GT 1b functions as a novel endogenous agonist of toll‐like receptor 2 inducing neuropathic pain
2020; Springer Nature; Volume: 39; Issue: 6 Linguagem: Inglês
10.15252/embj.2019102214
ISSN1460-2075
AutoresHyoungsub Lim, Jaesung Lee, Byunghyun You, Jae Hoon Oh, Hyuck Jun Mok, Yoo Sung Kim, Bo‐Eun Yoon, Byung Gon Kim, Seung Keun Back, Jong‐Sang Park, Kwang Pyo Kim, Ronald L. Schnaar, Sung Joong Lee,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle6 February 2020free access Transparent process GT1b functions as a novel endogenous agonist of toll-like receptor 2 inducing neuropathic pain Hyoungsub Lim Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Jaesung Lee Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Byunghyun You Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Jae Hoon Oh Department of Chemistry, Seoul National University, Seoul, Korea Search for more papers by this author Hyuck Jun Mok Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin, Korea Search for more papers by this author Yoo Sung Kim Department of Molecular Biology, Dankook University, Cheonan, Korea Search for more papers by this author Bo-Eun Yoon Department of Molecular Biology, Dankook University, Cheonan, Korea Search for more papers by this author Byung Gon Kim Department of Brain Science and Neurology, Ajou University School of Medicine, Suwon, Korea Search for more papers by this author Seung Keun Back Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon, Korea Search for more papers by this author Jong-Sang Park Department of Chemistry, Seoul National University, Seoul, Korea Search for more papers by this author Kwang Pyo Kim Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin, Korea Search for more papers by this author Ronald L Schnaar Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Sung Joong Lee Corresponding Author [email protected] orcid.org/0000-0002-4102-002X Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Hyoungsub Lim Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Jaesung Lee Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Byunghyun You Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Jae Hoon Oh Department of Chemistry, Seoul National University, Seoul, Korea Search for more papers by this author Hyuck Jun Mok Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin, Korea Search for more papers by this author Yoo Sung Kim Department of Molecular Biology, Dankook University, Cheonan, Korea Search for more papers by this author Bo-Eun Yoon Department of Molecular Biology, Dankook University, Cheonan, Korea Search for more papers by this author Byung Gon Kim Department of Brain Science and Neurology, Ajou University School of Medicine, Suwon, Korea Search for more papers by this author Seung Keun Back Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon, Korea Search for more papers by this author Jong-Sang Park Department of Chemistry, Seoul National University, Seoul, Korea Search for more papers by this author Kwang Pyo Kim Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin, Korea Search for more papers by this author Ronald L Schnaar Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Sung Joong Lee Corresponding Author [email protected] orcid.org/0000-0002-4102-002X Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea Search for more papers by this author Author Information Hyoungsub Lim1, Jaesung Lee1, Byunghyun You1, Jae Hoon Oh2, Hyuck Jun Mok3, Yoo Sung Kim4, Bo-Eun Yoon4, Byung Gon Kim5, Seung Keun Back6, Jong-Sang Park2, Kwang Pyo Kim3, Ronald L Schnaar7 and Sung Joong Lee *,1 1Department of Neuroscience and Physiology, Dental Research Institute, BK21-Plus, School of Dentistry, Seoul National University, Seoul, Korea 2Department of Chemistry, Seoul National University, Seoul, Korea 3Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin, Korea 4Department of Molecular Biology, Dankook University, Cheonan, Korea 5Department of Brain Science and Neurology, Ajou University School of Medicine, Suwon, Korea 6Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon, Korea 7Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA *Corresponding author. Tel: +82 2 880 2309; Fax: +82 2 740 5107; E-mail: [email protected] EMBO J (2020)39:e102214https://doi.org/10.15252/embj.2019102214 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Spinal cord microglia contribute to nerve injury-induced neuropathic pain. We have previously demonstrated that toll-like receptor 2 (TLR2) signaling is critical for nerve injury-induced activation of spinal cord microglia, but the responsible endogenous TLR2 agonist has not been identified. Here, we show that nerve injury-induced upregulation of sialyltransferase St3gal2 in sensory neurons leads to an increase in expression of the sialylated glycosphingolipid, GT1b. GT1b ganglioside is axonally transported to the spinal cord dorsal horn and contributes to characteristics of neuropathic pain such as mechanical and thermal hypersensitivity. Spinal cord GT1b functions as an TLR2 agonist and induces proinflammatory microglia activation and central sensitization. Pharmacological inhibition of GT1b synthesis attenuates nerve injury-induced spinal cord microglia activation and pain hypersensitivity. Thus, the St3gal2-GT1b-TLR2 axis may offer a novel therapeutic target for the treatment of neuropathic pain. Synopsis Peripheral nerve injury upregulates GT1b in the injured sensory neurons, which is released in the spinal cord and functions as an endogenous agonist of microglial TLR2. Nerve injury upregulates St3gal2 and subsequent GT1b synthesis in injured DRG neurons. GT1b functions as an endogenous agonist of TLR2 to activate microglia. Antagonizing St3gal2-GT1b-TLR2 signaling attenuates neuropathic pain. Introduction Neuropathic pain is a chronic pathological pain due to an injury or dysfunction in the peripheral or central nervous system. The clinical symptoms of neuropathic pain include spontaneous burning pain, enhanced pain in response to noxious stimuli (hyperalgesia), and pain in response to normally innocuous stimuli (allodynia) (Jensen & Finnerup, 2014). Studies over the last two decades have implicated spinal cord microglia activation as etiological factors that contribute to pain hypersensitivity at the spinal cord level (central sensitization) and consequent neuropathic pain (Inoue & Tsuda, 2009). In this regard, elucidating the molecular mechanisms by which peripheral nerve injury induces spinal cord microglia activation is critical to understanding the pathogenic mechanisms of neuropathic pain. Previously, we and others reported that toll-like receptor 2 (TLR2) signaling is a significant contributor to spinal cord microglia activation due to peripheral nerve injury (Kim et al, 2007; Stokes et al, 2013), which suggested a presence of certain TLR2 endogenous agonist in the spinal cord. However, there is still no information as to the TLR2 endogenous agonist that drives spinal cord microglia activation after nerve injury. The neuronal membrane consists of highly heterogeneous lipid components that are distinct from those in non-neuronal cells. Specifically, neurons are enriched in glycosphingolipids or gangliosides, accounting for 10-12% of the total membrane lipid content. Gangliosides localized in the outer leaflet of the membrane are constituents of lipid rafts and are implicated in neuronal communication with the extracellular microenvironment or contribute to neuron–glia interactions (Schnaar, 2016). Four major gangliosides (GM1, GD1a, GD1b, and GT1b) constitute up to 97% of gangliosides in the human brain (Tettamanti et al, 1973; Ando, 1983). Of note, the ganglioside profile is highly plastic, changing in brain development and pathophysiology (Olsen & Faergeman, 2017). For example, GD1a and GM1 levels increase at early brain developmental stages and contribute to neural differentiation and axonal outgrowth (Olsen & Faergeman, 2017). An increase of GT1b at a later stage contributes to axon–myelin stability of differentiated neurons (Olsen & Faergeman, 2017). Ganglioside composition also changes in several neuropathological conditions. For example, Alzheimer's disease is associated with increased brain levels of GM1 and GM2, whereas there is a decrease of GM1 content in Parkinson's disease (Molander-Melin et al, 2005; Wu et al, 2012). Intriguingly, in patients with peripheral neuropathy or polyneuropathy, a significant increase of "b-series" gangliosides (GD1b, GT1b, and GQ1b) was detected in the cerebrospinal fluid (CSF) (Trbojevic-Cepe et al, 1991). In animal models, GM1 administration into spinal cord or GM3 synthase depletion exerted strong analgesic effects on the nerve injury-induced neuropathic pain and diabetic neuropathy, respectively (Mao et al, 1992; Menichella et al, 2016), whereas intraplantar injection of GT1b produced nociceptive behavior (Watanabe et al, 2011). Taken together with the reports of ganglioside profile change in different neuropathological conditions and the relationship between CSF/spinal cord ganglioside levels and peripheral neuropathy, these findings suggest that ganglioside profile changes may also be implicated in the central sensitization process that occurs after nerve damage and that underlies neuropathic pain development. To test this hypothesis, here we investigated the ganglioside profile in the spinal cord in a nerve injury-induced mouse model of neuropathic pain. We found that GT1b is aberrantly upregulated in injured sensory neurons, transported to the spinal cord, and contributes to neuropathic pain. As to underlying mechanism, we discovered that GT1b in the spinal cord functions as an endogenous agonist of TLR2, which activates microglia and triggers the induction of proinflammatory genes that contribute to central sensitization and the neuropathic pain phenotype. Results Peripheral nerve injury upregulates GT1b in the spinal cord To investigate the ganglioside profile in an animal model of peripheral nerve injury-induced neuropathic pain, we isolated spinal cord tissues of L5 spinal nerve-transected or sham-operated mice and searched for differentially regulated gangliosides using LC-MRM/MS analysis. Among the four major gangliosides (GM1, GD1a, GD1b, and GT1b), we found that GT1b levels significantly increased in the spinal cord of nerve-injured mice compared to sham-operated mice (Fig 1A). Because GT1b is increased in the CSF of patients with peripheral neuropathy (Trbojevic-Cepe et al, 1991), we next examined the level of GT1b in the interstitial fluid (ISF) of the spinal cord. In these studies, we implanted a microdialysis probe in the spinal cord dorsal horn of the nerve-injured rat and collected ISF. Five days after the nerve injury, the concentration of GT1b in the ISF increased 8.4-fold compared to the sham control, as assessed using ELISA (Fig 1B). Likewise, the GT1b level in the CSF increased to 132 ± 20 ng/ml in the nerve-injured mice compared to 70 ± 9 ng/ml in the sham-operated mice (Fig 1C). These data demonstrate that peripheral nerve injury increases GT1b levels in spinal cord tissue, where it is released into the ISF and CSF. Figure 1. GT1b is upregulated and released in the spinal cord after nerve injury A. A LC-MRM/MS analysis of ganglioside GT1b species in pooled spinal cord tissues of Sham-operated or nerve-injured mice at 3 dpi. Representative traces of GT1b species (d18:1–18:0) are shown, and the intensity of the GT1b species is presented as the fold induction compared to the corresponding control. GM3-d3 served as internal standard for normalization. d18:1–18:0 means the ceramide structure (carbon number: double bond number), and d3 means deuterated form. B, C. GT1b level in ISF (B) and CSF (C) was analyzed by ELISA. Data are represented as mean ± SEM (Student's t-test, *P < 0.05, ***P < 0.001). Download figure Download PowerPoint GT1b induces pain hypersensitivity and spinal cord microglia activation To examine the effects of GT1b increase in the spinal cord on the nociceptive processing, we assayed several nociceptive behaviors in mice upon intrathecal GT1b injection. Figure 2A and B shows that we observed