Optogenetic inhibition of inflammasome in microglia to relieve neuropathic pain 2330993

Abstract Introduction The NLRP3 inflammasome has emerged as a central hub that converts diverse cellular stress signals into IL-1β—driven neuroinflammation in microglia under neuropathic pain. However, because NLRP3 functions as a multiprotein inflammasome complex, strategies that target NLRP3 alone face inherent limitations in controlling the spatial and temporal dynamics of inflammasome assembly and signaling. Methods To overcome these limitations, we developed NLRP3-TRAP (Temporally Restricted Assembled inflammasome Platform), an optogenetic system that enables spatiotemporally precise trapping of assembled NLRP3 inflammasomes using light. NLRP3 was tethered to a CIBN optogenetic module and co-expressed with a blue-light—responsive CRY2 variant (E281A, A9) in human microglial cells (HMC3). We evaluated multiple NLRP3—CIBN fusion designs to identify configurations compatible with efficient CRY2-mediated clustering. Using this optimized platform, we precisely analyzed NLRP3 clustering kinetics and light-dependent binding dynamics. Results Applying NLRP3-TRAP in LPS/ATP-stimulated HMC3 cells, blue-light illumination attenuated downstream inflammasome signaling, as evidenced by reduced levels of cleaved caspase-1 and mature IL-1β. For in vivo translation, NLRP3-TRAP were encapsulated into PLGA nanoparticles that are preferentially internalized by spinal microglia after intrathecal injection. In a spared nerve injury mouse model, repeated non-invasive LED stimulation produced reduction of pain behavior during post-injury days 4—12 compared with no-light controls, indicating a robust attenuation of neuropathic pain—like mechanical hypersensitivity. This behavioral improvement was accompanied by reduced microglial activation and decreased IL-1β expression in the spinal cord. Conclusion Together, these findings establish nano optogenetic modulation of microglial NLRP3 inflammasomes as a powerful strategy to dissect neuroimmune circuits and enable circuit-level anti-inflammatory interventions for neuropathic pain. Funding Source IBS-R001-Y4-2026-a00 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)