The Journal of Immunology

Involvement of Absent in Melanoma 2 in Inflammasome Activation in Macrophages Infected with Listeria monocytogenes

Kohsuke Tsuchiya, Hideki Hara, Ikuo Kawamura, Takamasa Nomura, Takeshi Yamamoto, Sylvia Daim, Sita R. Dewamitta, Yanna Shen, Rendong Fang, and Masao Mitsuyama

Listeria monocytogenes invades the cytoplasm of macrophages and induces the activation of caspase-1 and the subsequent maturation of IL-1b and IL-18. Although apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain (ASC), an adaptor protein of nucleotide-binding oligomerization domain (Nod)-like receptors, has been shown to play an essential role in inducing this cellular response to L. monocytogenes, the mechanism has not been fully elucidated. In this study, we demonstrate the role of absent in melanoma 2 (AIM2), a recently described receptor of cytosolic DNA, in the activation of caspase-1 upon infection with L. monocytogenes. Secretion of IL-1b and IL-18 from Nod-like receptor family, pyrin domain containing 3 (NLRP3) and Nod- like receptor family, caspase-activating and recruitment domain containing 4 (NLRC4) knockout macrophages in response to L. monocytogenes was only slightly decreased compared with the levels secreted from wild-type macrophages, whereas secretion from ASC knockout macrophages was completely impaired, suggesting that receptors other than NLRP3 and NLRC4 also take part in inflammasome activation in an ASC-dependent manner. To identify such receptors, the abilities of several receptor candidates (NLRP2, NLRP6, NLRP12, and AIM2) to induce the secretion of IL-1b in response to L. monocytogenes were compared using the inflammasome system reconstructed in HEK293 cells. Among these receptor candidates, AIM2 conferred the highest responsiveness to the bacterium on HEK293 cells. Knockdown of AIM2 significantly decreased the secretion of IL-1b and IL-18 from L. mono- cytogenes-infected macrophages. These results suggest that AIM2, in cooperation with NLRP3 and NLRC4, plays an important role in the activation of caspase-1 during L. monocytogenes infection. The Journal of Immunology, 2010, 185: 1186–1195.

