Engineered IL1R1-CD3 Tregs modulate neuroimmune responses and attenuate neuropathic pain in a rat CCI model.

Neuropathic pain is a chronic and disabling disorder driven by persistent neuroimmune activation in addition to aberrant neuronal signaling. Regulatory T cells (Tregs) play a central role in restraining inflammation; however, their therapeutic potential in neuropathic pain is limited by poor stability and survival within cytokine-rich inflammatory environments, particularly those dominated by interleukin-1 (IL-1) signaling. This study investigated whether redirecting IL-1 signaling through an engineered IL1R1-CD3 fusion receptor could enhance Treg persistence and restore their immunoregulatory capacity in a rat model of chronic constriction injury (CCI). CD4⁺CD25⁺ Tregs were isolated from rats and transduced with a lentiviral construct encoding an IL1R1-CD3ζ-IRES-GFP fusion receptor. Engineered Tregs were evaluated in vitro for migratory capacity, proliferation, and apoptosis in response to IL-1 stimulation. Neuropathic pain was induced by sciatic nerve CCI, followed by adoptive transfer of unmodified or IL1R1-CD3–engineered Tregs. Mechanical hypersensitivity was assessed behaviorally, while flow cytometry and molecular analyses were used to characterize immune cell infiltration, inflammatory mediator production, and intracellular signaling pathways within the spinal cord. Endogenous Tregs were unable to adequately control CCI-induced neuroinflammation: depletion aggravated mechanical hypersensitivity, and transfer of unmodified Tregs failed to confer analgesic benefit due to IL-1–induced apoptosis. In contrast, IL1R1-CD3 Tregs preserved IL-1–dependent chemotaxis, exhibited enhanced proliferative responses, and resisted apoptosis under inflammatory conditions, resulting in preferential accumulation within the spinal cord. Adoptive transfer of IL1R1-CD3 Tregs significantly alleviated mechanical hypersensitivity and reduced macrophage infiltration while promoting an anti-inflammatory phenotype. Microglial activation and production of pro-inflammatory mediators, including TNF, IL-1β, IL-6, and iNOS, were markedly suppressed, accompanied by attenuation of NF-κB and MAPK signaling. Furthermore, IL1R1-CD3 Tregs limited neutrophil recruitment and diminished neutrophil-derived cytokines, matrix metalloproteinases, and oxidative stress. By reinterpreting IL-1 signaling, IL1R1-CD3–engineered Tregs exhibit enhanced persistence and exert immunoregulatory effects across multiple innate immune populations, leading to attenuation of neuropathic pain. These findings suggest that signal-reprogrammed Treg therapy may represent a potential strategy for modulating IL-1–driven neuroinflammation and may provide a conceptual framework for future translational studies targeting chronic pain.