Elsevier

Neuroscience

Volume 372, 21 February 2018, Pages 225-236
Neuroscience

Research Article
Anti-nociceptive Role of CXCL1 in a Murine Model of Peripheral Nerve Injury-induced Neuropathic Pain

https://doi.org/10.1016/j.neuroscience.2017.12.048Get rights and content

Highlights

  • L5Tx induced significantly reduced chemokine responses in both CD4 KO and CD40 KO mice.

  • L5Tx induced a significant increase in CXCL1 expression at the protein level in CD40 KO mice but not WT or CD4 KO mice.

  • Administration of CXCL1 reduced L5Tx-induced mechanical hypersensitivity in WT mice.

  • CD40 KO mice expressed significantly higher levels of Ly6G than WT mice in the lumbar spinal cord post-L5Tx.

Abstract

Both spinal cord infiltrating CD4+ T lymphocytes and microglial CD40 contribute to the maintenance of neuropathic pain-like behaviors induced by spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we sought to investigate the involvement of multiple chemokines in microglial CD40-mediated and CD4+ T lymphocytes-mediated L5Tx-induced sensory hypersensitivity. Spinal cord chemokine expression in CD4 knockout (KO), CD40 KO, and wild type (WT) BALB/c mice was determined at the protein level via multiplex assays and at the RNA level via quantitative real-time PCR. In WT mice, L5Tx induced significant increases in CCL2, CCL3, and CCL5 expression (protein and RNA) up to day 21 post-L5Tx, while CD4 KO mice displayed blunted, predominantly non-significant, responses in these chemokines at protein levels post-L5Tx. L5Tx also induced increased expression of these chemokines in CD40 KO mice; however, the overall protein levels of these chemokines were significantly lower than those in WT mice. Further, L5Tx induced a significant increase in CXCL1 at the protein level and in CXCR2 at RNA level only in CD40 KO mice. Intrathecal administration of CXCL1 in WT mice significantly reduced L5Tx-induced mechanical hypersensitivity. CD40 KO mice also displayed higher levels of Ly6G (neutrophil marker) RNA expression in the lumbar spinal cord post-L5Tx. Altogether, our data suggest that CD4+ T lymphocytes and microglial CD40 mediate their pro-nociceptive effects in part by promoting selected chemokine responses, and more importantly, CXCL1 can play an anti-nociceptive role in peripheral nerve injury-induced neuropathic pain, which is possibly mediated by infiltrating neutrophils.

Introduction

Chronic pain affects approximately 100 million adults in the United States, with an estimated annual cost up to $635 billion (Gaskin and Richard, 2012). Neuropathic pain, defined as pain caused by a lesion or disease of the somatosensory system, is one of the most devastating kinds of chronic pain (Jensen et al., 2011). Unfortunately, treatment is still suboptimal. Delineating the mechanisms leading to neuropathic pain is essential to facilitate the development of more effective treatments. There is ample evidence that the development of neuropathic pain is closely related to immune responses. The role of one group of immune molecules, chemokines, has attracted more attention in recent years. Chemokines are a family of small secreted chemoattractant proteins that stimulate the migration and activation of various types of leukocytes (such as T cells, B cells, and monocytes) and play a central role in inflammatory responses. Chemokines have been implicated in various inflammatory diseases in both the peripheral and the central nervous systems (Charo and Ransohoff, 2006, Callewaere et al., 2007), as well as in the development of neuropathic pain (White et al., 2007, Gao and Ji, 2010). For example, reduced sensory hypersensitivity has been observed in CCL2 (monocyte attractant protein-1 (MCP-1)) receptor (CCR2) knockout (KO) mice following sciatic nerve ligation (Abbadie et al., 2003) and in mice treated with CCL2 neutralizing antibodies following spinal nerve ligation (Gao et al., 2009). Similarly, mice either lacking CCL5 (regulated on activation, normal T cell expressed and secreted (RANTES)) or intraperitoneally administered with a selective CCL5 receptor antagonist, Met-RANTES, showed reduced sensory hypersensitivity following partial sciatic nerve ligation (Liou et al., 2012, Liou et al., 2013). Furthermore, our recent study indicated that CCL5 is a key chemokine mediator in CGRP-induced mechanical hypersensitivity following spinal nerve L5 transection (L5Tx) (Malon and Cao, 2016). In addition, CX3CL1 (Fractalkine)/CX3CR1 signaling has also been examined extensively as a critical pathway that facilitates the communication between sensory neurons and resident microglia in the development of neuropathic pain (Zhang et al., 2013, Clark and Malcangio, 2014). Together, these studies highlight the importance of chemokines in the development of neuropathic pain.

