Title: Proteasome Inhibitors Prevent Caspase-1-Mediated Disease in Rodents Challenged with Anthrax Lethal Toxin
Abstract: NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases. NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases. Innate immunity plays a critical role in controlling microbial infections. Activation of the inflammasome, an integral part of the innate immune response, has been linked to cell death and morbidity triggered by Salmonella, Francisella, Listeria, and Staphylococcus.1Mariathasan S Newton K Monack DM Vucic D French DM Lee WP Roose-Girma M Erickson S Dixit VM Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.Nature. 2004; 430: 213-218Crossref PubMed Scopus (1406) Google Scholar, 2Mariathasan S Weiss DS Dixit VM Monack DM Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis.J Exp Med. 2005; 202: 1043-1049Crossref PubMed Scopus (335) Google Scholar, 3Mariathasan S Weiss DS Newton K McBride J O'Rourke K Roose-Girma M Lee WP Weinrauch Y Monack DM Dixit VM Cryopyrin activates the inflammasome in response to toxins and ATP.Nature. 2006; 440: 228-232Crossref PubMed Scopus (2333) Google Scholar The inflammasome has also been linked to macrophage killing triggered by lethal toxin (LT), a major virulence factor released by the Gram-positive bacterium Bacillus anthracis.4Boyden ED Dietrich WF Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin.Nat Genet. 2006; 38: 240-244Crossref PubMed Scopus (649) Google Scholar, 5Fink SL Bergsbaken T Cookson BT Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms.Proc Natl Acad Sci USA. 2008; 105: 4312-4317Crossref PubMed Scopus (313) Google Scholar, 6Muehlbauer SM Evering TH Bonuccelli G Squires RC Ashton AW Porcelli SA Lisanti MP Brojatsch J Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis.Cell Cycle. 2007; 6: 758-766Crossref PubMed Scopus (67) Google Scholar, 7Wickliffe KE Leppla SH Moayeri M Anthrax lethal toxin-induced inflammasome formation and caspase-1 activation are late events dependent on ion fluxes and the proteasome.Cell Microbiol. 2008; 10: 332-343Crossref PubMed Scopus (51) Google Scholar, 8Collier RJ Young JA Anthrax toxin.Annu Rev Cell Dev Biol. 2003; 19: 45-70Crossref PubMed Scopus (475) Google Scholar In fact, when injected into small animals, LT alone is sufficient to reproduce the majority of the symptoms of the anthrax disease. 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105: 7803-7808Crossref PubMed Scopus (299) Google Scholar Nalp1b is highly polymorphic in mice, and macrophages from strains expressing a dominant allele of Nalp1b are susceptible to rapid necrosis after LT exposure.4Boyden ED Dietrich WF Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin.Nat Genet. 2006; 38: 240-244Crossref PubMed Scopus (649) Google Scholar, 12Alileche A Serfass ER Muehlbauer SM Porcelli SA Brojatsch J Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response.PLoS Pathog. 2005; 1: e19Crossref PubMed Scopus (69) Google Scholar, 13Reig N Jiang A Couture R Sutterwala FS Ogura Y Flavell RA Mellman I van der Goot FG Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin.Cell Microbiol. 2008; 10: 1190-1207Crossref PubMed Scopus (23) Google Scholar Conversely, murine macrophages expressing a recessive Nalp1b allele undergo slow, caspase-1 independent apoptosis in response to LT challenge.6Muehlbauer SM Evering TH Bonuccelli G Squires RC Ashton AW Porcelli SA Lisanti MP Brojatsch J Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis.Cell Cycle. 2007; 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Rats are divided into susceptible strains that are rapidly killed after LT challenge, and strains that are resistant to rapid disease progression.19Nye SH Wittenburg AL Evans DL O'Connor JA Roman RJ Jacob HJ Rat survival to anthrax lethal toxin is likely controlled by a single gene.Pharmacogenomics J. 2008; 8: 16-22Crossref PubMed Scopus (17) Google Scholar In contrast to mice, however, the in vivo LT susceptibility of rat strains closely mimics the in vitro susceptibility of their corresponding macrophages.19Nye SH Wittenburg AL Evans DL O'Connor JA Roman RJ Jacob HJ Rat survival to anthrax lethal toxin is likely controlled by a single gene.Pharmacogenomics J. 2008; 8: 16-22Crossref PubMed Scopus (17) Google Scholar Backcrossing experiments using susceptible and resistant rat strains suggest that the (in vivo and in vitro) LT susceptibility in rats is controlled by a single dominant gene.19Nye SH Wittenburg AL Evans DL O'Connor JA Roman RJ Jacob HJ Rat survival to anthrax lethal toxin is likely controlled by a single gene.Pharmacogenomics J. 2008; 8: 16-22Crossref PubMed Scopus (17) Google Scholar After LT administration, rats develop pulmonary edema, and die from a vascular collapse, a hallmark of human anthrax.20Sherer K Li Y Cui X Li X Subramanian M Laird MW Moayeri M Leppla SH Fitz Y Su J Eichacker PQ Fluid support worsens outcome and negates the benefit of protective antigen-directed monoclonal antibody in a lethal toxin-infused rat Bacillus anthracis shock model.Crit Care Med. 2007; 35: 1560-1567Crossref PubMed Scopus (22) Google Scholar, 21Cui X Moayeri M Li Y Li X Haley M Fitz Y Correa-Araujo R Banks SM Leppla SH Eichacker PQ Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats.Am J Physiol Regul Integr Comp Physiol. 2004; 286: R699-R709Crossref PubMed Scopus (110) Google Scholar, 22Kuo SR Willingham MC Bour SH Andreas EA Park SK Jackson C Duesbery NS Leppla SH Tang WJ Frankel AE Anthrax toxin-induced shock in rats is associated with pulmonary edema and hemorrhage.Microb Pathog. 2008; 44: 467-472Crossref PubMed Scopus (45) Google Scholar Due to greater similarities with human anthrax, the rat model of LT-induced disease may be superior to the murine model. We hypothesized that macrophage killing and cytokine release are critical events in the death of susceptible rats. Due to the correlation in rats between in vivo and in vitro LT susceptibility, we proposed that LT-mediated macrophage killing controls the rapid disease progression seen in susceptible rats. Due to the correlation between the murine and rat systems, we hypothesized that the single dominant gene controlling rat susceptibility is the rat homologue to murine Nalp1b. If true, caspase-1 activation would also control macrophage killing. We found that caspase-1 and proteasome inhibitors prevented caspase-1 activation and macrophage cytolysis mediated by LT. Furthermore, the proteasome inhibitor NPI-0052 prevented disease progression and mortality in LT-treated susceptible rats. Proteasome inhibitors also increased survival in BALB/c mice challenged with LT. Taken together, we demonstrated that caspase-1 activation not only controls macrophage killing, but also disease progression in susceptible rats challenged with LT. Our findings suggest that drugs controlling the inflammasome and caspase-1 are potential therapeutics for inflammatory diseases mediated by microbial pathogens. Male Fischer (F344), Brown-Norway (BN), Lewis (LEW), and Wistar (WKY) rats (200 to 300 g) were obtained from Taconic Farms (Hudson, NY). Female BALB/c mice (6 to 8 weeks old) were obtained from the National Cancer Institute (Bethesda, MD). Boc-D-CMK and MG132 were obtained from Calbiochem (San Diego, CA). Salinosporamide-A (NPI-0052) and bortezomib (Velcade) were generous gifts from Nereus Pharmaceuticals (San Diego, CA) and Millenium Pharmaceuticals (Cambridge, MA), respectively. Tumor necrosis factor-α, Camptothecin, and cycloheximide were purchased from Biovision (Mountainview, CA). Endotoxin-free recombinant lethal factor (LF) and protective antigen (PA) were kindly provided by the Northeast Biodefense Protein Expression Core (Albany, NY). Rat bone marrow-derived macrophages (BMMs) were generated as previously described.23Boltz-Nitulescu G Wiltschke C Holzinger C Fellinger A Scheiner O Gessl A Forster O Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.J Leukoc Biol. 1987; 41: 83-91PubMed Google Scholar, 24Perez-Perez GI Shepherd VL Morrow JD Blaser MJ Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide.Infect Immun. 