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 non-canonical 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 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 Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella-derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli-derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1β 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.

Yang J ，Hwang I ，Lee E ，Shin SJ ，Lee EJ ，Rhee JH ，Yu JW ... -  《Frontiers in Immunology》

Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.

Pereira MS ，Morgantetti GF ，Massis LM ，Horta CV ，Hori JI ，Zamboni DS ... -  《-》

NLRP3 recruitment by NLRC4 during Salmonella infection.

NLRC4 and NLRP3, of the NOD-like receptor (NLR) family of intracellular proteins, are expressed in innate immune cells and are thought to nucleate distinct inflammasome complexes that promote caspase-1 activation, secretion of the proinflammatory cytokines IL-1β and IL-18, and a form of cell death termed pyroptosis. We show that NLRP3 associates with NLRC4 in macrophages infected with Salmonella typhimurium or transfected with flagellin. The significance of the interaction between the NLRC4 NACHT domain and NLRP3 was revealed when Nlrc4(S533A/S533A) bone marrow-derived macrophages (BMDMs) expressing phosphorylation site mutant NLRC4 S533A had only a mild defect in caspase-1 activation when compared with NLRC4-deficient BMDMs. NLRC4 S533A activated caspase-1 by recruiting NLRP3 and its adaptor protein ASC. Thus, Nlrc4(S533A/S533A) Nlrp3(-/-) BMDMs more closely resembled Nlrc4(-/-) BMDMs in their response to S. typhimurium or flagellin. The interplay between NLRP3 and NLRC4 reveals an unexpected overlap between what had been considered distinct inflammasome scaffolds.

Qu Y ，Misaghi S ，Newton K ，Maltzman A ，Izrael-Tomasevic A ，Arnott D ，Dixit VM ... -  《-》

Caspase-11 activation in response to bacterial secretion systems that access the host cytosol.

Casson CN ，Copenhaver AM ，Zwack EE ，Nguyen HT ，Strowig T ，Javdan B ，Bradley WP ，Fung TC ，Flavell RA ，Brodsky IE ，Shin S ... -  《-》

The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.

Zhao Y ，Yang J ，Shi J ，Gong YN ，Lu Q ，Xu H ，Liu L ，Shao F ... -  《-》