Previously, in studying the involvement of adaptive immunity in the development of neuropathic pain, we showed that there is a transient infiltration of CD4+ T cells, predominantly type 1 helper T cells, into the lumbar spinal cord following L5Tx, and CD4+ cell adoptive transfer experiments indicated the contribution of CD4+ T lymphocytes in the maintenance phase of L5Tx-induced mechanical hypersensitivity (Cao and DeLeo, 2008, Draleau et al., 2014). To investigate the potential role of the infiltrating CD4+ T cells within the spinal cord, we examined the role of microglial CD40 (interaction between microglial CD40 and CD40 ligand expressed by activated T cells provides a pathway for the interaction between infiltrating CD4+ T cells and resident microglia) and demonstrated that microglial CD40 also plays a critical role in the maintenance of L5Tx-induced mechanical hypersensitivity (Cao and DeLeo, 2008, Draleau et al., 2014). In addition, our results showed a greater reduction of L5Tx-induced mechanical hypersensitivity in CD40 KO mice and in the CD40 KO-WT chimeric mice (mice that do not have CD40 expression in the central nervous system (CNS)) than in CD4 KO mice, suggesting that microglial CD40 plays a more substantial role in neuropathic pain compared to infiltrating CD4+ T cells. We suspect this is partially due to CD40’s greater influence on microglial activation (Cao et al., 2012a), and studying CD40-mediated responses may reveal novel treatment strategies.

In the current study, we sought to further investigate the involvement of chemokines in both the roles of CD4+ T cell-mediated and microglial CD40-mediated mechanisms in the development of neuropathic pain. A multiplex assay and qRT-PCR were used to assess the expression of various chemokines in the lumbar spinal cord following L5Tx in both CD4 KO and CD40 KO mice. Our results pointed to a potential anti-nociceptive effect of CXCL1, which may be due to an increased recruitment of neutrophils in the spinal cord following injury.

Section snippets

Animals

Male and female adult wild type (WT) BALB/c mice (7–8 weeks old) were purchased from the Frederick Animal Facility (Frederick, MD, USA) of the Charles River Laboratories (Wilmington, MA, USA). All mice were allowed to habituate to the institutional animal facility for at least one week before experimental use. As previously described in detail (Cao and DeLeo, 2008, Cao et al., 2009), breeding pairs for BALB/c-CD4 KO mice and BALB/c-CD40 KO mice were originally obtained from Dr. William T. Lee

Lumbar spinal cord chemokine expression post-L5Tx in both CD4 KO and CD40 KO mice

Adult male and female CD4 KO, CD40 KO, and WT BALB/c mice were randomly assigned to either L5Tx or sham groups, as well as individual time groups. At selected times, days 0 (naïve animals), 3, 7, 14, and 21 post-surgery, the lumbar spinal cord from each mouse was harvested and processed for a chemokine multiplex assay performed by technicians at the Quansys Biosciences. Out of the 8 chemokines measured, four chemokines, CCL2, CCL3, CCL5, and CXCL1 displayed significant increases in one or more

Discussion

Following up on our previous findings regarding the contribution of CD4+ T cells and microglial CD40 in the maintenance of L5Tx-induced neuropathic pain, the current study investigated the roles of chemokines in CD4+ T cell-mediated and microglial CD40-mediated L5Tx-induced neuropathic pain-like behaviors by evaluating the RNA and protein levels of chemokines in the lumbar spinal cord following L5Tx in WT, CD4 KO, and CD40 KO mice. Due to the observed differential responses in CXCL1 expression

Conclusions

Altogether, our results support previously published data on chemokines’ pro-nociceptive role in neuropathic pain. However, more importantly our results suggested an anti-nociceptive role of CXCL1 in periphery nerve injury-induced neuropathic pain, which is potentially mediated by the infiltrating neutrophils. Further investigation into the underlying mechanisms of the protective function of CXCL1 may reveal novel drug targets for neuropathic pain treatment.

Acknowledgments

This work was supported by the National Institutes of Health [NIH/NIDA K01DA023503 (PI Cao), 2008-2012; NIH/NIGMS COBRE award, P20GM103643 (PD, Meng; sub-project PI, Cao), 2012-2016; NIH/NINDS R01NS098426 (PI Cao), 2016-2021). The funding agency had no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the article for publication. LC designed the study, performed animal surgeries, behavioral analysis and

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