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Comparative Study
. 2010 Apr 21;30(16):5754-66.
doi: 10.1523/JNEUROSCI.5007-09.2010.

SOCS3-mediated Blockade of JAK/STAT3 Signaling Pathway Reveals Its Major Contribution to Spinal Cord Neuroinflammation and Mechanical Allodynia After Peripheral Nerve Injury

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Free PMC article
Comparative Study

SOCS3-mediated Blockade of JAK/STAT3 Signaling Pathway Reveals Its Major Contribution to Spinal Cord Neuroinflammation and Mechanical Allodynia After Peripheral Nerve Injury

Elisa Dominguez et al. J Neurosci. .
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Abstract

Neuropathic pain after peripheral nerve injury, associated with local neuroinflammation in the spinal cord, is a severe incapacitating condition with which clinical treatment remains challenging. Inflammatory molecules signal through various intracellular transduction pathways, activation of which may amplify and cause spreading of the inflammatory response. We showed recently that spinal nerve lesion leads to rapid activation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signal transduction pathway in dorsal spinal cord microglia in relation with enhanced levels of spinal interleukin-6 (IL-6) protein. Here, we selectively inactivated JAK/STAT3 signaling in rat dorsal spinal cord glia through local, lentiviral-mediated production of the suppressor of cytokine signaling SOCS3, a physiologic inhibitory protein of JAK/STAT3, and analyzed its consequences in a preclinical model of neuropathic pain. The targeted blockade of JAK/STAT3 activity prevented the abnormal expression of IL-6, CC chemokine ligand CCL2, and activating transcription factor ATF3 induced in the spinal cord by chronic constriction injury of the sciatic nerve (CCI) and substantially attenuated mechanical hypersensitivity (allodynia) in rats. In naive rats, intrathecal administration of a proalgesic cytokine IL-6 rapidly activated microglial JAK/STAT3 and induced downstream changes closely resembling CCI-evoked alterations. We identified downstream mechanisms through which JAK/STAT3 pathway activation leads to the spreading of neuroinflammation. Our findings reveal that JAK/STAT3 signaling plays a major role in spinal cord plasticity and mechanical allodynia associated with peripheral nerve injury.

