Summary Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of IL-1β and IL-18. While wild type Salmonella typhimurium infection is lethal to mice, a strain that persistently expresses flagellin was cleared by the cytosolic flagellin detection pathway via NLRC4 activation of caspase-1; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1 induced pyroptotic cell death, released bacteria from macrophages and exposed them to uptake and killing by reactive oxygen species in neutrophils. Similarly, caspase-1 cleared unmanipulated Legionella and Burkholderia by cytokine-independent mechanisms. This demonstrates for the first time that caspase-1 clears intracellular bacteria in vivo independent of IL-1β and IL-18, and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C T ) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-Otetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4−/− (Ipaf−/−), and caspase-1−/− derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7−/− macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.

Rationale: Caspase-1 processes interleukin 1␤ (IL-1␤) and IL-18 but may also contribute to apoptosis. In this context, caspase-1 knockout mice have been shown to be protected from endotoxin-induced mortality, whereas IL-1␤ knockout mice are not protected. Objectives: We therefore sought to delineate the mechanisms responsible for the differential responses between caspase-1 and IL-1␤ knockout mice. Methods: Caspase-1 knockout, IL-1␤ knockout, and IL-1␤/IL-18 double knockout mice were compared with wild-type mice for survival after intraperitoneal challenge with live Escherichia coli. Measurements and Main Results: Caspase-1 knockout animals were protected from bacterial challenge, whereas wild-type, IL-1␤ knockout, and IL-1␤/IL-18 double knockout animals were not. Wild-type animals and both IL-1␤ knockout and IL-1␤/IL-18 double knockout mice demonstrated significant splenic B lymphocyte apoptosis, which was absent in the caspase-1 knockout mice. Importantly, IL-1␤/IL-18 double knockout mice were protected from splenic cell apoptosis and sepsis-induced mortality by the caspase inhibitor zVAD-fmk. Furthermore, wild-type but not caspase-1 knockout splenic B lymphocytes induced peritoneal macrophages to assume an inhibitory phenotype. Conclusion: Taken together, these findings suggest that caspase-1 is important in the host response to sepsis at least in part via its ability to regulate sepsis-induced splenic cell apoptosis.Keywords: apoptosis; caspase inhibition; septic shock; spleen More than 500,000 people develop sepsis annually and 175,000 of them die in the United States alone (1). Septic shock activates numerous proinflammatory mediators, which can result in multiple organ injury (2, 3). In addition, executioner cysteineaspartate proteases (caspases) play a key role in the disassembly of cells during septic shock via various proapoptotic stimuli. Pharmacologic blockade of caspase activation improves organ function and survival in animal models of sepsis and ischemia reperfusion injury (4). Interleukin 1␤ (IL-1␤) is one of the major proinflammatory cytokines known to be produced in sepsis (5-8). It is synthesized as an inactive 31-kD precursor that requires a unique cysteine protease, IL-1␤Ϫconverting enzyme (caspase-1), to generate biologically active 17-kD IL-1␤ (9, 10).(Received in original form April 20, 2006; accepted Although caspase-1 plays no part in the spontaneous apoptosis of monocytes and macrophages (11), its activation via intracellular pathogens can induce macrophage apoptosis (12, 13) and its deletion has been linked to survival in animal models of endotoxin shock (14). This protective effect could logically be attributed to caspase-1's role in activating the precursor, pro-IL-1␤. Unexpectedly, however, active IL-1␤ does not regulate survival from endotoxin shock, because IL-1␤ knockout animals are not protected from endotoxin-induced death (15). This difference may hold an important key to understanding the role of caspase-1 in host responses. Importantly, prior caspase-1 knockout experiment...

IL-1β is critical for TH17 cell survival, expansion, and effector function in vivo during autoimmune responses, including EAE. However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis is poorly defined. In the present study, we uncovered a novel T cell-intrinsic inflammasome that drives IL-1β production during TH17-mediated EAE pathogenesis. TCR activation induced pro-IL-1β expression, while ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on TH17 but not TH1 cells, and ATP-treated TH17 cells showed enhanced survival compared to ATP-treated TH1 cells, suggesting autocrine action of TH17-derived IL-1β. Together, these data reveal a critical role for IL-1β produced by a TH17 cell-intrinsic ASC-NLRP3-Caspase-8 inflammasome during CNS inflammation.

Receptor interacting protein-2 (RIP2) is a caspase recruitment domain (CARD)-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation. Although RIP2 has also been linked to caspase-1 activation, RIP2 knockout animals fail to show a defect in caspase-1-mediated processing of proIL-1β to its active form. Therefore, RIP2 function in binding to caspase-1 remains poorly understood. We hypothesized that caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IκB kinase-γ thus inducing NF-κB activation. We further hypothesized that ASC, which also interacts with caspase-1 via its CARD, may interfere with the caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of caspase-1 and proIL-1β processing. RIP2 transient transfection induced transcription of an NF-κB reporter gene. This RIP2-induced NF-κB activity and caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for caspase-1 as a scaffold for RIP2, caspase-1 knockout macrophages were suppressed in their ability to activate NF-κB, and septic caspase-1 knockout animals produced less IL-6, a functional marker of NF-κB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their caspase-1 activity while enhancing their NF-κB signal. These data suggest that ASC may direct caspase-1 away from RIP2-mediated NF-κB activation, toward caspase-1-mediated processing of proIL-1β by interfering with the RIP2 caspase-1 interaction.