isteria monocytogenes, a Gram-positive rod-shaped bacte- In response to L. monocytogenes, innate immune cells, such as rium, often causes food-borne infections in immunocom- macrophages and dendritic cells, produce a wide variety of proin- L promised hosts, including newborns and the elderly. As flammatory cytokines that are involved in host defense against this a facultative intracellular parasitic bacterium, L. monocytogenes in- pathogen. Although the expression of many of these cytokines is in- vades a variety of host cells, such as hepatocytes, fibroblasts, and duced due to recognition of bacterial cells or bacterial products by epithelial cells, and multiplies in the cytoplasm of these cells (1–4). TLRs (9–13), some are induced independently of TLRs and instead are Professional phagocytes, such as macrophages, are also permissive induced in response to sensing by intracellular surveillance mecha- for intracellular growth of L. monocytogenes. After being engulfed nisms that are not yet fully understood. Upon infection of macro- by macrophages and trapped in the phagosome, L. monocytogenes phages with L. monocytogenes, LLO-dependent bacterial entry into survives by disrupting the phagosomal membrane by secreting a cy- the cytoplasm but not TLR adaptors, MyD88 and Toll/IL-1R domain- tolysin, listeriolysin O (LLO), and other virulence factors, and then containing adapter inducing IFN-b, is necessary for the induction of escapes into the cytoplasm. Indeed, L. monocytogenes strains lack- the expression of type I IFNs (14–17). Cytoplasmic entry of bacteria ing functional LLO are avirulent and are not able to grow in macro- is also required for the induction of caspase-1 activation and subse- phages or escape from the phagosome (5–8). quent maturation of IL-1b and IL-18 upon infection with L. mono- cytogenes (18–20). Besides the delivery of bacterial cells into the cytoplasm of macrophages, LLO has been shown to play a role in Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto, Japan enhancing the activation of caspase-1, possibly by modulating some Received for publication March 31, 2010. Accepted for publication May 13, 2010. cellular signaling either directly or indirectly (21, 22). Address correspondence and reprint requests to Dr. Kohsuke Tsuchiya, Department Recent studies have revealed that nucleotide-binding oligomeri- of Microbiology, Kyoto University Graduate School of Medicine, Yoshida Konoe- zation domain (Nod)-like receptors (NLRs), which are cytoplasmic cho, Sakyo-ku, Kyoto, 606-8501, Japan. E-mail address: [email protected]. proteins typically composed of C-terminal leucine-rich repeats, ac.jp a central nucleotide-binding domain, and a variable N-terminal ef- The online version of this article contains supplemental material. fector domain, such as a caspase-activating and recruitment domain Abbreviations used in this paper: AIM2, absent in melanoma 2; ASC, apoptosis- (CARD), play central roles incaspase-1 activationin response tovar- associated speck-like protein containing a caspase-activating and recruitment do- main; BHI, brain heart infusion; BMM, bone marrow-derived macrophage; CARD, ious exogenous and endogenous stimuli. These stimuli may include caspase-activating and recruitment domain; IFNAR1, IFN-a/b receptor 1; ILO, ATP, pore-forming toxins, ureic acid crystals, alum, bacterial flagel- ivanolysin O; KO, knockout; LDH, lactate dehydrogenase; LLO, listeriolysin O; MOI, multiplicity of infection; NLR, Nod-like receptor; NLRC, Nod-like receptor lins, and infection with microbial pathogens (23–29). NLR family, family, caspase-activating and recruitment domain containing; NLRP, Nod-like re- pyrin domain containing 3 (NLRP3) and NLR family, CARD do- ceptor family, pyrin domain containing; Nod, nucleotide-binding oligomerization main containing 4 (NLRC4) are the most thoroughly investigated domain; PEC, peritoneal exudate cell; siRNA, small interfering RNA; WT, wild-type. NLRs in this field, and a number of microbial pathogens have been Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 reported to induce caspase-1 activation via one of these two recep- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001058 The Journal of Immunology 1187 tors (23). Two studies reported that L. monocytogenes is predomi- a second recombination event. The in-frame deletion of the flaA gene nantly recognized by NLRP3 (24, 30). However, another study was confirmed by PCR using the primer set P5 and P6 (Table I) and se- D reported that caspase-1 activation induced upon infection with L. quencing of the PCR product. The constructed mutant ( flaA) was not motile on low-agar BHI (0.375%) at 22˚C (data not shown). monocytogenes is mediated by multiple receptors, including NLRP3, NLRC4, and other unidentified receptors (31). Moreover, Bacterial growth conditions Franchi et al. (32) showed that neither NLRP3 nor NLRC4 is nec- For the preparation of bacterial stocks, all of the bacterial strains were grown essary for the activation of caspase-1 in macrophages infected with overnight in BHI broth at 37˚C with shaking. One volume of the overnight L. monocytogenes. Therefore, the mechanism of caspase-1 activa- culture was added to 100 volumes of fresh BHI broth and cultured for tion upon infection with this pathogen remains controversial. Differ- a further 5 h. Bacterial cells were washed, suspended in PBS supplemented with 10% glycerol, and stored in aliquots at 280˚C. Bacterial stocks were ences between the findings of these studies might result from thawed and diluted in RPMI 1640 just before infection of macrophages. differences in the experimental procedures, such as the bacterial strain used, the procedure for preparation of bacteria and macro- Cells phages, the infection dose, and pretreatment of macrophages with Peritoneal exudate cells (PECs) of mice were obtained 3 d after i.p. injection LPS (24, 30–32). of 2 ml 3% thioglycolate medium (Eiken Chemical). After being washed In this study, we demonstrate that infection of macrophages with with RPMI 1640 media (Nacalai Tesque), PECs were suspended RPMI 1640 supplemented with 10% FCS (+FCS) and incubated in 48-well micro- L. monocytogenes at a multiplicity of infection (MOI) of 1, without 5 plates at a density of 2 3 10 cells per well at 37˚C in a humidified 5% CO2 any prestimulation, is sufficient for the induction of a significant environment for 3 h. After incubation, the cells were washed with RPMI level of caspase-1 activation and this response is only partially de- 1640 + FCS and adherent PECs were used for infection studies. Bone pendent on NLRP3 and NLRC4. Furthermore, we demonstrate that marrow cells were obtained from the tibiae of mice and cultured in RPMI m apoptosis-associated speck-like protein containing a CARD (ASC), 1640 + FCS containing gentamicin (10 g/ml; Wako Pure Chemical In- dustries, Osaka, Japan) and recombinant mouse M-CSF (100 ng/ml; R&D a key inflammasome component that bridges some NLRs and pro- Systems, Minneapolis, MN) for 5 d. Then, adherent bone marrow-derived caspase-1 (23), is essential in the induction of caspase-1 activation. macrophages (BMMs) were collected, suspended in fresh RPMI 1640 + In addition, our data suggest that absent in melanoma 2 (AIM2), FCS, and plated at 2 3 105 cells per well in 48-well microplates. BMMs a recently described intracellular DNA receptor (33–36), plays were used the following day for infection studies. an important role in the activation of caspase-1 upon infection with Infection and stimulation of cells L. monocytogenes. To infect macrophages, bacteria were added to the culture of adherent PECs or BMMs at the indicated MOI. After 30 min, gentamicin (final concen- Materials and Methods tration of 5 mg/ml) was added to the culture to inhibit the growth of bac- Reagents teria. However, in Fig. 1F, gentamicin was added to the macrophage cul- ture 90 min after the addition of bacteria, and the cells were incubated for Ultra pure LPS was purchased from InvivoGen (Toulouse, France). Nigericin an additional 60 min, making a total infection time of 2.5 h. To stimulate was purchased from Sigma-Aldrich (St. Louis, MO). Penicillin G and chlor- cells with cytosolic DNA, Poly(dA)·Poly(dT) (Sigma-Aldrich) or Listeria amphenicol were purchased from Nacalai Tesque (Kyoto, Japan). genomic DNA was introduced into adherent PECs or HEK293 cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA). Mice FemaleC57BL/6micewerepurchasedfromJapanSLC(Hamamatsu,Japan). ELISA NLRP3 knockout (KO) mice generated by Prof. Ju¨rg Tschopp (University Levels ofcytokinesinculturesupernatants were determined by two-site sand- of Lausanne, Lausanne, Switzerland) (26), NLRC4 KO mice generated wich ELISA. Culture supernatants were stored at 280˚C prior to cytokine by Dr. Vishva Dixit (Genentech, South San Francisco, CA) (37), ASC KO measurement. The ELISA kits for IL-1a and IL-1b were purchased from mice generated by Prof. Shun-ichiro Taniguchi (Shinshu University, Nagano, eBioscience (San Diego, CA) and BD Biosciences (San Diego, CA), Japan) (38), caspase-1 KO mice generated by Dr. Keisuke Kuida (Millen- respectively. For the titration of IL-18, a pair of biotin-labeled and unlabeled a b nium Pharmaceuticals, Cambridge, MA), and IFN- / receptor 1 (IFNAR1) mAbs specific to IL-18 (Medical & Biological Laboratories, Nagoya, Japan) KO mice generated by Prof. Michel Aguet (Ecole Polytechnique Federale de were used. Lausanne, Lausanne, Switzerland) were kindly provided as gifts. Mice were maintained in specific pathogen-free conditions and used at 7–9 wk of age. Western blot analysis All of the experimental procedures performed on mice were approved by the 3 6 Animal Ethics and Research Committee of Kyoto University Graduate PECs were cultured on six-well microplates at a density of 1 10 cells School of Medicine, Kyoto, Japan. per well in RPMI 1640 + FCS at 37˚C for 3 h. The culture medium was replaced with Opti-MEM (Invitrogen), and adherent cells were infected with Bacterial strains L. monocytogenes at a MOI of 1. Culture supernatants were collected 24.5 h postinfection, and the cells were lysed with RIPA buffer (Nacalai Tesque). The parental wild-type (WT) L. monocytogenes strain used in this study The culture supernatants were concentrated 20-fold using 20% (w/v) tri- D was L. monocytogenes EGD (serovar 1/2a). Three isogenic mutants, hly, chloroacetate (Nacalai Tesque). The precipitates and cell lysates were sub- D D hly::hly, and hly::ilo, were constructed from WT L. monocytogenes jected to SDS-PAGE and subsequently transferred to polyvinylidene strain EGD using the homologous recombination method described in difluoride membranes by electroblotting. The membranes were immunoblot- a previous study (21). An isogenic deletion mutant of the flaA gene in L. ted with anti–caspase-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), monocytogenes strain EGD was also constructed using the homologous anti–IL-1b Ab (R&D Systems), or anti–b-actin Ab (Sigma-Aldrich). recombination method. A 1875-bp fragment located upstream of the flaA gene was amplified from the chromosomal DNA of WT L. monocytogenes Detection of lactate dehydrogenase release EGD by PCR with the P1 and P2 primer set (Table I). A 2012-bp fragment located downstream of the flaA gene was similarly amplified with the P3 Lactate dehydrogenase (LDH) activity was measured using an LDH cy- and P4 primer set (Table I). The two amplified fragments were ligated into totoxicity detection kit (TaKaRa BIO, Shiga, Japan). The percentage of the plasmid vector pHS-MCS, which carries a thermosensitive replication LDH release was calculated by using the following equation: percentage of origin (21). The resulting plasmid was introduced into competent cells of release = 100 3 (experimental LDH release 2 spontaneous LDH release)/ WT L. monocytogenes EGD by electroporation. Transformants were se- (maximal LDH release 2 spontaneous LDH release). To determine the lected initially at 30˚C on brain heart infusion (BHI) agar plates (Eiken maximal LDH release, cells were treated with 1% Triton X-100. Chemical, Tokyo, Japan) supplemented with erythromycin (5 mg/ml; Nacalai Tesque). Transformants were then grown on BHI agar plates with RT-PCR analysis erythromycin at 42˚C to select only those transformants with chromosomal integration of the plasmid. Cointegrates were subsequently grown in BHI Total cellular RNA of adherent PECs was extracted using the NucleoSpin broth (Eiken Chemical) without antibiotic selection at 30˚C to facilitate the RNA II kit (Macherey-Nagel, Du¨ren, Germany), according to the manu- isolation of derivatives in which integrated plasmids were resolved by facturer’s instructions. The collected RNA (0.2 mg) was treated with 1188 ROLE OF AIM2 IN LISTERIA INFECTION

