A Peripheral Pro-Inflammatory Axis Governs Sex-Specific Resolution of Neuropathic Pain

Introduction: Neuropathic pain remains a significant clinical challenge due to its complex pathophysiology and resistance to conventional treatments. Emerging evidence suggests that immune cells and cytokine signaling play crucial roles in modulating pain trajectories, with differences potentially existing between males and females. However, the specific immune-related cellular and molecular mechanisms that drive the transition from persistent pain to resolution remain poorly understood. In this study, we aimed to elucidate the cellular contributors and signaling pathways that regulate pain resolution, focusing on immune system involvement and sex-specific differences.

Methods: Neuropathic pain models were developed in male and female mice, including chemotherapy-induced neuropathy and peripheral nerve crush injury. In chemotherapy-induced neuropathy, paclitaxel dosing was titrated to yield resolving or persistent pain trajectories. At multiple stages (initiation, pre-resolution, post-resolution, and persistence), RNA sequencing was conducted on peripheral tissues (hind paw and dorsal root ganglion), selecting differentially expressed genes (P-adj < 0.05, FC >2). Immune cell populations were quantified through CD45+ cell isolation and flow cytometry, while Cre/LoxP models, diphtheria toxin receptor-mediated ablation, and antibody inhibition dissected cell-specific contributions.

Results: Mechanically-evoked hypersensitivity was more chronic in females. RNA sequencing showed immune-related gene upregulation during resolution in males, absent in persistent pain or at any stage in females. A peripheral population of myeloid cells was essential for resolution in males. Flow cytometry showed no quantitative differences in immune cell populations between sexes, suggesting qualitative immune responses contribute to male-specific resolution. The pro-inflammatory cytokine CCL2 was associated with resolution in males. Global CCR2 deletion or antibody inhibition impaired resolution in the chemotherapy-induced neuropathy model, while keratinocyte-specific CCL2 deletion impaired resolution in both models in males. Peripheral administration of recombinant CCL2, but not anti-inflammatory IL-4 or IL-10, resolved persistent pain.

Conclusion : Our findings reveal sex-specific immune mechanisms in neuropathic pain resolution, with peripheral myeloid cells and CCL2-CCR2 signaling pivotal for resolution in males. Females exhibited prolonged pain independent of these pathways, suggesting that mechanistic differences underlie sex-specific outcomes and may guide development of targeted treatments for chronic neuropathic pain.