Professor and Vice Chair University of California, Irvine Irvine, California, United States
Disclosure(s):
Melissa Lodoen, PhD: No financial relationships to disclose
Introduction/Rationale: 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.
