F4/80 cells around the site of injury (Fig 6D). Influence of hMSCs on MG/MF polarization Injection of hMSCs on pod 1 influenced MG/MF levels on pod 14. Quantification experi- ments revealed that hMSC-treated animals exhibited significantly greater Aif1 (a pan-MG/MF PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 12 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 5. Human mesenchymal stem/stromal cells (hMSCs) increased Ccl2 expression and reduced inflammatory responses. (A) All tested chemokine and receptor primers were detected at the expected rates in mouse samples (Ms). Ccl2 (B) and Ccr2 (C) expressions in the spinal cord were quantified via real time-PCR after spinal cord injury (SCI) and hMSC injection. Ccl2 levels were significantly increased in hMSC-treated mice (n = 19 mouse) on postoperative days (pod) 3 and 14 relative to the levels observed in HBSS-treated mice (n = 13 mouse) on the same pod. Temporary increases in Ccr2 expression were observed in HBSS-treated mice, with levels � � decreasing by pod 14. However, increases in Ccr2 expression remained significant in hMSC-treated mice on pod 14. Data are expressed as mean ± SEM. : P < 0.01 + + (Dunnett’s post hoc test), n.s.: not significant. (D) CCL2 cells (green) were co-labeled with NeuN cells (red) in both groups on pod 3. Blue indicates nuclear staining + + + using DAPI. (E) Slight CCR2 immunoreactivity (green) was observed in Ly-6G (pink with arrows) and Iba1 (arrows with dashed lines) cells. Moreover, some large PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 13 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury cells were labeled with CCR2 antibody (arrowhead). Blue indicates nuclear staining using DAPI. (F) No significant differences in the number of positive cells were + + observed between the hMSCs- and HBSS-treated groups (n = 3 mouse at each group). (G) Large CCR2 cells (red; arrows) co-localized with NeuN reactions (green) but not with GFAP reactions (pink). Blue indicates nuclear staining using DAPI. (H-K) The transplantation of hMSCs influenced inflammation-induced gene expression in the spinal cord on pod 3. Animals treated with hMSCs (n = 10 mouse) exhibited significant decreases in Il1b (H), Elane (I), and Mpo (J) expression in the spinal cord on pod 3 relative to HBSS-treated animals (n = 5 mouse). (K) Conversely, Zc3h12a expression was significantly greater in hMSC-treated mice (n = 9 mouse) than in HBSS-treated mice (n = 8 mouse) on pod 14. The gene expression levels are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as mean ± SEM. : P < 0.05 (Dunnett’s post hoc test), n.s.: not significant. https://doi.org/10.1371/journal.pone.0230080.g005 marker, also known as Iba1) expression than did HBSS-treated animals on pod 14 (Fig 7A). Immunostaining for Iba1 in the spinal cord revealed that Iba1 reactions clustered at both the peri-injury and injection sites in hMSC-treated mice; no such clustering was observed around the site of injection in the HBSS-treated group (Fig 7B). Fluorescent staining revealed that F4/ + + 80 MG/MF-like cells were co-expressed with Ym1 cells around the injection site, suggestive of M2-type AAMs (Fig 7C). Moreover, the number of F4/80 MG/MF-like cells was greater in hMSC-treated mice than in HBSS-treated mice. Expression of the AAM markers Arg1 (Arginase1) and Chil3, which encodes Ym1, was sig- nificantly lower in the hMSCs-treated group than in the HBSS-treated group on pod 3. How- ever, levels of gene expression had increased by 15- and 2-fold, respectively, in the hMSCs- treated group on pod 14 (Fig 7D and 7E). Moreover, the expression of the anti-inflammatory cytokine IL-4 had increased by 3-fold in hMSCs-treated animals by pod 14 (Fig 7F). Influence of hMSCs on axonal regeneration To determine the effect of hMSCs on axonal regeneration, we examined gene expression and protein localization corresponding to CRMP2 (encoded by Dpysl2) and GAP43 in the mouse spinal cord after SCI. Expression of Dpysl2 remained nearly unchanged in HBSS-treated mice Fig 6. Human mesenchymal stem/stromal cells (hMSCs) increased levels of CCL5 expression during the chronic phase. Expression of mouse (A) Ccl5 and (B) Ccr5 in the spinal cord was quantified via real-time