Differential Regulation of Caspase-1 Activation via NLRP3/NLRC4 Inflammasomes Mediated by Aerolysin and Type III Secretion System during Aeromonas This information is current as veronii Infection of October 2, 2021. Andrea J. McCoy, Yukiko Koizumi, Naomi Higa and Toshihiko Suzuki J Immunol 2010; 185:7077-7084; Prepublished online 29

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Regulation of Caspase-1 Activation via NLRP3/ NLRC4 Inflammasomes Mediated by Aerolysin and Type III Secretion System during Aeromonas veronii Infection

Andrea J. McCoy,1 Yukiko Koizumi, Naomi Higa, and Toshihiko Suzuki

Aeromonas spp. are Gram-negative bacteria that cause serious infectious disease in humans. Such bacteria have been shown to induce apoptosis in infected macrophages, yet the host responses triggered by macrophage death are largely unknown. In this study, we demonstrate that the infection of mouse bone marrow-derived macrophages with Aeromonas veronii biotype sobria triggers activation of caspase-1 with the ensuing release of IL-1b and pyroptosis. Caspase-1 activation in response to A. veronii infection requires the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain and both the NLRP3 and NLRC4 inflammasomes. Furthermore, caspase-1 activation requires aerolysin and a functional type III secretion system in A. veronii. Aerolysin-inducing caspase-1 activation is mediated through the NLRP3 inflammasome, with aerolysin- Downloaded from mediated cell death being largely dependent on the NLRP3 inflammasome. In contrast, the type III secretion system activates both the NLRP3 and NLRC4 inflammasomes. Inflammasome-mediated caspase-1 activation is also involved in host defenses against systemic A veronii infection in mice. Our results indicated that multiple factors from both the bacteria and the host play a role in eliciting caspase-1 activation during A. veronii infection. The Journal of Immunology, 2010, 185: 7077–7084.

he genus Aeromonas is responsible for a significant Aeromonads produce a vast repertoire of virulence factors, http://www.jimmunol.org/ number of animal and human infections. Aeromonas spp. including cytotoxins and enzymes. Studies have also identified T are opportunistic human pathogens that cause intestinal functional or putative type III secretion systems (T3SS) in some infections, of which the most common is gastroenteritis with pathogenic Aeromonas species (6, 7). T3SS are known to be criti- diarrhea. Such bacteria also cause extraintestinal infections, includ- cal virulence factors of many Gram-negative bacterial pathogens, ing wound, soft tissue, skin, and blood infections and septice- acting as a molecular syringe that injects a large number of mia (1). Studies identifying the leading cause of wound infec- effectors into the cytosol of host cells. Once in the cells, these tions in tidal wave survivors after the tsunami in Thailand as well effectors manipulate various cellular signaling pathways and as pathogen recovery in flood waters following Hurricane Katrina promote bacterial virulence (8). It has also been reported that by guest on October 2, 2021 in New Orleans, LA, highlight the importance of Aeromonas spp. Aeromonas species induce apoptosis in infected macrophages (9, as emerging pathogens (2–4). Among the 21 species that have 10). Initiation of host cell death is thought to be employed by the been differentiated using DNA–DNA hybridization, A. caviae, bacteria as a strategy for preventing eradication and clearance by A. veronii biotype sobria, and A. hydrophila are most commonly macrophages. However, the mechanisms responsible for the cell associated with human infections and account for .85% of all death of infected macrophages and host responses remain un- clinical isolates (1, 5). known. The innate immune system recognizes a vast array of pathogen- associated molecular patterns via membrane-associated TLRs and cytosolic nucleotide-binding oligomerization domain-like recep- Department of Molecular Bacteriology and Immunology, Graduate School of Med- tors (NLRs) (11). All TLRs and some NLRs, including Nod1 and