EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12276 ..................................................................................................................................................................
MicroRNA-155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury J. Yi*, D. Wang†, X. Niu*, J. Hu*, Y. Zhou* & Z. Li‡
Abstract *The Upper Limb Orthopedic Department of East Award, The First Affiliated Hospital of Sun YatSen University, Guangzhou, China; †Department of Microsurgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China; and ‡Department of Orthopaedics, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
Received 24 November 2014; Accepted in revised form 15 January 2015 Correspondence to: Z. Li, No.3 Affiliated Hospital of Zhongshan University, No 600 Tianhe Road, 510080 Guangzhou, China. E-mail: [email protected]
Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Mir-155 has been reported to be involved in both innate and adaptive immune responses. But the role of Mir-155 in spinal cord injury is still unknown. In our current study, Mir155 deficiency displays increased myelin sparring and enhanced SC repair process. The number of T cells, B cells and neutrophils are all significantly lower in Mir155/ group than that in WT group after SCI. IL-17A-producing cells and the expression of IL-17A are markedly lower in Mir-155/ mice than that in WT mice. We also found higher production of IL-17 by WT CD4+ T cells than Mir155/ CD4+ T cells in vitro. In our further DC-T cell coculture system, Mir-155 deficiency in DCs results in significantly less IL-17 production from T cells. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency is partly dependent on increased expression of SOCS1. In conclusion, our present work provides evidence to support the concept that Mir-155 deficiency suppresses Th17 cell differentiation and improves locomotor recovery after SCI.
Introduction Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue [1, 2]. The inflammation following SCI is considered an important process that promotes secondary damage to neuronal tissue in the spinal cord after traumatic injury and regulates the pathological progress during SCI [3–6]. A novel small non-coding RNA species known as microRNAs (miRNAs, miRs) is involved in biological control at multiple levels. They regulate gene expression essential for cell development and function through mRNA degradation or translational inhibition. Evidences indicated that miRNAs played important roles in immune system. Mir-155 has been found apparently upregulated in several activated immune cells, including T lymphocytes, B lymphocytes, macrophages and dendritic cells (DCs). It is upregulated by a broad range of inflammatory mediators during innate immune response [7, 8]. Previous studies have indicated that Mir-155 involved in both innate and adaptive immune responses [9, 10]. However, the role of Mir-155 in spinal cord injury is still unknown.
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In our current study, we demonstrated a crucial role for Mir-155 deficiency in suppressing Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency was partly dependent on increased expression of SOCS1.
Materials and methods Animal model. Animal facilities and protocols were approved by the Laboratory Animal Care and Use Committee of Sun Yat-Sen University. Three-month-old male wild-type (WT) C57 mice (Sun Yat-Sen University) and Mir-155/ mice (Jackson Laboratory Maine, USA) were anesthetized with isoflurane, and complete laminectomy was performed at the level of the 10th thoracic vertebra under a surgical microscope, after exposing the dorsal surface of the dura mater and taking the utmost care in avoiding any dural tear. A contusion SCI was produced using a commercially available SCI device (Infinite Horizons Impactor; Precision Systems and Instrumentation LLC, Fairfax, VA, USA) with an impact force of 60
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kilodynes [11]. There are five groups. They are Sham, WT, Mir-155/, WT + Anti-IL-17 and Mir-155/ + rmIL17 groups. For neutralization of endogenous IL-17A, 0.2 mg of neutralizing rabbit anti-mouse IL-17A (Biolegend, Houston, USA) was administered i.p. 5 min prior to reperfusion. In Mir-155/ + rmIL-17 group, mouse in vivo i.p. injections included recombinant mouse IL-17 [rmIL-17 (R&D Systems Newyork, USA), 20 lg/mouse 5 min prior to SCI. Assessment of locomotor behaviour. Hind-limb motor function was evaluated using the Basso Mouse Scale (BMS) open field locomotor test, in which the scores range from 0 points (no ankle movement) to nine points (complete functional recovery) [12]. BMS scores were recorded at 1, 3, 7, 14, 28 and 42 days after SCI, by two independent examiners blinded to the experimental conditions (HN, SW). Immunohistochemistry. The spinal cords were dissected out and post-fixed in the same fixative for a few hours. The tissue samples were immersed in 10% sucrose in 0.1 mol/l PBS at 4 °C for 24 h and 30% sucrose in 0.1 mol/l PBS for 24 h. Segments of the SC (cord segments T8 to T12) were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cut on a cryostat into serial axial or sagittal frozen sections 10 lm thick. The sections were serially mounted on glass slides, and fixed with 2% paraformaldehyde in 0.1 mol/l PBS for 5 min, rinsed in PBS and stored at 80 °C. Luxol fast blue (LFB) staining was used to evaluate the spared myelin and extent of demyelization as previously described [13]. For semi-quantitative analysis of demyelination, 10 axial sections randomly selected at a distance up to 5 mm cephalad and caudal to the epicentre were stained with LFB and examined 42 days after SCI. The LFB-positive area in the ventrolateral funiculus was analysed and calculated as the percentage cross-sectional area of residual tissue, as