L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis.

Macrophage adhesion and stretching have been shown to induce interleukin (IL)-1ß production, but the mechanism of this mechanotransduction remains unclear. Here we specify the molecular link between mechanical tension on tissue-resident macrophages and activation of the NLRP3 inflammasome, which governs IL-1ß production. NLRP3 activation enhances antimicrobial defense, but excessive NLRP3 activity causes inflammatory tissue damage in conditions such as pulmonary fibrosis and acute respiratory distress syndrome. We find that the actin-bundling protein L-plastin (LPL) significantly enhances NLRP3 assembly. Specifically, LPL enables apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) oligomerization during NLRP3 assembly by stabilizing ASC interactions with the kinase Pyk2, a component of cell-surface adhesive structures called podosomes. Upon treatment with exogenous NLRP3 activators, lung-resident alveolar macrophages (AMs) lacking LPL exhibit reduced caspase-1 activity, IL-1ß cleavage, and gasdermin-D processing. LPL-/- mice display resistance to bleomycin-induced lung injury and fibrosis. These findings identify the LPL-Pyk2-ASC pathway as a target for modulation in NLRP3-mediated inflammatory conditions.

Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus.

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key "switch" in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the "S5A" mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host-pathogen defense interactions.