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).