described previously [14]. Primary antibodies were antimouse GAP-43, anti-mouse NF-H, anti-mouse CD3, antimouse CD45R and anti-mouse (clone 7/4) neutrophil. After incubation, sections were washed four times with PBS-T, before incubation with an appropriate fluorescent secondary antibody diluted in PBS containing 5% BSA for an hour at room temperature. After incubation, sections were again washed four times with PBS-T, before being mounted with fluorescent mounting medium and coverslipped. A negative control was included for each stain, for which incubation with primary antibodies was omitted. Images of stained sections were captured using Olympus Digital DP70 photomicroscope, and DP Controller software. For analysis of glial cells, images of the injury site and areas distal (caudal and rostral) to the injury were taken. Using ImageJ software Houston, USA, densitometry measurements were made by thresholding the images and measuring the fraction of the immunopositive areas. For analysis of immune cells, images were taken from five frames surrounding the injury site of each section. Cells
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were counted manually and were calculated as the average number of cells per frame (150 lm2) [15]. Western blotting. The protein levels were determined by Western blotting. Protein extracted from cells or tissue was separated on 10% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). After being blocked with 5% nonfat milk in TBS for 3 h, the membranes were incubated with indicated primary antibodies (0.2 lg/ml) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000) for 3 h. b-actin was used as a loading control for comparison between samples. Nucleofection. Nucleofection was performed with Mouse T Cell Nucleofectorâ Kit and Nucleofector device (Amaxa, Koelin, Germany). First, 1 9 107 naive CD4+ T cells were resuspended in 100 ll nucleofectorâ solution. 2.5 lg pmaxGFPâ Vector or 100 pmol oligonucleotides (including SOCS1-TPMir155 and control-TPMir155) were added into the solution and mixed gently. Then, the mixtures were gently transferred to electroporation cuvettes and placed in the Nucleofector device. Cells were nucleofected in the X-01 program. Finally, transfected cells were transferred to a 12-well plate with 1.5 ml prepared Mouse T Cell Nucleofectorâ Medium in each plate and incubated in a humidified 37 °C/5% CO2 incubator until analysis. SOCS1-TPMir155 from Gene Tools, LLC (Corvallis, OR, USA) was used to interfere Mir-155 and suppressors of cytokine signalling 1 (SOCS1) interaction, with control-TPMir155 as the matched control. CD4+ T cell activation and polarization. Four hours after nucleofection, CD4+ T cells were activated by 5 lg/ml plate-bound anti-CD3 and 2 lg/ml soluble anti-CD28. For propagation under Th17 condition, 2.5 ng/ml rTGFb1 and 30 ng/ml rIL-6 were provided. All antibodies used were purchased from eBioscience (San Diego, CA, USA). All cytokines used were purchased from Peprotech (Rocky Hill, NJ, USA). Real-time PCR. Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology, Dalian, China) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in duplicate, and changes in relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. Flow cytometry analysis. For IL-17-producing cells detection, the damaged SC around the epicentre of the lesion (6 mm in length) was surgically dissected out with 175 U/ ml collagenase for 1 h at 37 °C. Cells were washed in DMEM containing 10% foetal bovine serum and filtered through a 40 lM nylon cell strainer under centrifugation to
286 Mir-155 Deficiency Suppresses Th17 Cell Differentiation J. Yi et al. .................................................................................................................................................................. remove tissue debris and obtain a single-cell suspension, as described previously [16]. From this point on, a cell-count was performed before every staining in every sample to maintain a cell density of 1.0 9 106 cells/100 ll. For Th17 cell analysis in vitro, 4 days after transfection and activation, cells were collected and used for detecting Th17 cells differentiation. Cells were stimulated for 4 h with 25 ng/ml phorbol myristate acetate (PMA) and 1 lg/ml ionomycin in the presence of 2 lM monensin in the last 2 h. Cells were collected and stained with FITC-labelled anti-mouse CD4 for 30 min at 4 °C. Then, cells were fixed and permeabilized according to the manufacturer’s protocol. Next, cells were incubated with PE-labelled antimouse IL-17A in the dark for 20 min. Finally, stained cells were resuspended in 200 ll washing buffer and analysed by FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). All reagents used were purchased from eBioscience. Statistical analysis. All data were presented as means SEM. The two-tailed Student t-test was applied for statistical analysis with P < 0.05 being considered statistically significant. Data were analysed using Prism software (GraphPad Software, Inc. California, USA).
Results Mir-155 deficiency promotes locomotor function after SCI
To investigate the therapeutic effects of Mir-155 deficiency on spinal cord repair, we compared locomotor improvement of Mir-155/ and WT mice. At day 7 after SCI, the Mir-155/ group showed a marked improvement in BMS locomotor score compared with the control group, and this difference continued up to A
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Figure 1 Mir-155 deficiency promotes locomotor function after SCI. A, Analysis of the locomotor Basso Mouse Scale (BMS) score after SCI; B, Quantification of LFB-positive spared myelin areas in the ventrolateral funiculus at the lesion site; C, Quantification of GAP-43-positive areas at day 42 after SCI; D, Quantification of NF-H-positive areas at day 42 after SCI. n = 6 for each group. *P < 0.05.
