(k, l) WT,Casp1/, orIl1b/Il18/mice were infected intraperitoneally with (k)L

(k, l) WT,Casp1/, orIl1b/Il18/mice were infected intraperitoneally with (k)L. pyroptosis, IL-1 Salmonella, cell death Macrophages make use of a compartmentalized dual detection system to respond to bacterial infection. Toll-like receptors (TLRs) respond to extracellular or vacuolar stimuli while Nod-like receptors (NLRs) respond to cytosolic perturbations. In general, the TLRs orchestrate downstream immune responses by inducing the transcription, translation and release of specific cytokines including IL-6 and IL-12. However, in the case of IL-1 and IL-18 the TLRs and NLRs function in concert. TLRs induce the expression of proIL-1 and proIL-18, following which NLR-dependent activation of caspase-1 regulates their proteolytic processing and release1. Many pathogens are controlled by caspase-1-mediated innate immune responses and these Rabbit Polyclonal to C1R (H chain, Cleaved-Arg463) are associated with the downstream effects of the pleiotropic cytokines IL-1 and IL-182. IL-1 promotes inflammation, including vasodilation and immune cell extravasation, and more recently has been implicated in the generation of TH17 responses. While IL-18 is best known for its activity in promoting interferon- (IFN-) production in TH1 cells, NK cells and cytotoxic T lymphocytes (CTLs), it can also enhance TH2 cell development and promote local inflammatory responses. Mice deficient in NLRP3 (NLR family, pyrin domain made up of 3, also known as cryopyrin) or caspase-1 are more susceptible to influenza contamination and this effect is primarily attributable to IL-1 signaling37. In contrast, bacteria such asShigellaare controlled by caspase-1-dependent IL-18 secretion, and not IL-1 secretion8. Caspase-1 activation is also involved in autoinflammatory diseases since mutations in NLRP3 that cause spontaneous caspase-1 activation result in a spectrum of cryopyrin-associated periodic fever syndromes. These syndromes can be effectively treated with IL-1 receptor antagonist, indicating that the symptoms are mostly IL-18 impartial2. Thus, a clear pathway from caspase-1 to IL-1 or IL-18 is usually observed in the clearance of several pathogens as well as in autoinflammatory syndromes. In vitro, in addition to processing IL-1 and IL-18, caspase-1 activation triggers a form of cell death called pyroptosis9. Like apoptosis, pyroptosis requires the proteolytic activation of specific caspases: caspase 3 and 7 for apoptosis and caspase-1 for pyroptosis. In contrast to apoptosis, which is often anti-inflammatory, pyroptosis is predicted to be proinflammatory due to the rapid loss of cell membrane integrity and release of cytosolic contents. Although certain mechanistic aspects have been characterizedin vitro, the functional role for pyroptosis remains unknown. Pathogenic bacteria can modulate host cell signaling pathways by delivering virulence factors to the cytosol of host cells. To this end, Type III secretion systems (T3SS) are one of the most complex and versatile mechanisms used by many Gram unfavorable pathogens10. However, the mammalian innate immune system has developed mechanisms to detect T3SS activity1,11. SPI1 T3SS, which promotes epithelial cell invasion forSalmonella typhimurium, can be readily detected by caspase-1in vitrovia NLRC4- (NLR family, CARD domain made up of 4; previously called Ipaf) mediated detection of inadvertently translocated flagellin or PrgJ rod protein1,11,12.In vivo, this detection can delay the kinetics of bacterial replication, but is not sufficient to prevent death of wild type (WT) mice13,14.S. typhimuriumexpress neither SPI1 nor flagellin during the systemic phase of contamination. Instead, the bacteria Chebulinic acid express a Chebulinic acid different T3SS, SPI2, which promotes replication within macrophages15. The SPI2 T3SS rod protein, SsaI, is not detected by Chebulinic acid NLRC412. Thus, by repressing both PrgJ and flagellin,S. Chebulinic acid typhimuriumis able to evade NLRC4 detection during the Chebulinic acid systemic phase of contamination12,16,17. Here we examine the consequences of NLRC4 and caspase-1 activation around the innate immune response to contamination. While WTS. typhimuriumcause a lethal systemic contamination in mice, anS. typhimuriumstrain that fails to evade NLRC4 is usually efficiently cleared. This clearance does not require the cytokines IL-1 and IL-18. Instead, clearance is usually associated with pyroptotic macrophage cell death. With the lysis of macrophages, pathogens are released into the extracellular space and become exposed to uptake and efficient killing by neutrophils through the activity of reactive oxygen species. == Results == == NLRC4 detects flagellin expressingS. typhimurium == In vivo, WTS. typhimurium(ST-WT) represses flagellin expression and we hypothesized that this increases virulence by enabling evasion of the intracellular flagellin detector, NLRC4. We sought to test this hypothesis by generating anS. typhimuriumstrain that persistently expresses flagellin (fliC). This strain, termed ST-FliCON, contains a plasmid in whichfliCis expressed by a SPI2 promoter. To explore whether ST-FliCONactivates NLRC4, we infected bone marrow derived macrophages (BMDM) with ST-WT or ST-FliCONand exhibited that ST-FliCONinduced IL-1 secretion and caspase-1 processing, whereas ST-WT did not (Fig. 1a, b). These responses required SPI2 T3SS within the bacteria (Fig. 1c) and were dependent on NLRC4 within macrophages (Fig. 1b, 1d). == Physique 1. Characterization of flagellin-expressingS. typhimurium. == (ad) BMDM were infected with ST-WT or ST-FliCONunder conditions where SPI2 T3SS is usually expressed and SPI1 T3SS is not expressed at the indicated multiplicity of contamination (MOI) for one hour followed by treatment with gentamicin. Total contamination time.