FIGURE 1. Involvement of NLRP3 and NLRC4 in inflammasome activation during infection with L. monocytogenes. A–C,Ad- herent PECs (A, B)orBMMs(C)fromWT, ASC KO, NLRP3 KO, or NLRP4 KO mice were left uninfected (2)orinfected(+)with WTor mutant L. monocytogenes strains at the indicated MOI for 24.5 h. D, Adherent PECs from each mouse strain were left uninfected or infected with WT L. monocytogenes at a MOI of 1 for 24.5 h. Culture supernatants were concentrated by trichloroacetate precip- itation. The precipitates and cell lysates were subjected to Western blot analysis. E, Adher- ent PECs from each mouse strain were left uninfected (0% control) or infected with WT L. monocytogenes at a MOI of 1. At the indicated time points, LDH release was de- termined. F, Adherent PECs from WT or NLRP3 KO mice were untreated (2)orpre- treated (+) with 10 ng/ml LPS overnight. The cells were left uninfected (2)orinfectedwith WT L. monocytogenes at a MOI of 1 or 50 for 2.5 h. G, Adherent PECs from WT mice were infected with WT or mutant L. monocyto- genes strains at a MOI of 1 for 24.5 h. IL-1b (A, C, F, G) and IL-18 (B) in the culture supernatants were assessed by ELISA. ELISA data are presented as the mean of trip- licate assays and the SD. All of the experi- ments were repeated at least twice and similar results were obtained. pp , 0.05.

RNase-free DNase (Promega, Madison, WI) to eliminate contaminating and 5 mg/ml gentamicin. For experiments, HEK293 cells were plated in DNA before being subjected to reverse transcription using Superscript 24-well microplates at a density of 2 3 105 cells per well and incubated VILO (Invitrogen). Quantitative real-time RT-PCR was then performed overnight. The cells were transfected with a total of 1 mg DNA per well on an ABI PRISM 7000 (Applied Biosystems, Foster City, CA) using using Transfectin Lipid Reagent (Bio-Rad Laboratories, Hercules, CA) Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) with primers according to the manufacturer’s instructions. After 38.5 h, the culture described in Supplemental Table I. Results were analyzed with ABI medium was replaced with gentamicin-free medium, and the transfected PRISM 7000 SDS software. Gene-specific transcript levels were normal- cells were left uninfected or infected with 105 CFU of WT L. monocyto- ized to the amount of b-actin mRNA. Extraction of total RNA from the genes. Gentamicin (20 mg/ml) was added to the culture 1.5 h postinfection, spleen or ovary was carried out using a Sepasol-RNA I Super kit (Naca- and the cells were incubated for an additional 8 h. We then confirmed that lai Tesque). The collected RNA was treated with RNase-free DNase and L. monocytogenes was able to invade HEK293 cells and proliferate in the again purified using NucleoSpin RNA II. Purified RNA was subjected cytoplasm (data not shown). to reverse transcription using Superscript VILO. Conventional PCR (nonquantitative PCR) was performed using GoTaq Green Master Mix RNA interference (Promega). PECs were transfected with Stealth small interfering RNA (siRNA) Reconstruction of the inflammasome system in HEK293 cells duplexes (Invitrogen) at a final concentration of 20 nM using siPORT Amine (Ambion, Austin, TX). Briefly, 0.6 ml siPORT was diluted in 20 ml The full-length open reading frames of ASC, procaspase-1, pro-IL-1b, pro- Opti-MEM, incubated at room temperature for 10 min, and then combined IL-18, NLRP2, NLRP6, NLRP12, and AIM2 were amplified from cDNA with siRNA diluted in 20 ml Opti-MEM. The mixture was incubated at preparations, which were obtained from the spleen or ovary of B6 mice by room temperature for 10 min, then transferred to a well in a 48-well PCR using KOD Plus enzyme (TOYOBO, Osaka, Japan) and the primer microplate. PECs were suspended in Macrophage-SFM (Invitrogen) at sets indicated in Supplemental Table II. The amplified fragments were a density of 1 3 106 cells per milliliter, and 0.2 ml of the suspension digested with restriction enzymes (Supplemental Table II) and inserted was plated in a well containing the siPORT–siRNA complex. The cells into the pFLAG-CMV2 vector (Sigma-Aldrich). The resulting plasmids were incubated at 37˚C for 48 h. Then, the siPORT–siRNA complex and were designated pFLAG-ASC, pFLAG-procaspase-1, pFLAG-pro-IL-1b, nonadherent cells were washed out with RPMI 1640 + FCS, and the pFLAG-proIL-18, pFLAG-NLRP2, pFLAG-NLRP6, pFLAG-NLRP12, transfected cells were used in experiments. The sense siRNA sequences and pFLAG-AIM2, respectively. HEK293 cells obtained from the Amer- were: AIM2-1, 59-UUA UCU UCU GGA CUU UAA ACA GCC C-39; ican Type Culture Collection (Rockville, MD) were maintained in DMEM AIM2-2, 59-UAA AGU CAU UGU CAC UGC GGG UGG C-39. Negative supplemented with 10% FCS, 6 mM L-glutamine, 1 mM sodium pyruvate, control siRNA was purchased from Invitrogen. The Journal of Immunology 1189

Table I. Primers used for the construction of DflaA

Primers Primer Sequences Restriction Enzymes P1 59-TAG ATA TAA TGG TAC CCG ATG TGT ATC CAC CTG TCG-39 KpnI P2 59-TAG ATA TAA TCC CGG GTT GTG TTT CCC TCC TAC ATT-39 XmaI P3 59-TAG ATA TAA TCC CGG GTA ACA TAT CAA ATT CAT TTA-39 XmaI P4 59-TAG ATA TAA TGA CGT CGA TCT GCG GTA CTG AAA CCG-39 AatII P5 59-CAG AAG ATC TGG AAC ATA AAG GTG C-39 P6 59-GAC GAG AGG CTA AGG GTA AAC AAT G-39 Restriction enzyme sites are underlined.

Isolation of L. monocytogenes genomic DNA discrepancies in the significance of NLRP3 in the activation of An overnight culture of L. monocytogenes in BHI broth was centrifuged, caspase-1. To further examine the involvement of the recognition and the pellet was suspended in an equal volume of 10 mM Tris buffer (pH of flagellin by NLRC4 in inflammasome activation in L. monocyto- 8) containing 50 mM EDTA, 20 mg/ml lysozyme, and 100 mg/ml RNase A genes-infected macrophages, macrophages from WT mice were and incubated at 37˚C for 1 h. After being mixed with a 1:6 volume of 5 M NaCl and a 1:7.5 volume of 10% cetyltrimethylammonium bromide/0.7 M NaCl solution, the bacterial lysates were incubated at 65˚C for 10 min. DNA was extracted from the mixture using chloroform/isoamyl alcohol and phenol/chloroform/isoamyl alcohol. Statistical analysis For comparisons between two groups, the Student t test was used when the variances of the groups were judged to be equal by the F test. Multi- group comparisons of mean values were made according to ANOVA and the Fisher’s protected least significant difference post hoc test after the confirmation of homogeneity of variances among the groups using Bar- tlett’s test. Statistical significance was determined as p , 0.05.

