Background & Aims

Neuropathic pain (NP) is the most prevalent and incapacitating form of chronic pain. The current treatments for NP are largely ineffective, underscoring the need for a comprehensive understanding of its cellular and molecular pathogenesis to advance therapeutic development. A significant emphasis has been placed on investigating the involvement of Fractalkine (CX3CL1) signaling between neurons and immune cells in the progression of NP. Several studies suggest that the CX3CL1 signaling may contribute to the pathogenesis of NP by inducing microglia activation and neuroinflammation, while other studies showed that CX3CR1-KO mice had increased allodynia after spared nerve injury and intra-neural injection of CX3CL1 significantly delayed the development of allodynia in wide-type animals. We aimed to address this controversy and determine the neuroprotective vs. pathogenic roles of CX3CL1 signaling in the context of NP using unique transgenic mice and transcriptomic analysis.

Methods

We studied the activation of immune cells by using CX3CR1 KO mice (CX3CR1GFP/GFP) expressing EGFP in microglia, macrophages/monocytes, dendritic cells, and NK cells. We developed a unique transgenic mouse model that over-expressed CX3CL1 (CX3CL1-Tg/CX3CR1GFP/+) and investigated the impact of enhancing CX3CL1 signaling on NP and nerve injury-induced differentially expressed genes (DEGs). Chronic constriction injury (CCI) was performed on wild-type (WT) and CX3CL1 overexpressing mice. Mechanical allodynia and thermal hyperalgesia were evaluated using the von Frey Filament test and the Hargreaves test. The total RNA and total protein of cytokines and chemokines in the sciatic nerve, dorsal root ganglion, and spinal cord were quantified through RT-PCR, Western blot, and ELISA respectively. CCI-induced DEGs were analyzed using RNA sequencing in the injured sciatic nerve at post-CCI day 28 in WT and CX3CL1-Tg mice.

Results

We demonstrated that enhancing CX3CL1 signaling reduced neuroinflammation and significantly alleviated NP likely through two distinct pathways: the CX3CR1-dependent pathway and the TGF-?/Smad2/3-dependent pathway, respectively. CCI of the sciatic nerve led to the activation of microglia and macrophages, cleavage and consumption of full-length CX3CL1, and a decrease in the expression of its downstream TGF-? family proteins. Blocking CX3CL1/CX3CR1 signaling in CX3CR1-KO mice exacerbated CCI-induced neuroinflammation. Conversely, overexpression of CX3CL1 in CX3CL1-Tg mice significantly enhanced TGF-?/Smad2/3 signaling, inhibited microglia activation, reduced pro-inflammatory cytokines, and alleviated CCI-induced hyperalgesia. Moreover, CX3CL1 overexpression effectively reversed CCI-induced DEGs and substantially restored neuro-immune functions that are deranged by CCI, providing a permissive environment for neural repair and regeneration and resolution of NP.

Conclusions

Our findings support that CX3CL1 signaling exerts a multifaceted impact, restraining the production of proinflammatory cytokines by immune cells, fostering neural repair and regeneration, diminishing both central and peripheral sensitizations, and ultimately alleviating NP through diverse pathways. This novel conceptual framework encapsulates pivotal intermediary mechanisms that could influence the delicate balance between neuroprotective and pathogenic outcomes in neuroimmune interactions related to NP. Importantly, our findings imply that the development of agonists and antagonists specifically targeting the C-terminus and N-terminus of CX3CL1 holds promise not only for providing the necessary tools for deepening our understanding of the molecular mechanisms underlying NP but also for unlocking translational applications, potentially leading to clinical treatments for this condition.

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Presenting Author

Jianguo Cheng

Poster Authors

Jianguo Cheng

MD, PhD

Cleveland Clinic

Lead Author

Qingyuan Fan

MD

National Institutes of Health

Lead Author

Jihye Kim

PhD

Cleveland Clinic

Lead Author

Jie Zhang

MD

Cleveland Clinic

Lead Author

Quanri Zhang

PhD

Cleveland Clinic

Lead Author

Riqiang Yan

PhD

University of Connecticut

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology