Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling

Bacteria-released components can modulate host innate immune response in the absence of direct host cell-bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated noncanonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium-derived OMVs caused a robust NLRC4-mediated, but not NLRP3-mediated, caspase-1 activation and interleukin-1b secretion in macrophages. Notably, OMV-associated flagellin is essential for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while nonflagellated Escherichia coli-released OMVs induced NLRC4-independent noncanonical inflammasome activation leading to NLRP3-mediated interleukin-1bsecretion. Intriguingly, flagellated Salmonella–derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and nonflagellated Escherichia coli–derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1bproduction after intraperitoneal challenge with Salmonella-released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection.