The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila

Microbes and Infection 14 (2012) 1123e1132 www.elsevier.com/locate/micinf

Original article

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila Nanako Katagiri a, Takeo Shobuike a,*, Bin Chang b, Akiko Kukita a, Hiroshi Miyamoto a a

Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan b Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo 162-8640, Japan Received 16 November 2011; accepted 20 March 2012 Available online 28 March 2012

Abstract Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Legionella pneumophila; Macrophage; Cellular response; Cell death; Caspases

1. Introduction Inhibitors of apoptosis proteins (IAPs) inhibit apoptosis by reducing the activity of caspases [1]. The antiapoptotic properties of IAPs are attributed to their characteristic Nterminal baculoviral IAP repeat (BIR) domains, which consist of approximately 70 amino acids. Human nucleotide oligomerization domain-like receptor (NLR) family apoptosis inhibitory protein (NAIP), a member of the IAP family, protects cells from apoptosis both in vivo and in vitro [2,3]. Human NAIP contains three sequential BIR domains at its Nterminus (designated as “BIR1e3”). DNA damage causes the release of mitochondrial cytochrome c into the cytosol. Cytochrome c triggers the activation of the apoptotic protease-

* Corresponding author. Tel.: þ81 952 34 2251; fax: þ81 952 34 2252. E-mail address: [email protected] (T. Shobuike).

activating factor-1 (APAF-1) apoptosome, which, in turn, mediates the activation of the initiator caspase, caspase-9. Activated caspase-9 processes the executioners caspase-3 and -7. Mature caspase-3 and -7 cleave many substrates, leading to apoptosis [4]. Human NAIP prevents apoptosis by binding, via its BIR3 domain, to procaspase-9 prior to its autoproteolysis upon apoptosome formation [5]. BIR2 also inhibits caspase-3 and -7 [6]. In contrast with the other IAPs, human NAIP features a nucleotide-binding oligomerization domain (NOD) followed by a C-terminal leucine-rich repeat (LRR). The NOD exhibits an ATP/dATP binding site that facilitates interaction between human NAIP and caspase-9 [7]. The LRR domain of human NAIP regulates the activity of BIR domains by interfering with their interaction with caspases [7]. There are >20 NOD-LRRcontaining proteins in mammals, several of which are known to participate in innate immunity by acting as cytosolic bacterial sensors [8].

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2012.03.006

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In murine bone marrow-derived macrophages (mBMMs), Naip5, one of four murine homologs of human NAIP, is involved in host defense against Legionella pneumophila, a gram-negative intracellular bacterial pathogen [9,10]. Infection of the human lung with L. pneumophila causes a severe pneumonia known as Legionnaires’ disease [11], the pathogenesis of which is closely linked to the intracellular growth of the bacterium in alveolar macrophages [12]. After ingestion into the phagosome of human macrophages, the bacteria use a type IVB secretion system (called “Icm/Dot transporter”) to secrete a variety of proteins (called “effectors”) into the host cell cytoplasm, thereby altering host cell processes and establishing an intracellular niche supporting bacterial growth [13,14]. In contrast to humans, macrophages from the C57BL/6 mouse strain restrict the intracellular growth of L. pneumophila [10]. Thus, the mechanism responsible for the restriction of intracellular bacterial growth has been extensively investigated using L. pneumophilainfected C57BL/6 BMMs [15e19]. The restriction of the intracellular growth of L. pneumophila in C57BL/6 BMMs was found to be dependent on caspase-1 activation [15]. Caspase-1 activation leads to the processing of pro-interleukin (IL)-1b, secretion of mature IL1b, and cell death (called “pyroptosis”) via mechanisms as yet unknown [20,21]. In C57BL/6 BMMs, caspase-1 activation is mediated by murine Naip5 (mNaip5) and by NLR family and CARD domain containing 4 (Nlrc4) in response to L. pneumophila flagellin (FlaA), through the activation of the Nlrc4 inflammasome [16e19]. Recent reconstitution studies identified direct binding of mNaip5, Nlrc4, and FlaA to form a hetero-oligomeric complex [22,23]. Thus, both pyroptotic cell death and the restriction of L. pneumophila growth are prevented in L. pneumophila-infected mBMMs, where mNaip5, Nlrc4, caspase-1, or bacterial flagellin are defective [15e19]. In addition, cytotoxic pore formation, observed in L. pneumophila-infected C57BL/6 BMMs, is also dependent on Nlrc4- and flagellin-mediated caspase-1 activation [24]. Human NAIP is the only functional NAIP in humans, while in the murine genome, several Naip-related sequences are repeated at the D1 region of chromosome 13, among which, at least four Naip (Naip1, Naip2, Naip5, and Naip6) appear to be functional [8,25]. Besides L. pneumophila flagellin, mNaip5 interacts with intracellular flagellin of Salmonella typhimurium, Yersinia enterocolitica, Photorhabdus luminescens, and Pseudomonas aeruginosa [23]. mNaip6 and mNaip2 recognize bacterial flagellin and type III secretion system rod proteins, respectively [22,23]. It is difficult to define the relationship between human NAIP and mNaip paralogs, because the putative amino acid sequence of human NAIP is equally similar (approximately 70%) to each murine paralog. mNaip2 inhibits apoptosis in PC12 neuronal cells [26], but, until date, no other mNaip, including Naip5, has been implicated in protecting cells from apoptosis. The role of human NAIP in the host response to L. pneumophila infection is unclear, whereas a number of molecular genetic studies have established that C57BL/6 Naip5 contributes to this process in C57BL/6 BMMs. Intracellular

