Fas-Mediated Inflammatory Response in Listeria monocytogenes Infection Ryosuke Uchiyama, Shin Yonehara and Hiroko Tsutsui This information is current as J Immunol published online 15 March 2013 of October 1, 2021. http://www.jimmunol.org/content/early/2013/03/15/jimmun ol.1203059

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 15, 2013, doi:10.4049/jimmunol.1203059 The Journal of Immunology

Fas-Mediated Inflammatory Response in Listeria monocytogenes Infection

Ryosuke Uchiyama,* Shin Yonehara,† and Hiroko Tsutsui*

The molecular mechanisms of Fas (CD95/Apo-1)-mediated apoptosis are increasingly understood. However, the role of Fas- mediated production of proinflammatory such as IL-18 and IL-1b in bacterial infection is unclear. We demonstrate the importance of Fas-mediated signaling in IL-18/IL-1b production postinfection with Listeria monocytogenes without the contribution of caspase-1 inflammasome. IL-18/IL-1b production in L. monocytogenes–infected peritoneal exudate cells from Fas-deficient mice was lower than those from wild type mice, indicating that Fas signaling contributes to production. L. monocytogenes infection induced expression on NK cells, which stimulates Fas expressed on the infected macro- phages, leading to the production of IL-18/IL-1b. This was independent of caspase-1, caspase-11, and nucleotide-binding domain and leucine-rich repeat–containing receptors (NLRs) such as Nlrp3 and Nlrc4, but dependent on apoptosis-associated speck-like

containing a caspase recruitment domain. Wild type cells exhibited caspase-8 activation, whereas Fas-deficient cells did Downloaded from not. L. monocytogenes–induced caspase-8 activation was abrogated by inhibitor for intracellular reactive oxygen species, N-acetyl- L-cysteine. L. monocytogenes–infected macrophages produced type-I IFNs such as IFN-b1, which was required for Il18 expression. Thus, Fas signaling regulates innate inflammatory cytokine production in L. monocytogenes infection. The Journal of Immunology, 2013, 190: 000–000.

as (CD95/Apo-1), a member of the TNF receptor family, is tory cytokine production via Fas signaling in bacterial infection http://www.jimmunol.org/ critically important for the induction of apoptosis (1, 2). In are not clear. F the immune system, Fas-induced apoptosis plays a pivotal Listeria monocytogenes is a Gram-positive intracellular bacte- role in the exclusion of self-reactive T cells or tumor cells (1, 2). rium that often causes food-borne infections in immunocompro- This is because functional defects of Fas or the Fas ligand (FasL) mised hosts (11). In mice infected with L. monocytogenes, IFN-g– in mice result in the development of lymphadenopathy or systemic dependent Th1 immunity against L. monocytogenes is generated, lupus erythematosus–like autoimmune disease (1, 2). In addition, and this is critically important for protection against systemic lis- Miwa et al. (3) and our group have shown that innate inflamma- teriosis (12). Murine listeriosis is therefore widely used as an an- b imal model for research into Th1 immunity (12). Upon infection

tory cytokines such as IL-18 and IL-1 are produced from neu- by guest on October 1, 2021 trophils and macrophages via Fas signaling without the induction with L. monocytogenes, inflammatory cytokines such as TNF-a of apoptosis (4). Furthermore, it has been shown that the pro- and IL-12 are produced via pattern recognition receptors (PRRs) duction of other cytokines or is also induced by Fas on infected macrophages or dendritic cells. TLRs are one of the stimulation (5–10). These data indicate the possibility that Fas well-known types of PRRs that are localized on the plasma signaling plays important roles in the innate immune system in membrane of macrophages (13). In addition, intracellular PRRs, response to infection by pathogenic microbes; however, the pre- such as nucleotide-binding domain and leucine-rich repeat–con- cise molecular mechanisms and physiological roles of inflamma- taining receptors (NLRs) and absent in melanoma 2 (AIM2)–like receptor, act as cytoplasmic sensors of invaded pathogen inside cells, and induce the production of IL-1 family cytokines such as *Department of Microbiology, Hyogo College of Medicine, Nishinomiya, 663-8501, IL-18/IL-1b (14). In particular, IL-18 was first discovered as a Japan; and †Laboratory of Molecular and Cellular Biology, Graduate School of Bio- studies, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan potent IFN-g–inducing factor (15, 16), and also contributes to the Received for publication November 7, 2012. Accepted for publication February 15, IFN-g production in response to L. monocytogenes infection (17). 2013. IL-18 and IL-1b are produced as inactive proform This work was supported by Japan Society for the Promotion of Science KAKENHI in the cytoplasmic region of cells. The proforms of IL-18 and Grant 20790334 and by a Grant-in-Aid for Researchers, Hyogo College of Medicine, IL-1b are cleaved to yield active cytokines by proteases such as 2010. caspase-1, which is activated by a protein complex called the R.U. carried out the experiments and drafted the manuscript; R.U., S.Y., and H.T. inflammasome (14, 18, 19). It is well known that inflammasome planned the experiments and analyzed data; and H.T. supervised the study. activation is induced by the recognition of pathogen-associated Address correspondence and reprint requests to Dr. Ryosuke Uchiyama, Department of Microbiology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, molecular patterns (PAMPs) or damage-associated molecular Hyogo 663-8501, Japan. E-mail address: [email protected] patterns by intracellular NLRs or AIM2-like receptor (18, 19). The online version of this article contains supplemental material. In the case of L. monocytogenes infection, it has been shown that Abbreviations used in this article: AIM2, absent in melanoma 2; Asc, apoptosis- inflammasome and caspase-1 activation are induced in an Nlrp3- associated speck-like protein containing a caspase recruitment domain; BMDM, bone or AIM2-dependent manner, and that these are required for the marrow–derived macrophage; FasL, Fas ligand; ICSBP, IFN consensus sequence binding protein; LLO, listeriolysin O; MOI, multiplicity of infection; Nac, N-ace- production of active IL-18/IL-1b (20–24). tyl-L-cysteine; NLR, nucleotide-binding domain and leucine-rich repeat–containing Fas is also a well-known factor for caspase activation (1, 2). In receptor; PEC, peritoneal exudate cell; PRR, pattern recognition receptor; ROS, addition, it has been shown that upon infection with L. mono- reactive oxygen species. cytogenes, the expression of Fas or FasL is induced and Fas sig- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 naling affects the regulation of L. monocytogenes infection.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203059 2 IL-18 PRODUCTION VIA Fas SIGNALING IN LISTERIAL INFECTION