icine, University of the Ryukyus, Okinawa, Japan 1 Nod2, activate the transcription of numerous genes including Current address: Naval Medical Research Center, Silver Spring, MD. cytokines and chemokines. Some cytokines, such as IL-1b, are Received for publication June 30, 2010. Accepted for publication September 29, expressed as proforms and require proteolytic cleavage for mat- 2010. uration. The cysteine protease caspase-1 is responsible for the This work was supported in part by Grant-in-Aid for Postdoctoral Fellowships for b Foreign Researchers 20-08458 (to A.J.M.), Grant-in-Aid for the Japan Society for the proteolytic processing and secretion of IL-1 and IL-18 as well Promotion of Science Fellows 22-3784 (to Y.K.), and Grant-in-Aid for Scientific as for the induction of pyroptosis, a form of proinflammatory Research on Priority Areas 21022042 (to T.S.) from the Japanese Ministry of Edu- necrotic cell death induced by infection with certain pathogenic cation, Culture, Sports, and Technology in Japan; by the Uehara Memorial Founda- tion; and by the Novartis Foundation (Japan) for the Promotion of Science (to T.S.). bacteria (12). Caspase-1 activation is mediated by the assembly of Address correspondence and reprint requests to Dr. Toshihiko Suzuki, Department of a supramolecular complex termed inflammasomes (13). Bacterial, Molecular Bacteriology and Immunology, Graduate School of Medicine, University viral, and fungal pathogens as well as noninfectious stimuli are of the Ryukyus, 207 Uehara, Nishiharacho, Okinawa 903-0125, Japan. E-mail ad- thought to trigger different inflammasomes, each defined by a dress: [email protected] particular NLR and the apoptosis-associated speck-like protein The online version of this article contains supplemental material. containing a caspase recruitment domain (ASC). The NLRP3 in- Abbreviations used in this paper: ASC, apoptosis-associated speck-like protein con- taining a caspase recruitment domain; Av, Aeromonas veronii; BMM, bone marrow- flammasome is activated in response to several stimuli, such as derived macrophage; hpi, hour(s) postinfection; LDH, lactate dehydrogenase; n.d., bacterial pore-forming toxins and noninfectious crystals (13), none detected; NLR, nucleotide-binding oligomerization domain-like receptor; potassium efflux (14), the generation of reactive oxygen species T3SS, type III secretion system; WT, wild-type. (15), and lysosomal disruption (16). In contrast, the NLRC4 in- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 flammasome is activated in response to bacterial flagellin deliv- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002165 7078 INFLAMMASOME ACTIVATION BY AEROMONAS VERONII INFECTION ered into the cytosol of infected cells during Salmonella and homogenates, and cytokine levels in serum were measured using an ELISA Legionella infections (17–20). A flagellin-independent activation assay. of the NLRC4 inflammasome has also been reported in response Statistical analysis to Shigella and Pseudomonas infections, and, recently, the rod protein of the T3SS was shown to contribute to flagellin-in- The statistical analyses were performed using unpaired two-tailed Student t tests. Differences were considered significant at a p value of ,0.05. dependent NLRC4 inflammasome activation (21–23). In this work, we investigated the role of NLR inflammasome activation in bone marrow derived macrophages (BMMs) during A. Results b veronii biotype sobria (A. veronii) infection and demonstrate that Infection with A. veronii induces caspase-1 activation, IL-1 aerolysin and T3SS are required to elicit caspase-1 activation and secretion, and pyroptosis the subsequent secretion of IL-1b and pyroptosis. Not surpris- Pathogenic aeromonads reportedly have the capacity to avoid ingly, aerolysin triggers the activation of the NLRP3 inflamma- phagocytosis and induce cytotoxicity in macrophages (6, 9, 10). How- some. However, T3SS of A. veronii triggers both NLRP3 and ever, whether bacterial infection by A. veronii induces caspase-1 NLRC4 inflammasome activation. Our data indicated that multiple