significant decreases in thresholds to mechanical and thermal stimuli in GT1b-injected mice, but not in vehicle-injected mice. GT1b decreased the pain thresholds in a dose-dependent manner. We next examined intrathecal GT1b effects on spinal cord microglia activation. Within 1 day of the intrathecal GT1b injection, we recorded a robust increase in Iba-1-immunoreactivity (IR) in the spinal cord, with even greater Iba-1-IR at day 3 (Fig 2C). Intrathecal GT1b administration also upregulated gene expression of Il1b, Tnf, and Bdnf, by 25-, 6-, and 1.6-fold, respectively (Fig 2D). Note that each of these genes is expressed in activated spinal cord microglia after nerve injury and has been implicated in the central sensitization process that contributes to nerve injury-induced neuropathic pain (Kawasaki et al, 2008b; Lu et al, 2009). These data demonstrate that GT1b induces spinal cord microglia activation and subsequent pain hypersensitivity in vivo. To test whether GT1b directly activates microglia, we also examined GT1b effect on microglia in vitro. In primary cultured microglia, GT1b induced production of IL-1β and BDNF, nitric oxide, and intracellular reactive oxygen species, which were confirmed by ELISA, Griess assay, and CM-H2DCFDA dye, respectively (Fig 2E–G). GT1b stimulation also induced p38 MAP kinase activation (Fig 2H), a key signaling event that has been implicated in proinflammatory gene expression in microglia (Ji & Suter, 2007). Taken together, these findings suggest that the nerve injury-induced GT1b upregulation in spinal cord directly activates spinal cord microglia and thereby induces pain hypersensitivity in mice. Figure 2. Intrathecal GT1b injection induced pain hypersensitivity and spinal cord microglia activation A, B. Mechanical allodynia (A) and thermal hyperalgesia (B) were assessed after intrathecal injection of GT1b (1 μg/5 μl, n = 6; 5 μg/5 μl, n = 7; 25 μg/5 μl, n = 6) or vehicle (n = 5; one-way ANOVA, *P < 0.05; ***P < 0.001, #P < 0.05; ##P < 0.01; ###P < 0.001 vs. vehicle). Data are expressed as mean ± SEM. C. GT1b (25 μg/5 μl, n = 3/time point) or the vehicle (saline, n = 3) was intrathecally administrated, and lumbar spinal cord sections were immunostained with Iba-1 antibody. Vehicle-injected mice served as controls, and representative images are shown (scale bar, 100 μm). The fluorescence intensity of Iba-1-IR in spinal cord dorsal horn and CM-H2DCFDA was measured and presented as the fold induction compared to the corresponding control. Data are represented as mean ± SEM (Student's t-test, **P < 0.01). D. L4-5 spinal cord tissues were removed after intrathecal injection of GT1b (25 μg/5 μl) or vehicle injection, and total RNA was isolated from the tissues. Transcript levels were measured by real-time RT–PCR and presented as the fold induction compared to the corresponding level measured in control. Data are represented as mean ± SEM (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001, vs. control). E, F. IL-1β, BDNF (E), and nitrite production (F) were measured in the conditioned media from primary microglia stimulated with GT1b. Data are expressed as mean ± SEM (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001, vs. control, #P < 0.05; ###P < 0.001). G. Intracellular reactive oxygen species production in primary microglia was measured using CM-H2DCFDA after GT1b treatment. Data are expressed as mean ± SEM (Student's t-test, **P < 0.01; scale bar, 100 μm). H. Glial cells were stimulated with 10 μg/ml of GT1b at the indicated time points, and p38 MAPK activation was determined using Western blot analysis. Data are expressed as mean ± SEM (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001, vs. control). Download figure Download PowerPoint GT1b is transported from the injured sensory neurons and released to the spinal cord dorsal horn after nerve injury To determine whether the increased spinal cord levels of GT1b is derived from damaged sensory neurons, we examined the expression levels of GT1b in the dorsal root ganglion (DRG). We