Bacillus anthracis spores and lethal toxin induce IL-1β viafunctionally distinct signaling pathways

Tae Jin Kang1, Subhendu Basu1, Lei Zhang1, Karen E. Thomas2, Stefanie N. Vogel2, LesBaillie3, and Alan S. Cross1

1Center for Vaccine Development, Department of Medicine, University of Maryland School ofMedicine, Baltimore, USA2Department of Microbiology and Immunology, University of Maryland School of Medicine,Baltimore, USA3Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, USA

AbstractPrevious reports suggested that lethal toxin (LT)-induced caspase-1 activity and/or IL-1βaccounted for Bacillus anthracis (BA) infection lethality. In contrast, we now report thatcaspase-1-mediated IL-1β expression in response to BA spores is required for anti-BA hostdefenses. Caspase-1−/− and IL-1β−/− mice are more susceptible than wild-type (WT) mice to lethalBA infection, are less able to kill BA both in vivo and in vitro, and addition of rIL-1β tomacrophages from these mice restored killing in vitro. Non-germinating BA spores inducedcaspase-1 activity, IL-1β and nitric oxide, by which BA are killed in WT but not in caspase-1−/−

mice, suggesting that the spore itself stimulated inflammatory responses. While spores inducedIL-1β in LT-susceptible and -resistant macrophages, LT induced IL-1β only in LT-susceptiblemacrophages. Cooperation between MyD88-dependent and -independent signaling pathways wasrequired for spore-induced, but not LT-induced, IL-1β. While both spores and LT inducedcaspase-1 activity and IL-1β, LT did not induce IL-1β mRNA, and spores did not induce celldeath. Thus different components of the same bacterium each induce IL-1β by distinct signalingpathways. Whereas the spore-induced IL-1β limits BA infection, LT-induced IL-1β enables BA toescape host defenses.

KeywordsAnthrax; Bacillus anthracis; Interleukin-1β; Lethal toxin; Spores

IntroductionDespite the identification of Bacillus anthracis (BA) as the cause of anthrax, its pathology inhumans is still ill-defined. Studies in animals have shown that the interaction of the sporewith the macrophages of the host is the key event in the infective process [1, 2]. Following

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Full correspondence: Dr. Alan S. Cross, Center for Vaccine Development, Department of Medicine, University of Maryland Schoolof Medicine, 685 W. Baltimore Street, HSF I-480, Baltimore, MD 21201, USA, Fax: +1-410-706-6205,across@medicine.umaryland.edu.Current address: Dr. Tae Jin Kang, College of Pharmacy, Sahmyook Univeristy, 26-21, Kongnung 2-dong Nowon-gu, Seoul139-742, Korea

Conflict of interest: The authors declare no financial or commercial conflict of interest.

Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2008/38141_s.pdf

NIH Public AccessAuthor ManuscriptEur J Immunol. Author manuscript; available in PMC 2013 June 13.

Published in final edited form as:Eur J Immunol. 2008 June ; 38(6): 1574–1584. doi:10.1002/eji.200838141.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

uptake by macrophages, spores germinate in the phagolysosome, resulting in the emergenceof an encapsulated vegetative bacterium expressing a tripartite toxin composed of protectiveantigen (PA), lethal factor (LF) and edema factor [3]. Toxin expression, which occurs within3 h of germination, is thought to contribute to intracellular survival by suppressingsuperoxide and nitric oxide (NO) production, key antibacterial killing mechanisms of themacrophage [4, 5]. Later in the disease, once the bacterium has escaped from themacrophage, lethal levels of toxin induce the development of the cytokine-independent,shock-like death associated with anthrax [6].

Recent data suggest that the spore, rather than being an inert structure, may actually interactwith the innate immune system to promote germination and subsequent infection [7, 8].While >106 spores/mL are required to elicit TNF-α and other cytokines, IL-1β is easilydetected following exposure of macrophages to levels of BA spores likely to be encounteredinitially in the environment (≤103 spores/mL) [9]. This suggests that spore-induced IL-1βand TNF-α may be generated by engagement of distinct receptor signaling pathways.

The product of IL-1β gene expression is an inactive form of IL-1β, pro-IL-1β, which mustbe processed by a cytosolic cysteine protease, caspase-1, to the active 17-kDa IL-1βcytokine [10]. Previous investigators have observed that the administration of purified lethaltoxin (LT) rapidly activates caspase-1 resulting in the extracellular release of IL-1β andIL-18 [11]. Similarly, Popov et al. [12] noted the induction of IL-1β by germination-proficient BA and concluded that the organisms' lethal activity could be due to caspase-1-mediated apoptosis. Each of these studies, therefore, concluded that caspase-1 might be apotential therapeutic target for the treatment of anthrax infection. Given our observation thatlow concentrations of BA spores induce IL-1β prior to germination and LT expression, weexamined whether the ability to induce IL-1β played a unique role in the pathogenesis ofanthrax infection.

Using a strain of BA that is impaired in its ability to germinate in the presence ofmacrophages, we now report that the spore itself induces IL-1β mRNA and protein but notcell death, and this cytokine is required for anti-BA host defenses. The MyD88 adaptorprotein is required for pro-IL-1β expression; however, phosphorylation of STAT1 throughthe MyD88-independent induction of IFN-β is necessary for optimal caspase-1-processingof the inactive IL-1β precursor to the active cytokine. In contrast, LT activates caspase-1 byan IFN-β-, STAT1-independent signaling pathway. Our finding in this study that BA spores,unlike LT, induce IL-1β via cooperation between both MyD88-dependent and -independentpathways, indicates that BA spores and LT have distinct differences in their ability to induceIL-1β and the functional responses that result.

ResultsCritical role of caspase-1 in host defenses against BA infection

To assess the importance of caspase-1 activity in host defenses against BA infection, wecompared the lethality of germination-proficient BA Sterne 34F2 (toxin+, capsule−) sporesin caspase-1+/+ and caspase-1−/− mice. The LD50 of this BA strain for C3H/HeN(caspase-1+/+) mice was 1×107 CFU. At a challenge dose of 1 × 106 spores i.p., all thecaspase-1−/− mice succumbed, while all of the background-matched caspase-1+/+ micesurvived (p=0.0111; Fig. 1A). Further, caspase-1+/+ mice had a progressive clearing of thechallenge dose of BA from spleen, lungs and liver over 72 h, while caspase-1−/− mice failedto do so (Fig. 1B), suggesting an inability of caspase-1−/− phagocytes to kill the organism.Similarly, at a challenge dose of 1×106 spores i.p., IL-1β−/− mice also were more susceptibleto infection with the Sterne 34F2 strain of BA (7/7 WT mice survived versus 0/7 IL-1β−/−

mice, p=0.0059; Fig. 1C).

