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 transcriptomics 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 protein-1 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.

Differential Effects of Regulatory T Cells in the Meninges and Spinal Cord of Male and Female Mice with Neuropathic Pain.

Immune cells play a critical role in promoting neuroinflammation and the development of neuropathic pain. However, some subsets of immune cells are essential for pain resolution. Among them are regulatory T cells (Tregs), a specialised subpopulation of T cells that limit excessive immune responses and preserve immune homeostasis. In this study, we utilised intrathecal adoptive transfer of activated Tregs in male and female mice after peripheral nerve injury to investigate Treg migration and whether Treg-mediated suppression of pain behaviours is associated with changes in peripheral immune cell populations in lymphoid and meningeal tissues and spinal microglial and astrocyte reactivity and phenotypes. Treatment with Tregs suppressed mechanical pain hypersensitivity and improved changes in exploratory behaviours after chronic constriction injury (CCI) of the sciatic nerve in both male and female mice. The injected Treg cells were detected in the choroid plexus and the pia mater and in peripheral lymphoid organs in both male and female recipient mice. Nonetheless, Treg treatment resulted in differential changes in meningeal and lymph node immune cell profiles in male and female mice. Moreover, in male mice, adoptive transfer of Tregs ameliorated the CCI-induced increase in microglia reactivity and inflammatory phenotypic shift, increasing M2-like phenotypic markers and attenuating astrocyte reactivity and neurotoxic astrocytes. Contrastingly, in CCI female mice, Treg injection increased astrocyte reactivity and neuroprotective astrocytes. These findings show that the adoptive transfer of Tregs modulates meningeal and peripheral immunity, as well as spinal glial populations, and alleviates neuropathic pain, potentially through different mechanisms in males and females.