detected basal levels GT1b-IR in sensory neurons of sham-operated control mice, which was further upregulated in ipsilateral L5 DRG neurons after nerve injury (Fig 3A). The specificity of GT1b antibody was tested in previous studies using purified gangliosides and knockout mice (Gong et al, 2002; Schnaar et al, 2002). To examine whether sensory neuron-derived GT1b is transported to the spinal cord, we ligated and transected the L5 dorsal root after L5 spinal nerve ligation in rats (Fig 3B). Similar to mice, GT1b was upregulated in the injured sensory neurons of the rat (Fig 3C). Figure 3D shows that dorsal rhizotomy followed by L5 spinal nerve ligation resulted in the accumulation of GT1b at the dorsal root transection site, indicating that GT1b is intra-axonally transported from the DRG to the spinal cord (Fig 3D). Notably, the rhizotomy abrogated the nerve injury-induced spinal cord microglia activation (Fig 3E). Taken together, these data indicate that nerve injury induces GT1b upregulation in injured sensory neurons and that GT1b axonally transported to the dorsal horn contributes to microglial activation. Figure 3. GT1b is upregulated in DRG neurons and transported to spinal cord after nerve injury A. L5 DRG tissue sections of sham-operated or nerve-injured mice (3 dpi) were immnostained with GT1b antibody. Representative images are shown (n = 3/group; scale bar, 100 μm). B. Schematic drawing of neuroanatomy and surgery. C, D. Rat L5 DRG (C) and L5 dorsal root sections (D) with concurrent rhizotomy and L5 SNL (3 dpi) were immunostained with GT1b antibody. The dotted line denotes the transection site. Representative images are shown (n = 3; scale bar, 100 μm). E. L4-5 spinal cord sections from rats with rhizotomy and SNL-injured rats with or without rhizotomy (3 dpi) were immunostained with Iba-1 antibody. Representative images are shown (n = 3/group; scale bar, 500 μm). F. The GT1b level was measured using ELISA in conditioned media of F11 differentiated cells after 1-h exposure to high concentration of potassium. Data information: The fluorescence intensity of GT1b-IR (A, C) and Iba-1-IR (E) was measured and presented as the fold induction compared to the corresponding control in each section. Data are represented as mean ± SEM (Student's t-test, **P < 0.01; ***P < 0.001, ###P < 0.001). Download figure Download PowerPoint Although we attempted to localize GT1b in the spinal cord by immunostaining, our results were inconsistent, making it difficult to document the accumulation of GT1b in the spinal cord, possibly due to its release after axonal transport. For this reason, we turned to a more functional assessment of sensory neuron-derived GT1b. Specifically, using the F11 sensory neuronal cell line in vitro, we asked whether GT1b is released in a neuronal activity-dependent manner. F11 cells were differentiated with dibutyryl cAMP and then depolarized for 1 h with 50 mM potassium. Figure 3F demonstrates the depolarization-induced accumulation of GT1b in the conditioned medium, indicating that GT1b can be released in a neuronal activity-dependent manner. Taken together, we conclude that the nerve injury-induced increase in spinal cord GT1b results from its increase in injured DRG neurons, followed by intra-axonal transport of GT1b to and its release in the ipsilateral dorsal horn. Nerve injury-induced St3gal2 upregulation in sensory neurons is upstream of GT1b We next investigated the molecular mechanisms by which nerve injury increases GT1b in sensory neurons. GT1b is synthesized from a precursor molecule, GD1b, by enzymatic addition of single sialic acid functional group. As two enzymes, ST3Gal-II and ST3Gal-III, have been implicated (Sturgill et al, 2012), we examined St3gal2 and St3gal3 expression in DRG neurons after nerve injury. Figure 4A shows that peripheral nerve injury increased St3gal2 mRNA expression by 80% in the L5 DRG. St3gal3 mRNA did not change in the DRG, and neither St3gal2 nor St3gal3 mRNA increased in the spinal cord (Fig 4A and B). We also recorded an increase of