Results Involvement of NLRP3, NLRC4, and ASC in caspase-1 activation upon infection with L. monocytogenes To clarify whether NLRP3 and NLRC4 are involved in the activation of caspase-1 upon infection with L. monocytogenes, adherent PECs from WT mice, NLRP3 KO mice, and NLRC4 KO mice were infected with the bacterium at a MOI of 1. The culture supernatants were collected 24.5 h postinfection, and the levels of IL-1b and IL-18 were determined. Although the secretion of these cytokines from NLRP3 KO macrophages and NLRC4 KO macrophages in response to L. monocytogenes infection seemed to be lower than that from WT macrophages, IL-1b and IL-18 were detected at a sig- nificant level even in the absence of NLRP3 or NLRC4 (Fig. 1A,1B). By contrast, ASC KO macrophages did not secrete IL-1b or IL-18 postinfection with L. monocytogenes (Fig. 1A,1B). Similar results were obtained when BMMs from each mouse strain were infected at a MOI of 1 or 2 (Fig. 1C and data not shown). To determine the level of activation of caspase-1 directly, the mature form of caspase-1 (p10 fragment) in the culture supernatants was detected by Western blotting using an anti–caspase-1 Ab. Caspase-1 p10, as well as ma- ture IL-1b, were detected in the culture supernatants of macro- phages from WT mice, NLRP3 KO mice, and NLRC4 KO mice FIGURE 2. Screening of the receptors involved in caspase-1 activation infected with L. monocytogenes, although they were not detected during L. monocytogenes infection. A, Total RNA was isolated from the in the culture supernatants of infected ASC KO macrophages spleen or ovary, and the expression of each receptor was detected by RT- (Fig. 1D). Moreover, LDH release induced upon infection with L. PCR using the primer sets indicated in Supplemental Table I. The amplifi- monocytogenes was significantly lower in caspase-1 KO macro- cation was performed for 38 cycles. B–D, HEK293 cells were cotransfected phages and ASC KO macrophages but not NLRP3 KO macro- with empty vector (333.5 ng), pFLAG-pro-IL-1b (300 ng), pFLAG-pro-IL- phages than that in WT macrophages (Fig. 1E). These results in- 18 (300 ng), pFLAG-procaspase-1 (50 ng), or pFLAG-ASC (15 ng). The dicated that ASC is indispensable for the activation of caspase-1 cells were also transfected with empty vector, pFLAG-AIM2, pFLAG- NLRP2, pFLAG-NLRP6, or pFLAG-NLRP12 (1.5 ng each). After 38.5 h, upon infection with L. monocytogenes, whereas NLRP3 and 5 NLRC4 are only partly involved. It should be noted that, as shown the transfected cells were left uninfected or infected with 10 CFU WT L. monocytogenes (B, C) or stimulated with 400 ng Poly(dA)·Poly(dT) (D). in a previous report (24), NLRP3 was essential for the secretion of b Gentamicin was added to the culture 1.5 h after the addition of bacteria, IL-1 and IL-18 when macrophages were pretreated with LPS and the cells were incubated for an additional 8 h. IL-1b (B, D) and IL-18 and subsequently infected with L. monocytogenes at a MOI of (C) in the culture supernatants were assessed by ELISA. ELISA data are 50 for 2.5 h (Fig. 1F and data not shown). This suggests that differ- presented as the mean of triplicate assays and the SD. All of the experiments ences in the infection protocol might be the cause of the were repeated at least twice and similar results were obtained. 1190 ROLE OF AIM2 IN LISTERIA INFECTION infected with WT L. monocytogenes or its isogenic mutant strains also transfected with AIM2, NLRP2, NLRP6, NLRP12, or an deficient in the flagellin gene (flaA) (Table I) or the LLO gene (hly). empty vector. Transfected cells were then infected with L. WT L. monocytogenes and DflaA similarly induced the secretion of monocytogenes, and levels of IL-1b and IL-18 in the culture super- IL-1b and IL-18, whereas Dhly did not (Fig. 1G and data not shown), natants were determined. Among these transfected cells, AIM2- suggesting that sensing of flagellin by NLRC4 is not important for transfected cells secreted the highest levels of IL-1b and IL-18 post- the activation of caspase-1 during infection with L. monocytogenes infection with L. monocytogenes (Fig. 2B,2C). Although even strain EGD. HEK293 cells transfected with other receptors or the empty vector exhibited an increased secretion of IL-1b and IL-18 when infected Screening of the receptors involved in caspase-1 activation in with L. monocytogenes (versus uninfected control), the increase in L. monocytogenes infection the concentration was only ∼500 pg/ml. However, the increase in the The data presented in Fig. 1A–D strongly suggested that, in addition concentration of IL-1b and IL-18 in the culture supernatants of to NLRP3 and NLRC4, some ASC-dependent receptors are in- AIM2-transfected cells infected with L. monocytogenes was volved in inducing caspase-1 activation during L. monocytogenes .1500 pg/ml. These results suggested that AIM2 may play a role infection. To identify these receptors, we selected four receptor in sensing L. monocytogenes. We also observed that only AIM2- molecules (NLRP2, NLRP6, NLRP12, and AIM2) as candidates transfected cells were responsive to Poly(dA)·Poly(dT), a canonical using the following criteria: 1) ability to interact with ASC via its ligand for AIM2 (Fig. 2D) (33–36). It is important to note that we pyrin domain (39–43); 2) ability to induce caspase-1 activation (40– could not confirm whether NLRP2, NLRP6, and NLRP12 were func- 43); and 3) expression in the spleen, a major target organ for L. tioning in the inflammasome system reconstructed in HEK293 cells, monocytogenes in which innate and adaptive immune responses because ligands for these receptors (i.e., a positive control) are not are actively induced upon infection in vivo (Fig. 2A). Although known. However, we continued to investigate the function of AIM2 NLRP2 was not expressed in the spleen, we did not omit this NLR in macrophages. because previous reports have shown that NLRP2 is upregulated in the human monocytic cell line THP-1 after stimulation with LPS AIM2 plays an important role in inducing activation of (44) and mediates an innate immune response to Fusobacterium caspase-1 in macrophages infected with L. monocytogenes nucleatum in gingival epithelial cells (45). To test whether To examine whether AIM2 is involved in the induction of caspase-1 candidate receptors take part in the activation of caspase-1 in L. activation during infection of macrophages with L. monocytogenes, monocytogenes-infected cells, we reconstructed the inflammasome we performed a knockdown of AIM2 in macrophages using two system in HEK293 cells. HEK293 cells were cotransfected siRNAs, AIM2-1 and AIM2-2, targeting independent sequences in with ASC, procaspase-1, pro-IL-1b, and pro-IL-18. The cells were the AIM2 transcript. The efficiency of the knockdown was verified