Kang et al. Page 2

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

We previously observed that murine peritoneal macrophages were capable of killing BA invitro through an NO-mediated bactericidal mechanism that targets the emerging vegetativebacilli [8, 9, 13, 14]. Therefore, we determined whether the susceptibility of caspase-1−/−

mice to lethal infection was due to an inability of its macrophages to kill the BA.Macrophages from caspase-1−/− mice had a markedly impaired ability to kill BA Sterne34F2 in vitro (Fig. 2A); however, addition of rIL-1β, a product of caspase-1 activity,restored the bactericidal activity of the caspase-1−/− macrophages in a dose-dependentmanner (Fig. 2A). In contrast to the compensatory effect of rIL-1β, addition of rIL-18,another cytokine that is processed to active cytokine from an inactive precursor bycaspase-1, failed to restore macrophage bactericidal activity (Fig. 2A). Thus, the defect inhost defenses of caspase-1−/− mice against BA appeared to be related to a lack of IL-1β.

As anticipated, addition of exogenous rIL-1β also restored the microbicidal activity ofmacrophages from IL-1β−/− mice (Fig. 2B). Of interest, at the highest dose of rIL-1β added,there was a decrease in microbicidal activity, as may occur in biological systems withcomplex regulatory mechanisms. Thus, the failure of caspase-1-deficient mice andmacrophages to control infection is mediated by IL-1β and is dependent on the cysteineprotease caspase-1.

BA spore-induced IL-1β requires MyD88 and caspase-1 activationWe previously demonstrated that while LPS stimulation of murine macrophages induced theMyD88-dependent expression of IL-1β mRNA and its inactive gene product, pro-IL-1β,cooperation with a MyD88-independent pathway was required for expression of activeIL-1β cytokine [15]. Therefore, we next asked whether BA spores, like LPS, could directlyinduce IL-1β mRNA and caspase-1 activity that processed pro-IL-1β gene product to themature cytokine. To ensure that we were examining the initial interaction betweenmacrophages and BA spores rather than vegetative bacilli, we used a congenic mutant of theSterne 34F2 strain, ΔgerH, that germinates poorly within macrophages [14, 16]. Thus,during macrophage cell culture there is limited expression of either LT or edema toxin (ET).Since the non-germinating ΔgerH BA spore is not killed by macrophages [14], it was usedin all experiments to examine intracellular pathway utilization, except for the macrophagekilling assays, where the germination-proficient Sterne 34F2 spore was employed.

Infection of macrophages with BA spores induced IL-1β mRNA after 2 h and requiredMyD88 (Supporting Information Fig. 1). Similar to LPS, BA spores induced caspase-1activity in macrophage cultures from caspase-1+/+ mice; however, as expected, such activitywas absent in macrophages from caspase-1−/− mice (Fig. 3A). Further, administration of BAspores to mice induced robust caspase-1 activity in vivo in liver and spleen (Fig. 3B) as wellas the ability to generate IL-1β protein (Fig. 3C). No germination of ΔgerH spores wasevident either by direct inspection or by susceptibility of spores in culture supernatants toheating.

Since macrophages kill the emerging vegetative bacilli within macrophages in an NO-dependent manner [8, 13], we measured whether BA could induce NO, a key microbicidaleffector mechanism of murine macrophages, in the absence of caspase-1. While ΔgerH BAspores induced the expression of NO in macrophages of caspase-1+/+ mice, there wasminimal NO generated following the same stimulation in macrophages of caspase-1−/− mice(Fig. 3D). However, when macrophages from caspase-1−/− mice were cultured with rIL-1β,there was an increased production of NO in response to BA spores (Fig. 3E), and this wasconsistent with the increased killing observed following the addition of rIL-1β tomacrophages from caspase-1−/− mice (Fig. 2A). In contrast to another report that LFsuppresses the release of NO from macrophages [5], our data showed that spores induced theproduction of NO by macrophages, and caspase-1 activity was required for NO release.

Kang et al. Page 3

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Thus, BA induces NO at a stage in the infection cycle prior to LT expression, and caspase-1is required.

BA killing requires spore-induced IFN-β to mediate STAT1 and caspase-1 activationActivation of the JAK-STAT pathway by either type I IFN (IFN-α and IFN-β) or IFN-γacting on the IFN-α/β or IFN-γ receptors, respectively, causes phosphorylation anddimerization of STAT1, which then translocates to the nucleus where it binds to ISRE andinduces inflammatory gene expression [17]. STAT1 activation is required for caspase-1activity [18], and ultimately, IL-1β protein expression [15]. In the absence of STAT1, therewas a decrease in caspase-1 activity (Fig. 4A), IL-1β protein production (Fig. 4B) and NOactivity (Fig. 4C) following macrophage stimulation with either BA spores or LPS.Consistent with these data, macrophages from STAT1−/− mice were unable to kill BA;however, exogenous IL-1β restored macrophage bactericidal activity (Fig. 4D).

Since macrophages have a relatively limited capacity to phosphorylate STAT1 through theinduction of IFN-γ, we studied the potential role of IFN-β in the microbicidal activity ofmacrophages. IFN-β is induced in a MyD88-independent fashion through either Toll-likereceptor (TLR) 3 or TLR4 due to the selective engagement of the Toll/IL-1R domain-containing adaptor inducing IFN-β by these TLR [19, 20].

Consequently, given the importance of STAT1 activation to caspase-1 activity, we nextdetermined whether BA spores were capable of inducing phosphorylation of STAT1. Usingmacrophages derived from WT and IFN-β−/− mice, we found that in the absence of IFN-β,there was no phosphorylation of STAT1 (Fig. 5A); however, addition of ΔgerH BA sporesto WT macrophages resulted in phosphorylation of serine 727, but not of tyrosine 701residues of STAT1, whereas LPS induced the phosphorylation of STAT1 at both sites (Fig.5A). Macrophages from IFN-β−/− mice also had diminished caspase-1 activity (Fig. 5B) andIL-1β protein expression (Fig. 5C) after stimulation with BA spores or LPS. As was the casewith macrophages from STAT1−/− mice, here, too, the inability of macrophages from IFN-β−/− mice to kill BA was reconstituted by the addition of rIL-1β (Fig. 5D). Further, IFN-β−/−

mice challenged with 1×106 CFU also were more susceptible to infection with the Sterne34F2 strain of BA (4/5 WT C57BL/6 mice survived versus 1/5 IFN-β−/− mice, p<0.05).Thus, like LPS, BA spores require IFN-β and phosphorylation of STAT1 for caspase-1activity, and this pathway has a critical role in host defenses against BA.