PCR in a time-dependent fashion after spinal cord injury (SCI) and hMSC injection. Mice treated with hMSCs (n = 19 mouse) exhibited significant increases in Ccl5 expression relative to HBSS-treated mice (n = 13 mouse) on postoperative day (pod) 14. However, no significant differences in Ccr5 expression were observed between the two groups during the experimental period. Data are expressed the relative level (fold) to compare with pod 0 � � (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. : P < 0.01 (Dunnett’s post hoc test), n.s.: + + not significant. (C) CCL5 immunoreactivity (green) was observed at the peri-injury site on pod 14 and merged with NeuN (red) and F4/80 (pink) cells. Mice treated + + + with hMSCs exhibited greater CCL5 immunoreactivity than did HBSS-treated mice. (D) Similar CCR5 reactions (green) were merged with F4/80 (red) cells in both experimental groups. Blue indicates nuclear staining using DAPI. https://doi.org/10.1371/journal.pone.0230080.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 14 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 7. Human mesenchymal stem/stromal cells (hMSCs) increased levels of alternatively activated macrophages (AAM) and anti-inflammatory cytokines. (A) The transplantation of hMSCs significantly increased the levels of Aif1 expression on postoperative day (pod) 14 (n = 9 mouse) relative to those observed in the HBSS- � � treated animals (n = 8 mouse). Data are expressed as the mean ± SEM. : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. (B) Iba1-positive MG/MF were + + clustered around the site of hMSC injection on pod 14 (n = 3 mouse in each group). (C) F4/80 cells (green) exhibited greater overlap with Ym1 cells (red) around the injection site in the hMSC-treated group than in the HBSS-treated group. Nuclear staining was performed using DAPI. Transplantation of hMSCs significantly increased the expression of the AAM markers Arg1 (D) and Chil3 (E), as well as that of the anti-inflammatory cytokine IL-4 (F) on pod 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. � � � : P<0.05, : P < 0.01 (Dunnett’s post hoc test), n.s.: not significant. https://doi.org/10.1371/journal.pone.0230080.g007 during the experimental period. However, Dpysl2 expression increased on pod 14 and was sig- nificantly higher in hMSC-treated mice than in HBSS-treated mice (Fig 8A). Similar tenden- cies were observed for Gap43 expression, which was significantly higher in hMSCs-treated + + animals on pod 14 (Fig 8B). CRMP2 and GAP43 immunoreactions were detected in the peri-injury region and clearly merged with NeuN neurons on pod 14 (Fig 8C and 8D). CCL5 promotes AAM activity and axonal regeneration To confirm whether increases in Ccl5 expression following hMSC injection contribute to MG/ MF polarization and axonal regeneration, mouse recombinant CCL5 was injected into the spi- nal cord after SCI on pod 7. On the following day, we examined the gene expression of AAM and axonal regeneration markers. Aif1 expression remained unchanged following CCL5 injec- tion (Fig 9A). However, CCL5-treated mice tended to increase the expression of Chil3 and Arg1 and exhibited significantly greater Chil3 expression than did vehicle-treated mice (p = 0.004) (Fig 9B and 9C), suggesting that CCL5 contributes to MG/MF polarization. CCL5-injected mice also exhibited increases in Dpysl2 (p = 0.097) and Gap43 (p = 0.033) expression (Fig 9D and 9E), suggesting that CCL5 also contributes to axonal regeneration. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 15 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 8. Human mesenchymal stem/stromal cells (hMSCs) promoted axonal regeneration. The transplantation of hMSCs increased the expression of the axonal regeneration markers Dpysl2 (A) and Gap43 (B). Mice treated with hMSCs (n = 9 mouse) exhibited significantly greater gene expression than did the HBSS-treated mice (n = 8 mouse) on postoperative day (pod) 14. All data are expressed the relative level (fold) to compare with pod 0 (basal level, n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are expressed as the mean ± SEM. : P < 0.05 (Student’s t-test), n.s.: not significant. (C) CRMP2 immunoreactivity (green) + + was observed in NeuN neurons (red) at the peri-injury site on pod 14. (D) GAP43 immunoreactivity (green) was merged with NeuN neurons (red) at the peri-injury site on pod 14 (n = 3 mouse in each group). Blue indicates nuclear staining using DAPI. https://doi.org/10.1371/journal.pone.0230080.g008 Discussion Human-MSC therapy has shown promise in supporting regeneration and/or recovery from diverse tissue injuries, including neuronal injuries [3–6]. Although we have reported that implanted hMSCs suppress neural injuries in mice by modulating MG/MF after brain ische- mia and SCI [7, 8], the mechanism and form of communication between hMSCs and MG/MF remain unclear. In the present study, we examined the influences of hMSCs on chemokine expressions because chemokines guide the migration of immune cells, including MF. Using our SCI model, we especially focused on hMSC-induced changes in two MF-related chemo- kine axes: CCL2/CCR2 and CCL5/CCR5. We here xenografted hMSCs into mouse spinal cord PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 16 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Fig 9. Microglia/macrophage (MG/MF) polarization and axonal regeneration were enhanced by CCL5. To determine whether increases in CCL5 following hMSC transplantation contribute to MG/MF polarization and axonal regeneration, recombinant mouse CCL5 (rmCCL5, n = 6 mouse) or vehicle (n = 6 mouse) was injected into the spinal cord on postoperative day (pod) 7. We examined levels of gene expression for MG/MF polarization (A-C) and axonal regeneration (D and E) 24 hours after injection. There were no obvious differences in the levels of the pan-macrophage marker Aif1 (A). Expression of the M2-type AAM marker Chil3 (B) significantly increased following the injection of rmCCL5 (p<0.01). Although Arg1 expression (C) also tended to increase, these increases were nonsignificant. Expression of the axonal regeneration marker Gap43 (E) significantly increased following the injection of rmCCL5 (p<0.05), while Dpysl2 (D) expression tended to increase (p = 0.097). All data are expressed the relative level (fold) to compare with vehicle treated animals (n = 6 mouse) after normalization against Gapdh as a housekeeping gene. Data are � � � expressed as the mean ± SEM. : P < 0.01, : P < 0.05 (Dunnett’s post hoc test). https://doi.org/10.1371/journal.pone.0230080.g009 without administration of immunosuppressants to not influence an immune response. Many studies were given immunosuppressants for decrease of the rejection by xenografting. How- ever, we have previously reported that injection of the hMSCs into immunocompetent mouse brain did not change the fate of cells to compare with immunodeficient mice [8]. These are supported and reproduced by many studies [10]. The Ccl2 and Ccr2 expressions drastically increased on pod1, and CCL2 immunoreactions were recognized in neurons in the acute phase following SCI. While previous studies have reported that CCR2 is mainly expressed in monocytes and infiltrated MF [34], CCR2 re- + + actions had barely merged with F4/80 MG/MF; however, CCR2 reactions overlapped greatly with Ly-6G granulocytes, which were likely neutrophils, at the epicenter of the injury on pod 1. Aberrations in the CCL2/CCR2 axis are reportedly associated with neuroinflamma- tory disease. [35–39]. CCR2 gene-deficient (KO) animals exhibit decreases in lesion size and recruitment of monocytes into the lesion following traumatic brain injury (TBI) [18] and SCI, [40]. Moreover, CCR2 neutrophils are activated under inflammatory conditions, thereby exacerbating the inflammatory response [41, 42]. According to these studies, most CCR2 / Ly-6G granulocytes on pod 1 exhibited co-localization of iNOS, suggesting an increase of inflammation. In the present study, the hMSC implantation resulted in improvements in locomotor activ- ity and tended to decrease lesion size; these findings were consistent with our previous report [7]. While we initially expected that the implanted hMSCs would diminish Ccl2 expression, PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 17 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury the levels of Ccl2, and not those of Ccr2, increased following hMSC transplantation. However, + + + the numbers of Ly-6G and CCR2 /Ly-6G cells did not change significantly with hMSC transplantation. Interestingly, CCR2 immunoreactions after hMSC transplantation were + + observed in NeuN -neurons as well as Ly-6G cells. Some recent studies have speculated that, in addition to its role in chemotaxis, CCL2 exerts neuroprotective and immunomodulatory effects [18, 19]. CCL2 aids in the migration of neural precursor cells and promotes neuroregeneration in patients with TBI [39] and stroke [43]. Kwon et al. suggested that the overexpression of CCL2 derived from neurons promotes axonal regeneration via AAM activation after CNS injury [44]. Investigating the condition of inflam- mation and neutrophil activation, we found that the transplantation of hMSCs decreased the gene expression levels of Il1b, Elane and Mpo; these observations indicated reduced inflamma- tion and neutrophil activation. Recent findings are consistent with our results: neutrophils polarized after infiltration can exert both beneficial and deleterious effects following tumori- genesis [45–47], stroke [48] and TBI [49]. Further, we found that mice treated with hMSCs exhibited persistent Ccl2 expression for 2 weeks as well as increases the expression of Zc3h12a, which encodes MCPIP1 and is known to induce by CCL2 and IL-1β, on pod14. MCPIP1 plays a role in anti-inflammation via the down-regulation of NF-κB signaling [50], and MCPIP1-KO mice reportedly exhibit significant increases in infarct volume after brain ischemia [51]. Although further studies are required to determine the precise roles of chronic increases in CCL2, the enhancement of Ccl2 expression via hMSC transplantation may contribute to anti- inflammation. We also examined the expression of Ccl5 after inducing SCI: it drastically increased by pod 14. While CCL5 binds to three receptors, CCR1, CCR3, and CCR5, we focused on CCR5 because Ccr5 expression remained significantly elevated through pod 14. Our findings vary in their consistency with previous research, as the role of the CCL5/CCR5 axis in CNS injuries is controversial in the literature. CCL5 deletion reduces infarct volume in cerebral ischemia [52], and CCR5 blockade improves locomotor function in a murine model of SCI [53]. These obser- vations suggest that CCL5 acts as a deleterious chemokine. Conversely, CCR5-KO mice exhibit increases in infarct volume in models of ischemia [54] and accelerated motor neuronal death in models of hypoglossal nerve injury [55]. Application of CCL5 to cultured neural cells signif- icantly suppressed cell death and increased the expression of some growth factors [21]. More- over, CCL5 decreased IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), and iNOS gene expression in lipopolysaccharide-treated primary MG cultures [55]. The present study found that the hMSC implantation significantly increased Ccl5 expres- sion by pod 14 but observed no changes in Ccr5 expression. Previous investigations have reported that CCL5 expression in neurons, MF, and astrocytes of the spinal cord of models of neuropathic pain and ischemia increased from pod 3 [21, 56]. We observed that CCL5 and CCR5 localized to neurons and MG/MF; this was not affected by hMSC implantation. How- ever, the intervention increased the expressions of pan-MG/MF (Aif1) and M2-type AAM (Arg1 and Chil3) markers. Moreover, the animals that received the hMSC implantation exhib- ited increases in Il4 expression by pod 14. IL-4 is a major activator of AAM polarization [57]. Interestingly, CCL2, CCL5, and insulin-like growth factor 1 (IGF-1) are also reportedly involved in the differentiation of MG/MF toward AAMs in osteoblasts [24], suggesting that greater hMSC-induced Il4 and Ccl5 expression by might stimulate MG/MF migration and polarization. Axonal extension and reconstruction of neuronal networks are important events in the recovery of spinal function. Research has suggested that AAMs promote axonal growth and overcome the effects of inhibitory substrates [58]. Several studies have also indicated that the implantation of AAMs into the injured spinal cord induces axonal regrowth and functional PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 18 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury improvement [59, 60]. In the present study, implantation of hMSCs increased the expression of the genes encoding axonal regenerative markers, including Dpysl2 and Gap43, on pod 14, and corresponding immunoreactions were observed in the neurons at the peri-injury site. To obtain direct evidence for the contribution of the increased Ccl5 expression to axonal exten- sion, we injected rmCCL5 into the spinal cord on pod 7 and determined the gene expression of M2-type AAM as well as the levels of axonal regenerative markers. We observed signifi- cantly greater Chil3 and Gap43 expression in rmCCL5-treated mice, which also tended to exhibit increases in Arg1 and Dpysl2 expression; however, no obvious changes in Aif1 expres- sion were observed following CCL5 injection. These results suggest that the CCL5 may par- tially participate in AAM polarization and axonal extension. In the present study, we examined the role of hMSCs after SCI by focusing on two represen- tative MF-related chemokine axes, CCL2/CCR2 and CCL5/CCR5, both of which exhibited acute or chronic increases following SCI. hMSC implantation upregulated the levels of these chemokines. We also confirmed improvements in the clinical symptoms of SCI following hMSC injection. While the CCL2/CCR2 axis contributes to the enhancement of inflammation, hMSC-mediated increases in CCL2 did not alter the number of granulocytes. Moreover, hMSCs might induce MG/MF polarization to the M2-type AAM and drive axonal generation. While chemokines could participate in these functions, the direct mechanisms by which the communication between hMSCs and the recipient tissues yield these phenomena remain to be elucidated. Future studies should examine mice subjected to chemokine KO or gene silencing to determine whether hMSCs influence CCL2/CCR2 and CCL5/CCR5 axes in post-SCI neuroregeneration. Conclusions In conclusion, the post-SCI implantation of hMSCs enhances the expression of Ccl2 and Ccl5 and improves locomotor activity. Moreover, the implantation of hMSCs enhances MG/MF polarization to AAM and the gene expression of axonal regenerative markers. The function of hMSCs might be partially mediated by the chemokine axes. Supporting information S1 Checklist. (DOCX) Acknowledgments We would like to thank Editage (www.editage.jp) for English language editing. Author Contributions Conceptualization: Kazumichi Yagura, Tomomi Tsumuraya, Atsushi Sato. Data curation: Kazumichi Yagura, Hirokazu Ohtaki, Naoto Kawada, Keisuke Suzuki, Motoyasu Nakamura. Formal analysis: Kazumichi Yagura, Hirokazu Ohtaki, Kazuyuki Miyamoto. Funding acquisition: Hirokazu Ohtaki, Atsushi Sato, Yutaka Hiraizumi. Investigation: Kazumichi Yagura, Hirokazu Ohtaki, Tomomi Tsumuraya, Atsushi Sato. Methodology: Kazumichi Yagura, Hirokazu Ohtaki. PLOS ONE | https://doi.org/10.1371/journal.pone.0230080 March 10, 2020 19 / 23 PLOS ONE CCL2 and CCL5 might contribute to inflammatory suppression and axonal extension after spinal cord injury Project administration: Kazumichi Yagura, Hirokazu Ohtaki, Masahiko Izumizaki, Yutaka Hiraizumi, Kazuho Honda. Resources: Hirokazu Ohtaki, Atsushi Sato, Yutaka Hiraizumi. Software: Kazumichi Yagura, Hirokazu Ohtaki. Supervision: Hirokazu Ohtaki, Koji Kanzaki, Kenji Dohi, Masahiko Izumizaki, Yutaka Hirai- zumi, Kazuho Honda. Validation: Kazumichi Yagura, Hirokazu Ohtaki, Tomomi Tsumuraya, Atsushi Sato, Kazuyuki Miyamoto, Naoto Kawada, Keisuke Suzuki, Motoyasu Nakamura, Koji Kanzaki, Masahiko Izumizaki, Yutaka Hiraizumi, Kazuho Honda. Visualization: Kazumichi Yagura, Hirokazu Ohtaki. Writing – original draft: Kazumichi Yagura, Hirokazu Ohtaki. Writing – review & editing: Kazumichi Yagura, Hirokazu Ohtaki, Koji Kanzaki, Masahiko Izumizaki, Yutaka Hiraizumi, Kazuho Honda. References 1. Rolls A, Shechter R, Schwartz M. The bright side of the glial scar in CNS repair. Nat Rev Neurosci. 2009; 10(3):235–41. https://doi.org/10.1038/nrn2591 PMID: 19229242. 2. Nesathurai S. Steroids and spinal cord injury: revisiting the NASCIS 2 and NASCIS 3 trials. J Trauma. 1998; 45(6):1088–93. https://doi.org/10.1097/00005373-199812000-00021 PMID: 9867054. 3. Uccelli A, Milanese M, Principato MC, Morando S, Bonifacino T, Vergani L, et al. 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