6 weeks after SCI (Fig 1A). Next, we used LFB staining to evaluate the sparing of myelin sheaths around axons at 42 days after SCI. The area of spared myelin in the Mir155/ group was markedly greater than that in the control group at day 42 (Fig 1B). Furthermore, a significantly larger GAP-43-positive area was shown in the HG-treated group at day 42 after SCI (Fig 1C). And the expression of NF-H in Mir-155/ group was also significantly higher than that in WT group at 42 days after SCI (Fig 1D). Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro
To determine whether Mir-155 deficiency affected the inflammatory response after SCI, we evaluated inflammatory cell infiltration at 42 days after SCI. Recruitment of T cells, macrophages and neutrophils to the injured spinal cord was assessed around the site of injury in Mir-155/ and WT mice. The numbers of T cells, macrophages and neutrophils were significantly lower in Mir-155/ mice than in WT mice (Fig 2A). Next, we detected IL-17A expression in Mir155/ and WT mice at 42 days after SCI. Results showed that the IL-17A-producing cells and the expression of IL17A, IL-6 and IL-23 were markedly lower in Mir-155/ mice than that in WT mice (Fig 2B–D). To determine whether the enhanced T cell activity in WT mice was a result of intrinsic differences in CD4+ T cells, we compared the cytokine profiles of WT and Mir155/ total CD4+ T cells in response to ex vivo stimulation with anti-CD3 and anti-CD28. We found higher production of IL-17 by WT CD4+ T cells than Mir155/ CD4+ T cells (Fig 3A). This indicates that the enhanced cytokine production of IL-17 that we observed in WT mice was in part a result of intrinsic differences in CD4+ T cells. To further determine the role of Mir-155 on Th17 differentiation, WT and Mir-155/ na€ıve CD4+ T cells were differentiated into Th17 cells. IL-17 production in Mir-155/ T cells was significantly reduced compared with WT-T cells (Fig 3B). Polarization of T cells to Th1, Th2 or Th17 phenotypes is a critical feature of cellmediated immunity and is influenced by production of cytokines by DC. Therefore, we tested whether Mir-155 modulates the expression of Th17-polarizing cytokine by DCs. For this, we stimulated bone marrow-derived DCs from WT and Mir-155/ mice with the TLR ligand LPS and analysed cytokine expression. In Mir-155/ DCs, decreased levels of Th17-polarizing cytokines were observed in response to LPS stimulation (Fig 3C). In our further DC-T cell coculture system, Mir-155 deficiency in DCs resulted in significantly less IL-17 production from T cells (Fig 3D). To further investigate the role of IL-17 in SCI, WT or Mir-155/ mice with SCI were treated with anti-IL-17A antibody or recombinant mouse IL-17A (rmIL-17). The
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Figure 2 Mir-155 deficiency suppresses IL17A production in vivo. A, Vales represent the mean cell number in regions surrounding the injury site of WT and mir-155/ mice 42 days after SCI; B, IL-17A-producing cells in injured spinal cord were analysed by FACS; C, IL-17A protein expression was analysed by Western blot; D, IL-17A mRNA expression was analysed by Q-PCR. n = 6 for each group. *P < 0.05.
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Figure 3 Mir-155 deficiency suppresses Th17 differentiation in vitro. A, Total CD4+ T cells were isolated from the spleens of WT and Mir-155/ mice and stimulated with anti-CD3 and anti-CD28. Supernatants from culture were harvested 72 h after initiation of cultures and analysed for IL-17; B, Naive CD4+ T cells were isolated from the spleens of WT and Mir-155/ mice and stimulated with anti-CD3 and anti-CD28 under Th17-skewing condition. Supernatants from culture were harvested 72 h after initiation of cultures and analysed for IL-17 by ELISA and Q-PCR; C, Bone marrow-derived DCs were isolated from WT and Mir-155/ mice and were cultured with 100 ng/ml LPS. Cell-free supernatants were measured for Th17-polarizing cytokine by ELISA. D, CD4+ T cells isolated from WT mice were cultured with CD11c+ DCs derived from WT and Mir-155/ mice. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IL-17. Data are representative of three to five independent experiments. *P < 0.05.
BMS locomotor score and the area of spared myelin in WT + Anti-IL-17 group was markedly greater than that in the WT group at day 42 (Fig 4A–B). The increased BMS locomotor score and spared myelin in Mir-155/ mice were both abrogated with additional rmIL-17 administration (Fig 4A–B). Furthermore, the increased GAP-43- and NF-H-positive areas induced by Mir-155 deficiency were also reversed by rmIL-17 treatment (Fig 4C).
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Mir-155 deficiency suppressed th17 cell differentiation via upregulation of SOCS1
Three transcripts, including SOCS1, SMAD2 and SMAD5, had been reported to be targets of Mir-155. But we found that SOCS1 was the only one that was enhanced by Mir-155 deficiency (the expression of SMAD2 and SMAD5 was not shown) (Fig 5A).
288 Mir-155 Deficiency Suppresses Th17 Cell Differentiation J. Yi et al. .................................................................................................................................................................. To further investigate the role of SOCS1 in the regulated differentiation of Th17, target protector (TP) was used. SOCS1-TPMir155 interfere Mir-155-SOCS1 interaction by binding to the binding site of Mir-155 in the 30 - untranslated region of SOCS1, without interfering Mir-155 interaction with other target mRNAs. So, when SOCS1-TPMir155 was transfected, a dramatic increase of SOCS1 protein was observed (Fig. 5B). Furthermore, the percentages of Th17 cells in CD4+ T cells as well as the
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Figure 4 Mir-155 deficiency promotes locomotor function partly dependent on reduced IL-17 production. A, Analysis of the locomotor Basso Mouse Scale (BMS) score after SCI; B, Quantification of LFBpositive spared myelin areas in the ventrolateral funiculus at the lesion site; C, Quantification of GAP-43-and NF-H-positive areas at day 42 after SCI. n = 6 for each group. *P < 0.05.
expression of IL-17A, IL-6 and IL-23 in cell culture supernatant were decreased when SOCS1-TPMir155 was transfected (Fig. 5C–D).