Since we previously observed that ingestion of BA by macrophages was required for NOgeneration and BA killing [8], we examined whether phagocytosis of BA spores bymacrophages was involved in the induction of IFN-β expression. We therefore exposedmacrophages to either LPS or ΔgerH BA spores, in the absence or presence of cytochalasinD, which prevents phagocytosis by disrupting the actin cytoskeleton. While both LPS andΔgerH BA spores induced IFN-β mRNA, BA spores were not able to induce IFN-β mRNAexpression in macrophages in the presence of cytochalasin D (Supporting Information Fig.2), unlike the case with LPS. This is consistent with our earlier finding that uptake of BAspores by macrophages was required for iNOS mRNA expression [8].

BA spores and LT induce IL-1β by distinct signaling pathwaysPrevious investigators reported that BA LT activates caspase-1 resulting in the extracellularrelease of IL-1β and cell death [11, 12]. LT induced cell death only in macrophages whoseactivation of caspase-1 occurred by Nalp1b. Nalp1b, a member of the NOD receptor family,is a key determinant of mouse macrophage susceptibility to LT [21], and macrophagesresistant to the lethal action of LT lacked this particular NOD-like receptor (NLR) protein.The association of NLR proteins and caspase-1 in a multiprotein complex has been termed

Kang et al. Page 4

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

an “inflammasome” [22]. Since we find that BA spores induce caspase-1 activity and IL-1βsecretion but no cell death in macrophages from both LT-sensitive and LT-resistant mice,we decided to compare the mechanisms by which LT and BA spores induce IL-1β secretion.

When examined in LT-resistant C57BL/6 mouse macrophages at 2 h, BA spores, but notLT, induced the secretion of IL-1β protein, as previously reported [23] (data not shown).Further, only the BA spores induced the expression of IL-1β mRNA at 2 h (SupportingInformation Fig. 3).

Since BA spores required a MyD88-independent signaling pathway to induce IL-1β proteinsecretion (Fig. 4 and 5), we examined whether LT similarly required signaling through thispathway. Macrophages from STAT1−/− (Fig. 6, upper panels) and IFN-β−/− (Fig. 6, lowerpanels) mice were treated with either LT or BA spores and IL-1β secretion, and caspase-1activity determined. There was an approximately threefold difference in the amount of IL-1βinduced by BA spores in the LT-susceptible (STAT1+/+ mice on a 129S background) versusthe LT-resistant mice (IFN-β+/+ mice on a C57BL/6 background), while there was a morethan tenfold difference in LT-induced IL-1β secretion between the two WT strains with thediffering backgrounds. STAT1+/+ mice, on an LT-sensitive (129S) background, had robustIL-1β secretion and caspase-1 activity in response to LT, and these responses were notdiminished in STAT1−/− mice (Fig. 6A and B, upper panels). Similarly, while at 24 h therewas less IL-1β secretion and caspase-1 activity in response to LT in IFN-β+/+ mice on anLT-resistant (C57BL/6) background, there was no decrease in these responses inmacrophages from IFN-β−/− mice (Fig. 6A and B, lower panels).

The lack of difference in LT-induced IL-1β secretion and caspase-1 activity between eachpair of WT and knockout mice is in contrast to the striking differences observed for BAspore-induced activities. Thus the requirement for a MyD88-independent signaling pathwayfor BA spore-induced IL-1β, but not for LT-induced IL-1β, further suggests that BA sporeand LT induce the caspase-1 activation and IL-1β secretion by distinct signaling pathways.

Since LT induces IL-1β secretion in LT-susceptible macro-phages in the absence of IL-1βmRNA induction, we hypothesized that the IL-1β in these culture supernatants was a resultof cell death, as previously suggested [11,12, 24]. We reasoned that if BA spores and LTinduced IL-1β by the same pathway, then we would observe a similar ability to cause celldeath in LT-susceptible, but not in LT-resistant macrophages. Thus we compared theinduction of cell death and IL-1β protein secretion in murine macrophages from 129S mice,known to be sensitive to anthrax LT, to murine macrophages from C57BL/6 and A/J mice,which are known to be resistant to LT [21]. A/J mice were included because this strain ofmouse is more susceptible to lethal infection with BA than other strains [25, 26].

As expected, LT induced cell death in macrophages from LT-susceptible (129S) mice asassessed by by annexin V staining (Fig. 7A), trypan blue dye exclusion (Fig. 7B) andcaspase-3 activity (data not shown), whereas it did not do so in macrophages from LT-resistant (C57BL/6) and LT-resistant, but lethal infectionsusceptible (A/J) mice, even at 24h. LT caused significantly more cell death than did the BA spores, and its level increasedover 24 h following the initial treatment (data not shown). These findings are similar toresults obtained when macrophages were treated with staurosporine, a potent inhibitor ofphospholipid/Ca++-dependent protein kinase and inducer of cell death [27]. In contrast, themacrophages treated with spores from the BA Sterne strain (including the germination-deficient Sterne mutant, ΔgerH), exhibited much reduced cell death. Thus, unlike LT, BAspores are less likely to induce cell death following their addition to macrophages from LT-susceptible mice. These data are in agreement with previous suggestions that LT-associatedIL-1β secretion is more likely a consequence of cell death than of new IL-1β synthesis [23].

Kang et al. Page 5

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

The addition of BA spores to cultures of macrophages from C57BL/6,129S and A/J miceshowed robust production of IL-1β by spores, but most was cell-associated (Fig. 7C). Incontrast, LT induced IL-1β production in 129S macrophages but minimally in C57BL/6 andA/J macrophages, and most of the LT-induced IL-1β in 129S cells was secreted. Thisfinding suggests that the induction of IL-1β by BA spores does not require Nalp1b. TheELISA assay detects both the 17-kDa and 33-kDa forms of IL-β, but since we, like others,do not find 17-kDa IL-1β in macrophage culture lysates by western blot (data not shown), itis likely that the cell-associated IL-1β is primarily measuring the pro-IL-1β (33-kDa) form.Thus, these data are consistent with the observation that unlike BA spores, LT inducesrelatively little pro-IL-1β.