FIGURE 3. AIM2 is involved in the activation of caspase-1 upon infection of macrophages with L. monocytogenes. Adherent PECs were transfected with siRNAs (AIM2-1, AIM2-2, or control siRNA). A, Knockdown of AIM2 was verified by real-time RT-PCR. B, Knockdown of AIM2 resulted in a significant decrease in IL-18 secretion in response to 400 ng Poly(dA)·Poly(dT). C, Adherent PECs transfected with each siRNA were left uninfected or infected with WT L. monocytogenes at a MOI of 1 for 24.5 h. Culture supernatants and cell lysates were subjected to Western blot analysis as mentioned in Fig. 1. D–F, AIM2 knockdown macrophages were infected with WT L. monocytogenes at a MOI of 1 for 24.5 h. IL-1b (D), IL-18 (E), and IL-1a (F) in the culture supernatants were assessed by ELISA. G, AIM2 knockdown macrophages were stimulated with 10 ng/ml LPS overnight. Then, the cells were additionally stimulated with 20 mM nigericin for 3 h, and IL-1b in the culture supernatants was assessed by ELISA. ELISA data are presented as the mean of triplicate assays and the SD. All of the experiments were repeated at least twice and similar results were obtained. pp , 0.05. The Journal of Immunology 1191 by real-time RT-PCR, and the expression of AIM2 was success- fully inhibited by both siRNAs (Fig. 3A). These siRNAs did not inhibit the expression of ASC, procaspase-1, and pro-IL-18, as de- termined by real-time RT-PCR (data not shown). Moreover, knock- down of AIM2 significantly decreased the secretion of IL-18 from macrophages stimulated with Poly(dA)·Poly(dT) (Fig. 3B). Secre- tion of caspase-1 p10, IL-1b, and IL-18 from macrophages infected with L. monocytogenes into the culture supernatants was signifi- cantly decreased by knockdown of AIM2 (Fig. 3C–E), whereas se- cretion of IL-1a and TNF-a induced by L. monocytogenes (Fig. 3F and data not shown) and secretion of IL-1b induced by LPS plus nigericin (Fig. 3G) were not affected. Moreover, knockdown of AIM2 did not affect intracellular growth of L. monocytogenes (Fig. 5A). Taken together, these findings suggested that AIM2 plays an important role in inducing activation of caspase-1 upon infection with L. monocytogenes.

Listeria genomic DNA can be a ligand for AIM2 AIM2 has recently been shown to recognize cytosolic DNA. There- fore, we proposed that DNA released from bacteria or organelles, such as the nucleus, damaged during the course of infection with L. monocytogenes could be responsible for the activation of cas- pase-1. We tested whether Listeria genomic DNA is able to induce the activation of caspase-1. Listeria genomic DNA was purified from bacterial culture, and part of the preparation was treated with DNase I (Fig. 4A). Untreated Listeria DNA clearly induced the secretion of IL-18 from macrophages, whereas the DNase I- treated sample did not, suggesting that Listeria genomic DNA is capable of inducing caspase-1 activation. Moreover, secretion of FIGURE 4. L. monocytogenes genomic DNA activates the AIM2 in- IL-18 induced by Listeria genomic DNA was decreased by knock- flammasome. A, Genomic DNA was prepared from L. monocytogenes and down of AIM2. These results indicated that Listeria genomic electrophoresed on a 0.9% agarose gel. DNase I treatment was carried out DNA is recognized by AIM2, thereby inducing the secretion of overnight at a concentration of 1 U per 1 mgDNA.B, Adherent PECs were the caspase-1–dependent cytokine. stimulated with 400 ng of purified Listeria DNA or DNase-treated Listeria DNA for 24 h. IL-18 in the culture supernatants was assessed by ELISA. Dead bacteria in the cytoplasm induce the secretion of C, AIM2 was knocked down in adherent PECs as mentioned in Fig. 3, and caspase-1–dependent cytokines in an AIM2-dependent manner the cells were stimulated with Listeria DNA for 24 h. IL-18 in the culture supernatants was assessed by ELISA. ELISA data are presented as the Even if it is assumed that DNA released from L. monocytogenes mean of triplicate assays and the SD. All of the experiments except for into the cytoplasm is responsible for the induction of caspase-1 those in A were repeated at least twice and similar results were obtained. activation upon infection of macrophages, the question regarding pp , 0.05. how DNA is released from the bacterium remains to be answered. After analyzing the kinetics of cytokine secretion and bacterial multiplication in host macrophages, we noticed a remarkable in- the secretion of IL-1b and IL-18 from uninfected macrophages crease in secretion of IL-1b and IL-18 at .9.5 h postinfection at (Fig. 5C) and never enhanced DNA-induced IL-18 secretion (data which stage the number of intracellular L. monocytogenes was not shown). Accordingly, it is suggested that even a small number reduced rather than increased (Fig. 5A,5B, and data not shown). of L. monocytogenes can induce the activation of caspase-1 if This result was consistent with our previous data that the activa- artificially lysed in the cytoplasm. Moreover, secretion of IL-1b tion of caspase-1 is detectable at .10 h postinfection with the and IL-18 from infected macrophages occurred immediately after LLO-expressing L. monocytogenes strain (22) and raised the pos- the addition of penicillin G (Fig. 5B and data not shown), and sibility that DNA or other caspase-1–activating agents might be secretion was significantly decreased by knockdown of AIM2 liberated from dead bacteria into the cytoplasm. To investigate (Fig. 5D and data not shown). Taken together, these findings in- whether L. monocytogenes killed in the cytoplasm were able to dicate that L. monocytogenes is unable to proliferate and survive induce caspase-1 activation, a bactericidal antibiotic, penicillin G, in macrophages for .12.5 h for unknown reasons and, at the was added to the culture 3.5 h postinfection. At that time, most of stationary phase-like period, dead bacteria in the cytoplasm might bacteria should have escaped into the cytoplasm (21). Penicillin G liberate their contents, including DNA, which then induces the rapidly killed intracellular bacteria, as described in a previous re- activation of caspase-1 via recognition by AIM2. port (46), and the number of intracellular L. monocytogenes at its peak was markedly fewer compared with the number of organisms AIM2 expression is upregulated upon infection with in the absence of penicillin G (Fig. 5A). However, although the L. monocytogenes addition of penicillin G resulted in a decrease in the secretion of Because AIM2 is an IFN-inducible gene (47) and L. monocyto- IL-1b and IL-18 in response to L. monocytogenes, the antibiotic genes induces the expression of type I IFNs in host macrophages did not severely impair secretion (Fig. 5C and data not shown). In (15, 16), it could be expected that the expression of AIM2 might contrast, chloramphenicol, a bacteriostatic antibiotic, almost com- be upregulated upon infection with this pathogen. Indeed, the ex- pletely impaired the secretion of IL-1b and IL-18 (Fig. 5C and pression of AIM2 was significantly higher in infected macro- data not shown). In our experiments, penicillin G never induced phages compared with that in the uninfected control (Fig. 6A). 1192 ROLE OF AIM2 IN LISTERIA INFECTION

FIGURE 5. Relationship between intracellular growth of L. monocytogenes and the secretion of IL-1b from infected macrophages. A, B, and D, Adherent PECs were transfected with each siRNA (AIM2-1 or control sequence) and infected with WT L. monocytogenes at a MOI of 1. Penicillin G (100 U/ml) was added to some wells 3.5 h postinfection (indicated by arrows). At the indicated time points, the number of intracellular bacteria was determined by a CFU assay (A). Culture supernatants were also collected, and IL-1b in the culture supernatants was assessed by ELISA (B, D). C, Adherent PECs were left uninfected of infected with WT L. monocytogenes at a MOI of 1. Penicillin G (100 U/ml) or chloramphenicol (20 mg/ml) was added to some wells 3.5 h postinfection. Culture supernatants were collected 24.5 h postinfection, and IL-1b in the culture supernatants was assessed by ELISA. The multiplicity index is the CFU at the indicated times divided by the CFU at 1.5 h postinfection. CFU and ELISA data are presented as the mean of triplicate assays and the SD. All of the experiments were repeated at least twice and similar results were obtained. pp , 0.05.

Furthermore, we found that the upregulation of AIM2 requires the Discussion type I IFN signal, because the expression of AIM2 in IFNAR1 KO In this study, we showed that AIM2 plays an important role in macrophages was not altered by infection with L. monocytogenes the activation of caspase-1 and the subsequent secretion of caspase- (Fig. 6A). It has been reported that the type I IFN signal is required 1–dependent cytokines, IL-1b and IL-18, upon infection of macro- for the activation of caspase-1 during infection with L. monocy- phages with L. monocytogenes. However, our findings are conflicting togenes (48). We also obtained a similar result that IL-18 secretion with data presented in previous reports (24, 30). Our observations induced by L. monocytogenes was significantly lower in IFNAR1 suggested that NLRP3 and NLRC4 are also involved in the secretion KO macrophages than that in WT macrophages (infection of un- of IL-1b and IL-18 in response to L. monocytogenes, although these treated macrophages at a MOI of 1) (data not shown). Therefore, it is NLRs play a more minor role. Hence, we conclude that AIM2, in conceivable that the inferior ability of IFNAR1 KO macrophages to cooperation with NLRP3 and NLRC4, plays an important role in the activate caspase-1 in response to L. monocytogenes might be attrib- activation of caspase-1 during L. monocytogenes infection. utable to the lower expression of AIM2, which is upregulated by the In the first report to investigate the role of NLRs in L. mono- type I IFN signal. Finally, it is important to point out the relationship cytogenes infection, the NLRP3–ASC pathway was shown to be between this study and our previous study in which LLO was shown essential for the activation of caspase-1 in macrophages infected to play a role in enhancing the activation of caspase-1 (22). In our with L. monocytogenes (24). In that study, macrophages were pre- previous study, the LLO-expressing L. monocytogenes strain but not treated with LPS and infected at a MOI of 50. In the current study, we the L. monocytogenes strain expressing another cytolysin, ivanoly- performed a similar experiment, and a consistent result was ob- sin O (ILO), instead of LLO, induced the activation of caspase-1 tained (Fig. 1F). It is noteworthy that Il-1b secretion in this con- efficiently, even though these bacterial strains invaded and multi- text was more rapidly induced compared with induction upon plied in the macrophage cytoplasm in a similar way. This suggested infection of untreated macrophages with L. monocytogenes at a that the activation of caspase-1 is dependent on not only the entry of low MOI. Therefore, it appeared that the activation of caspase-1 the bacterium into the cytoplasm but also on the distinct activity of in these two experiments was induced in a different manner. Be- LLO as a signaling ligand. To test whether LLO is involved in the cause LPS facilitates the formation of the NLRP3–inflammasome upregulation of AIM2, in this study we analyzed the expression of by upregulating the expression of NLRP3 through the activation of AIM2 in macrophages infected with each L. monocytogenes strain. NF-kB (49, 50), LPS pretreatment might enhance the contribution The LLO-expressing L. monocytogenes strain, but not the cytolysin- of NLRP3 to the induction of caspase-1 activation upon infection deficient strain or the ILO-expressing strain, significantly enhanced with L. monocytogenes. In another report by Warren et al. (31) the expression of AIM2 (Fig. 6B), suggesting that some LLO- the recognition of flagellin by NLRC4 was shown to be important dependent signal might be necessary for maximum activation of for the activation of caspase-1 in macrophages infected with L. caspase-1, at least in part, through the upregulation of AIM2. monocytogenes. It is known that the expression of flagellin is strictly The Journal of Immunology 1193

