NLRP3 is its own gatekeeper: a group hug of NLRP3 monomers controls inflammation.

A recent study by Hochheiser et al. describes the cryo-electron microscopy (cryoEM) structure of an autoinhibited nucleotide-binding domain-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) decamer that assembles via LRR interactions and is further stabilized by the small-molecule NLRP3-specific inhibitor CRID3 binding into a cleft within the NACHT domain. The study provides a springboard for the development of novel NLRP3-based therapies.

CRID3, a blocker of apoptosis associated speck like protein containing a card, ameliorates murine spinal cord injury by improving local immune microenvironment.

BACKGROUND: After spinal cord injury (SCI), destructive immune cell subsets are dominant in the local microenvironment, which are the important mechanism of injury. Studies have shown that inflammasomes play an important role in the inflammation following SCI, and apoptosis-associated speck-like protein containing a card (ASC) is the adaptor protein shared by inflammasomes. Therefore, we speculated that inhibiting ASC may improve the local microenvironment of injured spinal cord. Here, CRID3, a blocker of ASC oligomerization, was used to study its effect on the local microenvironment and the possible role in neuroprotection following SCI. METHODS: Murine SCI model was created using an Infinite Horizon impactor at T9 vertebral level with a force of 50 kdynes and CRID3 (50 mg/kg) was intraperitoneally injected following injury. ASC and its downstream molecules in inflammasome signaling pathway were measured by western blot. The immune cell subsets were detected by immunohistofluorescence (IHF) and flow cytometry (FCM). The spinal cord fibrosis area, neuron survival, myelin preservation, and functional recovery were assessed. RESULTS: Following SCI, CRID3 administration inhibited inflammasome-related ASC and caspase-1, IL-1ß, and IL-18 activation, which consequently suppressed M1 microglia, Th1 and Th1Th17 differentiation, and increased M2 microglia and Th2 differentiation. Accordingly, the improved histology and behavior have also been found. CONCLUSIONS: CRID3 may ameliorate murine SCI by inhibiting inflammasome activation, reducing proinflammatory factor production, restoring immune cell subset balance, and improving local immune microenvironment, and early administration may be a promising therapeutic strategy for SCI.

Development of Fluorescent and Biotin Probes Targeting NLRP3.

Extracellular signals drive the nucleation of the NLRP3 inflammasome which leads to the release of cytokines and causes inflammatory events. Hence, the inflammasome has gained enormous momentum in biomedical basic research. The detailed mechanisms of inflammasome generation and regulation remain to be elucidated. Our study was directed toward the design, convergent synthesis, and initial biochemical evaluation of activity-based probes addressing NLRP3. For this purpose, probes were assembled from a CRID3/MCC950-related NLRP3-binding unit, a linker portion and a coumarin 343 fluorophore or biotin. The affinity of our probes to NLRP3 was demonstrated through SPR measurements and their cellular activity was confirmed by reduction of the interleukin 1ß release from stimulated bone marrow-derived macrophages. The initial characterizations of NLRP3-targeting probes highlighted the coumarin probe 2 as a suitable tool compound for the cellular and biochemical analysis of the NLRP3 inflammasome.

The Supportive Role of NSC328382, a P2X7R Antagonist, in Enhancing the Inhibitory Effect of CRID3 on NLRP3 Inflammasome Activation in Rats with Dextran Sodium Sulfate-Induced Colitis.

PURPOSE: The NLRP3 inflammasome is a substantial component of the inflammation process. The complex pathogenesis of and the implication of a vast number of components in the inflammasome-activation pathway prompted us to search for compounds that have a wide therapeutic index and act at the level of multiple cellular targets. Although CRID3 blocks NLRP3 with high specificity in the laboratory, clinical trials of the compound reported weaker potency. METHODS: We used NSC328382, a P2X7R antagonist, as an adjunctive therapy and generated a strategy to potentiate the effects of CRID3 in rats with DSS-induced colitis. RESULTS: NSC328382/CRID3 combined therapy exhibited a significantly increased efficacy compared with either of the monotherapies. NSC328382/CRID3 was more efficient in 1) attenuating colon shortening and disease activity; 2) improving goblet cell density and both the macroscopic and microscopic scenario of the injured colon; 3) improving the antioxidant defense mechanisms of the inflamed colon against oxidative stress; and 4) mitigating the inflammation state by downregulating the proinflammatory cytokines. Pyroptotic cell death was also conspicuously restrained. Additionally, NSC328382 interrupted the MyD88/NF-κB axis. Moreover, NSC328382/CRID3 exhibited the ability to alter Th1/Th2 dominance. CONCLUSION: The clinical application of NSC328382/CRID3 may result in the generation of a novel approach for the treatment of IBDs.