Zenewicz et al. (25) showed that FasL expressed on T cells in Reagents response to L. monocytogenes infection induced downregulation N-acetyl-L-cysteine (Nac) was obtained from Wako Pure Chemical Industries of the immune response against L. monocytogenes by the induc- (Osaka, Japan). The ELISA kits for IL-18 and IFN-g were obtained from tion of apoptosis in infected cells. In contrast, Jensen et al. (26) MBL (Nagoya, Japan) and R&D Systems, respectively. The ELISA kits for showed the protective role of Fas-induced apoptosis in response to IL-1b, IL-6, TNF-a, and IL-12p40 were purchased from eBioscience (San L. monocytogenes infection in the livers of infected mice. These Diego, CA). Neutralizing Ab against murine FasL (MFL-1, hamster IgG) was provided by Dr. N. Kayagaki (Genentech) (30). Normal hamster IgG was results indicate that the physiological roles of Fas signaling in the purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Neutralizing innate immune response against L. monocytogenes infection re- Ab against IFN-aR1 and its control IgG were purchased from eBioscience. main controversial, and it is not clear whether Fas contributes to Rabbit anti-asialo GM1 Ab and normal rabbit IgG were purchased from the production of innate inflammatory cytokines in response to L. Wako Pure Chemical Industries. Pig complement was obtained from Cedarlane (Burlington, ON, Canada). Cytochalasin D and Bafilomycin A1 monocytogenes infection. were purchased from Sigma-Aldrich (St. Louis, MO). Costar Transwell In this study, we have investigated the contribution of Fas in the 3.0 mM filters were obtained from Corning (Corning, NY). induction of IL-18/IL-1b production in response to L. mono- In vitro infection of L. monocytogenes and ELISA cytogenes infection. Using peritoneal exudate cells (PECs) from Fas2/2 mice, we have shown that Fas signaling is involved in the PECs were infected with L. monocytogenes in vitro in the absence of production of IL-18/IL-1b from PECs infected with L. mono- antibiotics. After incubation for 1 h, gentamicin (Life Technologies) was added (5 mg/ml final concentration), and cells were incubated for 18 h. cytogenes in vitro. NK cells expressed FasL in response to L. Culture supernatants were collected and stored at 280˚C until cytokine monocytogenes infection, which stimulated Fas on infected mac- measurement by ELISA. Alternatively, adherent PECs were stimulated rophages, leading to IL-18/IL-1b production. In this process, with 0.1 mg/ml LPS (Sigma-Aldrich) for 6 h. After washing the cells with apoptosis-associated speck-like protein containing a caspase re- medium three times, cells were stimulated with L5178Y or FasL/L5178Y. Downloaded from cruitment domain (Asc) and intracellular reactive oxygen species After incubation for 18 h, cytokine levels in the supernatant were assessed by ELISA. (ROS) were required for cytokine production, and we found that ROS was required for caspase-8 activation in L. monocytogenes– In vivo infection of L. monocytogenes and CFU assay infected macrophages. Finally, type-I IFN produced by L. mono- Mice (at age 7 wk) were infected i.v. with 5.0 3 104 CFU L. mono- cytogenes–infected macrophages was required for the gene ex- cytogenes. At 3 d postinfection, livers and spleens were collected and pression of Il18. Collectively, these results indicate a new aspect of homogenized in 5 ml PBS. The homogenates were serially diluted 10-fold, http://www.jimmunol.org/ Fas signaling in the innate inflammatory response against L. mono- and colony counting was performed using tryptic soy agar plates. In ad- dition, sera were collected from infected mice, and the concentration of IL- cytogenes infection. 18 was analyzed using ELISA. Materials and Methods Quantitative real-time RT-PCR Animals Total cellular RNA was extracted using the RNeasy (Qiagen, Valencia, CA) according to the manufacturer’s protocols supplemented with DNase treat- Female mice of the C57B/6j (B/6) strain were purchased from Oriental ment (Qiagen) to eliminate contaminating DNA. RNA (0.2 mg) was reverse 2/2 a 2/2 Yeast Co. (Tokyo, Japan). Nlrc4 and Ifn r1 mice with the B/6 transcribed using SuperScript III reverse transcriptase and random primers background were kindly provided by Dr. Vishva M. Dixit (Genentech, (Life Technologies) in the presence of RNase Out recombinant RNase in- by guest on October 1, 2021 CA) and Prof. S. Nagata (Kyoto University, Kyoto, Japan), respectively, 2/2 2/2 2/2 hibitor (Life Technologies). Quantitative real-time RT-PCR was performed and were maintained in our laboratory. Fas , Caspase-1 , Asc , and on an Applied Biosystems 7500 Fast real-time PCR system (Life Technol- Nlrp32/2 mice on the B/6 background were maintained in our laboratory gld/gld ogies) using TaqMan Gene Expression Master Mix (Life Technologies). The (27, 28). FasL mice on B/6 background were purchased from Japan following mouse primers provided from Applied Biosystems Ex- SLC (Osaka, Japan). In this study, female mice were used at age 7–10 wk pression Assay (Life Technologies) were used: Fas, Mm01204974_m1; Il18, in the experiments. All mice were maintained under specific pathogen-free Mm00434225_m1; Il1b, Mm00434228_m1; Fasl, Mm00438864_m1; Ifnb1, conditions, and they received humane care as outlined in the Guide for the Mm00439552_s1; Gapdh, Mm99999915_g1. Gene-specific transcript levels Care and Use of Experimental Animals of the Hyogo College of Medicine. were normalized to the amount of gapdh mRNA, and the expression levels of Bacterium all genes are shown in terms of fold increase, which indicates the expres- sion levels compared with that of control gene expression. L. monocytogenes 43251 (American Type Culture Collection, Manassas, VA) used for these experiments was maintained in our laboratory as de- FACS analysis L. monocytogenes scribed previously (29). For the preparation of stocks, For the detection of FasL expression on NK cells, nonadherent cells were was grown overnight in brain-heart infusion broth (BD Biosciences, incubated with anti-CD16/32 Ab (eBioscience) to avoid nonspecific Ab Franklin Lakes, NJ) at 37˚C with shaking. One volume of the overnight reactions (Fc blocking). Cells were stained with PerCP-conjugated anti- culture was added to 100 vol fresh broth medium and cultured for a further NK1.1 Ab (eBioscience) and FITC-conjugated anti-FasL Ab (clone MFL3; 6 h. Bacterial cells were washed, suspended in PBS supplemented with 2 BioLegend, San Diego, CA). Cells were fixed with 1% paraformaldehyde 10% glycerol, and stored in aliquots at 80˚C. Bacterial stocks were in PBS and then analyzed using a FACSCalibur flow cytometer (BD thawed and diluted in PBS just before the experiments. Biosciences). Cells Depletion of NK cells PECs of mice were obtained 3 d after i.p. injection of 2 ml thioglycolate For the depletion of NK cells, PECs were incubated in culture medium in medium (EIKEN Chemical, Tokyo, Japan). After washing with RPMI 1640 medium, PECs were seeded into a 48-well plate at 5 3 105 cells/well, then the presence of anti-asialo GM1 Ab or control Ab at 4˚C for 30 min and incubated at 37˚C for 2 h in culture medium that consisted of RPMI 1640 then treated with complement at 37˚C for 30 min (31). After washing with medium supplemented with 10% FCS, 2-ME (Nacalai Tesque, Kyoto, Ja- RPMI medium three times, cells were counted and seeded into plates for in vitro experiments. pan), and L-glutamine (Life Technologies, Carlsbad, CA). To obtain adherent PECs, we washed each well with medium three times, and the remaining cells Detection of active caspase-8 were used for experiments. A FasL-expressing cell line (FasL/L5178Y) and its parental control cell (L5178Y) were kindly provided by Dr. N. Kayagaki Supernatants of cells stimulated with FasL or infected with L. mono- (Genentech) and maintained in our laboratory (4). FasL/L5178Y was se- cytogenes were concentrated using trichloroacetate (28). Cell lysates were lected by adding G418 (0.5 mg/ml; Nacalai Tesque) to the culture medium. prepared as previously described (28). Bone marrow cells from caspase-112/2 B/6 mice were provided from Dr. N. These samples were subjected to SDS-PAGE and subsequently trans- Kayagaki (Genentech). Bone marrow–derived macrophages (BMDMs) were ferred to polyvinylidene difluoride membranes by electroblotting. The prepared from bone marrow cells of normal or caspase-112/2 mice using membranes were immunoblotted with anti–caspase-8 Ab ( recombinant murine M-CSF (R&D Systems, Minneapolis, MN). Technology, Danvers, MA) and b-actin (Sigma-Aldrich). The Journal of Immunology 3

