OPTOGENETIC INHIBITION OF INFLAMMASOME IN MICROGLIAN TO RELIEVE NEUROPATHIC PAIN

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. 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. 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. 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. 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.