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

Abstract

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 non-canonical inflammasome pathway <i>via</i> 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 <i>via</i> flagellin delivery to the cytoplasm of host cells. <i>Salmonella typhimurium</i>-derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for <i>Salmonella</i> OMV-induced inflammasome response. Flagellated <i>Pseudomonas aeruginosa</i>-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated <i>Escherichia coli</i>-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient <i>Salmonella</i> OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that <i>Salmonella</i> OMV triggers NLRC4 inflammasome activation <i>via</i> OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling <i>via</i> OMV-bound lipopolysaccharide. Intriguingly, flagellated <i>Salmonella</i>-derived OMVs induced more rapid inflammasome response than flagellin-deficient <i>Salmonella</i> OMV and non-flagellated <i>Escherichia coli</i>-derived OMVs. Supporting these <i>in vitro</i> results, <i>Nlrc4</i>-deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with <i>Salmonella</i>-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.