Figures

Figure 1.
Figure 1.
Lentiviral-mediated production of tagged SOCS3 (SOCS3t) in rat primary glial cell cultures. Two days after transduction, the presence of SOCS3 mRNA and of the tag sequence V5 were assessed using conventional RT-PCR with 0.5 μg of total RNA extracted from primary glia transduced with LV–SOCS3t (35 ng of viral envelope protein p24) or LV–EGFP (control lentiviral vectors, 35 ng of p24) or untreated (controls). A, Socs3 and V5 mRNA levels were compared with housekeeping GAPDH mRNA levels for each condition. Western blots were performed with SOCS3- or V5-specific antibodies on total proteins extracted from glial cells, 2 d after transduction. α-Tubulin was used as a loading control. B, Addition of the V5 tag sequence allowed endogenous SOCS3 to be distinguished from transgene-derived SOCS3 protein. C, Whereas in control glia immunohistochemistry could not detect endogenous SOCS3-IR, SOCS3-IR (in red) was present in cells transfected with LV–SOCS3t (35 ng of p24, 2 d before experiments), fully overlapping with V5-immunolabeling (in green), thus confirming the transgene origin of SOCS3-IR in these cells. Scale bar, 50 μm.
Figure 2.
Figure 2.
Activation of the JAK/STAT3 transduction pathway. Unilateral CCI of the rat sciatic nerve resulted in the accumulation of the active, phosphorylated form of STAT3 (pSTAT3–Tyr705, in green) 2 d later in numerous cells of the superficial and medial laminae (I–IV) of the dorsal spinal cord, on the side that is ipsilateral to the lesion. A, pSTAT-IR was almost undetectable in the contralateral side of the dorsal spinal cord of CCI rats. B, pSTAT-IR was spatially distributed from approximately the mid-L6 segment to the end of the L4 segment of the spinal cord lumbar enlargement. C, Similarly, CCI injury induced microglial activation mainly in the ipsilateral side of the dorsal spinal cord, as indicated by specific microglial markers ITGAM or Iba1 (in red). Double-labeling experiments for pSTAT3 (in green) with either ITGAM or Iba1 revealed a large colocalization of pSTAT3 with both microglial markers. Colabeling with astrocytes marker GFAP antibodies showed almost no pSTAT-IR (in green) in astrocytes and weak pSTAT3-IR signal in only a very few neurons stained with NeuN antibodies. D, One week after CCI surgery, pSTAT3 labeling (in green) was still detectable but weaker than at 2 d after injury, in the ipsilateral dorsal spinal cord and was mainly present in microglial cells labeled with Iba1-IR (in red). Scale bars, 200 μm.
Figure 3.
Figure 3.
Upregulation of STAT3 target gene SOCS3 and of markers associated with spinal cord inflammatory state. Changes over time of SOCS3, ITGAM, IL-6, CCL2, and ATF3 mRNA levels were determined in the ipsilateral L4–L5 lumbar region of the dorsal spinal cord of CCI rats using semiquantitative real-time RT-PCR. Relative quantification (R.Q.) in arbitrary units (A.U.) corresponds to the ratio of specific mRNA over GAPDH mRNA. In each graph, the dotted line represents the relative quantification of respective mRNA determined in sham animals. Data are representative of different sets of operated rats. Each bar is the mean ± SEM of n = 4–5 rats. Nociceptive threshold of rats to mechanical stimulation was evaluated using von Frey filaments before CCI surgery and then at different postoperative times points. Fifteen grams were chosen as the cutoff threshold to prevent tissue injury. Each bar is the mean ± SEM of n = 8–12 rats. Sham values at each postoperative time point were pooled into one condition referred as 0 (white bars). *p < 0.05, CCI rats versus sham-operated rats at the same respective postoperative time.
Figure 4.
Figure 4.
Intrathecal injection of Il-6 resulted in changes reminiscent of CCI-induced alterations in the rat dorsal spinal cord. Vehicle-injected rats showed almost undetectable pSTAT3-IR. In contrast, we observed bilateral accumulation of pSTAT3-IR (pSTAT3–Tyr705, in green) in the superficial layers of the dorsal spinal cord 15 min and 3 h after intrathecal acute injection of IL-6 (1 μg in 25 μl). At both time points, pSTAT3 labeling colocalized mainly with Iba1 microglial marker, showing some colocalization with the NeuN neuronal marker in only a very few cells and almost no colocalization with GFAP-labeled astrocytes. Scale bars, 200 μm.
Figure 5.
Figure 5.
Effects of transducing primary glial cells or BV2 microglia with LV–SOCS3t on JAK/STAT3 pathway activity and inflammatory state markers. A, Stimulation of primary glia with IL-6 (50 ng/ml) resulted in rapid (15 min) pSTAT3 accumulation (i.e., JAK/STAT3 activation, Western blot). This effect was prevented in cells transduced 48 h earlier with LV–SOCS3t (350 or 35 ng/ml p24). Data are shown as mean ± SEM of three independent experiments. #p < 0.001, IL-6-treated versus untreated cell cultures; *p < 0.001, IL-6-treated LV–SOCS3t-transduced cells versus IL-6-treated uninfected cells. B, C, In both primary glial cells (B) and BV2 microglial cell line (C), IL-6-induced production (after 3 h incubation with IL-6) of inflammatory markers (IL-6, CCL2, TNFα) was efficiently inhibited in cells transduced 48 h before with LV–SOCS3t. IL-6 can also induce ATF3 production in BV2 microglia, this effect being significantly prevented in LV–SOCS3t-transduced cells. Each bar is the mean ± SEM (n = 4 for each group). #p < 0.05, IL-6-treated cells versus control cell cultures; *p < 0.05, IL-6-treated LV–SOCS3t-infected cells versus IL-6-treated uninfected cells. R.Q., Relative quantification; A.U., arbitrary unit.
Figure 6.
Figure 6.
Effects of LV–SOCS3t delivery into the rat dorsal spinal cord on JAK/STAT3, MAPK p38, and ERK pathways activities. A, CCI surgery resulted in pSTAT3 accumulation 2 d later in control or LV–EGFP-infected rats (i.e., JAK/STAT3 activation; Western blots) in the dorsal spinal cord region ipsilateral to the side of the lesion. CCI-evoked pSTAT3 accumulation was completely prevented in rats injected with LV–SOCS3t (2 μl, i.e., 70 ng of p24). Each bar is the mean ± SEM of three independent experiments (n = 3–4 for each group). #p < 0.001, non-injected (n.i.) or LV–EGFP-injected (LV-E) CCI rats versus sham-operated (sh.o.) rats; *p < 0.001, LV–SOCS3t-injected (LV-S) CCI rats versus non-injected or LV–EGFP-injected CCI rats. B, The levels of phosphorylated forms of p38 and ERK MAPK were also increased in the ipsilateral dorsal spinal cord of CCI rats 3 d after surgery (#p < 0.01, #p < 0.001, respectively, vs control). In LV–SOCS3t-injected CCI rats, the level of p38 tend to decrease (although this change was statistically not significant) and pERK levels were unchanged, remaining comparable with those found in control CCI rats (because no difference was observed in pERK1 and pERK2 protein levels under different experimental conditions, only levels of ERK1 protein are represented in quantification graph).
Figure 7.
Figure 7.
Effects of LV–SOCS3t delivery into the rat dorsal spinal cord on pain behavior, local spinal cord inflammation, and glial activity. A, CCI induced mechanical allodynia in control (non-injected) or LV-EGFP-injected rats (2 μl, i.e., 70 ng of p24). This effect was not seen in sham-operated rats and was potently attenuated in LV–SOCS3t-treated rats. Each point is the mean ± SEM of n = 8–10 animals. #p < 0.001, non-injected or LV–EGFP-injected CCI rats versus sham-operated rats; *p < 0.01 LV–SOCS3t-injected CCI rats versus control or LV–EGFP-injected CCI rats. B, LV–SOCS3t-mediated blockade of JAK/STAT3 signaling in the spinal cord effectively prevented the upregulation of IL-6, CCL2, or ATF3 mRNA associated with CCI, 15 and 21 d after surgery. B, However, the high levels of ITGAM mRNA (revealing microglial activation) were unaffected by local STAT3 blockade. C, At these time points, astrocyte activation is also associated with CCI. The upregulated expression of astrocyte activity marker GFAP, observed 21 d after CCI in control rats, was attenuated in LV–SOCS3t-injected CCI animals. IL-1β, whose expression is induced after CCI, particularly in activated astrocytes, was also attenuated in LV–SOCS3t-injected CCI rats. Western blot experiments, showing low levels of GFAP protein in LVSOCS3t-injected CCI rats compared with CCI controls, further confirmed the reduced astrocyte activation in rats with locally inhibited JAK/STAT3 signaling. Data represent the mean ± SEM of n = 4–6 rats for each experimental group. #p < 0.05, non-injected or LV–EGFP-injected CCI rats versus sham operated rats; *p < 0.05, LV–SOCS3t-injected versus LV–EGFP-injected CCI rats. For Western blot experiments, each bar is the mean ± SEM of three independent experiments (n = 3 for each group). R.Q. (A.U.), Relative quantification in arbitrary units; n.i., non-injected CCI rats; LV-E, LV–EGFP-injected CCI rats; LV-S, LV–SOCS3t-injected CCI rats; sh.o., sham-operated rats.
Figure 8.
Figure 8.
Viral vector-derived transgene production in the dorsal horn of the spinal cord after local intraparenchymal microinjection of LV–SOCS3t or LV–EGFP. A, Local production of transgene-derived mRNA was monitored at different time points after LV–SOCS3t injection using conventional or real-time RT-PCR amplification of the tag (V5) sequence (top) or SOCS3 (bottom), respectively. Data from conventional RT-PCR are representative of different sets of injected rats. Each bar is the mean ± SEM (n = 4, except at the 6 month postinjection time point, n = 2). R.Q. (A.U.), Relative quantification in arbitrary units. Control, Non-injected animals. B, The presence of transgene-derived SOCS3 protein was confirmed 1 week after LV–SOCS3t injection (Western blot for V5 antigen). Blots are representative of three independent experiments with distinct set of animals. NS, Nonspecific immunolabeling. C, Single injection of 2 μl (70 ng of p24) of viral suspension resulted in transgene (EGFP) expression strictly restricted to the injected dorsal horn and spreading rostrocaudally through 4–5 mm (i.e., in the region of the spinal cord in which pSTAT3 was highly accumulating; also see Fig. 2.) x, Microinjection point.

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