growth of L. pneumophila is supported by a variety of human cells [12,27,28], and caspase-1 activation is not triggered by L. pneumophila infection in primary human monocytes [29]. A reconstitution study in 293T cells revealed that the NLRC4 inflammasome is activated by human NAIP in response to purified Chromobacterium violaceum CprI and not L. pneumophila FlaA [23]. Conversely, gene silencing of NAIP by small interfering RNA (siRNA) or flagellin deficiency of L. pneumophila enhances bacterial growth in human THP-1 macrophages, primary monocyte-derived macrophages, alveolar macrophages, and A549 lung epithelial cells [27]. Overexpression of NAIP reduces L. pneumophila growth in A549 cells [27]. However, it is still unknown whether human NAIP per se mediates caspase-1 activation and promotes cell death in L. pneumophila-infected cells. In human lung and spleen tissue, human NAIP is predominantly expressed in macrophage cells [30], suggesting that it is involved in their survival. This study demonstrates the dual role of human NAIP, and also of mNaip5, in macrophage survival, leading to cell death caused by L. pneumophila infection and protecting from apoptosis induced by DNA damage. In particular, we demonstrated for the first time that human NAIP promotes caspase-1 activation in response to L. pneumophila infection, resulting in the inhibition of intracellular bacterial growth. 2. Materials and methods 2.1. Generation of RAW264.7 cells expressing human NAIP RAW264.7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). C57BL/6 Naip5 cDNA was obtained from RNA isolated from C57BL/6 BMM. Human NAIP cDNA was generated from RNA isolated from peripheral blood obtained from a healthy volunteer. Each cDNA was amplified by polymerase chain reaction (PCR) and then cloned into the pEGFP-C1 vector (Clontech Laboratories, Mountain View, CA, USA) to be translated as the following fusion proteins with green fluorescent protein (GFP): EGFPmNaip5 and EGFP-NAIP. The PCR primers for mNaip5 were the following: 50 -CACCTTTCCCCTTTA 0 0 GAAAATGGC-3 and 5 -TGAACTGTCAGTAGTCTTCAG GAGCATC-30 . The PCR primers for human NAIP were the following: 50 -GCTAGCCGCCATGGCCACCCAGCA 0 and 50 -GCTCATCAAGCACATGGTTAG-30 . GAAAG-3 RAW264.7 cells were transfected with each vector by electroporation and then cultured with 500 mg/ml G418 antibiotic solution for 2 weeks to select the clones in which the fusion gene was stably integrated into the chromosomes. Cell lysates of each clone were prepared and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto Immobilon-P Transfer Membranes (Millipore, Billerica, MA, USA) and probed with rabbit anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidaseconjugated anti-rabbit goat antibody. Immobilon Western

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Chemiluminescent HRP Substrate (Millipore) was used for antibody detection. Clones exhibiting the highest expression of the fusion gene were used in this study. 2.2. Cell viability Cells were cultured in 5% FBS-containing medium with or without camptothecin (Wako Pure Chemical Industries, Osaka, Japan), and 2  104 cells/well were incubated in 96well plates at 37  C in 5% CO2 for 24 h. Cell viability was quantified by the evaluation of the production of formazan dye using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, CCK-8 solution (10 ml per 100 ml of medium per well) was added, the plates were incubated at 37  C for 1 h, and the absorbance of each well was read at 450 nm using a microplate reader (Wallac 1420 ARVO MX Multilabel Counter; Perkin Elmer Japan, Yokohama, Japan). 2.3. Caspase activity Caspase activity was quantified by spectrophotometric detection of the chromophore p-nitroaniline ( pNA) at 405 nm after cleavage from labeled substrates: Ac-YVAD-pNA for caspase-1 and Ac-DEVD-pNA for caspase-3 and -7. Cells grown in 6-well plates (4  106 cells/well) were treated with 0.5-mM camptothecin for 8 h at 37  C in 5% CO2. Cells were lysed and assayed for caspase activity using the Caspase Colorimetric Assay Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s instructions.