ST3Gal-II protein in the L5 DRG of mice, 1 day post-injury (dpi) (Fig 4C). Specificity of the ST3Gal-II antibody was confirmed by the absence of immunostaining in the L5 DRG of St3gal2−/− mice (Fig 4C). To examine the relationship between ST3Gal-II upregulation and the GT1b increase in L5 DRG after nerve injury, we next measured GT1b levels in St3gal2−/− mice. Although significantly reduced, we nevertheless record basal levels of GT1b in the sensory neurons of the St3gal2−/− mice. We assume that this reflects synthesis by ST3Gal-III, an alternative GT1b-synthesizing enzyme (Sturgill et al, 2012). Most importantly, in the St3gal2−/− mice, GT1b was not further upregulated by the nerve injury, over the basal level (Fig 4D). As expected, the nerve injury-induced spinal cord microglia activation, documented by Iba-1-IR, was reduced in St3gal2−/− compared to wild-type (WT) mice after the injury (Fig 4E). Furthermore, the nerve injury-induced mechanical allodynia and thermal hyperalgesia were also reduced in the St3gal2−/− compared to WT mice (Fig 4F and G). We conclude that St3gal2 is a critical contributor to the GT1b increase in sensory neurons, spinal cord microglia activation, and pain hypersensitivity after nerve injury. To conclusively demonstrate the contribution of sensory neuron-specific ST3Gal-II to GT1b increase in the spinal cord, we generated sensory neuron-specific St3gal2 conditional knockout mice (Scn10a-Cre/St3gal2f/f). In these mice, GT1b increase in the spinal cord due to nerve injury was inhibited (Fig 4H). Accordingly, nerve injury-induced spinal cord microglia activation and pain hypersensitivity were significantly reduced (Fig 4I and J). Taken together, these data suggest that nerve injury induces an increase in GT1b in axotomized sensory neurons secondary to upregulation of ST3Gal-II. Thus, ST3Gal-II lies upstream of GT1b in the activation of spinal cord microglia and the associated neuropathic pain phenotype. Figure 4. St3gal2 upregulation in sensory neurons is responsible for nerve injury-induced GT1b upregulation and subsequent spinal cord microglia activation and neuropathic pain A, B. St3gal2 and St3gal3 mRNA expression in the pooled L5 DRG (A) and spinal cord tissues (B) of sham-operated and L5 SNT-injured mice were measured using real time RT–PCR. Data are represented as mean ± SEM (n = 3/group, Student's t-test, **P < 0.01). C. L5 DRG sections were prepared from WT mice (n = 4/group) with or without L5 SNT (1 dpi) and immunostained with ST3Gal-II antibody. Representative images are shown (scale bar, 50 μm). D, E. L5 DRG (D) and L4-5 spinal cord (E) sections from WT (n = 3/group) and St3gal2−/− mice (n = 3/group) with or without L5 SNT were immunostained with GT1b or Iba-1 antibody, respectively. Representative images are shown (scale bar, 100 μm). F, G. Mechanical allodynia (F) and thermal hyperalgesia (G) were evaluated in sham-operated or L5 SNT-injured WT (sham, n = 5; SNT, n = 5) and St3gal2−/− (sham, n = 5; SNT, n = 5) mice. Data are represented as mean ± SEM (one-way ANOVA, **P < 0.01; ***P < 0.001, vs. sham control in WT, #P < 0.05; ##P < 0.01, vs. nerve-injured WT in each time point). H. GT1b level in spinal cord tissues was analyzed by ELISA. Three mg of pooled ipsilateral L4-5 spinal cord tissues of sham-operated or L5 spinal nerve-injured mice were used. Data are presented as mean ± SEM (Student's t-test, **P < 0.01, ##P < 0.01). I. L4-5 spinal cord sections of floxed-St3gal2 (St3gal2f/f, n = 3/group) and sensory neuron-specific St3gal2−/− mice (Scn10a-Cre/St3gal2f/f, n = 3/group) with L5 SNT at 3 dpi were immunostained with Iba-1 antibody, and representative images are shown (scale bar, 100 μm). J. Mechanical allodynia was performed in sham-operated or L5 SNT-injured St3gal2f/f (SNT, n = 6) and Scn10a-Cre/St3gal2−/− (sham, n = 5; SNT, n = 7) mice (one-way ANOVA, ***P < 0.001, vs. sham control, #P < 0.05; vs. nerve-injured St3gal2f/f at each time point). Data information: The fluorescence intensity of ST3Gal-II-IR (C) and Iba-1-IR (E, I) was measured