of AIM2 (54, 55). These mice would be useful in further clarifying the role of this DNA receptor during infection with L. monocyto- genes in vivo. In the current study, we hypothesized that Listeria DNA liber- ated from dead bacteria in the cytoplasm is recognized by AIM2. Indeed, L. monocytogenes in host macrophages exhibited a growth curve, which was very similar to that in broth media, and secretion of IL-1b and IL-18 became detectable from the stationary phase- like period (.9.5 h postinfection). We suppose that, in a macro- phage compartmentalized by the plasma membrane, continuous proliferation of L. monocytogenes might cause the exhaustion of nutrients and subsequent bacterial death, possibly accompanied by the breakdown of the cell wall due to the action of autolysins and other hydrolytic enzymes. From our results, it appeared that lysis of L. monocytogenes in the host cell cytoplasm resulting in the liberation of the bacterial cell contents, including DNA, was able to induce the activation of caspase-1, because the addition of pen- icillin G at 3.5 h postinfection resulted in the rapid secretion of moderate levels of IL-1b in an AIM2-dependent manner (Fig. 5). In support of this idea, lysis of cytosolic L. monocytogenes by an expression-controlled autolysin or penicillin G has been implied to FIGURE 6. L. monocytogenes induces the upregulation of AIM2 de- enhance the liberation of DNA from bacteria into the host cyto- pending on the type I IFN signal. A, Adherent PECs from WT or IFNAR1 plasm (46, 56). However, in addition to dead bacteria, organelles KO mice were left uninfected or infected with L. monocytogenes at a MOI of host macrophages, such as the nucleus, are also possible sour- of 1. After 3.5 h, the cells were lysed and total RNA was isolated from the ces of DNA if they are damaged in the course of infection. More- lysates and subjected to real-time RT-PCR to assess the level of AIM2 over, it is still possible that at the late stage of infection the expression. B, Adherent PECs were left uninfected or infected with the number of intracellular L. monocytogenes decreased due to other LLO-deficient L. monocytogenes strain (Dhly), the LLO-expressing strain (Dhly::hly), or the ILO-expressing strain (Dhly::ilo) at a MOI of 1. After reasons, such as host cell death or bactericidal host effector mol- 3.5 h, the level of AIM2 expression was assessed by real-time RT-PCR. ecules. Furthermore, we did not show any evidence that DNA is Data are presented as the mean of triplicate assays and the SD. All of the actually responsible for the AIM2-dependent caspase-1 activation experiments were repeated at least twice and similar results were obtained. induced upon infection with L. monocytogenes because of the pp , 0.05. technical difficulties in proving this. Therefore, further research is required to fully elucidate the basis for AIM2–inflammasome activation upon infection of macrophages with L. monocytogenes. inhibited at 37˚C in L. monocytogenes strain EGD but not in the In agreement with other reports (19, 20, 24, 30, 31), we showed strain used in their study (10403s) (51, 52). Accordingly, differences that LLO was indispensable for the secretion of IL-1b from in the regulation of flagellin expression among bacterial strains macrophages infected with L. monocytogenes (Fig. 1F), sug- should be considered when discussing the role of NLRC4 in induc- gesting that bacterial entry into the cytosol is a key event in this ing caspase-1 activation upon infection with L. monocytogenes. context. However, in our study, we found that delivery of the bac- More recently, Meixenberger et al. (30) reported that NLRP3 terium into the cytosol alone was not sufficient for inducing inflam- is critical for IL-1b secretion from human PBMCs infected with masome activation, because chloramphenicol, which was added to L. monocytogenes, whereas AIM2 is not involved. One possible the macrophage culture after most bacteria had escaped into the explanation for the discrepancy between our data and this pre- cytosol, almost completely impaired the secretion of IL-1b in vious report may be the differences between human PBMCs and response to L. monocytogenes (Fig. 5C). Furthermore, we pre- primary mouse macrophages. For example, it has been reported viously showed that not only the entry of bacteria into the cytoplasm that monocytes isolated from human PBMCs constitutively release but also the unique activity of LLO as a signaling ligand is necessary ATP, which enables the cells to secrete bioactive IL-1b in response for the efficient activation of caspase-1 (22). In the current study, we to LPS alone in a NLRP3-dependent manner (53). By contrast, LPS showed that the expression of AIM2 was upregulated upon infection alone did not induce the secretion of IL-1b from adherent PECs with the LLO-expressing strain but not the ILO-expressing strain obtained from WT mice (data not shown). (Fig. 6B). Therefore, the difference in expression of AIM2 may Our data clearly showed that AIM2 is involved in caspase-1 ac- explain, at least in part, why LLO is required for efficient inflam- tivation in response to L. monocytogenes. However, knockdown of masome activation upon infection with invasive L. monocytogenes. AIM2 did not result in overall impairment of the secretion of We have recently found that, compared with the LLO-expressing caspase-1–dependent cytokines. One possible explanation for this strain, the ILO-expressing strain only moderately induced the ex- is that AIM2 function in macrophages transfected with targeting pression of type I IFNs (data not shown), which transduce a signal siRNA might remain at a low level, because DNA-induced IL-18 essential for AIM2 upregulation upon L. monocytogenes infection secretion was also not completely abrogated (Fig. 3B). In addition, through a common receptor composed of IFNAR1 (Fig. 6A). These because NLRP3 and NLRC4 appear to be partly involved in the data, together with the results of further analysis, will be presented secretion of IL-1b and IL-18 in response to L. monocytogenes,it in our future paper (Hara et al., manuscript in preparation). is also possible that these NLRs partly compensate for the knock- AIM2 has also been shown to play a role in inflammasome ac- down of AIM2. AIM2 KO mice were recently generated, and one tivation during infection with DNAviruses (35, 55). It is therefore of group has shown that the activation of caspase-1 is partially in- interest whether AIM2 is also involved during infection with other duced in AIM2 KO macrophages infected with L. monocytogenes, pathogens, especially intracellular parasitic bacteria, such as Shi- suggesting a compensatory role for other receptors in the absence gella and Francisella. A recent report has shown that activation of 1194 ROLE OF AIM2 IN LISTERIA INFECTION caspase-1 upon infection with Francisella depends on AIM2 (54). 16. Stockinger, S., B. Reutterer, B. Schaljo, C. Schellack, S. Brunner, T. Materna, Thus, broad pathogens that deliver DNA into the host cytoplasm M. Yamamoto, S. Akira, T. Taniguchi, P. J. Murray, et al. 2004. IFN regulatory factor 3-dependent induction of type I IFNs by intracellular bacteria is mediated may likewise be sensed by AIM2. by a TLR- and Nod2-independent mechanism. J. Immunol. 173: 7416–7425. In conclusion, in this study, we found that AIM2, in cooperation 17. 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