Therefore, we hypothesized that most of the secreted IL-1β following LT is from pre-formed stores. To confirm this hypothesis, we treated macrophages from 129S mice witheither LT or BA spores in the presence or absence of cycloheximide (Fig. 7D). In theabsence of cycloheximide, each of the agonists induced IL-1β secretion; however, whereascycloheximide inhibited BA spore-induced IL-1β secretion, the presence of this proteinsynthesis inhibitor had little effect on LT-induced IL-1β secretion. These data are consistentwith the notion that LT-induced IL-1β secretion is due to LT-induced caspase-1 activityacting on pre-existing pools of pro-IL-1β that are released upon LT-induced cell death. Incontrast, the induction of IL-1β following BA spores is due primarily to IL-1β mRNAexpression and spore-induced caspase-1 activity acting on the newly synthesized pro-IL-1βprotein.

DiscussionThese data strongly support the hypothesis that the expression of IL-1β protein in responseto BA spores is critical to host defenses against BA. In the absence of caspase-1 or IL-1βthere is an increased susceptibility to lethal infection with BA spores. Indeed, to ourknowledge, with the exception of differences in leptin expression, the increasedsusceptibility of IL-1β−/− mice to lethal BA infection is the only phenotype attributed tothese mice. Further, we find that the BA spore-induced regulation of IL-1β proteinexpression involves cooperation between a MyD88-dependent component (induction of pro-IL-1β), and a MyD88-independent component by which IFN-β-induced STAT1 activation isrequired for optimal caspase-1 processing of the inactive pro-IL-1β to the active cytokine. Inthe absence of any of these critical components (IL-1β expression, caspase-1 activity, IFN-β, STAT1 activation, or NO production), the host is unable to kill the BA. Since IL-1βexpression is induced both by non-germinating spores prior to LT expression and by the LTexpressed by vegetative bacilli, its induction by the two agonists may occur by distinctproximal signaling pathways and may serve different roles during each stage of infection.

We recently reported that LPS induction of IFN-β via a MyD88-independent pathway ofTLR4 led to the activation of STAT1 at both tyrosine 701 and serine 727 residues, and thephosphorylated STAT1 was necessary for optimal caspase-1 activity to process the pro-IL-1β to active IL-1β [15, 19]. Thus, our findings in this study that BA spores induce IL-1βvia cooperation between both MyD88-dependent and -independent pathways are similar toour findings with LPS. Since then, a similar role for IFN-β was reported for Francisellatularensis [28]. Unlike the case with LPS, however, BA spores do not appear to use TLR4[9, 29], nor are they recognized by TLR2, as one of us reported for F. tularensis [30].Further, in the present study we show that unlike the case with LPS, the BA spore must beingested by the macrophage for IFN-β mRNA to be expressed (Supporting Information Fig.2). This is consistent with our earlier finds that ingestion of BA spores is required for iNOSexpression [8], as well as reports from other laboratories that uptake of bacteria is aprerequisite for cytokine signaling [31, 32]. We recently reported that while macrophages

Kang et al. Page 6

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

killed Sterne 34F2 spores in vitro by a mechanism that required NO formation, they wereunable to kill non-germinating BA spores and we therefore concluded that the macrophageskilled the emerging vegetative bacilli [8].

Previous investigators have added germination-proficient BA spores to macrophage anddendritic cell cultures under conditions that favored germination to bacillary forms and theexpression of both LT and anthrolysin O, both of which have been associated with cell death[33–35]. This has made it difficult to differentiate between the effects of BA spores andvegetative bacilli on macrophage cytokine responses. In contrast, in our studies we observedthat live BA spores induced IL-1β mRNA and protein in macrophages under conditions inwhich there was little, if any, germination. In the absence of germination, it is unlikely thatLT and/or anthrolysin O were expressed in adequate quantities to induce IL-1β. Thus theIL-1β response in our studies is likely attributable to the spore itself, and not to the ability ofLT to induce IL-1β [2, 11, 12, 23]. This is not surprising since peptidoglycan derived from anon-toxin-producing species of Bacillus also induced IL-1β [36].

Our data show that at relatively low doses, BA spores induce IL-1β mRNA and protein aswell as caspase-1 activity, each of which is required for BA killing both in vitro and in vivo,but minimal cell death (Fig. 6, Supporting Information Fig. 3). In contrast, we confirm thefindings of others [23] that LT induces IL-1β protein, and cell death, but little IL-1β mRNAexpression (Supporting Information Fig. 3). While the administration of a high dose of LT(100 μg) induced IL-1β in vivo, the lack of LT-induced IL-1β mRNA expression led to thespeculation that LT liberated pre-formed IL-1β from lysed cells [23]. Consistent with thathypothesis, we show that LT-stimulated 129S macrophages pre-treated with cycloheximideare still able to robustly secrete IL-1β, unlike the case with BA spore stimulation (Fig. 7E).

Multiple reports document the inconsistent ability of LT to induce cytokines [2, 11, 23, 36–38]. One potential explanation for these variable results is the use of inbred strains of micewith variable sensitivity to LT-induced macrophage necrosis. LT has been shown to inducemacrophage cell death in several mouse strains [5, 21, 23, 33, 36–40]via a p38-dependentpathway [39]. Susceptibility of mouse macrophages to LT was linked to the extremelypolymorphic gene locus NALP1, which encodes Nalp1b, an intracellular receptor in theNLR family. The NLR family of intracellular receptors forms a multiprotein complex(“inflammasome”) with various adaptor proteins that leads to the activation of caspase-1[22] and the processing and secretion of IL-1β [23].

Boyden and Dietrich [21] showed LT-activated caspase-1 at an early (2 h) time point only inmacrophages from the susceptible mouse strain, 129S1, as well as from transgenicNalp1b129S1 mice, but not in macrophages from LT-resistant mouse strains C57BL/6 orDBA2. In contrast to the variable induction of IL-1β protein and cell death in LT-resistant(A/J and C57BL/6) and -susceptible (129S) mice, we find that BA spores consistentlyinduced IL-1β mRNA and protein in the absence of cell death in macrophages from bothLT-susceptible and -resistant strains of mice (Fig. 6 and 7; Supporting Information Fig. 3).Further, another pathway of macrophage cell death required TLR4-mediated activation ofprotein kinase R by germination-proficient BA [41]; however, we find that BA spores caninduce IL-1β in the absence of TLR4 [9]. The activation of caspase-1 by BA spores maytherefore require NLR different from the Nalp1b required by LT. For example, Francisella-induced caspase-1 activation requires the adaptor protein ASC [28], and flagellin fromSalmonella and Legionella signals through an Ipaf inflammasome [42]. We are currentlyexamining those possibilities for BA.