activation in macrophages remains uncertain. We examined caspase- factors from both the bacteria and the host are involved in caspase- 1 activation in infected BMMs and observed that upon infection 1 activation during proinflammatory responses. with WT A. veronii, BMMs underwent a rapid cell death with membrane swelling (Fig. 1A), a morphological feature of necrotic Materials and Methods cell death. During A. veronii infection both caspase-1 activation Bacterial strains and IL-1b secretion were induced, with processing and secretion b Downloaded from The wild-type (WT) A. veronii biotype sobria AeG1 was isolated from of IL-1 being dependent on caspase-1 (Fig. 1B,1C). LDH, an a clinically fatal case of septicemia and described elsewhere (24). Isogenic indicator of necrotic cell death, was also released into culture A. veronii mutants, ΔaerA (aerolysin mutant), ΔaopB (mutant of AopB, an supernatants during infection (Fig. 1D). LDH release from cas- essential component of T3SS), and the ΔaerAΔaopB double mutant were pase-1–deficient BMMs was diminished at early times of A. ver- constructed using allele replacement strategies and the suicide vector onii infection but reached a level comparable to that of WT BMMs pYAK1, provided by Dr. T. Iida (Osaka University, Osaka, Japan). The WT Salmonella enterica serovar Typhimurium SL1344 was provided by Dr. by 3 hpi (Fig. 1D), indicating that the effect of caspase-1 on LDH http://www.jimmunol.org/ Okada (Kitasato University, Tokyo, Japan). Bacterial strains were grown in release is partial and that caspase-1–independent cell death Luria-Bertani broth. mechanisms may also be engaged. Taken together, these data in- Mice and preparation of macrophages dicate that A. veronii infection leads to macrophage cell death via pyroptosis, a proinflammatory form of cell death. C57BL/6 mice were purchased from Japan SLC (Tokyo, Japan) as WT mice. C57BL/6 background caspase-1–deficient, NALP3-deficient (Nlrp32/2) A. veronii-inducing caspase-1 activation is dependent on both 2 2 2 2 2 2 (25), NLRC4-deficient (Nlrc4 / ) (26), and ASC (Asc / or Pycard / )- NLRP3 and NLRC4 deficient mice (27) were housed in a pathogen-free facility. BMMs were prepared from the femurs and tibias of the above mice and cultured for Host cell recognition of various pathogens leading to caspase-1 5–6 d in 10% FCS-RPMI 1640 supplemented with 30% mouse L cell su- activation is mediated by the formation of a large molecular by guest on October 2, 2021 pernatant. All animal studies were performed in accordance with proto- platform termed an inflammasome (28). The inflammasome com- cols approved by the Animal Care and Use Committee of the University of the Ryukyus (Okinawa, Japan). plex includes an NLR family member and the adaptor ASC. To demonstrate that the same occurs during A. veronii infection, Reagents we examined caspase-1 activation and IL-1b processing/secretion LPS (O55:B5) and cytochalasin D were purchased from Sigma-Aldrich (St. from NLRP3-, NLRC4-, and ASC-deficient BMMs infected with Louis, MO). The following Abs were obtained commercially: rabbit anti- WT A. veronii. To our surprise, caspase-1 activation was abrogated mouse caspase-1 (sc-514; Santa Cruz Biotechnology, Santa Cruz, CA), in BMMs deficient in ASC but not in NLRP3- or NLRC4-deficient b goat anti-mouse IL-1 (AF-401-NA; R&D Systems, Minneapolis, MN), BMMs (Fig. 2A). In contrast, small amounts of processed mature and rabbit anti-mouse IL-18 (5180R-100; BioVision, Mountainview, CA). IL-1b were detected in ASC-deficient cells postinfection with A. Bacterial infection, lactate dehydrogenase assay, and ELISA veronii. The cleavage of proIL-18, another substrate of active BMMs were seeded at a density of 5 3 105 cells per well in 24-well plates caspase-1, was also detected in a caspase-1–independent manner containing 10% FCS-RPMI 1640. LPS (1 mg/ml)-primed cells were (data not shown). However, the secretion of IL-1b, as examined infected with A. veronii at a multiplicity of infection of ∼10 per cell. The using an ELISA, was significantly inhibited in ASC-deficient plates were centrifuged at 600 3 g for 10 min to synchronize the infection cells and partially but significantly inhibited in NLRP3-deficient stage and then incubated at 37˚C. At the indicated times postinfection, lactate dehydrogenase (LDH) activity in the culture supernatants was cells (Fig. 2B), suggesting that ASC and NLRP3 are involved in measured using a CytoTox 96 assay kit (Promega, Madison, WI) according eliciting IL-1b secretion in response to A. veronii infection. to the manufacturer’s protocol. Cytokines released in the culture super- Consistent with what we observed in Fig 1D, infection of natants were quantified using an ELISA (R&D Systems). The endpoint of NLRP3-, ASC-, and NLRC4-deficient BMMs with A. veronii the experiments was defined as 3 h postinfection (hpi), as Aeromonas are leads to a delay in LDH release over time (Fig. 2C), suggesting extracellular pathogens and multiply quickly in cell culture media. that NLRP3, NLRC4, and ASC are at least partially involved Immunoblots in cell death. BMMs were seeded at a density of 2 3 106 cells per well in 6-well plates Presently, NLRP3/NLRC4 double-knockout mice are unavail- and infected with bacteria. The cells were lysed and combined with the able. Therefore, to clarify how inflammasomes formed by NLRP3 supernatant precipitated with 10% TCA. The samples were loaded onto or NLRC4 contribute to caspase-1 activation by A. veronii,we 15% SDS-PAGE, and the cleaved form of caspase-1, IL-1b, was detected examined caspase-1 activation and IL-1 b release in the presence using anti–caspase-1, anti–IL-1b, or anti–IL-18 Ab, respectively. of a high concentration of extracellular potassium, which is known Mouse infections to inhibit NLRP3 inflammasome activation (14). High extracellular Seven-week-old age- and sex-matched mice were infected i.p. with 1 3 106 potassium concentration inhibited NLRP3-mediated caspase-1 CFU bacteria. Mice were sacrificed at 6 hpi and the tissues and sera were activation triggered by LPS plus ATP stimulation (Fig. 3A)but harvested. Bacterial load was determined by plating dilutions of tissue only partially inhibited caspase-1 activation and IL-1b processing/ The Journal of Immunology 7079 Downloaded from http://www.jimmunol.org/

FIGURE 1. Infection with A. veronii (Av) induces caspase-1 activation, IL-1b release, and pyroptosis in infected BMMs. BMMs from WT or caspase-1–deficient (Casp12/2) mice were primed with LPS (1 mg/ml; 3 h) and infected with WT A. veronii for the indicated times. A, Phase contrast images of WT BMMs either uninfected (2) or 1 hpi. Representative by guest on October 2, 2021 damaged macrophages are shown. Scale bar, 20 mm. B, The activation of caspase-1 and IL-1b processing in infected WT or caspase-1–deficient FIGURE 2. A. veronii (Av)-inducing caspase-1 activation and IL-1b BMMs was analyzed using immunoblotting with anti–caspase-1 or anti– release is dependent on ASC and partially dependent on NLRP3. BMMs 2 2 2 2 IL-1b Ab. C, IL-1b secretion from the infected BMMs into the culture from WT, NLRP3-deficient (Nlrp3 / ), ASC-deficient (Asc / ), or 2 2 supernatants at 3 hpi was analyzed using an ELISA. D, Culture super- NLRC4-deficient (Nlrc4 / ) mice were primed with LPS and infected natants from infected BMMs were analyzed for LDH release. Data are with WT A. veronii. A, The activation of caspase-1 and IL-1b processing in presented as the means 6 SD of triplicate samples and were compared infected WT, NLRP3-deficient, ASC-deficient, or NLRC4-deficient BMMs using a Student t test. **p , 0.01. Data are representative of at least two was analyzed using immunoblotting with anti–caspase-1 or anti–IL-1b Ab. independent experiments. n.d., none detected. B, IL-1b secretion into culture supernatants from infected BMMs at 3 hpi was analyzed using an ELISA. n.d., none detected. C, Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented 6 secretion in WT BMMs infected with A. veronii (Fig. 3B,3C), as the means SD of triplicate samples and were compared using the , , supporting the notion that NLRP3 and at least one other NLR Student t test. *p 0.05; **p 0.01. Data