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washed three times with phosphate-buffered saline and were stained with ethidium homodimer-1 (EthD-1) and calcein-AM using the LIVE/DEADÒ Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. Slides were analyzed using a confocal laser scanning microscope (LSM 5 Pascal; Carl Zeiss MicroImaging, Tokyo, Japan). The mean and standard deviation were derived by counting a minimum of three fields from three different wells per sample. 2.6. Lactase dehydrogenase (LDH) release Cytotoxicity was quantified by evaluation of the activity of LDH released from cells using the LDH Cytotoxicity Detection Kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. Cells in 96-well plates (1  105 cells/well) were infected with the bacterium at an MOI of 20 by centrifugation at 400  g for 10 min to synchronize the infection stage, followed by incubation for 30 min at 37  C in 5% CO2. The cells were then washed three times with phosphatebuffered saline and culture medium was added. Culture supernatant was collected at specific time points after infection and assayed for LDH, or cells in 96-well plates (2  104 cells/ well) were treated with camptothecin for 24 h. Because of a high background level, FBS was reduced to 5% in this assay. Percentage cytotoxicity was calculated using the following equation: 100ðexperimental LDH  spontaneous LDHÞ= ðmaximum LDH  spontaneous LDHÞ

2.4. Bacteria 2.7. Interleukin-1b secretion L. pneumophila bacteria were grown for 48 h on buffered charcoal-yeast extract alpha (BCYE alpha)-agar plates or for 22 h in buffered yeast extract alpha broth. The L. pneumophila strains used in experiments were as follows: wild-type JR32, Nagasaki 80-045, and an avirulent Dicm mutant 25D [31,32]. Mutant flaA was generated by allelic exchange as previously described [32]. Briefly, DNA fragments containing flagellinencoding flaA of the Nagasaki 80-045 strain were amplified by PCR using the following primer pair: 50 -GGCTCAAG TAATCAACAC-30 and 50 -CATCGCTGTACCTGTTTG-30 . The kanamycin-resistance (KmR) gene was inserted into flaA fragments and cloned into the pLAW344 vector. The plasmid was introduced into the Nagasaki 80-045 strain of L. pneumophila by electroporation to mediate allelic exchange. The KmR insertion into genomic flaA was verified by PCR. The insertion conferred truncation of flaA, resulting in the production of only the N-terminal portion (amino acids 1e192) of the FlaA.

For quantification of IL-1b secretion using the enzyme-linked immunosorbent assay, cells in 96-well plates (1  105 cells/well) were pretreated with 0.5 mg/ml of the synthetic bacterial lipopeptide tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4; InvivoGen, San Diego, CA, USA) for 3 h and then infected with the bacterium at an MOI of 20 by centrifugation as described above. At specific time points after infection, cytokine in the supernatant was measured using the Mouse IL-1b EnzymeLinked Immunosorbent Assay Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. We confirmed that pretreatment with Pam3CSK4 does not affect the cytotoxicity of L. pneumophila-infected macrophages. To block endogenous caspases, cells were incubated with specific inhibitors at 5, 10, and 20 mM for 2 h before infection: acetyl-Tyr-ValAla-Asp-chloromethylketone for caspase-1 (Ac-YVAD-CMK; Bachem AG, Bubendorf, Switzerland) and acetyl-Tyr-Val-AlaAsp-chloromethylketone for caspase-3 and -7 (Ac-DEVDCMK; Calbiochem, La Jolla, CA, USA).

2.5. Pore formation 2.8. Bacterial intracellular growth Cells grown on 8-well chamber slides were infected with L. pneumophila at a multiplicity of infection (MOI) of 40 by incubation for 4 h at 37  C in 5% CO2. The cells were then

We infected 1  106 cells in 24-well plates with L. pneumophila at an MOI of 0.03 by centrifugation as described above.

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Cells were lysed at 0, 24, and 48 h with 0.2% saponin for 10 min, lysates were plated on BCYE-alpha plates, and the number of colony-forming units was counted after incubation at 37  C for 3 days. For transient expression assays, RAW264.7 cells were transfected using Nucleofector (Lonza, Basel, Switzerland) according to the manufacturer’s protocol (Nucleofector solution T, Nucleofector program U-14) with 5 mg expression vectors encoding GFP, GFP-mNaip5, or GFP-NAIP per 3.3  106 cells. After incubation for 16 h at 37  C in 5% CO2 in 96-well plates (3  105 cells/well), cells were infected with L. pneumophila JR32 strain at an MOI of 0.1 as described above and then lysed at 0 and 48 h to determine the number of colony-forming units. 2.9. Statistical analyses Statistical analyses were performed using the Student’s t test. 3. Results 3.1. Human NAIP inhibits the apoptosis of camptothecin-treated macrophages To demonstrate the effect of human NAIP on the survival of macrophages, we first established murine RAW264.7 macrophages that stably expressed full-length human NAIP as

