In vitro investigation of neuroimmune signalling in neuropathic pain: A focus on Tregs, DRG sensory neurons, and microglia

Neuroimmune interactions are central to the development and maintenance of neuropathic pain (pain resulting from lesion or disease to the somatosensory nervous system) through both peripheral and central sensitisation. Emerging evidence suggests that the chronic nature of neuropathic pain may reflect a failure of immune mechanisms that normally resolve pain, positioning immune cells as promising therapeutic targets. Among these, regulatory T cells (Tregs) exhibit immunosuppressive, analgesic, neuroprotective, and tissue-reparative functions. This thesis investigated neuroimmune interactions between Tregs and two key cellular mediators of neuropathic pain, dorsal root ganglia (DRG) sensory neurons and microglia, in vitro. Using live-cell imaging, this work demonstrates for the first time that activated Tregs enhance neurite outgrowth of mouse DRG sensory neurons within 4 hours of co-culture and participate in bidirectional attraction with their neurites. This effect was blocked by the amphiregulin (Areg) receptor antagonist AG1478, identifying Areg as a key mediator. Paclitaxel (PTX), a chemotherapeutic drug known to cause chemotherapy-induced peripheral neurotoxicity and neuropathic pain, significantly inhibited DRG neurite outgrowth across multiple parameters within 200 minutes. Co-culturing PTX-treated neurons with Tregs restored neurite outgrowth to control levels, an effect blocked by AG1478 and the neuropeptide Y (NPY) antagonist BIBO3304, implicating both Areg and NPY in Treg-mediated neuroprotection. The Treg cytokine interleukin-35 (IL-35) also conferred partial neuroprotection. In primary cultures of mouse inflammatory microglia, in vitro IL-35 treatment induced a homeostatic shift, reducing phagocytosis, increasing motility, and altering morphology. These effects were most pronounced in cells from males, contributing to the established sexual dimorphism of microglia molecular physiology. Using microfluidic chambers to mimic in vivo DRG nerve terminal-microglia interactions in the spinal cord, PTX-treated DRG neurons altered ‘activated’ BV2 cell line microglia morphology via axon-glia signalling, an effect partially reversed by IL-35. Collectively, these findings underscore the value of in vitro models of pain-related cellular networks for guiding targeted therapies and identify Tregs and their mediators, Areg, NPY, and IL-35, as novel targets for neuroimmune modulation. This work suggests that Tregs modulate both DRG and microglial responses that converge to drive neuropathic pain, highlighting a potential avenue for Treg-based immunomodulatory therapy.