Description:
<jats:p>Activation of the NLRP3 inflammasome and subsequent maturation of IL‐1β have been implicated in acute lung injury (ALI), which is characterized by inflammation, IL‐1β release, alveolar epithelial cell injury, hyperplasia of alveolar type 2 cells, accumulation of fibroblasts, deposition of collagen, and scar formation. We investigated the role of vimentin, a type III intermediate filament, in this process using three well characterized murine models of ALI which require NLRP3 for inflammasome activation. It was demonstrated that in these ALI models, the pathophysiologic changes are attenuated in <jats:italic>Vim</jats:italic><jats:italic><jats:sup>−/−</jats:sup></jats:italic> mice. We hypothesized that vimentin directly interacts with NLRP3 for normal inflammasome activity. Therefore, we examined, by co‐immunoprecipitation, whether NLRP3 and vimentin interact in a human macrophage cell line, THP‐1, and mouse bone marrow derived macrophages (BMDM). Next, using bio‐layer interferometery we investigated whether NLRP3 and vimentin directly interact. Preliminary results indicated that full length vimentin directly interacts with NLRP3 with a K<jats:sub>D</jats:sub> around 240 nM. To investigate which region of vimentin interacts with NLRP3 we used peptide arrays. Our results indicate that the vimentin head domain has and a small region of the tail domain interact with NLRP3 while the central rod domain shows very little interaction. Since vimentin has a large number of amino acids which can be phosphorylated, the binding between phosphomimetic vimentin peptides and NLRP3 was also examined. Phosphomimetic peptides of the vimentin head domain inhibit binding to NLRP3 while increased binding was observed in the tail domain when T426D and S430D mutations were examined. To further demonstrate a functional interaction between vimentin and NLRP3, vimentin was deleted from BMDMs using crispr/Cas9 and vimentin domains were expressed in these Vim KO cells using lentiviral infection. These new cell lines were examined for IL‐1β release after activation with LPS and ATP. Additionally, localization of vimentin and vimentin domains near NLRP3 after activation was investigated using proximity ligation assays. Our data strongly indicate that NLRP3 and vimentin directly interact and that this interaction is important for NLRP3 inflammasome activity.</jats:p><jats:p><jats:bold>Support or Funding Information</jats:bold></jats:p><jats:p>This work was supported by National Heart, Lung, and Blood Institute (HL71643; HL128194)</jats:p>