Discussion This study revealed a crucial role for Mir-155 in regulating Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency was partly dependent on increased expression of SOCS1. Interleukin (IL)-17A is a member of the IL-17 family, which includes six structurally related isoforms: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17A was secreted by different cells, including Th17 cells, cd T cells, NK cells, NKT cells and neutrophils. Recent studies have identified IL-17 (Th17) originated from the subset of T cells, which plays a predominant role in the pathogenesis of autoimmune diseases [17–19]. While most nervous system disease pathogeneses are associated with autoimmune or immune inflammatory injury, the data clearly support the notion that both anti-inflammatory (Th2 or Tregs) and proinflammatory (Th1 or Th17) T cells were activated in the context of a sterile SCI [20]. Importantly, recent clinical trials with short duration IL-17 antagonistic therapy in established rheumatoid arthritis (RA) have provided the direct evidence in pathological role of IL-17 in RA and indicated that blockade of IL-17 in humans may represent a valid therapeutic approach [21]. In our experiment, Mir-155 deficiency alleviates Th17 response and limits Th17 differentiation. Furthermore, the alleviated SCI induced by Mir-155/ was abolished with
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Figure 5 Mir-155 deficiency suppressed th17 cell differentiation via upregulation of SOCS1. A, SOCS1 protein expression in regions surrounding the injury site of WT and Mir155/ mice 42 days after SCI was analysed by Western blot; B, 4 days after SOCS1TPMir155 and control-TPMir155 were transfected, the levels of the transcripts involved in Th17 cells differentiation were detected by Q-PCR; C, 4 days after SOCS1TPMir155 and control-TPMir155 were transfected, the percentages of Th17 were analysed by flow cytometry; D, 4 days after SOCS1-TPMir155 and control-TPMir155 were transfected, the level of IL-17A, IL-6 and IL23 in cell culture supernatant was quantified by ELISA. *P < 0.05 versus all other groups. n = 6.
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additional IL-17 administration. All of these demonstrated that decreased IL-17 expression contributes to the alleviated SCI induced by Mir-155/. Many direct targets of Mir-155 in DCs and T cells have been identified. The essential role of Mir-155 elucidated by targeting SOCS1 expression is not only confined to Treg cells, but also macrophages and DCs. Recently, it has been shown that IL-12 production by DCs is regulated by Mir155-mediated targeting of SOCS1 [22]. Consistent with previous studies, we found reduced expression of Th17polarizing cytokines in DCs from Mir-155/ mice with SCI. Altered expression of Th17-polarizing cytokine expression in DCs that influence the differentiation of Th17 responses could be an additional mechanism by which Mir-15/ mice are resistant to SCI. SOCS1 is a negative regulator of Janus kinase (JAK)/ STAT signalling pathway. Recently, the role of SOCS1 in Th17 cell differentiation and function was clarified by characterizing a mimetic of SOCS1, namely novel tyrosine kinase inhibitor peptide (Tkip). Tkip has been demonstrated to blocked IL-6-induced activation of STAT3, inhibited the development of Th17 and the production of IL-17A [23–25]. Furthermore, a total of 83 overlapping candidate target genes of Mir-155 were predicted through three different analysis programmes, and SOCS1 was at the top of the list [26]. In our current study, we showed that Mir-155 promoted Th17 cell differentiation and enhanced Th17 cell function by directly inhibiting SOCS1. These were in accordance with previous studies indicating the relationship of SOCS1 and Mir-155 [27, 28]. In conclusion, our present work provides evidence to support the concept that Mir-155 deficiency suppresses Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro via upregulation of SOCS1 expression. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in the immunoregulation, Mir-155 may be a novel therapeutic to protect SCI.
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5 Kato H, Kanellopoulos GK, Matsuo S et al. Neuronal apoptosis and necrosis following spinal cord ischemia in the rat. Exp Neurol 1997;148:464–74. 6 Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med 1997;3:73–6. 7 O’Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci USA 2007;104:1604–9. 8 Ruggiero T, Trabucchi M, De Santa F et al. LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages. FASEB J 2009;23:2898–908. 9 Thai TH, Calado DP, Casola S et al. Regulation of the germinal center response by microRNA-155. Science 2007;316:604–8. 10 Rodriguez A, Vigorito E, Clare S et al. Requirement of bic/ microRNA-155 for normal immune function. Science 2007;316:608–11. 11 Scheff SW, Rabchevsky AG, Fugaccia I, Main JA, Lumpp JJ. Experimental modeling of spinal cord injury: characterization of a force-defined injury device. J Neurotrauma 2003;20:179–93. 12 Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 2006;23:635–59. 13 Guerrero AR, Uchida K, Nakajima H et al. Blockade of interleukin-6 signalling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice. J Neuroinflammation 2012;9:40. 14 Chen KB, Uchida K, Nakajima H et al. Tumor necrosis factor-alpha antagonist reduces apoptosis of neurons and oligodendroglia in rat spinal cord injury. Spine (Phila Pa 1976) 2011;36:1350–8. 15 Hill F, Kim CF, Gorrie CA, Moalem-Taylor G. Interleukin-17 deficiency improves locomotor recovery and tissue sparing after spinal cord contusion injury in mice. Neurosci Lett 2011;487:363–7. 16 Saiwai H, Ohkawa Y, Yamada H et al. The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury. Am J Pathol 2010;176:2352–66. 17 Langrish CL, Chen Y, Blumenschein WM et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005;201:233–40. 18 Kotake S, Udagawa N, Takahashi N et al. IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103:1345–52. 19 Nakae S, Nambu A, Sudo K, Iwakura Y. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J Immunol 2003;171:6173–7. 20 Ankeny DP, Popovich PG. Mechanisms and implications of adaptive immune responses after traumatic spinal cord injury. Neuroscience 2009;158:1112–21. 21 Genovese MC, Van den Bosch F, Roberson SA et al. LY2439821, a humanized anti-interleukin-17 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a phase I randomized, double-blind, placebo-controlled, proof-of-concept study. Arthritis Rheum 2010;62:929–39. 22 Lu C, Huang X, Zhang X et al. miR-221 and miR-155 regulate human dendritic cell development, apoptosis, and IL-12 production through targeting of p27kip1, KPC1, and SOCS-1. Blood 2011;117:4293–303. 23 Flowers LO, Subramaniam PS, Johnson HM. A SOCS-1 peptide mimetic inhibits both constitutive and IL-6 induced activation of STAT3 in prostate cancer cells. Oncogene 2005;24:2114–20. 24 Jager LD, Dabelic R, Waiboci LW et al. The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis. J Neuroimmunol 2011;232:108–18.