a fusion protein with GFP, which we used in all experiments. As controls, RAW264.7 cells expressing GFP alone or GFPmNaip5 were also established (Fig. 1A). Camptothecin is a DNA-damaging agent that induces apoptosis in many cell types [33]. To test the antiapoptotic effect of human NAIP in RAW264.7 macrophages, the cells were treated with various concentrations of camptothecin for 24 h. Camptothecin inhibited the growth of RAW264.7 cells in a dose-dependent manner, and, as expected, expression of human NAIP reduced this antiproliferative effect (Fig. 1B). RAW264.7 cells expressing GFP alone exhibited 17% survival 24 h after the treatment with 0.5 mg/ml camptothecin, as determined by the evaluation of formazan production. In contrast, RAW264.7 cells expressing human NAIP exhibited significantly enhanced survival (50%). Cell death was confirmed by measuring the relative release of the cytosolic enzyme LDH, which indicates post-apoptotic necrosis (Fig. 1C). After treatment with 0.5 mg/ ml camptothecin, human NAIP-expressing RAW264.7 cells exhibited significantly lower LDH activity than GFPexpressing cells. Similar protective effects were observed in cells expressing C57BL/6 Naip5. To examine whether human NAIP inhibits caspase-3 and -7 in RAW264.7 cells, caspase activity was measured in camptothecin-treated cells (Fig. 1D). Caspase-3 and -7 activities increased approximately 20-fold in cells expressing GFP alone compared with the mock dimethyl sulfoxide-treated

Fig. 1. Human NAIP and murine NLR family apoptosis inhibitory protein 5 (mNaip5) suppress camptothecin-induced apoptosis in RAW264.7 macrophages. (A) Immunoblot of RAW264.7 macrophages expressing green fluorescent protein (GFP), GFP-mNaip5, or GFP-NAIP, analyzed with anti-GFP antibody. Each lane contains an equal amount of total protein. (B) Viability of cells treated with various concentrations of camptothecin for 24 h. Cell numbers relative to mock (dimethyl sulfoxide [DMSO])-treated cells are presented. (C) Cells were treated with 0.25 or 0.5 mg/ml camptothecin for 24 h and assayed for cytotoxicity. The error bars represent standard deviation of the mean of triplicate experiments. Data are representative of at least two independent experiments. (D) Lysates from RAW264.7 macrophages expressing GFP, GFP-mNaip5, or GFP-NAIP treated with 0.5 mg/ml camptothecin or an equivalent volume of DMSO for 8 h were assayed for caspase-1 and caspase-3 and -7 activity. The error bars represent standard error of three independent experiments. *p < 0.05, relative to cells expressing GFP alone.

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cells, indicating that these cells undergo apoptosis. Induction of caspase-3 and -7 activity decreased by approximately 50% upon expression of human NAIP. Caspase-1, as the negative control, was not activated in the cells. Expression of human NAIP in RAW264.7 cells did not affect viability or cause activation of caspases without the apoptotic agent. The results were comparable to those in cells expressing C57BL/6 Naip5. These results indicate that human NAIP suppresses apoptosis by inhibiting caspase-3 and -7 in RAW264.7 macrophages, consistent with the results in previous reports on other cell types [2]. Furthermore, our data revealed that C57BL/6 Naip5 is also involved in caspase-3 and -7 inhibition, which, in turn, prevents apoptotic macrophage death. 3.2. Human NAIP promotes cytotoxicity by pore formation in L. pneumophila-infected macrophages L. pneumophila infection triggers cytotoxic pore formation in C57BL/6 BMMs, which is required for Nlrc4 and caspase-1 function [24], and, presumably, mNaip5 function. To address the contribution of human NAIP to pore formation in L. pneumophila-infected macrophages, RAW264.7 cells expressing human NAIP were infected with the bacterium and then stained with a membrane-impermeable dye, EthD-1. Counterstaining was performed with calcein-AM, a membrane permeable dye. Mock-infected cells exclude

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EthD-1 (Fig. 2A). After 4 h of infection with L. pneumophila, RAW264.7 cells expressing either human NAIP or C57BL/6 Naip5 became permeable to EthD-1, indicating a substantial loss of membrane integrity, whereas cells expressing GFP alone maintained an intact plasma membrane (Fig. 2B). Note that the fluorescent signal of GFP was much lower than that of calcein-AM, but it was still detected in dead cells; therefore, the merged images of dead cells exhibited yellow coloration. These results indicate that both human NAIP and C57BL/6 Naip5 promote cytotoxicity by pore formation in macrophages infected with L. pneumophila. 3.3. Human NAIP promotes pyroptosis in L. pneumophila-infected macrophages Cytotoxicity was confirmed by measuring the relative release of LDH. At 2 h and 4 h after infection with wild-type L. pneumophila, LDH activity was detected in human NAIPexpressing RAW264.7 cells in a manner comparable to that of mNaip5-expressing cells (Fig. 3A). Cells expressing GFP alone exhibited LDH activity, but significantly less than human NAIP-expressing cells. In addition, cytotoxicity was not observed in the avirulent Dicm mutant 25D L. pneumophilainfected RAW264.7 cells, even when expressing human NAIP or C57BL/6 Naip5 (data not shown). This suggests that the L. pneumophila-induced death of RAW264.7 cells is dependent

Fig. 2. Human NAIP triggers pore formation in response to Legionella pneumophila infection. RAW264.7 macrophages expressing GFP, GFP-mNaip5, or GFPNAIP adherent to chamber slides were infected with wild-type L. pneumophila (Nagasaki 80-045 strain), or the isogenic DflaA mutant L. pneumophila, for 4 h at an MOI of 40. Cells were stained with ethidium homodimer-1 and calcein-AM. (A) Representative images of pore formation visualized by fluorescent microscopy (20 objective). Ethidium homodimer-1-stained macrophages (red) show permeabilized cells (top panels) and calcein-AM (green) show total cells (middle panels). Bottom panels show merged images. (B) Minimum 400 cells were examined for each experimental condition. The error bars represent standard deviation of the mean of triplicate experiments. Data are representative of at least two independent experiments. *p < 0.05, relative to cells expressing GFP alone.

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A

B

C

Fig. 3. Human NAIP promotes pyroptosis in response to L. pneumophila flagellin. (A) RAW264.7 macrophages expressing GFP, GFP-mNaip5, or GFP-NAIP were infected with wild-type L. pneumophila (Nagasaki 80-045 strain), or the isogenic DflaA mutant L. pneumophila, at an MOI of 20 and were assayed for cytotoxicity at 2 h and 4 h. (B) Cells were primed with Pam3CSK4 for 3 h to induce expression of pro-IL-1b and were left uninfected (UI) or infected with wild-type or DflaA L. pneumophila at an MOI of 20. Supernatants of the cultures infected for 2 h and 4 h were assayed for secretion of IL-1b. (C) Pam3CSK4-primed cells were left uninfected (UI) or infected with wild-type L. pneumophila at an MOI of 20 in the presence of a caspase-1 inhibitor (YVAD), a caspase-3 and -7 inhibitor (DEVD), or an equivalent volume of DMSO. Supernatants of the cultures infected for 4 h were assayed for secretion of IL-1b. The error bars represent standard deviation of the mean of triplicate experiments. Data are representative of at least two independent experiments. *p < 0.05, relative to cells expressing GFP alone.

on the bacterial type IVB secretion system (the Icm/Dot transporter), which is consistent with the findings of the other studies on C57BL/6 BMMs [16,17]. Pore formation in the cell membrane of C57BL/6 BMMs triggered by L. pneumophila is caspase-1 dependent and leads to cell death in a process called pyroptosis [24]. Thus, it is possible that L. pneumophila-induced cell death is associated with caspase-1 activity in human NAIP-expressing RAW264.7 cells. To address this possibility, we investigated the role of caspase-1 in the death of L. pneumophila-infected macrophages by estimating the secretion of IL-1b, which is dependent on caspase-1 activation. After L. pneumophila infection, RAW264.7 cells expressing human NAIP or C57BL/6 Naip5 secreted significant amounts of IL-1b, whereas no substantial secretion was observed in GFP-expressing cells (Fig. 3B). A caspase-1 inhibitor abrogated the secretion of IL-1b from infected cells in a dose-dependent manner, whereas an inhibitor of caspase-3 and -7 did not, indicating that this process is solely dependent on caspase-1 activation (Fig. 3C). These data indicate that human NAIP and C57BL/6 Naip5 are involved in caspase-1 activation and subsequent induction of pyroptosis in macrophages infected with L. pneumophila.

infected C57BL/6 BMMs [16e19]. Thus, we investigated whether the human NAIP-mediated death of infected cells is dependent on flagellin. As expected, cytotoxic pore formation and LDH release were not triggered by flagellin-deficient (DflaA) L. pneumophila, even in human NAIP-expressing RAW264.7 cells (Figs. 2A and 3A). Similar results were obtained in experiments with C57BL/6 Naip5-expressing RAW264.7 cells. Next, to examine whether human NAIP and L. pneumophila flagellin promote caspase-1 activation in combination, secretion of IL-1b was measured in RAW264.7 cells infected with the DflaA mutant (Fig. 3B). After infection with flagellin-deficient L. pneumophila, no secretion of IL-1b was substantially observed in RAW264.7 cells, even when expressing human NAIP or C57BL/6 Naip5. Because toll-like receptor 5, an extracellular sensor of flagellin, is not expressed in RAW264.7 cells [34], our data indicate that human NAIP, and also C57BL/6 Naip5, promote pyroptosis in L. pneumophila-infected RAW264.7 cells in response to cytosolic flagellin.

3.4. Cytosolic flagellin of L. pneumophila is required for human NAIP-promoted pyroptosis

It has been suggested that, in mBMMs, pyroptotic cell death in response to cytosolic L. pneumophila flagellin is associated with the restriction of intracellular bacterial growth [16e19]. To investigate whether human NAIP-mediated pyroptosis contributes to the restriction of the intracellular

L. pneumophila flagellin, a component of the flagella encoded by flaA, is required for the induction of pyroptosis in

3.5. Human NAIP restricts the intracellular growth of L. pneumophila in response to cytosolic flagellin

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growth of L. pneumophila, we counted the number of intracellular bacteria. We demonstrated that RAW264.7 macrophages support intracellular growth [35] and that the intracellular growth of the avirulent Dicm strain 25D was restricted (Fig. 4A), indicating that intracellular bacterial growth in RAW264.7 cells is dependent on the Icm/Dot transporter. Flagellin deficiency did not affect intracellular bacterial growth in RAW264.7 cells (Fig. 4B), indicating that endogenous mNaip5 does not restrict intracellular L. pneumophila growth. At 2 days after infection with wild-type L. pneumophila, the number of intracellular bacteria decreased by 90% upon expression of human NAIP (Fig. 4C), indicating a human NAIP-dependent restriction of the intracellular growth of L. pneumophila. This restriction was abrogated by infection with flagellin-deficient L. pneumophila (Fig. 4C), indicating that bacterial flagellin is required for the human NAIP-dependent restriction of the intracellular growth of L. pneumophila. Comparable results were obtained in experiments with C57BL/ 6 Naip5-expressing RAW264.7 cells. To exclude the possibility that the restriction occurred by chance in the particular clone of RAW264.7 cells, transient transfection assays were performed. RAW264.7 cells were transfected with expression vectors

106 105 104 103 102 0

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In this report, we demonstrated that human NAIP regulates caspase-1 activation-mediated cell death (called “pyroptosis”) in response to L. pneumophila infection in RAW264.7 macrophages. While a number of studies have established that C57BL/6 Naip5 contributes to the promotion of caspase-1 activation in response to L. pneumophila infection, this is the first report detailing the role of human NAIP in this process. Human NAIP also protects macrophages from DNA damage-induced apoptosis. These results demonstrate the importance of human NAIP in regulating the host response to cytosolic stimuli.

GFP mNaip5 NAIP

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Fold increase

Colony-forming units

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4. Discussion

B

WT Δicm

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encoding GFP, human NAIP, or mNaip5, and were infected with wild-type L. pneumophila for 2 days. Transient expression of human NAIP reduced bacterial growth in RAW264.7 cells (Fig. 4D). The mild effect of human NAIP on the restriction of bacterial growth could be attributable to transfection efficiency (w50%). Together, our data suggest that, in L. pneumophilainfected macrophages, human NAIP promotes pyroptosis in response to bacterial flagellin, which contributes to the restriction of the intracellular growth of L. pneumophila.

Colony-forming units

Colony-forming units

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* *

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102 0

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Fig. 4. Human NAIP restricts L. pneumophila growth in macrophages in response to bacterial flagellin. (A) The type IVB secretion system of L. pneumophila is required for intracellular growth in RAW264.7 macrophages. RAW264.7 cells were infected with wild-type L. pneumophila (JR32 strain), or the avirulent Dicm mutant 25D L. pneumophila harboring Dicm, at an MOI of 0.03. Bacterial growth was assayed by determining colony-forming units at the indicated time points. (B) Endogenous mNaip5 does not restrict intracellular growth of L. pneumophila in RAW264.7 macrophages. RAW264.7 cells were infected with wild-type L. pneumophila (Nagasaki 80-045 strain), or the isogenic DflaA mutant at an MOI of 0.02. (C) RAW264.7 macrophages expressing GFP, GFP-mNaip5, or GFP-NAIP were infected with wild-type or DflaA L. pneumophila at an MOI of 0.03. The error bars represent standard deviation of the mean of triplicate experiments. Data are representative of at least three independent experiments. (D) RAW264.7 macrophages were transiently transfected with expression vectors encoding GFP, GFPmNaip5, or GFP-NAIP. After 16 h, transfected cells were infected with L. pneumophila JR32 strain at an MOI of 0.1. Intracellular growth was calculated as (colony-forming units at 2 d)/(colony-forming units at 0 d). The error bars represent standard error of the mean of three independent experiments. *p < 0.05, relative to cells expressing GFP alone.

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Notably, no significant caspase-1 activation evaluated by IL-1b secretion in response to L. pneumophila infection was observed in RAW264.7 cells expressing GFP alone. Moreover, flagellin deficiency did not affect intracellular growth of L. pneumophila in RAW264.7 cells. These results support the permissiveness to L. pneumophila of mBMMs derived from BALB/c mice [10], from which RAW264.7 cells are generated. It has been suggested that the differences in amino acid sequences and expression levels of mNaip5 among mouse strains affect susceptibility to L. pneumophila in mBMMs [10,36]. Indeed, although the nucleotide sequence of endogenous mNaip5 cDNA was intact (data not shown), expression of endogenous mNaip5 could not be detected by Western blotting of cell lysates from unmodified RAW264.7 cells using commercially available antibodies, which could not distinguish mNaip5 from other mNaip proteins based on their size (1402 aa), except for mNaip2 (1444 aa) (data not shown). Therefore, the defects in response to L. pneumophila might be, at least in part, attributable to quantitative reduction of, but not qualitative changes in, endogenous mNaip5 in RAW264.7 cells. Taken together, these results suggest the effect of endogenous mNaip5 on the response of RAW264.7 cells to L. pneumophila infection is negligible, and restoration of caspase-1 activation and growth restriction are solely attributable to exogenous human NAIP or C57BL/6 Naip5 in RAW264.7 cells. While, in future, it will be necessary to determine whether human NAIP binds to FlaA in human NAIP-expressing RAW264.7 cells infected with L. pneumophila, our results suggest that human NAIP recognizes L. pneumophila flagellin, and subsequently promotes caspase-1 activation. Contrary to this, a reconstitution study in 293T cells revealed that the human NLRC4 inflammasome is activated by human NAIP in response to purified C. violaceum CprI, but not to purified L. pneumophila FlaA [23]. This discrepancy might be attributable to the difference in experimental conditions, infection of live L. pneumophila, or administration of purified FlaA. Considering that infection of L. pneumophila harboring a defect in the type IVB secretion system never leads to caspase-1 activation in mBMMs [15,16], other bacterial factors such as effector proteins might be required in addition to FlaA released from L. pneumophila for efficient activation of the Nlrc4 inflammasome in response to pathophysiological L. pneumophila infection of macrophages. The second possible interpretation for the discrepancy is that endogenous mNaip5 might potentially initiate the process, which is further augmented by exogenous human NAIP in RAW264.7 cells. We consider this to be unlikely because the effect of endogenous mNaip5 is negligible on the response of RAW264.7 cells to L. pneumophila infection as described above. Our results demonstrate that human NAIP and C57BL/6 Naip5 function in a similar manner in L. pneumophila-infected RAW264.7 macrophages: dependent on bacterial flagellin, they promote pore formation in the macrophage membrane and subsequent pyroptotic cell death and hamper intracellular L. pneumophila growth. In C57BL/6 BMMs, pyroptotic cell death in response to L. pneumophila flagellin is mediated by

mNaip5 and Nlrc4 via activation of the Nlrc4 inflammasome, which is involved in the restriction of intracellular L. pneumophila growth [16e19,22,23]. It is plausible that Nlrc4 inflammasome activation is promoted by human NAIP, as well as by C57BL/6 Naip5, in response to L. pneumophila flagellin in RAW264.7 cells. It is possible that human NAIP promotes activation of the human NLRC4 inflammasome, leading to pyroptosis in response to L. pneumophila flagellin in human macrophages and contributing to the restriction of intracellular bacterial growth. This agrees with a published study, which showed that either suppression of human NAIP or NLRC4 or flagellin-deficiency enhances L. pneumophila growth in human macrophages [27]. Conversely, the intracellular growth of L. pneumophila is supported by a variety of human cells [12,27,28] and L. pneumophila flagellin does not trigger caspase-1 activation in human cells [23,27,29]. A possible explanation for this discrepancy could be that human NAIP is intrinsically functional, but ineffective in caspase-1 activation in response to L. pneumophila infection in human cells. It is assumed that caspase-1 activity remains unaffected after L. pneumophila infection in human cells supporting bacterial growth, because previous reports in mBMMs [15e19] and our data indicate that the restriction of L. pneumophila growth correlates with caspase-1 activation. The absence of caspase-1 activation and restriction of intracellular L. pneumophila growth in humans might be attributed to certain defects in gene(s) involved in pyroptosis (such as human NAIP, NLRC4, or CASP1). In C57BL/6 BMMs, a defect in any one of these genes results in no restriction of L. pneumophila growth [15e19]. As an example, L. pneumophila multiplies in human U937-derived macrophages [28], where expression of NLRC4 is negligible [37]. Indeed, we could not observe L. pneumophila FlaAdependent responses in U937-derived macrophages (data not shown), which is in agreement with the previous report [23]. Considering that differences in the expression levels of mNaip5 among mouse strains affect the restriction of L. pneumophila growth in mBMMs [10,36], restoration of the factors could affect the susceptibility of human cells to L. pneumophila infection, as in our results. Indeed, transient overexpression of either human NAIP or NLRC4 has been shown to restrict L. pneumophila growth in human A549 cells, although the underlying mechanism has not been elucidated [27]. Moreover, our results are consistent with a recent report showing that a copy number variation in the NAIP gene is associated with cell death upon L. pneumophila infection [38]. This further suggests that higher amounts of the NAIP protein may confer host resistance to L. pneumophila. It is also possible that the host response to L. pneumophila infection is intrinsically different between mice and humans. In human monocytes, L. pneumophila downregulates apoptosisassociated speck-like protein (ASC) to prevent caspase-1 activation, while exogenous expression of the same enables cells to activate caspase-1 and to restrict L. pneumophila growth [29], neither of which are observed in mBMMs [15]. Thus, humans might possess distinct mechanisms for the suppression of pyroptosis in response to L. pneumophila

