Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

The innate immune system senses microbial pathogens through recognition of conserved entities collectively referred to as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). These entities interact with conserved pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and C-type lectin receptors (CLRs) that are expressed by immune cells and other cell types. Activation of PRRs by PAMPs is dictated by the availability and expression levels of PAMPs at different stages of infection and results in host responses which are vital for inflammation and immunity against pathogens (1). However, some pathogens, including Salmonella spp., a facultative intracellular pathogen, have evolved the ability to use these host responses for their own replication and establishment of infection (2).Flagellin, the monomeric protein constituting bacterial flagella, is one of the key Salmonella effector molecules which binds and activates membrane-bound TLR-5 as well as the cytosolic sensor NLRC4 and plays a major role in generating inflammatory responses (3–5). In macrophages, flagellin as well as the rod protein PrgJ, which are inadvertently released into the host cytosol by the type III secretion system (T3SS), are detected by the NAIPs. In mice, seven NAIPs are present of which NAIP1 senses the T3SS needle protein, NAIP2 detects the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin (6–9). Humans however encode a single functional NAIP which has been recently shown to broadly detect multiple T3SS proteins and flagellin (10). Ligand binding to the NAIPs leads to recruitment and oligomerization of NLRC4 (11, 12). Activation of the NAIP-NLRC4 inflammasome by these effectors and activation of the NLRP3 inflammasome by an as yet unidentified aconitase-regulated Salmonella effector (13–15) results in caspase-1-dependent pyroptosis and production of active IL-1β which promotes clearance of the bacterium and protects the host against Salmonella (13, 16, 17). It is believed that as infection progresses, Salmonella circumvents this host-protective response by suppressing the expression of flagellin to lower than the resting levels usually expressed by bacteria in culture (18). Down-regulation of flagellin is essential for the bacterium to establish successful infection. Previous work has shown that a Salmonella Typhimurium strain modified to constitutively express flagellin (ST-FliC^ON) and therefore unable to naturally down-regulate flagellin expression is avirulent and cleared successfully from the host compared to its wild-type (WT) counterpart (17). Despite this central role of flagellin in Salmonella pathogenesis, the molecular mechanisms that regulate the physiological switch of Salmonella from a flagellin-high to a flagellin-low phenotype and aid in establishment of an intracellular niche within macrophages in vivo are incompletely understood.Upon entry into macrophages, Salmonella resides in a vacuole called the Salmonella-containing vacuole (SCV) where it shuts down expression of the Salmonella pathogenicity island 1 (SPI-1) and concomitantly switches on expression of Salmonella pathogenicity island 2 (SPI-2), which is activated by the PhoP/PhoQ two-component system (19) and encodes genes required for intracellular replication. Prior work has shown that shutdown of SPI-1 in growth media that mimic conditions associated with the SCV such as acidic pH and low Mg²⁺ is also accompanied by repression of flagellin (20, 21). This is because low pH and low Mg²⁺ activate the PhoP/PhoQ system (20, 22, 23) and activated PhoP is believed to suppress expression of flagellin (21). A noteworthy issue relating to these early studies is that effects on PhoP/PhoQ-regulated genes were examined only during in vitro culture of bacteria in growth medium and not in the context of S. Typhimurium residence within macrophages. Therefore, the physiological contribution of these mechanisms to flagellin repression of intracellular Salmonella remains debatable. For example, contrary studies have shown that the effect of low pH on flagellin protein expression is observed only at a very low pH (pH = 3) and not at pH 5 (20) which is close to the physiologically relevant pH of the SCV (24, 25). Likewise, neither variation of extracellular Mg²⁺ nor reduced Mg²⁺ in the SCV was found to play a role in PhoP activation by Salmonella inside macrophages (26). Consequently, the regulatory mechanisms conventionally thought to repress flagellin expression by Salmonella remain controversial and there is scarce evidence to suggest that these factors are responsible for down-regulation of flagellin by bacteria residing within macrophages. Moreover, the physiological mechanisms that regulate repression of flagellin in vivo are unknown.In this study we describe a host innate immune circuit that regulates expression of Salmonella flagellin during both the early/extracellular and the later/intracellular phases of macrophage infection with this pathogen. We find that during early infection of macrophages with S. Typhimurium, rapid NLRC4 inflammasome-dependent macrophage pyroptosis is necessary and sufficient for releasing a host lysophospholipid stimulus that promotes synthesis and release of flagellin from Salmonella. Unexpectedly, these host factors regulate not only the initial increase in flagellin production but also the later down-regulation of flagellin by Salmonella inside macrophages. This later effect is mediated by a natural type 1 IFN-dependent host negative feedback response that represses expression of NLRC4 and the lysophospholipid biosynthetic enzyme calcium-independent phospholipase A2 (iPLA2) within cells, causing a decline in intracellular lysophospholipids over time, which promotes eventual down-regulation of flagellin by intracellular bacteria. Our data identify host NLRC4 inflammasome activity as a temporal and biphasic regulator of expression of its own bacterial ligand, flagellin. We also describe a physiologically relevant type 1 IFN-mediated host mechanism that controls switching of Salmonella from a flagellin-high to a flagellin-low phenotype within macrophages in vivo. These findings have important implications for understanding the intricate evolutionary adaptations that shape host–pathogen cross-talk.