are representative of at least two independent experiments. inflammasome are activated by A. veronii. In contrast, caspase-1 activation and IL-1b processing were completely inhibited during A. veronii infection of NLRC4-deficient BMMs in the presence of the pathogen’s ability to activate caspase-1 or to process/secrete high potassium concentrations (Fig. 3B,3C). Taken together, these IL-1b in infected BMMs (Fig. 4A,4B), suggesting that other results indicate that both NLRP3 and NLRC4 are required for the unidentified bacterial factors may be involved in caspase-1 acti- full activation of caspase-1 upon A. veronii infection. vation. Numerous publications have identified the requirement of A. veronii-inducing caspase-1 activation is mediated by a T3SS for activation of caspase-1 by the NLRC4 inflammasome. bacterial aerolysin and T3SS Consequently, we identified a T3SS in our strain of A. veronii. Not Previously, Gurcel et al. (29) demonstrated that only the pro- surprisingly, an A. veronii T3SS mutant (DaopB) unable to pro- duction of aerolysin was required to induce caspase-1 activation duce AopB, one of the essential components of the T3SS of A. during A. trota infection of fibroblasts. However, we recently veronii, induced caspase-1 activation, IL-1b secretion, and LDH demonstrated that A. hydrophila-mediated caspase-1 activation release comparable to that observed by WT A. veronii (Fig. 4A– and IL-1b secretion in BMM was orchestrated by three pore- C). We next sought to determine what combined effect the de- forming toxins (aerolysin, hemolysin, and MARTX) (30). Of letion of both aerolysin and the T3SS (DaerADaopB) might have these three pore-forming toxins we were only able to identify on caspase-1 activation. Unexpectedly, the A. veronii DaerADaopB a homologous aerolysin gene in our A. veronii strain (data not double mutant did not induce caspase-1 activation and IL-1b shown). Deletion of aerolysin (DaerA)inA. veronii did not alter secretion (Fig. 4A,4B). Moreover, LDH was not released post- 7080 INFLAMMASOME ACTIVATION BY AEROMONAS VERONII INFECTION

FIGURE 3. A. veronii-inducing caspase-1 acti- vation and IL-1b release are dependent on both NLRP3 and NLRC4 inflammasomes. LPS-primed BMMs from WT or NLRC4-deficient (Nlrc42/2) mice were treated with ATP or infected with WT A. veronii in the absence or presence of KCl (130 mM). A, The activation of caspase-1 and IL-1b processing in WT BMMs triggered by ATP (5 mM) were analyzed using immunoblotting with anti–caspase-1 or anti–IL-1b Ab. B, The activation of caspase-1 and IL-1b processing in infected WT or NLRC4-deficient BMMs. C, IL-1b secretion into culture supernatants from infected BMMs at 3 hpi was analyzed using an ELISA. Data are pre- sented as the means 6 SD of triplicate samples and compared using a Student t test. *p , 0.05.

Data are representative of at least two independent Downloaded from experiments. Av, A. veronii; n.d., none detected. http://www.jimmunol.org/ infection with the double mutant (Fig. 4C). Collectively, these These data suggest that phagocytosis of bacteria is not necessary data suggest that aerolysin and the T3SS of A. veronii are essential for A. veronii T3SS-inducing inflammasome activation. bacterial factors in the activation of caspase-1, secretion of IL-1b, and cytotoxicity in infected macrophages. A. veronii aerolysin triggers caspase-1 activation via the We further examined whether A. veronii needs to be intracellular NLRP3 inflammasome, whereas T3SS-mediated caspase-1 to trigger T3SS-mediated caspase-1 activation. Cytochalasin D is activation is induced via both the NLRP3 and the NLRC4 an inhibitor of phagocytosis that disrupts F-actin in host cells and inflammasomes has previously been shown to inhibit pyroptosis by Salmonella To further determine how aerolysin and the T3SS mediate inflam- pathogenicity island 1 T3SS (31, 32). In contrast to the effect seen masome activation, we examined caspase-1 activation in NLRP3- by guest on October 2, 2021 during Salmonella infection, caspase-1 activation was not affected and NLRC4-deficient BMMs during infection with our mutants. by cytochalasin D during infection of BMMs with the DaerA In the absence of the T3SS (DaopB mutant producing aerolysin mutant (T3SS+) as well as ATP stimulation (Supplemental Fig. 1). [Aer+]), caspase-1 activation and IL-1b processing/secretion were