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infection. Recently, in human U937-derived macrophages, NAIP was shown to recognize CprI, the C. violaceum type III secretion system needle protein, and subsequently promote activation of the Nlrc4 inflammasome [23]. Further investigation is necessary to determine whether the same is true of human NAIP-expressing RAW264.7 cells. In addition to the promotion of pyroptosis, our data demonstrate another functional similarity between human NAIP and C57BL/6 Naip5 in the inhibition of apoptosis. Expression of C57BL/6 Naip5, and also of human NAIP, inhibited apoptosis in RAW264.7 macrophages, where the activity of caspase-3 and -7 was significantly reduced. It is plausible that mNaip5 binds to caspase-3 and -7 and/or uses procaspase-9 sequestration to block the activation of APAF-1 apoptosome, as human NAIP does [5,6]. Human NAIP is predominantly expressed in macrophage cells of the lung and spleen [30], suggesting that human NAIP protects macrophage cells from apoptotic cell death in these human tissues. Overall, our data suggest that human NAIP and mNaip5 regulate cell survival in response to specific intracellular signals: they inhibit activation of APAF-1 apoptosome triggered by cytochrome c, which leads to apoptosis, and they promote activation of the Nlrc4 inflammasome as triggered by L. pneumophila flagellin, which leads to pyroptosis. Future studies are necessary to determine the molecular mechanism underlying this regulation. Acknowledgments This work was supported in part by KAKENHI (21659108 and 22591108) from Japan Society for the Promotion of Science. References [1] S.M. Srinivasula, J.D. Ashwell, IAPs: what’s in a name? Mol. Cell 30 (2008) 123e135. [2] P. Liston, N. Roy, K. Tamai, C. Lefebvre, S. Baird, G. Cherton-Horvat, R. Farahani, M. McLean, J.E. Ikeda, A. MacKenzie, R.G. Korneluk, Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes, Nature 379 (1996) 349e353. [3] D.G. Xu, S.J. Crocker, J.P. Doucet, M. St-Jean, K. Tamai, A.M. Hakim, J.E. Ikeda, P. Liston, C.S. Thompson, R.G. Korneluk, A. MacKenzie, G.S. Robertson, Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus, Nat. Med. 3 (1997) 997e1004. [4] M. Lamkanfi, T.D. Kanneganti, Caspase-7: a protease involved in apoptosis and inflammation, Int. J. Biochem. Cell Biol. 42 (2010) 21e24. [5] J. Davoodi, M.H. Ghahremani, A. Es-Haghi, A. Mohammad-Gholi, A. Mackenzie, Neuronal apoptosis inhibitory protein, NAIP, is an inhibitor of procaspase-9, Int. J. Biochem. Cell Biol. 42 (2010) 958e964. [6] J.K. Maier, Z. Lahoua, N.H. Gendron, R. Fetni, A. Johnston, J. Davoodi, D. Rasper, S. Roy, R.S. Slack, D.W. Nicholson, A.E. MacKenzie, The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7, J. Neurosci. 22 (2002) 2035e2043. [7] J. Davoodi, L. Lin, J. Kelly, P. Liston, A.E. MacKenzie, Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9, J. Biol. Chem. 279 (2004) 40622e40628. [8] K. Schroder, J. Tschopp, The inflammasomes, Cell 140 (2010) 821e832. [9] E. Diez, S.H. Lee, S. Gauthier, Z. Yaraghi, M. Tremblay, S. Vidal, P. Gros, Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila, Nat. Genet. 33 (2003) 55e60.

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