BA spores (or products) may gain access to the cytosolic sensors from the phagolysosomeeither through the elaboration of a cholesterol-dependent cytolysin, anthrolysin O, similar to

Kang et al. Page 7

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

other cholesterol-dependent cytolysin-elaborating bacteria, such as Listeria [33], or throughpannexin channels. Pannexin-1 is a hemichannel protein that interacts with the P2X7receptor, which has been proposed as a mechanistic link between bacterial stimuli and thecryopyrin inflammasome [43]. Thus, the mechanisms of IL--β induction and release by LTdiffer markedly from that by the spore and may reflect different functions. Whereas the BALT-induced IL-1β is associated with a mechanism that enables BA to incapacitate themacrophage and escape host defenses, we found that the early BA spore-induced IL-1βenables the host innate immune system to limit BA infection.

Based on previous studies in which antagonism of IL-1β or use of anti-oxidants protectedmice from the lethal effects of LT, Kalns et al. [44] delivered LT to mice via infection withlive, germination-proficient BA spores. Contrary to their expectation that mice lacking eitherresponsiveness to IL-1β or ability to generate free radicals (i.e. NO) would be protectedfrom lethal infection, they observed an accelerated death in both IL-1R receptor−/− andiNOS−/− mice, each associated with an increased bacterial load. Our findings that early hostdefenses against BA require both IL-1β and NO production, while the IL-1β induced by LTis associated with the later lethal events, provide an explanation for their observations, anddemonstrate that the BA spores and LT generate the IL-1β by different mechanisms.

Based on some of the preceding data with LT, it has been suggested that the IL-1β-generating complex may be a therapeutic target for anthrax infection [11, 12], and in thelater stages of infection, when significant amounts of LT are expressed, this may be areasonable hypothesis. However, such a strategy may be a “two-edged sword”: In this study,we showed that non-germinating BA spores induced an IL-1β response at a stage in itsreplicative cycle that precedes LT expression. Given our data showing the importance ofIL-1β expression to the capacity of the macrophage to kill the emerging vegetative bacilli,the premature inhibition of IL-1β activity may enable the organism to establish an importantfoothold within the host and disseminate; however, given that LT requires a specific NLR,targeting that/those NLR may be a more attractive therapeutic target for the toxemia ofanthrax than a broad-based inhibition of caspase-1.

Materials and methodsSpores

Germination-proficient BA Sterne 34F2 and a congenic germination-deficient mutant strain,ΔgerH, were used during this study [13, 14]. The spores were prepared as previouslydescribed [8]. Sterne spores were used for in vivo studies and killing assays, and ΔgerHspores were for in vitro cytokine induction.

Mice129S6/SvEvTac-Stat<tm1rds (STAT1−/−), 129S6/SvEvTac (STAT1+/+) and H2r-IL1βtm1Lvp

(IL-1β−/−) mice were purchased from Taconic Farms (Hudson, NY). A/J and C57BL/6Jmice were obtained from Jackson Laboratory (Bar Harbor, ME). C57BL/6J mice were usedas the control strain for IFN-β−/− mice (backcrossed more than eight times onto a C57BL/6background). These mice, kindly provided by Dr. Eleanor Fish (University of Toronto,Toronto, Canada), were bred at the University of Maryland School of Medicine. C3H/HeNCr caspase-1+/+ (Charles River Laboratories, Wilmington, MA) and caspase-1−/− micewere also used in this study. The latter mice were originally provided by Dr. Paul Jolicoeur(Montreal, Canada) and have been backcrossed onto a C3H background for more than tengenerations. MyD88−/− mice were originally provided by Dr. S. Akira (Osaka University,Osaka, Japan) and bred at the University of Maryland School of Medicine. Since they werebackcrossed more than eight times onto a C57BL/6 background, C57BL/6J mice were used

Kang et al. Page 8

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

as WT controls. All experiments were conducted with Institutional Animal Care and UseCommittee approval.

Mice and macrophage infectionCaspase-1+/+ or caspase-1−/− mice were injected with Sterne 34F2 strain spores i.p. andobserved for mortality. Results were accumulated from three separate experiments. At 48and 72 h following exposure, mice were sacrificed, and lungs, spleens and liver werecollected, homogenized and quantitatively cultured on L-agar. IL-1β−/− mice and WTcontrols (C57BL/6J) were similarly challenged with Sterne 34F2 strain spores i.p.Thioglycollateelicited peritoneal macrophages were collected, cultured and infected withspores at a multiplicity of infection (MOI) of 1, and macrophage-associated BA weredetermined as described previously [14].

ELISA for IL-1βMacrophages were stimulated with a TLR4 agonist (LPS derived from Escherichia coliO111:B4; List Laboratories Inc., Campbell, CA), BA spores, or LT (rPA and LF from BA,List Laboratories Inc.). Culture supernatants were assayed for mouse IL-1β by ELISA(DuoSet; R&D Systems). The monoclonal antibodies used in this assay were generated withrmIL-1β (17 kDa) as the immunogen. Using this assay, the lower limit of detection of IL-1βwas 3.9 pg/mL.

Caspase-1 assayAssay of caspase-1 activity in vivo was performed as previously described [45]. For assay ofcaspase-1 activity in vitro, 1 × 106 murine peritoneal macrophages were infected with sporesovernight at an MOI of 1. Extracts were centrifuged at 10 000 ×g for 5 min at 4°C and thecaspase-1 activity was measured [45]. Reactions with samples (enzyme) mixed withsubstrate (YVAD-pNA) were run. The total increase in the optical density at 405 nm versusthat of the sample-alone wells then was calculated. Caspase-1 activity was expressed asfollows: (maximum OD405/μg protein) × 10 000 [45].

Measurement of cell deathAnnexin V staining and caspase-3 assays were performed according to the manufacturer'sinstructions using an apopNexin-FITC apoptosis Detection Kit (Chemicon Inc., Temecula,CA) and caspase-3 kit (ApoTarget; BioSource Int. Inc., Rockville, MD), respectively.

