NLRP3 inflammasome assembly and activation are mediated by dynein-dependent microtubule transport during Toxoplasma gondii infection of human immune cells

IL-1β is a potent pro-inflammatory cytokine that plays a key role in innate immunity against infection. The NLRP3 inflammasome is a multi-protein complex that mediates the processing of pro-IL-1β into mature bioactive IL-1β during T. gondii infection of human peripheral blood monocytes. However, the cellular mechanisms that facilitate the assembly of NLRP3 inflammasome components into a complex in T. gondii-infected cells remain unknown. We utilized super-resolution imaging to visualize the transport of inflammasome components during T. gondii infection of human monocytic cells. NLRP3 co-localized with host cell microtubules and the microtubule-organizing center (MTOC), specifically in infected cells, and proximity-ligation assays (PLA) revealed close interaction between NLRP3 and α-tubulin during infection. Treatment of cells with an inhibitor of cytoplasmic dynein motor proteins to prevent dynein-dependent microtubule transport resulted in reduced NLRP3 interaction with α-tubulin, decreased caspase-1 cleavage, and reduced IL-1β processing and release, suggesting a key role for dynein-mediated transport of NLRP3 on microtubules for assembly and activation of the NLRP3 inflammasome. After inflammasome-mediated cleavage, bioactive IL-1β was released from T. gondii-infected human monocytes in a gasdermin D- and E-independent manner and in the absence of pore formation. To visualize and track IL-1β processing and release during infection, we expressed an IL-1β fluorescence resonance energy transfer (FRET) construct in human monocytic cells, in which mTurquoise and mNeonGreen were fused to the N- and C-termini of pro-IL-1β, respectively. Intact pro-IL-1β exhibited FRET signal, whereas cleavage of pro-IL-1β was detected through the loss of FRET. These studies revealed that T. gondii infection increased processing of IL-1β, which localized proximal to the plasma membrane and in plasma membrane-coated vesicles and protrusions. Collectively, these findings expand our understanding of the mechanisms of NLRP3 inflammasome assembly and IL-1β processing and release during T. gondii infection of human immune cells.