Centrosome-associated assembly and trafficking coordinate spatial activation of the NLRP3 inflammasome in primary human monocytes 2310149

Abstract

Abstract Introduction The spatial organization of NLRP3 inflammasome activation in human monocytes is poorly understood. By examining Toxoplasma gondii infection of primary human monocytes, we identified centrosome-linked and PIKfyve-dependent endosomal pathways that coordinate NLRP3 inflammasome assembly. Methods Inflammasome assembly and activation were analyzed in T. gondii-infected human monocytes using a combination of super-resolution and live-cell imaging along with biochemical and immunological assays. Pharmacological inhibitor experiments and inducible gene knock-out systems were used to gain mechanistic insights into pathways of inflammasome assembly during infection. Results Super-resolution imaging revealed that infection drives NLRP3 redistribution from diffuse cytosolic puncta to discrete pericentrosomal clusters coinciding with active caspase-1 in primary human monocytes. Dynamic live-cell imaging of NLRP3—mNeonGreen-expressing cells during infection captured directed movement of NLRP3 puncta toward the γ-tubulin-defined microtubule-organizing center and coalescence into a radial, spoke-like structure, indicating the centrosome as a principal scaffold for inflammasome organization. This configuration required the centrosomal kinase NEK7, and pharmacological disruption of the CPAP-β-tubulin scaffold abolished ASC assembly and downstream inflammasome activation under both T. gondii infection and canonical LPS + ATP stimulation. Consistent with the vesicle-like dynamics of NLRP3 assembly, inhibition or degradation of PIKfyve, a key regulator of early endosomal homeostasis, caused aberrant retention of NLRP3 within EEA1+ early endosomes and markedly reduced inflammasome activation and IL-1β cleavage while leaving NF-κB-dependent priming intact. Conclusion Collectively, these results delineate a coupled centrosomal-endosomal system that spatially governs NLRP3 inflammasome activation in primary human monocytes and reveals assembly checkpoints that determine inflammasome competence. Funding Source NIH Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)