Activation of NLRC4 by Flagellated Bacteria Triggers Caspase-1–Dependent and –Independent Responses To Restrict Legionella pneumophila Replication in Macrophages and In Vivo

Abstract

Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1−/− mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1–independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1–dependent and –independent responses for bacterial growth restriction in macrophages and in vivo.

Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo

“…Our data support the hypothesis that this pathway does not play a role in the restriction of L. pneumophila and L. gratiana infection in vivo (Figs. 4C, 6D, and 7B); this result is supported by previous observations that NLRP3 and ASC are dispensable for the restriction of L. pneumophila replication in vivo (18,19,35). Notably, caspase-11 facilitated the recruitment of neutrophils to the sites of infection via the induction of IL-1a (40).…”

“…The mechanisms underlying NLRC4-mediated host resistance to L. pneumophila infection are still unclear, but pyroptosis has been proposed to be directly involved in host resistance (22). Furthermore, the NLRC4 inflammasome was reported to trigger the restriction of bacterial replication by additional processes dependent on flagellin but independent of caspase-1 and caspase-11 (18). Another pathway for caspase-1 activation has been reported in addition to the flagellin-mediated activation of the NLRC4 inflammasome.…”

“…Additionally, studies with Salmonella Typhimurium and L. pneumophila revealed that the bacterial flagellin functioned as an agonist for the NAIP5/NLRC4 signaling platform (13)(14)(15)(16)(17). Moreover, this inflammasome was reported to be the key for restriction of L. pneumophila replication in mouse macrophages and in vivo (11,13,(16)(17)(18)(19). Once activated, this inflammasome promotes the formation of pores in the macrophage membrane (16,17,20); this process culminates in the induction of an inflammatory form of cell death termed pyroptosis (reviewed in Ref.…”

Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila

“…The transition from a replicative phase to a transmissive phase prior to egress coincides with the activation of several virulence traits, including motility and the production of flagellin (17). Cytosolic flagellin induces a proinflammatory response that leads to host cell death through the activation of Naip/Nlrc1 and caspase-1-mediated pyroptosis (59)(60)(61)(62). L. pneumophila may exploit this pathway for egress, whereby activation of the flagellar regulon at late stages of infection triggers host cell lysis and the release of bacteria.…”

The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice

“…pneumophila flagellin plays a crucial role in the immune response and growth restriction of L. pneumophila in mice (12,29,31). In murine macrophages, flagellin acts as an important PAMP that triggers inflammasome activation and immune signaling (11,12).…”