Nitrite measurementNO production was determined by the Griess reaction as previously described [8]. The limitof detection was 0.781 μM. The data were recorded and analyzed using SOFTmax version4.6 software (Molecular Devices, Menlo Park, CA).

Real-time PCRIFN-β and IL-1β mRNA were measured by real-time PCR from RNA harvested fromperitoneal macrophages obtained from C57BL/6 and MyD88−/− mice, as previouslydescribed [46]. Briefly, cells were lysed with RNA-STAT60 (Tel-Test Inc., Friendship, TX).RNA was extracted and precipitated with chloroform and isopropanol. The RNA wasreverse-transcribed using AMV reverse transcriptase (Promega) and poly-T priming. ThecDNA was quantified by realtime PCR using SYBR Green master mix (AppliedBiosystems) and the ABI prism 7900 HT cycler. Intron-spanning primers were designedusing the Primer Express 2.0 program (Applied Biosystems). Relative gene expression wascalculated using the ΔCt method and normalized to the housekeeping gene HPRT for IL-1βand GAPDH for IFN-β (1.8 ^ [CtHPRT-CtGene]). Statistical analysis was performed by one-

Kang et al. Page 9

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

way ANOVA with repeated measures, followed by Dunnett's multiple comparison post-hoctest with p≤0.05 chosen as the level of significance. The primers were: IFN-β: sense, 5′-CACTTGAAGAGCTATTACTGGAGGG-3′; anti-sense, 5′-CTCGGACCACCATCCAGG-3′; IL-1β: sense, 5′-ACAGAATATCAACCAACAAGTGATATTCTC-3′; antisense, 5′-GATTCTTTCCTTTGAGGCCCA-3′.

Western blotMacrophages stimulated with spores were lysed in lysis buffer (50 mM Tris buffer, pH 7.4,150 mM NaCl, 1% NP-40 and 2 mM DTT), resolved with 10% SDS-PAGE and transferredto a nitrocellulose membrane (Hybond™ECL™; Amersham Bioscience). Rabbit polyclonalantibodies to total STAT1 and to phospho-STAT1-serine 727 (Cell Signaling Technology,Beverly, MA), respectively, and mouse anti-phospho-STAT1-tyrosine 701 (Zymed, SanFrancisco, CA) were used as previously described [15].

Statistical analysisAll data were expressed as mean ± SD. Student's t-test was used to analyze the data forstatistical significance (GraphPad Prism), and significance was set at p<0.05.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThis work was supported by NIH NIAID Mid-Atlantic Regional Center of Excellence grant U54 AI-057168 (A.C.and S.N.V.), R21 AI-059093 (A.C.) and RO1 AI-18797 (S.N.V.).

References1. Dixon TC, Meselson M, Guillemin J, Hanna PC. Anthrax. N Engl J Med. 1999; 341:815–826.

[PubMed: 10477781]

2. Hanna PC, Acosta D, Collier RJ. On the role of macrophages in anthrax. Proc Natl Acad Sci USA.1993; 90:10198–10201. [PubMed: 8234277]

3. Guidi-Rontani C, Weber-Levy M, Labruyere E, Mock M. Germination of Bacillus anthracis sporeswithin alveolar macrophages. Mol Microbiol. 1999; 31:9–17. [PubMed: 9987105]

4. O'Brien J, Friedlander A, Dreier T, Ezzell J, Leppla S. Effects of anthrax toxin components onhuman neutrophils. Infect Immun. 1985; 47:306–310. [PubMed: 3917427]

5. Pellizzari R, Guidi-Rontani C, Vitale G, Mock M, Montecucco C. Anthrax lethal factor cleavesMKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha.FEBS Lett. 1999; 462:199–204. [PubMed: 10580119]

6. Moayeri M, Leppla SH. The roles of anthrax toxin in pathogenesis. Curr Opin Microbiol. 2004;7:19–24. [PubMed: 15036135]

7. Baillie L, Hibbs S, Tsai P, Cao GL, Rosen GM. Role of superoxide in the germination of Bacillusanthracis endospores. FEMS Microbiol Lett. 2005; 245:33–38. [PubMed: 15796976]

8. Raines KW, Kang TJ, Hibbs S, Cao GL, Weaver J, Tsai P, Baillie L, et al. Importance of nitricoxide synthase in the control of infection by Bacillus anthracis. Infect Immun. 2006; 74:2268–2276.[PubMed: 16552057]

9. Basu S, Kang TJ, Chen WH, Fenton MJ, Baillie L, Hibbs S, Cross AS. Role of Bacillus anthracisspore structures in macrophage cytokine responses. Infect Immun. 2007; 75:2351–2358. [PubMed:17339355]

Kang et al. Page 10

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

10. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, Flavell RA. Altered cytokineexport and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science. 1995;267:2000–2003. [PubMed: 7535475]

11. Cordoba-Rodriguez R, Fang H, Lankford CS, Frucht DM. Anthrax lethal toxin rapidly activatescaspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18. J Biol Chem.2004; 279:20563–20566. [PubMed: 15010463]

12. Popov SG, Popova TG, Grene E, Klotz F, Cardwell J, Bradburne C, Jama Y, et al. Systemiccytokine response in murine anthrax. Cell Microbiol. 2004; 6:225–233. [PubMed: 14764106]

13. Weaver J, Kang TJ, Raines KW, Cao GL, Hibbs S, Tsai P, Baillie L, et al. The protective role ofBacillus anthracis exosporium in macrophage-mediated killing by nitric oxide. Infect Immun.2007; 75:3894–3901. [PubMed: 17502390]

14. Kang TJ, Fenton MJ, Weiner MA, Hibbs S, Basu S, Baillie L, Cross AS. Murine macrophages killthe vegetative form of Bacillus anthracis. Infect Immun. 2005; 73:7495–7501. [PubMed:16239551]

15. Joshi VD, Kalvakolanu DV, Chen W, Kang TJ, Zhang L, Thomas TE, Vogel SN, Cross AS. A rolefor STAT1 in the regulation of lipopolysaccharide-induced interleukin-1β expression. J InterferonCytokine Res. 2006; 26:739–747. [PubMed: 17032168]

16. Weiner MA, Hanna PC. Macrophage-mediated germination of Bacillus anthracis endosporesrequires the gerH operon. Infect Immun. 2003; 71:3954–3959. [PubMed: 12819082]

17. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, et al. Targeteddisruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STATsignaling pathway. Cell. 1996; 84:431–442. [PubMed: 8608597]