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MicroRNA-155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury J. Yi*, D. Wang†, X. Niu*, J. Hu*, Y. Zhou* & Z. Li‡
Abstract *The Upper Limb Orthopedic Department of East Award, The First Affiliated Hospital of Sun YatSen University, Guangzhou, China; †Department of Microsurgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China; and ‡Department of Orthopaedics, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
Received 24 November 2014; Accepted in revised form 15 January 2015 Correspondence to: Z. Li, No.3 Affiliated Hospital of Zhongshan University, No 600 Tianhe Road, 510080 Guangzhou, China. E-mail: [email protected]
Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Mir-155 has been reported to be involved in both innate and adaptive immune responses. But the role of Mir-155 in spinal cord injury is still unknown. In our current study, Mir155 deficiency displays increased myelin sparring and enhanced SC repair process. The number of T cells, B cells and neutrophils are all significantly lower in Mir155/ group than that in WT group after SCI. IL-17A-producing cells and the expression of IL-17A are markedly lower in Mir-155/ mice than that in WT mice. We also found higher production of IL-17 by WT CD4+ T cells than Mir155/ CD4+ T cells in vitro. In our further DC-T cell coculture system, Mir-155 deficiency in DCs results in significantly less IL-17 production from T cells. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency is partly dependent on increased expression of SOCS1. In conclusion, our present work provides evidence to support the concept that Mir-155 deficiency suppresses Th17 cell differentiation and improves locomotor recovery after SCI.
Introduction Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue [1, 2]. The inflammation following SCI is considered an important process that promotes secondary damage to neuronal tissue in the spinal cord after traumatic injury and regulates the pathological progress during SCI [3–6]. A novel small non-coding RNA species known as microRNAs (miRNAs, miRs) is involved in biological control at multiple levels. They regulate gene expression essential for cell development and function through mRNA degradation or translational inhibition. Evidences indicated that miRNAs played important roles in immune system. Mir-155 has been found apparently upregulated in several activated immune cells, including T lymphocytes, B lymphocytes, macrophages and dendritic cells (DCs). It is upregulated by a broad range of inflammatory mediators during innate immune response [7, 8]. Previous studies have indicated that Mir-155 involved in both innate and adaptive immune responses [9, 10]. However, the role of Mir-155 in spinal cord injury is still unknown.
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In our current study, we demonstrated a crucial role for Mir-155 deficiency in suppressing Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency was partly dependent on increased expression of SOCS1.