1995; 63: 1183-1187PubMed Google Scholar Bone marrow cells were flushed from femora and tibiae of F344, LEW, BN, and WKY rats. Cells were differentiated into macrophages by incubation for 7 days in Dulbecco’s modified Eagle’s medium supplemented with 20% conditioned medium from a confluent culture of L929 fibroblasts as a source of CSF-1. After removal of nonadherent cells, macrophages were recovered by washing plates with cold PBS containing 5 mmol/L EDTA. BMMs were uniformly positive for ED1 (AbD Serotec, Raleigh, NC) staining. Cell viability was analyzed by the water-soluble tetrazolium (WST) assay, as described previously.15Squires RC Muehlbauer SM Brojatsch J Proteasomes control caspase-1 activation in anthrax lethal toxin-mediated cell killing.J Biol Chem. 2007; 282: 34260-34267Crossref PubMed Scopus (68) Google Scholar Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and propidium iodide exclusion assays were performed as previously described.25Akoachere M Squires RC Nour AM Angelov L Brojatsch J Abel-Santos E Identification of an in vivo inhibitor of Bacillus anthracis spore germination.J Biol Chem. 2007; 282: 12112-12118Crossref PubMed Scopus (54) Google Scholar, 26Diaz-Griffero F Hoschander SA Brojatsch J Bystander killing during avian leukosis virus subgroup B infection requires TVB(S3) signaling.J Virol. 2003; 77: 12552-12561Crossref PubMed Scopus (12) Google Scholar For analysis of caspase-1 activation, we used a fluorescent caspase-1 activity assay, FLICA, from Immunochemistry Technologies (Bloomington, MN). The assay was performed in 96-well plates, with 8 × 104 cells/well. Cells were incubated with LT and/or inhibitors, and stained in complete media with FLICA reagent (FAM-YVAD-FMK) for 2 hours. Cells were washed five times in PBS containing 10% fetal bovine serum, and fluorescence was measured on a Perkin Elmer Victor 3 multititer plate reader. For analysis of caspase-3 activation, we used a colorimetric caspase-3 assay as recommended by the manufacturer, R&D Systems (Minneapolis, MN). Electron microscopy was performed as described previously.27Alileche K Squires RC Muehlbauer SM MPL Brojatsch J Mitochondrial impairment mediates cytolysis in anthrax lethal toxin-treated murine macrophages.Cell Cycle. 2006; 5: 100-106Crossref PubMed Scopus (31) Google Scholar Western blotting was performed as previously described.6Muehlbauer SM Evering TH Bonuccelli G Squires RC Ashton AW Porcelli SA Lisanti MP Brojatsch J Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis.Cell Cycle. 2007; 6: 758-766Crossref PubMed Scopus (67) Google Scholar Membranes were probed with a polyclonal antibody to MEK-3 (Santa Cruz Biotechnology, Santa Cruz, CA; number Sc-960), monoclonal antibody to actin (Ac-40: Sigma, St. Louis, MO), and polyclonal antibody to rat interleukin (IL)-18 (MAB521; R&D Systems) at dilutions of 1:500. Polyclonal horseradish peroxidase-conjugated antibodies to rat and mouse immunoglobulins (Sc-2313 and 2314; Santa Cruz Biotechnology) were used as the secondary antibodies, and blots were developed by using Luminol Enhancer Substrate (Fisher Scientific, Pittsburgh, PA). Rats were injected intravenously with 150 μg/kg of LT or PBS. LT was prepared by diluting PA and LF in sterile PBS in a 4:1 ratio (PA:LF). Alternately, rats were injected with PBS only. Bortezomib and NPI-0052 were injected intravenously 60 minutes before LT injections. Bortezomib was dosed at 1 mg/kg, and infused in a solution of 0.35 mg/ml in physiological saline. NPI-0052 was dosed at 0.2 mg/kg and infused in a solution of 0.1 mg/ml in 5% Solutol (BASF Aktiengesellschaft, Germany). The final NPI-0052 solution contained 1% DMSO. In experiments using NPI-0052, control animals and rats treated with LT-only received injections of the NPI-0052 vehicle solution 60 minutes before LT injection. Blood collections were made by puncture of the inferior vena cava. In rat survival assays, animals were monitored continuously for up to 4 hours post-LT injection. Surviving rats were sacrificed at 4 hours post-LT or PBS injection. Histopathological studies were performed as previously described.28Goldman D Lee SC Casadevall A Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat.Infect Immun. 