18. Chin YE, Kitagawa M, Kuida K, Flavell RA, Fu XY. Activation of the STAT signaling pathwaycan cause expression of caspase 1 and apoptosis. Mol Cell Biol. 1997; 17:5328–5337. [PubMed:9271410]

19. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, et al. TLR4,but not TLR2, mediates IFN-β-induced STAT1α/β-dependent gene expression in macrophages.Nat Immunol. 2002; 3:392–398. [PubMed: 11896392]

20. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, et al. Role ofadaptor TRIF in the MyD88-independent Tolllike receptor signaling pathway. Science. 2003;301:640–643. [PubMed: 12855817]

21. Boyden ED, Dietrich WF. Nalp1b controls mouse macrophage susceptibility to anthrax lethaltoxin. Nat Genet. 2006; 38:240–244. [PubMed: 16429160]

22. Martinon F, Burns K, Tschopp J. The inflammasome: A molecular platform triggering activationof inflammatory caspases and processing of proIL-1β. Mol Cell. 2002; 10:417–426. [PubMed:12191486]

23. Moayeri M, Haines D, Young HA, Leppla SH. Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice. J Clin Invest. 2003; 112:670–682. [PubMed:12952916]

24. Popov SG, Villasmil R, Bernardi J, Grene E, Cardwell J, Wu A, Alibek D, et al. Lethal toxin ofBacillus anthracis causes apoptosis of macrophages. Biochem Biophys Res Commun. 2002;293:349–355. [PubMed: 12054607]

25. Welkos SL, Keener TJ, Gibbs PH. Differences in susceptibility of inbred mice to Bacillusanthracis. Infect Immun. 1986; 51:795–800. [PubMed: 3081444]

26. Harvill ET, Lee G, Grippe VK, Merkel TJ. Complement depletion renders C57BL/6 mice sensitiveto Bacillus anthracis Sterne strain. Infect Immun. 2005; 73:4420–4422. [PubMed: 15972541]

27. Miwa K, Asano M, Horai R, Iwakura Y, Nagata S, Suda T. Caspase 1-independent IL-1βreleaseand inflammation induced by the apoptosis inducer Fas ligand. Nat Med. 1998; 4:1287–1292.[PubMed: 9809553]

28. Henry T, Brotcke A, Weiss DS, Thompson LJ, Monack DM. Type I interferon signaling isrequired for activation of the inflammasome during Francisella infection. J Exp Med. 2007;204:987–994. [PubMed: 17452523]

29. Hughes MA, Green CS, Lowchyj L, Lee GM, Grippe VK, Smith MF Jr, Huang LY, et al. MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice:

Kang et al. Page 11

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Implications for Toll-like receptor signaling. Infect Immun. 2005; 73:7535–7540. [PubMed:16239556]

30. Katz J, Zhang P, Martin M, Vogel SN, Michalek SM. Toll-like receptor 2 is required forinflammatory responses to Francisella tularensis LVS. Infect Immun. 2006; 74:2809–2816.[PubMed: 16622218]

31. Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innateimmune response. Immunity. 2006; 24:93–103. [PubMed: 16413926]

32. Philpott DJ, Girardin SE. The role of Toll-like receptors and Nod proteins in bacterial infection.Mol Immunol. 2004; 41:1099–1108. [PubMed: 15476921]

33. Park JM, Ng VH, Maeda S, Rest RF, Karin M. Anthrolysin O and other gram-positive cytolysinsare Toll-like receptor 4 agonists. J Exp Med. 2004; 200:1647–1655. [PubMed: 15611291]

34. Brittingham KC, Ruthel G, Panchal RG, Fuller CL, Ribot WJ, Hoover TA, Young HA, et al.Dendritic cells endocytose Bacillus anthracis spores: Implications for anthrax pathogenesis. JImmunol. 2005; 174:5545–5552. [PubMed: 15843553]

35. Pickering AK, Osorio M, Lee GM, Grippe VK, Bray M, Merkel TJ. Cytokine response to infectionwith Bacillus anthracis spores. Infect Immun. 2004; 72:6382–6389. [PubMed: 15501768]

36. Vermeulen MW, Gray GR. Processing of Bacillus subtilis peptidoglycan by a mouse macrophagecell line. Infect Immun. 1984; 46:4476–4483.

37. Moayeri M, Martinez NW, Wiggins J, Young HA, Leppla SH. Mouse susceptibility to anthraxlethal toxin is influenced by genetic factors in addition to those controlling macrophage sensitivity.Infect Immun. 2004; 72:4439–4447. [PubMed: 15271901]

38. Erwin JL, DaSilva LM, Bavari S, Little SF, Friedlander AM, Chanh TC. Macrophage-derived celllines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin.Infect Immun. 2001; 69:1175–1177. [PubMed: 11160016]

39. Park JM, Greten FR, Li ZW, Karin M. Macrophage apoptosis by anthrax lethal factor through p38MAP kinase inhibition. Science. 2002; 297:2048–2051. [PubMed: 12202685]

40. Friedlander AM, Bhatnagar R, Leppla SH, Johnson L, Singh Y. Characterization of macrophagesensitivity and resistance to anthrax lethal toxin. Infect Immun. 1993; 61:245–252. [PubMed:8380282]

41. Hsu LC, Park JM, Zhang K, Luo JL, Maeda M, Kaufman RJ, Eckmann L, et al. The protein kinasePKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature. 2004;428:341–345. [PubMed: 15029200]

42. Lamkanfi M, Kanneganti TD, Franchi L, Nunez G. Caspase-1 inflammasomes in infection andinflammation. J Leukoc Biol. 2007; 82:220–225. [PubMed: 17442855]

43. Kanneganti TD, Lamkanfi M, Kim YG, Chen G, Park JH, Franchi L, Vandenabeele P, Nunez G.Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasomeindependent of Toll-like receptor signaling. Immunity. 2007; 26:433–443. [PubMed: 17433728]

44. Kalns J, Scruggs J, Millenbaugh N, Vivekananda J, Shealy D, Eggers J, Kiel J. TNR receptor 1,IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection. BiochemBiophys Res Commun. 2002; 292:41–44. [PubMed: 11890668]

45. Joshi VD, Kalvakolanu DV, Hebel JR, Hasday JD, Cross AS. Role of caspase 1 in murineantibacterial host defenses and lethal endotoxemia. Infect Immun. 2002; 70:6896–6903. [PubMed:12438367]

46. Toshchakov VU, Basu S, Fenton MJ, Vogel SN. Differential involvement of BB loops of Toll-IL-1resistance (TIR) domain-containing adapter proteins in TLR4- versus TLR2-mediated signaltransduction. J Immunol. 2005; 175:494–500. [PubMed: 15972684]

Abbreviations

BA Bacillus anthracis

LF lethal factor

LT lethal toxin

Kang et al. Page 12

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NLR NOD-like receptor

PA protective antigen

Kang et al. Page 13

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 1.Caspase-1 has a critical role in host defenses against BA infection in vivo. (A, C) Mice wereinfected i.p. with spores from BA Sterne 34F2 (106 CFU) and their survival was observedfor 7 days. (B) Liver, spleen and lungs from caspase-1+/+ (open bars) and caspase-1−/− (solidbars) mice were collected at day 2 and 3 post-infection, homogenized and plated on L-agarfor enumeration of CFU. Each point represents the mean and SD of at least four animalschallenged in each group.