FIGURE 4. Aerolysin and T3SS of A. veronii are essential for the induction of caspase-1 activation and cell death. BMMs from WT mice were primed with LPS and infected with WT A. veronii or its respective isogenic mutants, DaerA (T3SS intact), DaopB (aero- lysin intact), or double mutant DaerADaopB. A, Acti- vation of caspase-1 and IL-1b processing in infected WT BMMs was analyzed using immunoblotting with anti–caspase-1 or anti–IL-1b Ab. B,IL-1b secretion into culture supernatants from infected BMMs at 3 hpi was analyzed using an ELISA. n.d., none detected. C, Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented as the means 6 SD of triplicate samples. Data are representative of at least two independent experiments. The Journal of Immunology 7081 dependent on NLRP3 and ASC but not on NLRC4 (Fig. 5A,5C), inflammasomes. Initially, we observed an apparent absence of indicating that aerolysin triggers caspase-1 via the NLRP3 inflam- caspase-1 activation in ASC-deficient BMMs infected with the masome. Priming of macrophages by factors such as LPS is DaerA mutant despite a detectable amount of processed and se- reportedly necessary to activate the NLRP3 inflammasome (33). creted IL-1b (Fig. 5B,5C). However, upon longer exposure of However, BMMs infected either the DaopB mutant (Aer+) or the our blotted membrane, we detected small amounts of activated DaerA mutant (T3SS+) led to caspase-1 activation and the pro- caspase-1, indicative of weak activation of caspase-1 in ASC- cessing of IL-1b and IL-18 in the absence of LPS pretreatment deficient BMMs upon DaerA mutant infection (Fig. 5B). These (Supplemental Fig. 2). Thus, LPS priming is not necessary to acti- results suggest that NLRP3 and NLRC4 inflammasomes play in- vate the NLRP3 inflammasome in response to A. veronii aerolysin. dependent or additive roles in the full activation of caspase-1 in In contrast to the T3SS mutant, A. veronii DaerA (T3SS+) was response to the A. veronii T3SS and also suggest that an ASC- capable of inducing caspase-1 activation and IL-1b processing/ independent NLRC4–caspase-1 pathway is also activated. Col- secretion in NLRP3- and NLRC4-deficient BMMs (Fig. 5B, 5C). lectively, these findings suggest that caspase-1 activation after A. Similar to what we observed with WT A. veronii, active caspase-1 veronii infection is mediated by two inflammasome signaling and mature IL-1b were nearly absent when high extracellular pathways, the aerolysin–NLRP3 and T3SS–NLRP3/NLRC4 axes. potassium was added to NLRC4-deficient cells during infection Interestingly, these two pathways have unique roles in macro- (Fig. 5B,5C). Taken together, these data demonstrate that the phage cell death as measured by LDH release. In the absence of T3SS plays a role in the activation of both the NLRP3 and NLRC4 aerolysin, the functional T3SS (DaerA mutant) initiates cell death Downloaded from http://www.jimmunol.org/

FIGURE 5. A. veronii aerolysin triggers caspase-1 activation via the NLRP3 inflammasome and T3SS triggers caspase-1 activation via both the NLRP3 and

the NLRC4 inflammasome. BMMs from WT, NLRP3- by guest on October 2, 2021 (Nlrp32/2), ASC- (Asc2/2), or NLRC4-deficient (Nlrc42/2) mice were primed with LPS and infected with WT A. veronii or its respective isogenic mutants, DaerA (T3SS intact) or DaopB (aerolysin intact), in the absence or presence of 130 mM KCl. A, Activation of caspase-1 and IL-1b processing in infected WT, NLRP-deficient, ASC-deficient, or NLRC4-deficient BMMs postinfection with WT A. veronii or DaopB. B, Activation of caspase-1 and IL-1b processing in infected NLRP3-deficient, ASC-deficient, NLRC4-de- ficient, or KCl-treated NLRC4-deficient BMMs post- infection with DaerA mutant. C, IL-1b secretion into culture supernatants from the infected BMMs indicated in A and B at 3 hpi was analyzed using an ELISA. D, Culture supernatants from the infected BMMs in- dicated in A and B were analyzed for LDH release. Data are presented as the means 6 SD of triplicate samples. Data are representative of at least two in- dependent experiments. 7082 INFLAMMASOME ACTIVATION BY AEROMONAS VERONII INFECTION via activation of the NLRP3/NLRC4 inflammasome (Fig. 5D, right panel). However, a functional T3SS likely activates cell death via noncaspase-1–mediated pathways, as demonstrated by the considerable release of LDH when A. veronii DaerA infects NLRC4-deficient BMMs in the presence of high extracellular potassium (Fig. 5D, right panel) or caspase-1 deficient BMMs (Supplemental Fig. 3). In comparison, LDH release from NLRP3- and ASC-deficient BMMs was abrogated during infection with A. veronii DaopB (Aer+), suggesting that the well-known pore- forming toxin aerolysin is supported by caspase-1 activation for the induction of cell lysis. This notion is supported by the signifi- cantly diminished LDH released from caspase-1–deficient BMMs infected with A. veronii DaopB, which expresses only aerolysin toxins required to activate caspase-1 (Supplemental Fig. 3). Inflammasome activation is involved in host defense against A. veronii. We examined the in vivo role of caspase-1 activation in the innate immune response against A. veronii infection using a mouse sepsis model. Since A. veronii DaerA and DaopB mutants have a highly attenuated virulence in mice (data not shown), the A. veronii WT strain was used in the analysis. WT, caspase-1–de- Downloaded from ficient, or ASC-deficient mice were infected i.p. with 106 CFU of A. veronii. At 6 hpi, severe clinical symptoms, such as a hunched appearance and lethargy, were observed in all of the infected caspase-1– and ASC-deficient mice, whereas 40% (two of five mice) of the WT mice were affected. The bacterial burden in the spleens and livers of the caspase-1–deficient mice and in the http://www.jimmunol.org/ spleens of ASC-deficient mice was significantly higher than that of the WT mice (Fig. 6A), suggesting that caspase-1 activation via ASC contributes to the antibacterial activities upon A. veronii FIGURE 6. Caspase-1 and ASC are involved in host antibacterial ac- infection. Although the reduction in the level of IL-1b, as well as tivity against WT A. veronii infection in a mouse sepsis model. A, WT, TNF-a, was not significant in caspase-1– and ASC-deficient mice caspase-1–deficient, or ASC-deficient mice (n = 5) were infected i.p. with compared with WT mice (Fig. 6B,6C), the IL-18 level in the sera WT A. veronii, and spleen and liver homogenates were plated at 6 hpi to at 6 hpi was significantly reduced (Fig. 6D). These data suggest determine the bacterial CFU per organ. B–D, Sera from infected mice were a that the production of IL-18 rather than IL-1b by the activation of harvested and analyzed using an ELISA for the levels of circulating TNF- (B), IL-1b (C), or IL-18 (D). Data are presented as the means 6 SD and by guest on October 2, 2021 caspase-1 via ASC contributes at least in part to the antibacterial compared using a Student t test. *p , 0.05; **p , 0.01. n.d., none activities in a mouse sepsis model of infection with A. veronii. detected. Discussion Pathogenic bacteria express a variety of virulence factors, such as is involved in the cytotoxicity of aerolysin. Conversely, inflam- pore-forming toxins or virulence-associated secretion systems, that masome activation reportedly plays a role in promoting cell sur- disrupt host cell systems or manipulate cellular signaling pathways vival by activating membrane biogenesis in response to the aer- to allow the establishment of infection. Two members of the NLR olysin of A. salmonicida in epithelial cells (29). Although this family, NLRP3 and NLRC4, sense bacterial virulence factors or discrepancy may be attributable to differences in the experimental factor-mediated cellular events and form inflammasomes, being conditions, toxins, and cells that were used, our results suggest multiprotein complexes that induce caspase-1 activation. In this that NLRP3-mediated caspase-1 activation is required for the in- study, we demonstrated that the infection of BMMs with A. veronii duction of A. veronii aerolysin-mediated cell death in macro- induced caspase-1 activation via inflammasomes, which were phages. differentially regulated by NLRP3 and NLRC4 in response to Although the NLRC4 inflammasome is primarily activated by pore-forming aerolysin and the T3SS secretion system of A. ver- the detection of the T3SS of Salmonella, Shigella, Legionella, and onii. Pseudomonas (13), we have demonstrated the dual activation of Our data establish that aerolysin induces caspase-1 via the NLRP3 and NLRC4 inflammasomes in the detection of the A. NLRP3 inflammasome, consistent with the widely proposed notion veronii T3SS. Because A. veronii is flagellated bacterium, it is of pore-forming toxin-mediated NLRP3 activation. Notably, al- likely that at least flagellin contributes to the NLRC4-dependent though the aerolysin of Aeromonas is known to be a pore-forming activation of caspase-1. Similarly, a recent study revealed that the toxin that disrupts the cell membrane, based on our data for LDH T3SS of Yersinia pseudotuberculosis is also detected by both release from infected macrophages, the cytotoxicity induced by NLRP3 and NLRC4 during caspase-1 activation (35). Taken to- aerolysin-producing A. veronii is largely dependent on the NLRP3 gether, this raises the possibility that structural or functional dif- inflammasome. Indeed, aerolysin-producing but not T3SS-harbor- ferences in the T3SS of various bacteria are related to the ing A. veronii induced significant less LDH release in NLRP3-, recognition by NLRP3 or NLRC4. Both Salmonella pathogenicity ASC-, and caspase-1–deficient macrophages. This phenotype of island 1 and 2, the components of T3SS of Salmonella, trigger inflammasome- and caspase-1–dependent cell death resembles NLRC4 inflammasome, and interestingly caspase-1 activation via that of LPS-primed ATP stimulation (34), raising the possibility NLRP3 is also induced in a T3SS-independent manner in the that membrane pore formation by aerolysin is insufficient to in- infection with Salmonella (36). 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Differential regu- A. veronii infection remains unknown, further elucidation of the lation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in role of inflammasome activation during infection is likely to pro- Shigella-infected macrophages. PLoS Pathog. 3: e111. vide important insights into how the activation of different inflam- 23. Miao, E. A., D. P. Mao, N. Yudkovsky, R. Bonneau, C. G. Lorang, S. E. Warren, I. A. Leaf, and A. Aderem. 2010. Innate immune detection of the type III se- masomes contributes to host immune responses. cretion apparatus through the NLRC4 inflammasome. Proc. Natl. Acad. Sci. USA 107: 3076–3080. 24. Song, T., C. Toma, N. Nakasone, and M. Iwanaga. 2004. Aerolysin is activated Acknowledgments by metalloprotease in Aeromonas veronii biovar sobria. J. Med. Microbiol. 53: 477–482. We thank the members of the Suzuki Laboratory for their advice. We also 25. Martinon, F., V. Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout- thank R. Flavell and K. Kuida for providing the caspase-1–deficient mice, associated uric acid crystals activate the NALP3 inflammasome. Nature 440: Ju¨rg Tschopp for providing the NLRP3-deficient mice, Vishva Dixit for 237–241. providing the NLRC4-deficient mice, and Shun’ichiro Taniguchi for pro- 26. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee, M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of viding the ASC-deficient mice. We also thank Dr. T. Iida for providing the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218. some materials. 27. Yamamoto, M., K. Yaginuma, H. Tsutsui, J. Sagara, X. Guan, E. Seki, K. Yasuda, M. Yamamoto, S. Akira, K. Nakanishi, et al. 2004. ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated Disclosures signal adaptor molecules. Genes Cells 9: 1055–1067. 28. Franchi, L., T. Eigenbrod, R. Mun˜oz-Planillo, and G. Nun˜ez. 2009. 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