1994; 62: 4755-4761PubMed Google Scholar Rats were IV-injected with LT, NPI-0052, or both, and blood was collected as described above. Blood was kept on ice for 30 minutes to allow clotting, and then centrifuged at 4°C to separate the serum fraction. Sera were stored at −80°C, and submitted to the Albert Einstein College of Medicine Hormone Assay Core facility. The multiplex assay (RCYTO-80K-PMX24) was performed according to the manufacturer’s instructions (Millipore, Billerica, MA). Rat spleens were harvested immediately after sacrifice, and splenocytes were obtained via mechanical disruption and passage through a 70-μm cell strainer. Red blood cells were eliminated with RBC lysis buffer (Sigma). For each staining condition, 106 cells were resuspended in PBS and stained with the violet LIVE/DEAD viability dye (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Cells were washed and resuspended in flow cytometry buffer (PBS, 2% fetal bovine serum, 0.05% NaN3). Fc receptors were blocked with anti-rat CD32 (clone D34-485; BD Pharmingen, Carlsbad, CA). Surface markers were stained with Alexa Fluor 488-conjugated anti-CD3 and phosphatidylethanolamine-conjugated anti-CD11b/c (clones 1F4 and OX-42; Biolegend, San Diego, CA). After staining, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with flow cytometry buffer containing 0.1% saponin, and stained with Alexa Fluor 647-conjugated anti-CD68 (clone ED1; AbD Serotec, Raleigh, NC). Cells were acquired on an LSRII flow cytometer (BD Biosciences, Palo Alto, CA), and analyzed by using FlowJo software (Tree Star, Ashland, OR). Multiple comparisons of non-normally distributed data were performed by using the Kruskal-Wallis test followed by the Dunn’s multiple comparison test. For normally distributed data, comparisons were performed by using analysis of variance followed by the Student-Newman-Keuls test. Survival differences were calculated by using the Kaplan-Meier statistic. Statistics were performed with GraphPad Software (La Jolla, CA). P values <0.05 were considered significant. LT killing of murine macrophages is strain-specific and dependent on activation of the inflammatory proteins Nalp1b and caspase-1.4Boyden ED Dietrich WF Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin.Nat Genet. 2006; 38: 240-244Crossref PubMed Scopus (649) Google Scholar, 29Watters JW Dietrich WF Genetic, physical, and transcript map of the Ltxs1 region of mouse chromosome 11.Genomics. 2001; 73: 223-231Crossref PubMed Scopus (25) Google Scholar, 30Moayeri M Haines D Young HA Leppla SH Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice.J Clin Invest. 2003; 112: 670-682Crossref PubMed Scopus (258) Google Scholar, 31Moayeri M Martinez NW Wiggins J Young HA Leppla SH Mouse susceptibility to anthrax lethal toxin is influenced by genetic factors in addition to those controlling macrophage sensitivity.Infect Immun. 2004; 72: 4439-4447Crossref PubMed Scopus (83) Google Scholar Recent studies have indicated that LT susceptibility of rat macrophages is also strain-dependent and controlled by a single, dominant gene, potentially the rat homologue of murine Nalp1b.19Nye SH Wittenburg AL Evans DL O'Connor JA Roman RJ Jacob HJ Rat survival to anthrax lethal toxin is likely controlled by a single gene.Pharmacogenomics J. 2008; 8: 16-22Crossref PubMed Scopus (17) Google Scholar Based on the similar LT susceptibility pattern of murine and rat macrophages, we predicted that LT killing of susceptible rat macrophages was also caspase-1-dependent. Genetic backcrossing experiments in rats further suggested that a dominant gene not only controlled LT killing in vitro, but also LT-mediated disease progression.19Nye SH Wittenburg AL Evans DL O'Connor JA Roman RJ Jacob HJ Rat survival to anthrax lethal toxin is likely controlled by a single gene.Pharmacogenomics J. 2008; 8: 16-22Crossref PubMed Scopus (17) Google Scholar This in vitro and in vivo correlation in rats is clearly distinct from the murine system, where in vitro and in vivo LT susceptibilities do not correlate.30Moayeri M Haines D Young HA Leppla SH Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice.J Clin Invest. 2003; 112: 670-682Crossref PubMed Scopus (258) Google Scholar, 31Moayeri M Martinez NW Wiggins J Young HA Leppla SH Mouse susceptibility to anthrax lethal toxin is influenced by genetic factors in addition to those controlling macrophage sensitivity.Infect Immun. 2004; 72: 4439-4447Crossref PubMed Scopus (83) Google Scholar Based on these studies, we hypothesized that a dominant gene controls macrophage killing as well as vascular collapse in rats challenged with LT. To test this hypothesis we initially determined whether the inflammatory proteincaspase-1 controlled macrophage killing of susceptible rat macrophages. Based on the in vitro and in vivo correlation in rats, we further investigated whether inhibition of caspase-1 could prevent macrophage killing and disease in LT-treated rats. To determine whether LT activates the inflammatory caspase-1 in susceptible rat macrophages, we generated primary BMMs from susceptible and resistant rat strains. BMMs responded to a cytotoxic dose of LT (250 and 500 ng/ml of LF and PA, respectively)27Alileche K Squires RC Muehlbauer SM MPL Brojatsch J Mitochondrial impairment mediates cytolysis in anthrax lethal toxin-treated murine macrophages.Cell Cycle. 2006; 5: 100-106Crossref PubMed Scopus (31) Google Scholar in a strain-dependent fashion. F344 and BN-derived macrophages were rapidly killed within 2 to 4 hours by LT, whereas LEW and WKY BMMs were resistant to rapid LT-induced cytolysis (Figure 1A, and Supplemental Figure 1, C and D, at http://ajp.amjpathol.org). Next we tested whether LT killing of susceptible rat macrophages was caspase-1-dependent. The caspase-1 inhibitor Boc-D-CMK blocked caspase-1 activation and cytolysis of LT-treated F344 macrophages (Figure 1, B and C) indicating that caspase-1 activation was required for LT killing of susceptible rat BMMs. Consistent with caspase-1-dependence,6Muehlbauer SM Evering TH Bonuccelli G Squires RC Ashton AW Porcelli SA Lisanti MP Brojatsch J Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis.Cell Cycle. 2007; 6: 758-766Crossref PubMed Scopus (67) Google Scholar, 12Alileche A Serfass ER Muehlbauer SM Porcelli SA Brojatsch J Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response.PLoS Pathog. 2005; 1: e19Crossref PubMed Scopus (69) Google Scholar we detected significant processing of the caspase-1 substrates IL-18 and IL-1β in the lysates of F344 BMMs after LT exposure (Figure 1D and Supplemental Figure 1B at http://ajp.amjpathol.org). Caspase-1 activation in LT-treated F344 BMMs was further confirmed by using the fluorogenic activated caspase-1 specific stain, FLICA (Figure 1C). As caspase-1-mediated cell death has been linked to necrosis induction,5Fink SL Bergsbaken T Cookson BT Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms.Proc Natl Acad Sci USA. 2008; 105: 4312-4317Crossref PubMed Scopus (313) Google Scholar, 32Averette KM Pratt MR Yang Y Bassilian S Whitelegge JP Loo JA Muir TW Bradley KA Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.PLoS One. 2009; 4: e7913Crossref PubMed Scopus (47) Google Scholar, 33Bergsbaken T Cookson BT Innate immune response during Yersinia infection: critical modulation of cell death mechanisms through phagocyte activation.J Leukoc Biol. 2009; 86: 1153-1158Crossref PubMed Scopus (33) Google Scholar, 34Brough D Rothwell NJ Caspase-1-dependent processing of pro-interleukin-1beta is cytosolic and precedes cell death.J Cell Sci. 2007; 120: 772-781Crossref PubMed Scopus (189) Google Scholar, 35Lara-Tejero M Sutterwala FS Ogura Y Grant EP Bertin J Coyle AJ Flavell RA Galan JE Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis.J Exp Med. 2006; 203: 1407-1412Crossref PubMed Scopus (287) Google Scholar we tested for necrotic markers in LT-treated F344 macrophages. These showed several morphological features consistent with necrosis, including membrane impairment (as measured by propidium iodide uptake; Figure 1