Kang et al. Page 14

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 2.Caspase-1 is essential for host defenses against BA infection in vitro. Macrophages fromcaspase-1−/− (A) and IL-1β−/− (B) mice were pre-treated with various doses of rIL-1β orrIL-18 for 15 h, infected with Sterne 34F2 spores (MOI 1), and CFU were determined at 5 has described previously [10]. Microbial killing by these macrophages was compared to thatof macrophages from WT controls. Data are representative of at least two independentexperiments, each done in triplicate; *p<0.05, **p<0.01 compared to the killing by untreatedmacrophages from caspase-1−/− and IL-1β−/− mice.

Kang et al. Page 15

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 3.Caspase-1 activation is required for IL-1β and NO induction by BA spore. Macrophages(106) from caspase-1+/+ and caspase-1−/− mice were treated with LPS (100 ng/mL) andΔgerH spores (106 CFU) for 15 h, and lysates were assayed for caspase-1 activity (A), andsupernatants for IL-1β protein (C) and NO levels (D). (B) Caspase-1 activity in vivo wasalso determined using liver and spleen from infected mice. Livers and spleens of C3H/HeNmice were isolated at 16 h after treatment with LPS (75 μg) or spores (106 CFU),respectively, and tissue extracts prepared. Caspase-1 activity was calculated as described inMaterials and methods. (E) Macrophages from caspase-1−/− mice were pre-treated withrIL-1β, infected with Sterne 34F2 spores (MOI 1), and cell culture supernatants werecollected for NO level. Data are representative of at least two independent experiments, eachdone in triplicate; *p<0.05, **p<0.01.

Kang et al. Page 16

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 4.STAT1 activation by BA spores is required for caspase-1-mediated IL-1β and NOproduction, and BA killing in vitro. Macrophages (106) from STAT1+/+ and STAT1−/− micewere treated with LPS (100 ng/mL) and ΔgerH spores (106 CFU) for 15 h, and lysates wereassayed for caspase-1 activity (A), and supernatants for IL-1β protein (B) and NO levels(C). (D) Macrophages from STAT1+/+ and STAT1−/− mice were pre-treated with rIL-1β(0.2 ng/mL) for 15 h and infected with Sterne 34F2 spores. CFU were determined at 3 and 5h. Data are representative of at least three independent experiments, each done in triplicate;*p<0.05, **p<0.01 compared to STAT1−/− cells.

Kang et al. Page 17

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 5.BA spores induce IFN-β-mediated caspase-1 and STAT1 activation required for BA killingin vitro. Macrophages from IFN-β+/+ and IFN-β−/− mice (A) were treated with LPS (100 ng/mL), or ΔgerH spores (MOI 1) for 2 h. Cell lysates were assayed for STAT1 activation bywestern blot. Macrophages (106) from IFN-β+/+ and IFN-β−/− mice were treated with LPS(100 ng/mL) and ΔgerH spores (106 CFU) for 15 h, and lysates were assayed for caspase-1activity (B), and supernatants for IL-1β protein (C). (D) Macrophages (106) from IFN-β+/+

and IFN-β−/− mice were pre-treated with and without rIL-1β (0.2 ng/mL) for 15 h andinfected with Sterne 34F2 spores. CFU were determined at 5 h. Data are representative of atleast three independent experiments, each done in triplicate; *p<0.05, **p<0.01 compared toIFN-β−/− macrophages.

Kang et al. Page 18

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 6.The induction of caspase-1 activation and IL-1β secretion by LT is not dependent on IFN-βand STAT1 signaling pathway. Macrophages (2×106) from STAT1+/+, STAT1−/− (A, B;both on a 129S background, which is LT-susceptible), IFN-β+/+ and IFN-β−/− (C, D; both ona C57BL/6 background, which is LT-resistant) mice were treated with LPS (100 ng/mL),BA ΔgerH, Sterne 34F2 spores (MOI 1), LT (2.5 μg PA + 1.0 μg LF/mL) for 24 h, and thencell culture supernatants and lysates were collected, respectively, for determination of IL-1β(A, C) and caspase-1 activity (B, D); *p<0.05, **p<0.01 compared to cells from STAT1−/−

(A, B) or IFN-β−/− (C, D) mice.

Kang et al. Page 19

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 7.Cell lysis by LT, not spore, causes the release of IL-1β from cells. Macrophages (2×106)from C57BL/6, 129S and A/J mice were treated with BA ΔgerH, Sterne 34F2 spores (MOI1), LT (2.5 μg PA + 1.0 μg LF/mL) for 3 h, and then cells were analyzed by annexin Vstaining (A) and trypan blue staining (B). (C) Macrophages (2×106) from C57BL/6, 129Sand A/J mice were treated with LPS, BA ΔgerH, Sterne 34F2 spores, LT for 3 h, and thenculture supernatants and cell lysates were collected for total IL-1β (supernatants and lysate,open bars) and the secreted IL-1β (extracellular IL-1β, solid bars). (D) The effect ofcycloheximide (5 μg/mL) on IL-1β in culture supernatants (i.e. secretion) was determinedby pre-incubation cells with cycloheximide (open bar) for 30 min (untreated cells arerepresented by closed bars) prior to addition of stimulants for an additional 24 h.Staurosporine (5 μM) was used as a positive control for induction of cell death. *p<0.05,**p<0.01 compared to cells treated with cycloheximide. Data are representative of at leastthree independent experiments, each done in triplicate.

Kang et al. Page 20

Eur J Immunol. Author manuscript; available in PMC 2013 June 13.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript