SPINE Volume 40, Number 2, pp 95-101 ©2015, Lippincott Williams & Wilkins
BASIC SCIENCE
Enhanced Expression of Neurotrophic Factors in the Injured Spinal Cord Through Vaccination With Myelin Basic Protein-Derived Peptide Pulsed Dendritic Cells Yufu Wang, MD,* Jing Li, MD,† Pengyu Kong, MD,* Song Zhao, MD,* Hui Yang, MD,* Chao Chen, MSc,‡ and Jinglong Yan, MD*
Study Design. Vaccination of spinal cord injury (SCI) mice with myelin basic protein-derived peptide (A91) pulsed dendritic cells (DC) to enhance brain-derived neurotrophic factor and neurotrophin-3 (NT-3) expression in injured spinal cord. Objective. To investigate the effect of A91-pulsed DC (A91-DC) on expression of neurotrophic factor in injured spinal cord. Summary of Background Data. SCI leads to progressive secondary tissue degeneration, and no satisfactory treatment is currently available. Accumulating evidence indicates that administration of neurotrophic factors to injured spinal cord is partially successful at promoting nerve tissue repair. However, most of strategy can cause secondary injury and limiting their wide clinical application. Methods. Proliferation of T cells and the capability of CD4+ T cells to secret neurotrophic factors were first measured in vitro to demonstrate the stimulus action of the A91-DC. In SCI mice model, enzyme-linked immunosorbent assay and immunofluorescence was employed to investigate the brain-derived neurotrophic factor and NT-3 expression in injured spinal cord. Furthermore, the neuroprotective effect of A91-DC in injured spinal cord was examined through histology measurement.
From the *Department of Orthopedics Surgery, Second Hospital, Harbin Medical University, Harbin, P.R. China; †Department of Pathology and Center of Electron Microscope, School of Basic Medicine, Harbin Medical University, Harbin, P.R. China; and ‡Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada. Acknowledgment date: April 25, 2014. First revision date: August 3, 2014. Second revision date: October 1, 2014. Acceptance date: October 2, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). National Natural Science Foundation (No. 81401342) and Health and Family Planning Commission of Heilongjiang Foundation (no. 2014-325) funds were received to support this work. Relevant financial activities outside the submitted work: grant. Address correspondence and reprint requests to Jinglong Yan, MD, Department of Orthopedics Surgery, Second Hospital, Harbin Medical University, 246 Baojian Rd, Harbin, 150081, P.R. China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000694 Spine
Results. In this study, we demonstrated that A91-DC promoted the capability of T cells to secret neurotrophic factors and in the subacute phase of SCI. Moreover, vaccination with A91-DC enhanced the expression level of brain-derived neurotrophic factor and NT-3 and exerted neuroprotective effect in injured spinal cord. Conclusion. The findings of study demonstrate that the therapeutic strategy of vaccination A91-DC is a potential minimally invasive approach that could provide strong neurotrophic factor support after SCI. Key words: spinal cord injury, neurotrophic factors, dendritic cell, vaccine, T cell, brain-derived neurotrophic factor, neurotrophin-3, myelin basic protein-derived peptide, mini-invasive, neuroprotection. Level of Evidence: Spine 2015;40:95–101
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njury to the adult mammalian spinal cord results in extensive axonal degeneration and variable amounts of neuronal loss and often leads to severe functional deficits.1 Failure of neurons to spontaneously regenerate is thought to be the result of the nonpermissive environment of the lesion area, which is due to the lack of growth-promoting molecules and the presence of inhibitory molecules.2 A strategy that provides a supportive growth environment via the administration of neurotrophic factors is partially successful at promoting nerve tissue repair within the spinal cord.3,4 Accumulating evidence has demonstrated that a high concentration of neurotrophic factors in injured spinal cord is beneficial for axonal growth and neuronal survival in vitro and in vivo.5 Neurotrophic factors can be introduced to the injured spinal cord by direct injection, continuous infusion, or placement of growth-factor saturated gelfoam. However, these methods do not achieve long-term, localized, high-dose delivery of neurotrophic factors. Gene therapy is an alternative approach that has the capability of delivering a high concentration of localized growth factor to the site of injury, but it can cause secondary injury such as bleeding and infection, thereby limiting wide clinical application.6 Studies over the last few years have provided evidence indicating that T cells are a source of neurotrophic factors.7 www.spinejournal.com
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BASIC SCIENCE Furthermore, CD4+ T cells but not B cells or CD8+ T cells are neuroprotective in Central Nervous System (CNS). Thus, boosting the T-cell response specific for CNS antigens is considered a potential way of ameliorating spinal cord injury (SCI). A previous study has demonstrated that injection of mature dendritic cells (DCs) pulsed with spinal cord homogenate (SCH) enhanced the infiltration of CD4+ T cell in injured spinal cord and provided better tissue preservation.8 However, a potential caveat to using SCH is that DCs might induce pathological neuroinflammation and encephalomyelitis (EAE).9 It seems that using A91-derived peptide (A91), A91 amino acids 87-99 and replacing the lysine residue at position 91 with alanine, is more beneficial because it induces accumulation of T cells in the spinal cords but does not induce EAE.10 Therefore, A91 is more appropriate for DC vaccine to treat SCI. Compared with gene therapy and direct injection of neurotrophic factors, the DC vaccine is a minimally invasive therapeutic approach. The aim of this study was to determine whether vaccination with A91-pulsed DC (A91-DC) enhances expression of neurotrophic factors in the spinal cord via promoting the infiltration of T cells to secret neurotrophic factors.
MATERIALS AND METHODS Animals and Spinal Cord Injury Adult BALB/c mice (weighing 24–26 g) were purchased from the Animal Breeding Center of Harbin Medical University (Harbin, PR China). All surgical procedures and postoperative animal care were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, United States). In accordance with the clip compression model described previously,11 all mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 mL/ kg). The skin was incised along the midline of the back, and the paravertebral muscles of the thoracic-level (T8–T10) vertebrae were dissected out. Laminectomy was performed at the T9 level. A 3-se-long extradural compression with a vascular clip (with 10-g force) was performed around the exposed spinal cord to cause an acute compression injury.
Preparation of Mouse DCs DCs were obtained from bone marrow using a method described previously.12 At day 0, bone marrow cells obtained from femurs and tibias were cultured in RPMI-1640 medium (Gibco Invitrogen, Grand Island, NY) supplemented with 100 U/mL penicillin, and 100 μg/mL streptomycin, 2 mM L-glutamine, 1% nonessential amino acids, 50 mM b-mercaptoethanol, 1 mM pyruvate, and 10% heat-inactivated and filtered fetal calf serum (Gibco Invitrogen, Grand Island, NY). Cytokine recombinant murine granulocyte macrophage colonystimulating factor (rmGM-CSF, PeproTech, Rocky Hill, NJ) at a concentration of 200 U/mL was added. On day 3, an additional 2.5 mL of RPMI-1640 medium containing 200 U/mL rmGM-CSF was added to the plates. On days 6 and 8, half of the culture supernatant was collected and centrifuged, and the cell pellet was resuspended in 2.5 mL of fresh RPMI-1640 96
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containing 200 U/mL rmGM-CSF and returned to the original plate. On day 10, cells were ready for use. Some of these DCs were used as immature DC (iDC). Other cells were resuspended in fresh DC medium (without additional cytokines; 2 × 106 cells/mL) containing Myelin basic protein (MBP)derived peptide, A91 (100 mg/mL) (HD Bioscience, China). DCs that were pulsed with or without A91 were stimulated with lipopolysaccharide (LPS, 1 mg/mL), respectively, for 24 hours to obtain A91-DCs or mDC. All of the cells were kept on ice until they were injected. Just before injection, the cells were centrifuged and resuspended in (phosphate-buffered saline (PBS) 3 times (1 × 06 cells in 0.3 mL of PBS for intraperitoneal injection). Twenty-four hours after SCI, mice were injected peritoneally with PBS, iDC, or A91-DC.
Mixed Lymphocyte Reaction T lymphocytes were purified by using the method of nylon wool column filtration. DCs used in the mixed lymphocyte reaction (including iDC, mDC, and A91-DC) were irradiated (2000 rad) to inhibit DNA replication before use. T cells from injured mice were cultured in 96-well plates at 5 × 105 per well and stimulated with PBS (100 μL), iDC, mDC, or A91-DC (1 × 105/well) in quintuple wells. After incubation for 72 hours at 37°C and 5% CO2, 20 μL MTT (5 g/L) was added 4 hours before the termination of culture. Then, 50 μL DMSO was added to each well and oscillated for 10 minutes. The absorbance was measured at a wave length of 570 nm, using automatic enzyme-linked immunosorbent assay reader (Bio-Rad). In another experiment, T cells from each group (sham, iDC and A91-DC) killed 8 days after DC injection were cultured in 96-well plates at 4 × 105 per well and stimulated with phorbol 12-myristate 13-acetate. PBS was used as a control.
Enzyme-Linked Immunosorbent Assay Proteins were extracted from the spinal cord (at the T9 level) and spleen using a lysis buffer. Extracts were centrifuged for 10 minutes at 13,000× g, at 4°C and then the supernatants were collected. The supernatants were subject to detection using the brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) enzyme-linked immunosorbent assay kit (Boster Biological Technology, Wuhan, PR China) in accordance with the instructions of the manufacturer.
Immunofluorescence For immunofluorescence, the sections were blocked with 5% albumin bovine diluted in 0.1% Triton X-100/0.1M TBS for 60 minutes. Primary antibodies (1:200, Santa Cruz, CA) used for immunofluorescence staining were rat anti-CD4 IgG2b, rat anti-CD3 IgG2b, rabbit anti-BDNF, and anti-NT-3. All the antibodies were applied to sections overnight at 4°C. On the following day, the sections were incubated for 60 minutes at 37°C with FITC-labeled goat antirabbit IgG and Cy3-labeled goat antirat IgG (1:1000, Beyotime Institute of Biotechnology, PR China) and the slides were washed. These slides were incubated with diamidino-phenylindole (DAPI, 10mg/mL) and slides were washed, cover slipped, and examined using a laser January 2015
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BASIC SCIENCE scanning confocal fluorescence microscope (Zeiss 510 LSM, Germany). To examine the NF200 staining in the injured spinal cord, sagittal sections (representing bilateral and midsagittal areas of interest) were prepared from the lesion area (2.5 mm rostral and caudal to the lesion epicenter). The specificity of the immunofluorescence staining was confirmed by a control staining with primary IgG antibody.
Behavioral Analysis Motor function of the hindlimbs was analyzed using the Basso Mouse Scale open-field score.13 The evaluations were made by 2 blind observers for all analyzed groups. Behavioral analysis was performed at days 7, 14, 21, and 28 after treatment.
Statistical Analysis
Data are presented as mean values ± SD. Quantitative data from open-field locomotor scores were evaluated for statistical significance using 2-way analysis of variance. At different time points, 1-way analysis of variance, followed by SNK test was used to analyze the difference between each group. Data from OD values, cytokine concentrations, and quantity of cells were analyzed by 1-way analysis of variance (SPSS 16.0, Inc., Chicago, IL).
RESULTS A91-DC-Enhanced T-cell Proliferation The function of the A91-DCs to activate proliferation of T cells was examined in vitro. At 72 hours, the proliferative activity of T cells induced by A91-DC was the most powerful among the groups. The T cells induced by immature DC had a slight proliferative effect when compared with untreated T cells (Figure 1A). To authenticate the effect of A91-DC on activating T cells in SCI mice, we assessed the proliferative ability of T cells obtained from the spleen. The proliferation of T cells from SCI mice was significantly decreased compared with normal mice. T cells from mice injected peritoneally with A91-DC and mDC displayed a significantly stronger proliferative response than PBS- and iDC-treated mice (Figure 1B).
Enhanced Expression of Neurotrophic Factors• Wang et al
A91-DC-Enhanced Production of BDNF and NT-3 by T Cells In Vitro Because iDC cannot induce T-cell proliferation, it was not considered as a therapeutic DC in the following experiments. To investigate whether A91-DC could influence the secretion of neurotrophic factors via T cells, production of BDNF and NT-3 was measured on day 8 postinjury; T cells were isolated immediately and cultured for 72 hour in vitro. The production of BDNF and NT-3 in supernatant was measured. A significant decrease of BDNF and NT-3 protein expression was observed in T cells isolated from PBS-treated mice compared with normal mice. The secretion of BDNF and NT-3 from T cells obtained from mice treated with A91-DC was higher than that obtained from mice treated with PBS (Figure 2A). Figure 2B shows that CD3+ T cells in spleen secreted rich BDNF and NT-3 after treatment with A91-DC.
A91-DC-Enhanced Infiltration of CD4⫹ T Cells That Secreted BDNF and NT-3 at the Injury Site The spinal cords were taken from each experimental group on day 8 after injury. As shown in Figure 3A, very few CD4+ T cells were detected in the injured spinal cord in the PBS- and DC-treated group. However, significantly higher numbers of CD4+ T cells were observed in the A91-DC group than in the PBS group. Furthermore, colocalization of BDNF and NT-3 from spinal cord section revealed that the number of doublepositive CD4 and BDNF/NT-3 cells in A91-DC group was higher than that in 2 other groups (Figure 3B, C), suggesting these infiltrated CD4+ T cells secreted rich BDNF and NT-3 at the injury site.
A91-DC Treatment Increased the Production of BDNF and NT-3 in Injured Spinal Cord Protein expression of BDNF and NT-3 was assessed at day 8. The production of BDNF and NT-3 was significantly increased in the A91-DC group compared with PBS- and DC-treated group (Figure 4A). In addition, the longitudinal spinal cord sections were immunostained for BDNF or NT-3 to detect the expression of nerve growth factors. Significantly increased BDNF and NT-3 protein immunofluorescence was
Figure 1. Proliferative activity of T cells. A, MTT assays show influences on T-cell proliferation by iDC, mDC, and A91-DC. iDC, mDC, and A91-DC were plated in 96-well round-bottom culture plates as stimulators and coincubated with purified T cells as responders. B, The proliferative activity of T cells obtained from sham mice and SCI mice treated with PBS, mDC, or A91-DC were determined by MTT. Phorbol 12-myristate 13-acetate was used to stimulate T-cell proliferation in vitro. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with PBS-treated group or compared with sham group by analysis of variance. PBS indicates phosphate-buffered saline. Spine
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detected in the A91-DC-treated mice as compared with the DC- and PBS-treated mice (Figure 4B). Statistical analysis revealed a significantly higher level of immunofluorescence intensity of BDNF and NT-3 in mice receiving A91-DC than that in other 2 groups (Figure 4C).
The Increased Expression of Neurotrophic Factors by A91-DC-Promoted Neuroprotective Effect at the Injured Site
Figure 2. Enhanced production of BDNF and NT-3 in the spleen after treatment with A91-DC postinjury. A, The production of BDNF and NT-3, secreted by T cells, was measured at day 8 after injury using enzyme-linked immunosorbent assay. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with normal group, *P < 0.05, compared with PBS-treated group by analysis of variance. B, Colocalization of BDNF (green) or NT-3 (green) with T cells (red) in the spleen section of mice treated with A91-DC. Scale bar, 50 μm.
An open-field test using the Basso Mouse Scale locomotor rating was performed to investigate the functional recovery from SCI in each group. Typical paraplegia was observed in all animals after injury. Administration of A91-DC in the injured spinal cord induced significant locomotor recovery after SCI. The significant functional recovery associated with A91-DC was observed on day 14 (Figure 5A). Furthermore, the lesion area of spinal cord tissue at 14 days after injury as quantified on the basis of HE staining showed larger tissue damage in PBS- and DC-treated mice than that in the A91-DC-treated mice (Figure 5B, C) though A91DC-induced more T cells infiltration in injured spinal cord. The difference in the lesion area between PBS and DC was not statistically significant. HE staining also showed that in the white matter, vacuolation was widespread and was prominent around lesion area in the PBS- and DC-treated mice. However, A91-DC significantly decreased the area of vacuolation in white matter (Figure 5B). Histopathological grading of the white matter confirmed A91-DC-exerted protective effect on white matter (Figure 5D). In addition, NF-200 staining was used to detect the residual axons and neurons in the lesion area. Histological analysis revealed a significantly higher level of neurofilament immunoactivity in mice receiving A91-DC than those of the control group (Figure 5E, F).
Figure 3. Effect of A91-DC on CD4+ T cells at the injured site. A, Quantification of CD4+ T cells was analyzed at the injured site in each group at day 7 after injury. Scale bar, 50 μm. The number of CD4+ T cells was counted from 4 microscopic fields in the injured site for each sample. B, Infiltrated CD4+ T cells secreted rich BDNF and NT-3 at the injury site. Scale bar, 25 μm. C, Number of double positive CD4 and BDNF/NT-3 cells among 3 groups. Data are given as means ± SD, 5 mice per group; *P < 0.05 versus PBS group (analysis of variance). PBS indicates phosphatebuffered saline.
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Figure 5. Neuroprotective effect of A91-DC at the injured site. A, Effect of A91-DC on functional recovery from SCI mice. Data represent means ± SD, 5 mice per group, *P < 0.05, versus PBS group, analysis of variance for repeated measures. B, Representative photomicrographs illustrating spinal cord lesion areas defined by H&E staining (×40). C, The mean lesion area among the 3 experimental groups. D, Histopathological grading reflecting the vacuolation in the white injury. E, Neurofilament immunostaining in the spinal cord lesion 2 weeks after injury. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. F, The fold increase of immunofluorescence intensity of NF200 staining in the injury site. Data represent means ± SD, 5 mice per group, *P < 0.05, versus PBS group, analysis of variance.
Figure 4. Expression of BDNF and NT-3 at the injured site after treatment with A91-DC. A, BDNF and NT-3 levels were determined using enzyme-linked immunosorbent assay in PBS, mDC, and A91-DC groups at day 8 after spinal cord injury. B, Protein levels of BDNF and NT-3 were determined by immunohitochemical staining. Scale bar, 100 μm. C, Immunofluorescence intensity of BDNF and NT-3 was analyzed. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with PBS group by analysis of variance. PBS indicates phosphate-buffered saline.
DISCUSSION The findings of this study indicate that vaccination with A91derived peptide pulsed DC is a potential therapeutic approach that can efficiently increase BDNF and NT-3 in injured spinal cord through promoting T-cell activation. The increased Spine
neurotrophic factors induced by A91-DC are sufficient to lead to functional recovery of SCI. It has become clear that within the CNS, immune responses are carefully controlled to prevent injury to sensitive tissue with limited regenerative capacity.14,15 One of the mechanisms by which immune cells maintain CNS plasticity is their ability to express and control growth factor production. Under pathological conditions, the T cell is a source of neurotrophic factors, and the expression of these factors can be significantly increased by antigen activation.16 It has been reported that some nerve growth factors are produced and continuously released at the site of injury by activated T cells.17 In addition, T-cell–derived NTs may locally induce microglia to release a second neurotrophic factor. Several studies have shown that NTs induce secretion of other NTs, and that cytokines such as interleukin-1β and tumor necrosis factor α stimulate microglia neurotrophic factor release.18,19 Thus, the increased www.spinejournal.com
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BASIC SCIENCE neurotrophic factors and multiple cytokines induced by A91-DC can also stimulate local microglia or monocytes to secrete other neurotrophic factors that may constitute another source of BDNF and NT-3 in the lesion site. In several studies, SCH was used as a stimuli antigen to induce CNS-specific T cells because SCH contains multiple CNS antigens.8 In the current study, A91 was used to replace SCH because A91 does not induce potential EAE, which may lead to a better tissue protection. Immunization with A91 was showed to provide neuroprotection in CNS injury.20,21 However, the neuroprotective effect achieved by immunization strategy is related to the gene background of the animals (strains) and the kind of adjuvant.22 DC strategies contribute to reduce the dose of antigen required, while additionally limiting cross-presentation by other cell types.23 For stimulation of an immune response, DCs migrate to the spleen or draining lymph nodes. There, DCs prime the immune response to antigens and may also prime self-antigen-specific responses observed in autoimmunity. Therefore, we assessed the capability of A91-DC to induce T-cell proliferation. As expected, A91-DC is efficient at priming the proliferative response of T cells. A91-DC also significantly enhanced the capability of T cells to secret BDNF and NT-3, suggesting that A91-DC may constitute a rich neurotrophic pool outside of the nervous system. Furthermore, we demonstrated that activated T cells infiltrated in the injured spinal cord for a prolonged period (at day 8 postinjection), which means that these T cells may elicit continued neurotrophic factor expression during the repair process in cases of SCI. In this study, we observed that vaccination with A91-DC provides more protective effect at the lesion site. This neuroprotective effect is partially attributed to the immunological regulation of T cells, but increased BDNF and NT-3 expression also plays a key role. BDNF and NT-3 have both been shown to enhance neuronal survival and plasticity in vitro and in vivo.24 NT-3 and BDNF are also biologically active proteins that promote the survival of pure oligodendrocyte and myelination of regenerating axons.25 Moreover, BDNF and NT-3 contribute to enhanced protection of neurons from the potentially detrimental effects of inflammatory cytokines.26 The increased levels of BDNF and NT-3 in the spinal cord also contribute to tolerance against EAE that may potentially be induced by DC vaccination.27 In conclusion, A91-DC partially promoted the level of neurotrophic factors production in the subacute phase of SCI. The therapeutic strategy of vaccination with A91-DC could not only induce the beneficial immunomodulation of T cells but also provide strong neurotrophic factor support after SCI.
➢ Key Points Vaccination with MBP-pulsed peptide (A91) DCs is a potential minimally invasive approach that could promote BDNF and NT-3 expression via T cells in an injured spinal cord. 100
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This therapeutic strategy could provide adequate neurotropic factor to support some degree of SCI recovery. A91-DC is a potential strategy for treating SCI.
Acknowledgments Yufu Wang and Jing Li contributed equally to this work.
References
1. Rowland JW, Hawryluk GW, Kwon B, et al. Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg Focus 2008;25:E2. 2. Schwab ME. Repairing the injured spinal cord. Science 2002;295: 1029–31. 3. Varon S, Conner JM. Nerve growth factor in CNS repair. J Neurotrauma 1994;11:473–86. 4. Brock JH, Rosenzweig ES, Blesch A, et al. Local and remote growth factor effects after primate spinal cord injury. J Neurosci 2010;30:9728–37. 5. Blesch A, Tuszynski MH. Transient growth factor delivery sustains regenerated axons after spinal cord injury. J Neurosci 2007;27:10535–45. 6. Blesch A, Lu P, Tuszynski MH. Neurotrophic factors, gene therapy, and neural stem cells for spinal cord repair. Brain Res Bull 2002; 57:833–8. 7. Schwartz M, London A, Shechter R. Boosting T-cell immunity as a therapeutic approach for neurodegenerative conditions: the role of innate immunity. Neuroscience 2009;158:1133–42. 8. Wang Y, Wang K, Chao R, et al. Neuroprotective effect of vaccination with autoantigen-pulsed dendritic cells after spinal cord injury. J Surg Res 2012;176:281–92. 9. Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7:31–4. 10. Hauben E, Gothilf A, Cohen A, et al. Vaccination with dendritic cells pulsed with peptides of myelin basic protein promotes functional recovery from spinal cord injury. J Neurosci 2003;23:8808–19. 11. Marques SA, Garcez VF, Del Bel EA, et al. A simple, inexpensive and easily reproducible model of spinal cord injury in mice: morphological and functional assessment. J Neurosci Methods 2009;177:183–93. 12. Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 1999;223:77–92. 13. Basso DM, Fisher LC, Anderson AJ, et al. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 2006;23:635–59. 14. Yong VW, Rivest S. Taking advantage of the systemic immune system to cure brain diseases. Neuron 2009;64:55–60. 15. Graber JJ, Dhib-Jalbut S. Protective autoimmunity in the nervous system. Pharmacol Ther 2009;121:147–59. 16. Moalem G, Gdalyahu A, Shani Y, et al. Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun 2000;15:331–45. 17. Kerschensteiner M, Gallmeier E, Behrens L, et al. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999;189:865–70. 18. Heese K, Hock C, Otten U. Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem 1998;70:699–707. 19. Kruttgen A, Moller JC, Heymach JV Jr, et al. Neurotrophins induce release of neurotrophins by the regulated secretory pathway. Proc Natl Acad Sci U S A 1998;95:9614–9. 20. Martinon S, Garcia E, Gutierrez-Ospina G, et al. Development of protective autoimmunity by immunization with a neural-derived January 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140563_LR 100
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21.
22. 23. 24.
peptide is ineffective in severe spinal cord injury. PLoS One 2012;7:e32027. Garcia E, Silva-Garcia R, Mestre H, et al. Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury. J Neurosci Res 2012;90:656–63. Hendrix S, Nitsch R. The role of T helper cells in neuroprotection and regeneration. J Neuroimmunol 2007;184:100–12. Cohn L, Delamarre L. Dendritic cell-targeted vaccines. Front Immunol 2014;5:255. Alcala-Barraza SR, Lee MS, Hanson LR, et al. Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS. J Drug Target 2010;18:179–90.
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Enhanced Expression of Neurotrophic Factors• Wang et al
25. McTigue DM, Horner PJ, Stokes BT, et al. Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J Neurosci 1998;18:5354–65. 26. Flugel A, Matsumuro K, Neumann H, et al. Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis: inhibition of monocyte transendothelial migration. Eur J Immunol 2001;31:11–22. 27. Hammarberg H, Lidman O, Lundberg C, et al. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J Neurosci 2000;20:5283–91.
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Enhanced Expression of Neurotrophic Factors in the Injured Spinal Cord Through Vaccination With Myelin Basic Protein-Derived Peptide Pulsed Dendritic Cells Yufu Wang, MD,* Jing Li, MD,† Pengyu Kong, MD,* Song Zhao, MD,* Hui Yang, MD,* Chao Chen, MSc,‡ and Jinglong Yan, MD*
Study Design. Vaccination of spinal cord injury (SCI) mice with myelin basic protein-derived peptide (A91) pulsed dendritic cells (DC) to enhance brain-derived neurotrophic factor and neurotrophin-3 (NT-3) expression in injured spinal cord. Objective. To investigate the effect of A91-pulsed DC (A91-DC) on expression of neurotrophic factor in injured spinal cord. Summary of Background Data. SCI leads to progressive secondary tissue degeneration, and no satisfactory treatment is currently available. Accumulating evidence indicates that administration of neurotrophic factors to injured spinal cord is partially successful at promoting nerve tissue repair. However, most of strategy can cause secondary injury and limiting their wide clinical application. Methods. Proliferation of T cells and the capability of CD4+ T cells to secret neurotrophic factors were first measured in vitro to demonstrate the stimulus action of the A91-DC. In SCI mice model, enzyme-linked immunosorbent assay and immunofluorescence was employed to investigate the brain-derived neurotrophic factor and NT-3 expression in injured spinal cord. Furthermore, the neuroprotective effect of A91-DC in injured spinal cord was examined through histology measurement.
From the *Department of Orthopedics Surgery, Second Hospital, Harbin Medical University, Harbin, P.R. China; †Department of Pathology and Center of Electron Microscope, School of Basic Medicine, Harbin Medical University, Harbin, P.R. China; and ‡Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada. Acknowledgment date: April 25, 2014. First revision date: August 3, 2014. Second revision date: October 1, 2014. Acceptance date: October 2, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). National Natural Science Foundation (No. 81401342) and Health and Family Planning Commission of Heilongjiang Foundation (no. 2014-325) funds were received to support this work. Relevant financial activities outside the submitted work: grant. Address correspondence and reprint requests to Jinglong Yan, MD, Department of Orthopedics Surgery, Second Hospital, Harbin Medical University, 246 Baojian Rd, Harbin, 150081, P.R. China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000694 Spine
Results. In this study, we demonstrated that A91-DC promoted the capability of T cells to secret neurotrophic factors and in the subacute phase of SCI. Moreover, vaccination with A91-DC enhanced the expression level of brain-derived neurotrophic factor and NT-3 and exerted neuroprotective effect in injured spinal cord. Conclusion. The findings of study demonstrate that the therapeutic strategy of vaccination A91-DC is a potential minimally invasive approach that could provide strong neurotrophic factor support after SCI. Key words: spinal cord injury, neurotrophic factors, dendritic cell, vaccine, T cell, brain-derived neurotrophic factor, neurotrophin-3, myelin basic protein-derived peptide, mini-invasive, neuroprotection. Level of Evidence: Spine 2015;40:95–101
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njury to the adult mammalian spinal cord results in extensive axonal degeneration and variable amounts of neuronal loss and often leads to severe functional deficits.1 Failure of neurons to spontaneously regenerate is thought to be the result of the nonpermissive environment of the lesion area, which is due to the lack of growth-promoting molecules and the presence of inhibitory molecules.2 A strategy that provides a supportive growth environment via the administration of neurotrophic factors is partially successful at promoting nerve tissue repair within the spinal cord.3,4 Accumulating evidence has demonstrated that a high concentration of neurotrophic factors in injured spinal cord is beneficial for axonal growth and neuronal survival in vitro and in vivo.5 Neurotrophic factors can be introduced to the injured spinal cord by direct injection, continuous infusion, or placement of growth-factor saturated gelfoam. However, these methods do not achieve long-term, localized, high-dose delivery of neurotrophic factors. Gene therapy is an alternative approach that has the capability of delivering a high concentration of localized growth factor to the site of injury, but it can cause secondary injury such as bleeding and infection, thereby limiting wide clinical application.6 Studies over the last few years have provided evidence indicating that T cells are a source of neurotrophic factors.7 www.spinejournal.com
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BASIC SCIENCE Furthermore, CD4+ T cells but not B cells or CD8+ T cells are neuroprotective in Central Nervous System (CNS). Thus, boosting the T-cell response specific for CNS antigens is considered a potential way of ameliorating spinal cord injury (SCI). A previous study has demonstrated that injection of mature dendritic cells (DCs) pulsed with spinal cord homogenate (SCH) enhanced the infiltration of CD4+ T cell in injured spinal cord and provided better tissue preservation.8 However, a potential caveat to using SCH is that DCs might induce pathological neuroinflammation and encephalomyelitis (EAE).9 It seems that using A91-derived peptide (A91), A91 amino acids 87-99 and replacing the lysine residue at position 91 with alanine, is more beneficial because it induces accumulation of T cells in the spinal cords but does not induce EAE.10 Therefore, A91 is more appropriate for DC vaccine to treat SCI. Compared with gene therapy and direct injection of neurotrophic factors, the DC vaccine is a minimally invasive therapeutic approach. The aim of this study was to determine whether vaccination with A91-pulsed DC (A91-DC) enhances expression of neurotrophic factors in the spinal cord via promoting the infiltration of T cells to secret neurotrophic factors.
MATERIALS AND METHODS Animals and Spinal Cord Injury Adult BALB/c mice (weighing 24–26 g) were purchased from the Animal Breeding Center of Harbin Medical University (Harbin, PR China). All surgical procedures and postoperative animal care were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, United States). In accordance with the clip compression model described previously,11 all mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 mL/ kg). The skin was incised along the midline of the back, and the paravertebral muscles of the thoracic-level (T8–T10) vertebrae were dissected out. Laminectomy was performed at the T9 level. A 3-se-long extradural compression with a vascular clip (with 10-g force) was performed around the exposed spinal cord to cause an acute compression injury.
Preparation of Mouse DCs DCs were obtained from bone marrow using a method described previously.12 At day 0, bone marrow cells obtained from femurs and tibias were cultured in RPMI-1640 medium (Gibco Invitrogen, Grand Island, NY) supplemented with 100 U/mL penicillin, and 100 μg/mL streptomycin, 2 mM L-glutamine, 1% nonessential amino acids, 50 mM b-mercaptoethanol, 1 mM pyruvate, and 10% heat-inactivated and filtered fetal calf serum (Gibco Invitrogen, Grand Island, NY). Cytokine recombinant murine granulocyte macrophage colonystimulating factor (rmGM-CSF, PeproTech, Rocky Hill, NJ) at a concentration of 200 U/mL was added. On day 3, an additional 2.5 mL of RPMI-1640 medium containing 200 U/mL rmGM-CSF was added to the plates. On days 6 and 8, half of the culture supernatant was collected and centrifuged, and the cell pellet was resuspended in 2.5 mL of fresh RPMI-1640 96
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containing 200 U/mL rmGM-CSF and returned to the original plate. On day 10, cells were ready for use. Some of these DCs were used as immature DC (iDC). Other cells were resuspended in fresh DC medium (without additional cytokines; 2 × 106 cells/mL) containing Myelin basic protein (MBP)derived peptide, A91 (100 mg/mL) (HD Bioscience, China). DCs that were pulsed with or without A91 were stimulated with lipopolysaccharide (LPS, 1 mg/mL), respectively, for 24 hours to obtain A91-DCs or mDC. All of the cells were kept on ice until they were injected. Just before injection, the cells were centrifuged and resuspended in (phosphate-buffered saline (PBS) 3 times (1 × 06 cells in 0.3 mL of PBS for intraperitoneal injection). Twenty-four hours after SCI, mice were injected peritoneally with PBS, iDC, or A91-DC.
Mixed Lymphocyte Reaction T lymphocytes were purified by using the method of nylon wool column filtration. DCs used in the mixed lymphocyte reaction (including iDC, mDC, and A91-DC) were irradiated (2000 rad) to inhibit DNA replication before use. T cells from injured mice were cultured in 96-well plates at 5 × 105 per well and stimulated with PBS (100 μL), iDC, mDC, or A91-DC (1 × 105/well) in quintuple wells. After incubation for 72 hours at 37°C and 5% CO2, 20 μL MTT (5 g/L) was added 4 hours before the termination of culture. Then, 50 μL DMSO was added to each well and oscillated for 10 minutes. The absorbance was measured at a wave length of 570 nm, using automatic enzyme-linked immunosorbent assay reader (Bio-Rad). In another experiment, T cells from each group (sham, iDC and A91-DC) killed 8 days after DC injection were cultured in 96-well plates at 4 × 105 per well and stimulated with phorbol 12-myristate 13-acetate. PBS was used as a control.
Enzyme-Linked Immunosorbent Assay Proteins were extracted from the spinal cord (at the T9 level) and spleen using a lysis buffer. Extracts were centrifuged for 10 minutes at 13,000× g, at 4°C and then the supernatants were collected. The supernatants were subject to detection using the brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) enzyme-linked immunosorbent assay kit (Boster Biological Technology, Wuhan, PR China) in accordance with the instructions of the manufacturer.
Immunofluorescence For immunofluorescence, the sections were blocked with 5% albumin bovine diluted in 0.1% Triton X-100/0.1M TBS for 60 minutes. Primary antibodies (1:200, Santa Cruz, CA) used for immunofluorescence staining were rat anti-CD4 IgG2b, rat anti-CD3 IgG2b, rabbit anti-BDNF, and anti-NT-3. All the antibodies were applied to sections overnight at 4°C. On the following day, the sections were incubated for 60 minutes at 37°C with FITC-labeled goat antirabbit IgG and Cy3-labeled goat antirat IgG (1:1000, Beyotime Institute of Biotechnology, PR China) and the slides were washed. These slides were incubated with diamidino-phenylindole (DAPI, 10mg/mL) and slides were washed, cover slipped, and examined using a laser January 2015
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BASIC SCIENCE scanning confocal fluorescence microscope (Zeiss 510 LSM, Germany). To examine the NF200 staining in the injured spinal cord, sagittal sections (representing bilateral and midsagittal areas of interest) were prepared from the lesion area (2.5 mm rostral and caudal to the lesion epicenter). The specificity of the immunofluorescence staining was confirmed by a control staining with primary IgG antibody.
Behavioral Analysis Motor function of the hindlimbs was analyzed using the Basso Mouse Scale open-field score.13 The evaluations were made by 2 blind observers for all analyzed groups. Behavioral analysis was performed at days 7, 14, 21, and 28 after treatment.
Statistical Analysis
Data are presented as mean values ± SD. Quantitative data from open-field locomotor scores were evaluated for statistical significance using 2-way analysis of variance. At different time points, 1-way analysis of variance, followed by SNK test was used to analyze the difference between each group. Data from OD values, cytokine concentrations, and quantity of cells were analyzed by 1-way analysis of variance (SPSS 16.0, Inc., Chicago, IL).
RESULTS A91-DC-Enhanced T-cell Proliferation The function of the A91-DCs to activate proliferation of T cells was examined in vitro. At 72 hours, the proliferative activity of T cells induced by A91-DC was the most powerful among the groups. The T cells induced by immature DC had a slight proliferative effect when compared with untreated T cells (Figure 1A). To authenticate the effect of A91-DC on activating T cells in SCI mice, we assessed the proliferative ability of T cells obtained from the spleen. The proliferation of T cells from SCI mice was significantly decreased compared with normal mice. T cells from mice injected peritoneally with A91-DC and mDC displayed a significantly stronger proliferative response than PBS- and iDC-treated mice (Figure 1B).
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A91-DC-Enhanced Production of BDNF and NT-3 by T Cells In Vitro Because iDC cannot induce T-cell proliferation, it was not considered as a therapeutic DC in the following experiments. To investigate whether A91-DC could influence the secretion of neurotrophic factors via T cells, production of BDNF and NT-3 was measured on day 8 postinjury; T cells were isolated immediately and cultured for 72 hour in vitro. The production of BDNF and NT-3 in supernatant was measured. A significant decrease of BDNF and NT-3 protein expression was observed in T cells isolated from PBS-treated mice compared with normal mice. The secretion of BDNF and NT-3 from T cells obtained from mice treated with A91-DC was higher than that obtained from mice treated with PBS (Figure 2A). Figure 2B shows that CD3+ T cells in spleen secreted rich BDNF and NT-3 after treatment with A91-DC.
A91-DC-Enhanced Infiltration of CD4⫹ T Cells That Secreted BDNF and NT-3 at the Injury Site The spinal cords were taken from each experimental group on day 8 after injury. As shown in Figure 3A, very few CD4+ T cells were detected in the injured spinal cord in the PBS- and DC-treated group. However, significantly higher numbers of CD4+ T cells were observed in the A91-DC group than in the PBS group. Furthermore, colocalization of BDNF and NT-3 from spinal cord section revealed that the number of doublepositive CD4 and BDNF/NT-3 cells in A91-DC group was higher than that in 2 other groups (Figure 3B, C), suggesting these infiltrated CD4+ T cells secreted rich BDNF and NT-3 at the injury site.
A91-DC Treatment Increased the Production of BDNF and NT-3 in Injured Spinal Cord Protein expression of BDNF and NT-3 was assessed at day 8. The production of BDNF and NT-3 was significantly increased in the A91-DC group compared with PBS- and DC-treated group (Figure 4A). In addition, the longitudinal spinal cord sections were immunostained for BDNF or NT-3 to detect the expression of nerve growth factors. Significantly increased BDNF and NT-3 protein immunofluorescence was
Figure 1. Proliferative activity of T cells. A, MTT assays show influences on T-cell proliferation by iDC, mDC, and A91-DC. iDC, mDC, and A91-DC were plated in 96-well round-bottom culture plates as stimulators and coincubated with purified T cells as responders. B, The proliferative activity of T cells obtained from sham mice and SCI mice treated with PBS, mDC, or A91-DC were determined by MTT. Phorbol 12-myristate 13-acetate was used to stimulate T-cell proliferation in vitro. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with PBS-treated group or compared with sham group by analysis of variance. PBS indicates phosphate-buffered saline. Spine
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detected in the A91-DC-treated mice as compared with the DC- and PBS-treated mice (Figure 4B). Statistical analysis revealed a significantly higher level of immunofluorescence intensity of BDNF and NT-3 in mice receiving A91-DC than that in other 2 groups (Figure 4C).
The Increased Expression of Neurotrophic Factors by A91-DC-Promoted Neuroprotective Effect at the Injured Site
Figure 2. Enhanced production of BDNF and NT-3 in the spleen after treatment with A91-DC postinjury. A, The production of BDNF and NT-3, secreted by T cells, was measured at day 8 after injury using enzyme-linked immunosorbent assay. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with normal group, *P < 0.05, compared with PBS-treated group by analysis of variance. B, Colocalization of BDNF (green) or NT-3 (green) with T cells (red) in the spleen section of mice treated with A91-DC. Scale bar, 50 μm.
An open-field test using the Basso Mouse Scale locomotor rating was performed to investigate the functional recovery from SCI in each group. Typical paraplegia was observed in all animals after injury. Administration of A91-DC in the injured spinal cord induced significant locomotor recovery after SCI. The significant functional recovery associated with A91-DC was observed on day 14 (Figure 5A). Furthermore, the lesion area of spinal cord tissue at 14 days after injury as quantified on the basis of HE staining showed larger tissue damage in PBS- and DC-treated mice than that in the A91-DC-treated mice (Figure 5B, C) though A91DC-induced more T cells infiltration in injured spinal cord. The difference in the lesion area between PBS and DC was not statistically significant. HE staining also showed that in the white matter, vacuolation was widespread and was prominent around lesion area in the PBS- and DC-treated mice. However, A91-DC significantly decreased the area of vacuolation in white matter (Figure 5B). Histopathological grading of the white matter confirmed A91-DC-exerted protective effect on white matter (Figure 5D). In addition, NF-200 staining was used to detect the residual axons and neurons in the lesion area. Histological analysis revealed a significantly higher level of neurofilament immunoactivity in mice receiving A91-DC than those of the control group (Figure 5E, F).
Figure 3. Effect of A91-DC on CD4+ T cells at the injured site. A, Quantification of CD4+ T cells was analyzed at the injured site in each group at day 7 after injury. Scale bar, 50 μm. The number of CD4+ T cells was counted from 4 microscopic fields in the injured site for each sample. B, Infiltrated CD4+ T cells secreted rich BDNF and NT-3 at the injury site. Scale bar, 25 μm. C, Number of double positive CD4 and BDNF/NT-3 cells among 3 groups. Data are given as means ± SD, 5 mice per group; *P < 0.05 versus PBS group (analysis of variance). PBS indicates phosphatebuffered saline.
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Figure 5. Neuroprotective effect of A91-DC at the injured site. A, Effect of A91-DC on functional recovery from SCI mice. Data represent means ± SD, 5 mice per group, *P < 0.05, versus PBS group, analysis of variance for repeated measures. B, Representative photomicrographs illustrating spinal cord lesion areas defined by H&E staining (×40). C, The mean lesion area among the 3 experimental groups. D, Histopathological grading reflecting the vacuolation in the white injury. E, Neurofilament immunostaining in the spinal cord lesion 2 weeks after injury. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. F, The fold increase of immunofluorescence intensity of NF200 staining in the injury site. Data represent means ± SD, 5 mice per group, *P < 0.05, versus PBS group, analysis of variance.
Figure 4. Expression of BDNF and NT-3 at the injured site after treatment with A91-DC. A, BDNF and NT-3 levels were determined using enzyme-linked immunosorbent assay in PBS, mDC, and A91-DC groups at day 8 after spinal cord injury. B, Protein levels of BDNF and NT-3 were determined by immunohitochemical staining. Scale bar, 100 μm. C, Immunofluorescence intensity of BDNF and NT-3 was analyzed. Data are given as means ± SD, 5 mice per group, *P < 0.05, compared with PBS group by analysis of variance. PBS indicates phosphate-buffered saline.