Materials and methods Animal model. Animal facilities and protocols were approved by the Laboratory Animal Care and Use Committee of Sun Yat-Sen University. Three-month-old male wild-type (WT) C57 mice (Sun Yat-Sen University) and Mir-155/ mice (Jackson Laboratory Maine, USA) were anesthetized with isoflurane, and complete laminectomy was performed at the level of the 10th thoracic vertebra under a surgical microscope, after exposing the dorsal surface of the dura mater and taking the utmost care in avoiding any dural tear. A contusion SCI was produced using a commercially available SCI device (Infinite Horizons Impactor; Precision Systems and Instrumentation LLC, Fairfax, VA, USA) with an impact force of 60
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kilodynes [11]. There are five groups. They are Sham, WT, Mir-155/, WT + Anti-IL-17 and Mir-155/ + rmIL17 groups. For neutralization of endogenous IL-17A, 0.2 mg of neutralizing rabbit anti-mouse IL-17A (Biolegend, Houston, USA) was administered i.p. 5 min prior to reperfusion. In Mir-155/ + rmIL-17 group, mouse in vivo i.p. injections included recombinant mouse IL-17 [rmIL-17 (R&D Systems Newyork, USA), 20 lg/mouse 5 min prior to SCI. Assessment of locomotor behaviour. Hind-limb motor function was evaluated using the Basso Mouse Scale (BMS) open field locomotor test, in which the scores range from 0 points (no ankle movement) to nine points (complete functional recovery) [12]. BMS scores were recorded at 1, 3, 7, 14, 28 and 42 days after SCI, by two independent examiners blinded to the experimental conditions (HN, SW). Immunohistochemistry. The spinal cords were dissected out and post-fixed in the same fixative for a few hours. The tissue samples were immersed in 10% sucrose in 0.1 mol/l PBS at 4 °C for 24 h and 30% sucrose in 0.1 mol/l PBS for 24 h. Segments of the SC (cord segments T8 to T12) were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cut on a cryostat into serial axial or sagittal frozen sections 10 lm thick. The sections were serially mounted on glass slides, and fixed with 2% paraformaldehyde in 0.1 mol/l PBS for 5 min, rinsed in PBS and stored at 80 °C. Luxol fast blue (LFB) staining was used to evaluate the spared myelin and extent of demyelization as previously described [13]. For semi-quantitative analysis of demyelination, 10 axial sections randomly selected at a distance up to 5 mm cephalad and caudal to the epicentre were stained with LFB and examined 42 days after SCI. The LFB-positive area in the ventrolateral funiculus was analysed and calculated as the percentage cross-sectional area of residual tissue, as described previously [14]. Primary antibodies were antimouse GAP-43, anti-mouse NF-H, anti-mouse CD3, antimouse CD45R and anti-mouse (clone 7/4) neutrophil. After incubation, sections were washed four times with PBS-T, before incubation with an appropriate fluorescent secondary antibody diluted in PBS containing 5% BSA for an hour at room temperature. After incubation, sections were again washed four times with PBS-T, before being mounted with fluorescent mounting medium and coverslipped. A negative control was included for each stain, for which incubation with primary antibodies was omitted. Images of stained sections were captured using Olympus Digital DP70 photomicroscope, and DP Controller software. For analysis of glial cells, images of the injury site and areas distal (caudal and rostral) to the injury were taken. Using ImageJ software Houston, USA, densitometry measurements were made by thresholding the images and measuring the fraction of the immunopositive areas. For analysis of immune cells, images were taken from five frames surrounding the injury site of each section. Cells
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were counted manually and were calculated as the average number of cells per frame (150 lm2) [15]. Western blotting. The protein levels were determined by Western blotting. Protein extracted from cells or tissue was separated on 10% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). After being blocked with 5% nonfat milk in TBS for 3 h, the membranes were incubated with indicated primary antibodies (0.2 lg/ml) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000) for 3 h. b-actin was used as a loading control for comparison between samples. Nucleofection. Nucleofection was performed with Mouse T Cell Nucleofectorâ Kit and Nucleofector device (Amaxa, Koelin, Germany). First, 1 9 107 naive CD4+ T cells were resuspended in 100 ll nucleofectorâ solution. 2.5 lg pmaxGFPâ Vector or 100 pmol oligonucleotides (including SOCS1-TPMir155 and control-TPMir155) were added into the solution and mixed gently. Then, the mixtures were gently transferred to electroporation cuvettes and placed in the Nucleofector device. Cells were nucleofected in the X-01 program. Finally, transfected cells were transferred to a 12-well plate with 1.5 ml prepared Mouse T Cell Nucleofectorâ Medium in each plate and incubated in a humidified 37 °C/5% CO2 incubator until analysis. SOCS1-TPMir155 from Gene Tools, LLC (Corvallis, OR, USA) was used to interfere Mir-155 and suppressors of cytokine signalling 1 (SOCS1) interaction, with control-TPMir155 as the matched control. CD4+ T cell activation and polarization. Four hours after nucleofection, CD4+ T cells were activated by 5 lg/ml plate-bound anti-CD3 and 2 lg/ml soluble anti-CD28. For propagation under Th17 condition, 2.5 ng/ml rTGFb1 and 30 ng/ml rIL-6 were provided. All antibodies used were purchased from eBioscience (San Diego, CA, USA). All cytokines used were purchased from Peprotech (Rocky Hill, NJ, USA). Real-time PCR. Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology, Dalian, China) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in duplicate, and changes in relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. Flow cytometry analysis. For IL-17-producing cells detection, the damaged SC around the epicentre of the lesion (6 mm in length) was surgically dissected out with 175 U/ ml collagenase for 1 h at 37 °C. Cells were washed in DMEM containing 10% foetal bovine serum and filtered through a 40 lM nylon cell strainer under centrifugation to
286 Mir-155 Deficiency Suppresses Th17 Cell Differentiation J. Yi et al. .................................................................................................................................................................. remove tissue debris and obtain a single-cell suspension, as described previously [16]. From this point on, a cell-count was performed before every staining in every sample to maintain a cell density of 1.0 9 106 cells/100 ll. For Th17 cell analysis in vitro, 4 days after transfection and activation, cells were collected and used for detecting Th17 cells differentiation. Cells were stimulated for 4 h with 25 ng/ml phorbol myristate acetate (PMA) and 1 lg/ml ionomycin in the presence of 2 lM monensin in the last 2 h. Cells were collected and stained with FITC-labelled anti-mouse CD4 for 30 min at 4 °C. Then, cells were fixed and permeabilized according to the manufacturer’s protocol. Next, cells were incubated with PE-labelled antimouse IL-17A in the dark for 20 min. Finally, stained cells were resuspended in 200 ll washing buffer and analysed by FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). All reagents used were purchased from eBioscience. Statistical analysis. All data were presented as means SEM. The two-tailed Student t-test was applied for statistical analysis with P < 0.05 being considered statistically significant. Data were analysed using Prism software (GraphPad Software, Inc. California, USA).
Results Mir-155 deficiency promotes locomotor function after SCI
To investigate the therapeutic effects of Mir-155 deficiency on spinal cord repair, we compared locomotor improvement of Mir-155/ and WT mice. At day 7 after SCI, the Mir-155/ group showed a marked improvement in BMS locomotor score compared with the control group, and this difference continued up to A
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Figure 1 Mir-155 deficiency promotes locomotor function after SCI. A, Analysis of the locomotor Basso Mouse Scale (BMS) score after SCI; B, Quantification of LFB-positive spared myelin areas in the ventrolateral funiculus at the lesion site; C, Quantification of GAP-43-positive areas at day 42 after SCI; D, Quantification of NF-H-positive areas at day 42 after SCI. n = 6 for each group. *P < 0.05.
6 weeks after SCI (Fig 1A). Next, we used LFB staining to evaluate the sparing of myelin sheaths around axons at 42 days after SCI. The area of spared myelin in the Mir155/ group was markedly greater than that in the control group at day 42 (Fig 1B). Furthermore, a significantly larger GAP-43-positive area was shown in the HG-treated group at day 42 after SCI (Fig 1C). And the expression of NF-H in Mir-155/ group was also significantly higher than that in WT group at 42 days after SCI (Fig 1D). Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro
To determine whether Mir-155 deficiency affected the inflammatory response after SCI, we evaluated inflammatory cell infiltration at 42 days after SCI. Recruitment of T cells, macrophages and neutrophils to the injured spinal cord was assessed around the site of injury in Mir-155/ and WT mice. The numbers of T cells, macrophages and neutrophils were significantly lower in Mir-155/ mice than in WT mice (Fig 2A). Next, we detected IL-17A expression in Mir155/ and WT mice at 42 days after SCI. Results showed that the IL-17A-producing cells and the expression of IL17A, IL-6 and IL-23 were markedly lower in Mir-155/ mice than that in WT mice (Fig 2B–D). To determine whether the enhanced T cell activity in WT mice was a result of intrinsic differences in CD4+ T cells, we compared the cytokine profiles of WT and Mir155/ total CD4+ T cells in response to ex vivo stimulation with anti-CD3 and anti-CD28. We found higher production of IL-17 by WT CD4+ T cells than Mir155/ CD4+ T cells (Fig 3A). This indicates that the enhanced cytokine production of IL-17 that we observed in WT mice was in part a result of intrinsic differences in CD4+ T cells. To further determine the role of Mir-155 on Th17 differentiation, WT and Mir-155/ na€ıve CD4+ T cells were differentiated into Th17 cells. IL-17 production in Mir-155/ T cells was significantly reduced compared with WT-T cells (Fig 3B). Polarization of T cells to Th1, Th2 or Th17 phenotypes is a critical feature of cellmediated immunity and is influenced by production of cytokines by DC. Therefore, we tested whether Mir-155 modulates the expression of Th17-polarizing cytokine by DCs. For this, we stimulated bone marrow-derived DCs from WT and Mir-155/ mice with the TLR ligand LPS and analysed cytokine expression. In Mir-155/ DCs, decreased levels of Th17-polarizing cytokines were observed in response to LPS stimulation (Fig 3C). In our further DC-T cell coculture system, Mir-155 deficiency in DCs resulted in significantly less IL-17 production from T cells (Fig 3D). To further investigate the role of IL-17 in SCI, WT or Mir-155/ mice with SCI were treated with anti-IL-17A antibody or recombinant mouse IL-17A (rmIL-17). The
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Figure 2 Mir-155 deficiency suppresses IL17A production in vivo. A, Vales represent the mean cell number in regions surrounding the injury site of WT and mir-155/ mice 42 days after SCI; B, IL-17A-producing cells in injured spinal cord were analysed by FACS; C, IL-17A protein expression was analysed by Western blot; D, IL-17A mRNA expression was analysed by Q-PCR. n = 6 for each group. *P < 0.05.
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Figure 3 Mir-155 deficiency suppresses Th17 differentiation in vitro. A, Total CD4+ T cells were isolated from the spleens of WT and Mir-155/ mice and stimulated with anti-CD3 and anti-CD28. Supernatants from culture were harvested 72 h after initiation of cultures and analysed for IL-17; B, Naive CD4+ T cells were isolated from the spleens of WT and Mir-155/ mice and stimulated with anti-CD3 and anti-CD28 under Th17-skewing condition. Supernatants from culture were harvested 72 h after initiation of cultures and analysed for IL-17 by ELISA and Q-PCR; C, Bone marrow-derived DCs were isolated from WT and Mir-155/ mice and were cultured with 100 ng/ml LPS. Cell-free supernatants were measured for Th17-polarizing cytokine by ELISA. D, CD4+ T cells isolated from WT mice were cultured with CD11c+ DCs derived from WT and Mir-155/ mice. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IL-17. Data are representative of three to five independent experiments. *P < 0.05.
BMS locomotor score and the area of spared myelin in WT + Anti-IL-17 group was markedly greater than that in the WT group at day 42 (Fig 4A–B). The increased BMS locomotor score and spared myelin in Mir-155/ mice were both abrogated with additional rmIL-17 administration (Fig 4A–B). Furthermore, the increased GAP-43- and NF-H-positive areas induced by Mir-155 deficiency were also reversed by rmIL-17 treatment (Fig 4C).
