Session Title: Innate Immunity and Rheumatic Disease: Signaling Mechanisms
Session Type: Abstract Submissions (ACR)
Background/Purpose NLRP3 (NOD-like receptor family, pyrin domain containing 3) has a pivotal role in nucleating inflammasome, cytoplasmic multiprotein complexes that mediate the maturation of the proinflammatory cytokines interleukin-1β (IL-1b) by activating caspase-1. Mutations in the gene encoding NLRP3 cause a series of autoinflammatory disease, cryopyrin-associated periodic syndromes (CAPS). It has been reported that generation of reactive oxygen species (ROS) is one of the major NLRP3 inflammasome activating factor. However, the molecular mechanism of relationship between change of cellular redox state and NLRP3 inflammasome activation has not been elucidated. Here we show that cofilin-1, a redox sensitive actin binding protein, is involved in NLRP3 inflammasome activation.
Methods Mouse bone marrow derived macrophages (BMDMs) were obtained by differentiating bone marrow progenitors from tibial and femoral bone with M-CSF for 7 days. Inflammasome activation experiments were performed in two stages, initial LPS priming for 3 h and then inflammasome activation for 30 to 50 min by replacing the medium with RPMI 1640 medium supplemented with activators (ATP or nigericin). Inflammasome activation was analyzed by Western blotting of secreted interleukin-1b (IL-1b). The lysates from BMDMs or PT67 cells transiently transfected with expression constructs for the wild-type (WT) or various deleted forms of NLRP3 were immunoprecipitated with anti-cofilin-1 antibody.
Results When the NLRP3 inflammasome is activated, not only activated IL-1b but also inflammasome components, such as ASC and caspase-1 are secreted. We found that cofilin-1 is also secreted along with IL-1b from the LPS-primed BMDMs when the cells are treated with ATP, a NLRP3 inflammasome activator. In addition, knockdown of cofilin-1 reduces inflammasome activation in response to ATP, which suggests that cofilin-1 has an important role for the activation of the NLRP3 inflammasome. Cofilin-1 directly interacts to the nucleotide-binding domain (NBD) of NLRP3 protein. However, when the cells are stimulated with the NLRP3 inflammasome activators, ATP or nigericin, cofilin-1 is oxidized and dissociated from NLRP3. Indeed, the interaction of cofilin-1 with NLRP3 is increased significantly when the oxidation sites of cofilin-1 are substituted from cysteine to alanine. Finally, we found that NLRP3 inflammasome activation is attenuated when WT cofilin-1 is replaced with the oxidation-resistant mutant cofilin-1.
Conclusion Taken together, these results suggest that cofilin-1 is a key component in regulating the activation of the NLRP3 inflammasome in response to ROS. Furthermore, since cofilin-1 is an actin binding protein that depolymerizes and severs actin filaments, and is able to translocate into mitochondria during oxidative stress condition, our finding about the interaction of cofilin-1 with NLRP3 provides an important molecular mechanism for the mitochondrial localization of the NLRP3 inflammasome.
Y. H. Park,
D. L. Kastner,
J. J. Chae,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cofilin-1-is-a-ros-sensor-in-regulating-the-nlrp3-inflammasome/