The specific NLRP3 N-terminal domain is essential for NLRP3 activation and inflammasome formation in response to K ⁺ efflux.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton’s tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Signaling through the inflammasome is important for the inflammatory response. Low concentrations of intracellular K+ are associated with the specific oligomerization and activation of the NLRP3 inflammasome, a type of inflammasome involved in sterile inflammation. After NLRP3 oligomerization, ASC protein binds and forms oligomeric filaments that culminate in large protein complexes named ASC specks. ASC specks are also initiated from different inflammasome scaffolds, such as AIM2, NLRC4, or Pyrin. ASC oligomers recruit caspase-1 and then induce its activation through interactions between their respective caspase activation and recruitment domains (CARD). So far, ASC oligomerization and caspase-1 activation are K+-independent processes. Here, we found that when there is low intracellular K+, ASC oligomers change their structure independently of NLRP3 and make the ASCCARD domain more accessible for the recruitment of the pro-caspase-1CARD domain. Therefore, conditions that decrease intracellular K+ not only drive NLRP3 responses but also enhance the recruitment of the pro-caspase-1 CARD domain into the ASC specks.

Summary: NLRP3 is an inflammasome-forming protein that plays a key role in conditions ranging from infections to Alzheimer’s disease. NLRP3 is activated by many potassium (K ⁺ )-dependent or - independent stimuli related to pathogens or sterile insults. Molecularly, human NLRP3 assembles into a decameric ‘cage’ and its interaction with both the trans-Golgi network (TGN) and the microtubule organization centre (MTOC) has been proposed as critical for NLRP3 activation. However, the relative mechanistic requirement for K ⁺ -dependent versus - independent stimuli is unclear. Using spatially and dynamically high-resolution microscopy in human macrophages, we found that K ⁺ -dependent NLRP3 stimulation triggers two distinct activation pathways: ( i ) the decameric cage-dependent pathway, which was also exclusively required for the K ⁺ -independent stimulus, imiquimod; and ( ii ) a cage- and TGN/MTOC-independent pathway that was fully functional in an NLRP3 protein engineered to form monomers and unable to interact with membrane lipids. Collectively, our results delineate two parallel yet biologically distinct NLRP3 activation pathways.

The NLRP3 inflammasome contributes to a wide range of conditions from infections to Alzheimer’s disease. NLRP3 forms an inactive decameric cage, that upon interaction with the trans-Golgi network (TGN) and microtubule organization center (MTOC), leads to inflammasome activation, yet whether non-decamer NLRP3 species form functional inflammasomes remains unclear. Here, we design a NLRP3 exon 3 deletion variant that forms low molecular weight NLRP3 assemblies. Spatially and dynamically highly resolved microscopy in THP-1 and human macrophages shows that nigericin, a K ⁺ -dependent NLRP3 stimulus, can trigger two distinct activation pathways: (i) the rapidly engaged decameric cage-dependent pathway; and (ii) a decameric cage-independent, TGN/MTOC-distal, and slow-reacting pathway employed by low molecular weight NLRP3 species, that dominates in human neutrophils. Collectively, our results delineate two parallel yet biologically distinct NLRP3 activation pathways, thereby providing a framework to understand NLRP3-driven inflammation across a wide range of pathological context and cell types.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.