and presented as the fold induction compared to the corresponding control in each section. Data are represented as mean ± SEM (Student's t-test, **P < 0.01; ***P < 0.001, ##P < 0.01). Download figure Download PowerPoint GT1b is an endogenous agonist of microglial TLR2 We previously reported that TLR2 is required for nerve injury-induced spinal cord microglia activation (Kim et al, 2007). Here, we asked whether TLR2 is involved in the GT1b-mediated spinal cord microglia activation. We first examined GT1b effects in vitro. We prepared glial cells from WT and Tlr2−/− mice and found that GT1b-induced expression of several genes implicated in persistent pain was almost completely abrogated in glial cells from Tlr2−/− mice (Fig 5A). Next, we examined the contribution of Tlr2 in vivo. Figure 5B shows that spinal cord microglia activation after intrathecal GT1b, as assessed by Iba-1 immunostaining, was almost entirely abolished in the Tlr2−/− mice compared to WT mice. The GT1b-induced expression of Il1b, Tnf, and Bdnf in the spinal cord was also prevented in the Tlr2−/− mice (Fig 5C). We then tested the susceptibility of the Tlr2−/− mice to the intrathecal GT1b-induced neuropathic pain phenotype. Figure 5D and E shows that intrathecal GT1b provoked hypersensitivity to both mechanical and thermal stimuli in control mice, but this was almost completely prevented in the Tlr2−/− mice. We conclude that the pronociceptive effects of GT1b are mediated by TLR2, which is expressed mainly on spinal cord microglia in vivo (Lim et al, 2013). Figure 5. GT1b functions as an endogenous agonist of TLR2 to activate microglia A. Glial cells cultured from WT and Tlr2−/− mice were stimulated with 10 μg/ml of GT1b for 3 h, and transcript levels were measured using real-time RT–PCR. Data are represented as mean ± SEM (Student's t-test, **P < 0.01; ***P < 0.001, vs. control in WT, #P < 0.05; ###P < 0.001, vs. Tlr2−/− mice). B. Lumbar spinal cord sections prepared from WT (n = 3/group) and Tlr2−/− mice (n = 3/group) 1 day after intrathecal injection of GT1b (25 μg/5 μl in saline) were immunostained with Iba-1 antibody, and representative images are shown (scale bar, 100 μm). Vehicle-injected mice served as controls. The fluorescence intensity of Iba-1-IR was measured and presented as the fold induction compared to the corresponding control. Data are represented as mean ± SEM (Student's t-test, **P < 0.01, vs. control in WT, ##P < 0.01, vs. Tlr2−/− mice). C. Transcript levels were measured using real-time RT–PCR in L4-5 spinal cord tissues of WT and Tlr2−/− mice after intrathecal injection of GT1b (25 μg/5 μl, n = 3/group) or vehicle. Data are represented as mean ± SEM (n = 3/group, Student's t-test, *P < 0.05; **P < 0.01, vs. control in WT, #P < 0.05; ##P < 0.01, vs. in Tlr2−/− mice). D, E. Mechanical allodynia (D) and thermal hyperalgesia (E) were assessed in WT (vehicle, n = 5; GT1b, n = 5) and Tlr2−/− mice (vehicle, n = 6; GT1b, n = 7) after intrathecal injection of GT1b (25 μg/5 μl) or vehicle. Data are represented as mean ± SEM (one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001, vs. vehicle in WT, #P < 0.05; ##P < 0.01; ###P < 0.001, vs. WT GT1b group in each time point). F. Primary microglia cultured from Cx3cr1+/GFP and Tlr2−/−/Cx3cr1+/GFP mice were incubated with 5 μg/ml of GT1b-rhodamine in the presence or absence of LTA (n = 8/group), and representative images are shown (scale bar, 20 μm). The fluorescence intensity of GT1b-rhodamine was measured and presented as mean ± SEM (Student's t-test, *P < 0.05; ***P < 0.001). Download figure Download PowerPoint The TLR2 receptor mediates signaling by a variety tissue damage-associated molecules (Yu et al, 2006; Kim et al, 2013). Taken together with our demonstration that TLR2 contributes to nerve injury-induced microglia activation, we hypothesized that nerve injury must trigger exposure of a damage-associated TLR2 endogenous agonist. Based on the requirement of TLR2 in GT1b-induced microglia activation,
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