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Mir-155 deficiency suppressed th17 cell differentiation via upregulation of SOCS1
Three transcripts, including SOCS1, SMAD2 and SMAD5, had been reported to be targets of Mir-155. But we found that SOCS1 was the only one that was enhanced by Mir-155 deficiency (the expression of SMAD2 and SMAD5 was not shown) (Fig 5A).
288 Mir-155 Deficiency Suppresses Th17 Cell Differentiation J. Yi et al. .................................................................................................................................................................. To further investigate the role of SOCS1 in the regulated differentiation of Th17, target protector (TP) was used. SOCS1-TPMir155 interfere Mir-155-SOCS1 interaction by binding to the binding site of Mir-155 in the 30 - untranslated region of SOCS1, without interfering Mir-155 interaction with other target mRNAs. So, when SOCS1-TPMir155 was transfected, a dramatic increase of SOCS1 protein was observed (Fig. 5B). Furthermore, the percentages of Th17 cells in CD4+ T cells as well as the
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Figure 4 Mir-155 deficiency promotes locomotor function partly dependent on reduced IL-17 production. A, Analysis of the locomotor Basso Mouse Scale (BMS) score after SCI; B, Quantification of LFBpositive spared myelin areas in the ventrolateral funiculus at the lesion site; C, Quantification of GAP-43-and NF-H-positive areas at day 42 after SCI. n = 6 for each group. *P < 0.05.
expression of IL-17A, IL-6 and IL-23 in cell culture supernatant were decreased when SOCS1-TPMir155 was transfected (Fig. 5C–D).
Discussion This study revealed a crucial role for Mir-155 in regulating Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency was partly dependent on increased expression of SOCS1. Interleukin (IL)-17A is a member of the IL-17 family, which includes six structurally related isoforms: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17A was secreted by different cells, including Th17 cells, cd T cells, NK cells, NKT cells and neutrophils. Recent studies have identified IL-17 (Th17) originated from the subset of T cells, which plays a predominant role in the pathogenesis of autoimmune diseases [17–19]. While most nervous system disease pathogeneses are associated with autoimmune or immune inflammatory injury, the data clearly support the notion that both anti-inflammatory (Th2 or Tregs) and proinflammatory (Th1 or Th17) T cells were activated in the context of a sterile SCI [20]. Importantly, recent clinical trials with short duration IL-17 antagonistic therapy in established rheumatoid arthritis (RA) have provided the direct evidence in pathological role of IL-17 in RA and indicated that blockade of IL-17 in humans may represent a valid therapeutic approach [21]. In our experiment, Mir-155 deficiency alleviates Th17 response and limits Th17 differentiation. Furthermore, the alleviated SCI induced by Mir-155/ was abolished with
B A
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Figure 5 Mir-155 deficiency suppressed th17 cell differentiation via upregulation of SOCS1. A, SOCS1 protein expression in regions surrounding the injury site of WT and Mir155/ mice 42 days after SCI was analysed by Western blot; B, 4 days after SOCS1TPMir155 and control-TPMir155 were transfected, the levels of the transcripts involved in Th17 cells differentiation were detected by Q-PCR; C, 4 days after SOCS1TPMir155 and control-TPMir155 were transfected, the percentages of Th17 were analysed by flow cytometry; D, 4 days after SOCS1-TPMir155 and control-TPMir155 were transfected, the level of IL-17A, IL-6 and IL23 in cell culture supernatant was quantified by ELISA. *P < 0.05 versus all other groups. n = 6.
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additional IL-17 administration. All of these demonstrated that decreased IL-17 expression contributes to the alleviated SCI induced by Mir-155/. Many direct targets of Mir-155 in DCs and T cells have been identified. The essential role of Mir-155 elucidated by targeting SOCS1 expression is not only confined to Treg cells, but also macrophages and DCs. Recently, it has been shown that IL-12 production by DCs is regulated by Mir155-mediated targeting of SOCS1 [22]. Consistent with previous studies, we found reduced expression of Th17polarizing cytokines in DCs from Mir-155/ mice with SCI. Altered expression of Th17-polarizing cytokine expression in DCs that influence the differentiation of Th17 responses could be an additional mechanism by which Mir-15/ mice are resistant to SCI. SOCS1 is a negative regulator of Janus kinase (JAK)/ STAT signalling pathway. Recently, the role of SOCS1 in Th17 cell differentiation and function was clarified by characterizing a mimetic of SOCS1, namely novel tyrosine kinase inhibitor peptide (Tkip). Tkip has been demonstrated to blocked IL-6-induced activation of STAT3, inhibited the development of Th17 and the production of IL-17A [23–25]. Furthermore, a total of 83 overlapping candidate target genes of Mir-155 were predicted through three different analysis programmes, and SOCS1 was at the top of the list [26]. In our current study, we showed that Mir-155 promoted Th17 cell differentiation and enhanced Th17 cell function by directly inhibiting SOCS1. These were in accordance with previous studies indicating the relationship of SOCS1 and Mir-155 [27, 28]. In conclusion, our present work provides evidence to support the concept that Mir-155 deficiency suppresses Th17 differentiation after SCI in mice. Mir-155 deficiency displayed increased myelin sparring and enhanced SC repair process. Mir-155 deficiency suppressed th17 cell differentiation in vivo and in vitro via upregulation of SOCS1 expression. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in the immunoregulation, Mir-155 may be a novel therapeutic to protect SCI.
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