DISCUSSION The findings of this study indicate that vaccination with A91derived peptide pulsed DC is a potential therapeutic approach that can efficiently increase BDNF and NT-3 in injured spinal cord through promoting T-cell activation. The increased Spine
neurotrophic factors induced by A91-DC are sufficient to lead to functional recovery of SCI. It has become clear that within the CNS, immune responses are carefully controlled to prevent injury to sensitive tissue with limited regenerative capacity.14,15 One of the mechanisms by which immune cells maintain CNS plasticity is their ability to express and control growth factor production. Under pathological conditions, the T cell is a source of neurotrophic factors, and the expression of these factors can be significantly increased by antigen activation.16 It has been reported that some nerve growth factors are produced and continuously released at the site of injury by activated T cells.17 In addition, T-cell–derived NTs may locally induce microglia to release a second neurotrophic factor. Several studies have shown that NTs induce secretion of other NTs, and that cytokines such as interleukin-1β and tumor necrosis factor α stimulate microglia neurotrophic factor release.18,19 Thus, the increased www.spinejournal.com
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BASIC SCIENCE neurotrophic factors and multiple cytokines induced by A91-DC can also stimulate local microglia or monocytes to secrete other neurotrophic factors that may constitute another source of BDNF and NT-3 in the lesion site. In several studies, SCH was used as a stimuli antigen to induce CNS-specific T cells because SCH contains multiple CNS antigens.8 In the current study, A91 was used to replace SCH because A91 does not induce potential EAE, which may lead to a better tissue protection. Immunization with A91 was showed to provide neuroprotection in CNS injury.20,21 However, the neuroprotective effect achieved by immunization strategy is related to the gene background of the animals (strains) and the kind of adjuvant.22 DC strategies contribute to reduce the dose of antigen required, while additionally limiting cross-presentation by other cell types.23 For stimulation of an immune response, DCs migrate to the spleen or draining lymph nodes. There, DCs prime the immune response to antigens and may also prime self-antigen-specific responses observed in autoimmunity. Therefore, we assessed the capability of A91-DC to induce T-cell proliferation. As expected, A91-DC is efficient at priming the proliferative response of T cells. A91-DC also significantly enhanced the capability of T cells to secret BDNF and NT-3, suggesting that A91-DC may constitute a rich neurotrophic pool outside of the nervous system. Furthermore, we demonstrated that activated T cells infiltrated in the injured spinal cord for a prolonged period (at day 8 postinjection), which means that these T cells may elicit continued neurotrophic factor expression during the repair process in cases of SCI. In this study, we observed that vaccination with A91-DC provides more protective effect at the lesion site. This neuroprotective effect is partially attributed to the immunological regulation of T cells, but increased BDNF and NT-3 expression also plays a key role. BDNF and NT-3 have both been shown to enhance neuronal survival and plasticity in vitro and in vivo.24 NT-3 and BDNF are also biologically active proteins that promote the survival of pure oligodendrocyte and myelination of regenerating axons.25 Moreover, BDNF and NT-3 contribute to enhanced protection of neurons from the potentially detrimental effects of inflammatory cytokines.26 The increased levels of BDNF and NT-3 in the spinal cord also contribute to tolerance against EAE that may potentially be induced by DC vaccination.27 In conclusion, A91-DC partially promoted the level of neurotrophic factors production in the subacute phase of SCI. The therapeutic strategy of vaccination with A91-DC could not only induce the beneficial immunomodulation of T cells but also provide strong neurotrophic factor support after SCI.
➢ Key Points Vaccination with MBP-pulsed peptide (A91) DCs is a potential minimally invasive approach that could promote BDNF and NT-3 expression via T cells in an injured spinal cord. 100
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This therapeutic strategy could provide adequate neurotropic factor to support some degree of SCI recovery. A91-DC is a potential strategy for treating SCI.
Acknowledgments Yufu Wang and Jing Li contributed equally to this work.
References
1. Rowland JW, Hawryluk GW, Kwon B, et al. Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg Focus 2008;25:E2. 2. Schwab ME. Repairing the injured spinal cord. Science 2002;295: 1029–31. 3. Varon S, Conner JM. Nerve growth factor in CNS repair. J Neurotrauma 1994;11:473–86. 4. Brock JH, Rosenzweig ES, Blesch A, et al. Local and remote growth factor effects after primate spinal cord injury. J Neurosci 2010;30:9728–37. 5. Blesch A, Tuszynski MH. Transient growth factor delivery sustains regenerated axons after spinal cord injury. J Neurosci 2007;27:10535–45. 6. Blesch A, Lu P, Tuszynski MH. Neurotrophic factors, gene therapy, and neural stem cells for spinal cord repair. Brain Res Bull 2002; 57:833–8. 7. Schwartz M, London A, Shechter R. Boosting T-cell immunity as a therapeutic approach for neurodegenerative conditions: the role of innate immunity. Neuroscience 2009;158:1133–42. 8. Wang Y, Wang K, Chao R, et al. Neuroprotective effect of vaccination with autoantigen-pulsed dendritic cells after spinal cord injury. J Surg Res 2012;176:281–92. 9. Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7:31–4. 10. Hauben E, Gothilf A, Cohen A, et al. Vaccination with dendritic cells pulsed with peptides of myelin basic protein promotes functional recovery from spinal cord injury. J Neurosci 2003;23:8808–19. 11. Marques SA, Garcez VF, Del Bel EA, et al. A simple, inexpensive and easily reproducible model of spinal cord injury in mice: morphological and functional assessment. J Neurosci Methods 2009;177:183–93. 12. Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 1999;223:77–92. 13. Basso DM, Fisher LC, Anderson AJ, et al. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 2006;23:635–59. 14. Yong VW, Rivest S. Taking advantage of the systemic immune system to cure brain diseases. Neuron 2009;64:55–60. 15. Graber JJ, Dhib-Jalbut S. Protective autoimmunity in the nervous system. Pharmacol Ther 2009;121:147–59. 16. Moalem G, Gdalyahu A, Shani Y, et al. Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun 2000;15:331–45. 17. Kerschensteiner M, Gallmeier E, Behrens L, et al. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999;189:865–70. 18. Heese K, Hock C, Otten U. Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem 1998;70:699–707. 19. Kruttgen A, Moller JC, Heymach JV Jr, et al. Neurotrophins induce release of neurotrophins by the regulated secretory pathway. Proc Natl Acad Sci U S A 1998;95:9614–9. 20. Martinon S, Garcia E, Gutierrez-Ospina G, et al. Development of protective autoimmunity by immunization with a neural-derived January 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140563_LR 100
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BASIC SCIENCE
21.
22. 23. 24.
peptide is ineffective in severe spinal cord injury. PLoS One 2012;7:e32027. Garcia E, Silva-Garcia R, Mestre H, et al. Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury. J Neurosci Res 2012;90:656–63. Hendrix S, Nitsch R. The role of T helper cells in neuroprotection and regeneration. J Neuroimmunol 2007;184:100–12. Cohn L, Delamarre L. Dendritic cell-targeted vaccines. Front Immunol 2014;5:255. Alcala-Barraza SR, Lee MS, Hanson LR, et al. Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS. J Drug Target 2010;18:179–90.
Spine
Enhanced Expression of Neurotrophic Factors• Wang et al
25. McTigue DM, Horner PJ, Stokes BT, et al. Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J Neurosci 1998;18:5354–65. 26. Flugel A, Matsumuro K, Neumann H, et al. Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis: inhibition of monocyte transendothelial migration. Eur J Immunol 2001;31:11–22. 27. Hammarberg H, Lidman O, Lundberg C, et al. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J Neurosci 2000;20:5283–91.
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