Interleukin-35 alleviates neuropathic pain and induces an anti-inflammatory shift in spinal microglia in nerve-injured male mice

Immune cells are critical in promoting neuroinflammation and neuropathic pain and in facilitating pain resolution, depending on their inflammatory and immunoregulatory cytokine response. Interleukin (IL)-35, secreted by regulatory immune cells, is a member of the IL-12 family with a potent immunosuppressive function. In this study, we investigated the effects of IL-35 on pain behaviors, spinal microglia phenotype following peripheral nerve injury, and in vitro microglial cultures in male and female mice. Intrathecal recombinant IL-35 treatment alleviated mechanical pain hypersensitivity prominently in male mice, with only a modest effect in female mice after sciatic nerve chronic constriction injury (CCI). IL-35 treatment resulted in sex-specific microglial changes following CCI, reducing inflammatory microglial markers and upregulating anti-inflammatory markers in male mice. Spatial transcriptomic analysis revealed that IL-35 suppressed microglial complement activation in the superficial dorsal horn in male mice after CCI. Moreover, in vitro studies showed that IL-35 treatment of cultured inflammatory microglia mitigated their hypertrophied morphology, increased their cell motility, and decreased their phagocytic activity, indicating a phenotypic shift towards homeostatic microglia. Further, IL-35 altered microglial cytokines/chemokines in vitro, suppressing the release of IL-9 and monocyte-chemoattractant protein1 and increasing IL-10 in the supernatant of male microglial cultures. Our findings indicate that treatment with IL-35 modulates spinal microglia and alleviates neuropathic pain in male mice, suggesting IL-35 as a potential sex-specific targeted immunomodulatory treatment for neuropathic pain.