Nanotube Topography Inhibits Nlrp3 Inflammasome Activation By Reducing Microtubule Glutamylation

This study investigates how titanium nanotube topography (TNTs) modulates NLRP3 inflammasome activation via microtubule dynamics, addressing MARK4 and tubulin polyglutamylation in peri-implant immune responses. Bone marrow - derived macrophages (BMDMs) from C57BL/6J mice (Ethics approval #2024 - 124) were cultured on polished Ti (pTi) or anodized TNTs. NLRP3 activation was induced with LPS/ATP or nigericin. Microtubule dynamics were analyzed by immunofluorescence (tubulin acetylation/polyglutamylation) and drugs (nocodazole/paclitaxel). TTLL4/CCP5 siRNA knockdown and MARK4 adenoviral overexpression assessed their roles. Osteoclast differentiation was evaluated by TRAP/F - actin assays. A rat peri - implantitis model (SD rats, n = 5/group) with pTi/TNTs implants and LPS injections had osseointegration/inflammation evaluated by micro - CT, histology (H&E, TRAP), and immunofluorescence for NLRP3, MARK4, polyglutamylated tubulin. Data were analyzed by ANOVA (p < 0.05). TNTs disrupt microtubule stability and organization, diminish microtubule polyglutamylation levels and consequently inhibit NLRP3 inflammasome assembly and activation. Upregulation of microtubule glutamylation levels reverses TNT-mediated inhibition of NLRP3 inflammasome activation, while MARK4 downregulation further attenuates polyglutamylation and inflammasome hyperactivation. In vivo, TNT implants exhibited higher bone-implant contact, fewer osteoclasts, and reduced NLRP3/MARK4/polyglutamylation levels versus pTi. TNTs suppress NLRP3 inflammasome activation by destabilizing microtubules via MARK4 downregulation and reduced polyglutamylation, thereby enhancing osseointegration in peri-implantitis, offering a novel strategy to design implants with enhanced osteoimmunomodulatory properties.