Statistical analysis observations, we also found that the elevation of IL-18 was lower 2/2 gld/gld Results are expressed as means 6 SD. Student t test was used to determine in sera from Fas (Fig. 1D) and FasL (Supplemental Fig. the statistical significance of the values obtained, and p values ,0.05 were 1C) mice infected with L. monocytogenes than from normal mice. 2 2 considered statistically significant. Furthermore, Fas / mice were more susceptible to L. mono- cytogenes infection than normal mice in vivo in the livers (Fig. Results 1E). These results indicate that the FasL–Fas signaling pathway Involvement of Fas in IL-18/IL-1b production from PECs is involved in the production of IL-18/IL-1b in response to L. infected with L. monocytogenes monocytogenes infection in vitro and in vivo, and these inflam- matory responses possibly play a role in the primary regulation of To elucidate the contribution of Fas signaling in the production of L. monocytogenes infection. IL-18/IL-1b from L. monocytogenes–infected cells, we used in vitro b infection experiments using thioglycolate-induced PECs from Contribution of NK cells to IL-18/IL-1 production via Fas mice (12). We found that Fas gene expression was induced in signaling whole PECs infected with L. monocytogenes (Fig. 1A); therefore, It is known that thioglycolate-induced whole PECs include mac- we next infected PECs from normal, Fas2/2, or functional FasL- rophages and IFN-g–producing cells such as NK cells (31). It has deficient (FasLgld/gld) mice with L. monocytogenes. Postinfection been shown that activated macrophages express Fas on their cell with L. monocytogenes for 18 h, levels of cytokines in culture surface (6); in contrast, FasL is expressed on activated NK cells supernatants were analyzed by ELISA, and we found that the (32). To elucidate the cellular mechanisms of cytokine production production levels of IL-18/IL-1b in PECs from Fas2/2 (Fig. 1B) via Fas signaling upon infection with L. monocytogenes, whole and FasLgld/gld (Supplemental Fig. 1A) mice were lower than PECs were infected with L. monocytogenes, and the expression of Downloaded from those in normal mice without any reduction in the gene expression FasL was analyzed. As shown in Fig. 2A, Fasl gene expression levels of Il18 and Il1b (Fig. 1C). It has already been shown that was induced in nonadherent PECs in response to L. mono- IL-18 contributes to the production of IFN-g from PECs synergis- cytogenes infection. Furthermore, FasL was expressed on the cell tically with IL-12 (31); we also found a reduction of IFN-g in Fas2/2 surface of NK cells postinfection with L. monocytogenes (Fig. PECs, possibly because of the low production of IL-18 (Fig. 1B). 2B). These indicate the possibility that infection with L. mono-

In contrast, we did not find the low production of cytokines such cytogenes induced the expression of FasL on NK cells, which http://www.jimmunol.org/ as TNF-a, IL-6, and IL-12p40 in PECs from Fas2/2 (Fig. 1B) or stimulated Fas expressed on the infected macrophages, leading to FasL gld/gld (data not shown) mice, indicating that Fas signaling the production of IL-18/IL-1b. To examine this possibility, non- contributes to the production of IL-18/IL-1b upon infection with adherent cells or NK cells in whole PECs were depleted by L. monocytogenes. To confirm the involvement of Fas signaling washing the cells with RPMI medium or treating whole PECs with in the production of IL-18/IL-1b upon infection with L. mono- anti-asialo GM1 Ab and complements; then the cytokine levels cytogenes, whole PECs from normal mice were infected with were analyzed (Fig. 2C, 2D). In the absence of nonadherent cells L. monocytoges in the presence of anti-FasL neutralizing Ab or or NK cells, the production of IL-18/IL-1b was significantly re- control IgG. Supplemental Fig. 1B shows that blockade of FasL– duced, indicating the involvement of NK cells in production of Fas signaling resulted in the reduction of IL-18/IL-1b production cytokines via Fas signaling upon infection with L. monocytogenes. by guest on October 1, 2021 from L. monocytogenes–infected cells. In addition to these in vitro We also found that IFN-g production was clearly abolished in the

FIGURE 1. Contribution of Fas signaling in the production of IL-18/IL-1b from whole PECs upon in- fection with L. monocytogenes.(A) Whole PECs were infected with L. monocytogenes at a MOI of 5, and gene expression of Fas was analyzed by quantitative real-time RT-PCR. (B) Whole PECs from normal or Fas2/2 mice were infected with L. monocytogenes at a MOI of 5. After incubation for 18 h, cytokine levels in the supernatants were measured by ELISA. (C) Gene expression levels of normal or Fas2/2 PECs infected with L. monocytogenes at 8 h postinfection were ana- lyzed by quantitative real-time RT-PCR. (D) Mice (n = 5) were infected i.v. with 5.0 3 104 CFU L. mono- cytogenes. The serum concentration of IL-18 in normal and Fas2/2 mice at 3 d postinfection was analyzed using ELISA. (E) Livers and spleens from mice (n =5) infected with 5.0 3 104 CFU L. monocytogenes were removed at 3 d postinfection and homogenized in 5 ml PBS. The homogenates were inoculated on tryptic soy agar plates to determine the number of bacteria. Re- sults shown are representative of three experiments. *p , 0.05; error bars indicate SD. 4 IL-18 PRODUCTION VIA Fas SIGNALING IN LISTERIAL INFECTION

FIGURE 2. Contribution of NK cells to the pro- duction of IL-18/IL-1b from whole PECs infected with L. monocytogenes.(A) Whole PECs were in- fected with L. monocytogenes at a MOI of 5. After incubation for 18 h, Fasl gene expression in non- adherent PECs (Non ad-PEC) was analyzed using quantitative real-time RT-PCR. (B) FACS analysis of FasL on NK cells of whole PECs infected with L. monocytogenes. At 18 h postinfection, cells were gated for NK1.1; then expression of FasL on the cell surface was analyzed. (C) Adherent PECs (ad- PEC) were infected with L. monocytogenes in the presence or absence of nonadherent PECs; then cytokine production was analyzed by ELISA. (D) NK cells were depleted using anti-asialo GM1 Ab (aAsGM1) and complement (Comp.); then cells were infected with L. monocytogenes at an MOI of 5. Cytokine production was analyzed by ELISA. (E) Nonadherent PECs were added into culture wells containing adherent PECs infected with L. monocytogenes (MOI of 5) in the presence or ab- Downloaded from sence of Transwells. After incubation for 18 h, cytokine levels in the supernatant were determined using ELISA. Results shown are representative of three experiments. *p , 0.05; error bars indicate SD. http://www.jimmunol.org/ absence of NK cells upon infection with L. monocytogenes; this is tion of IL-18/IL-1b from macrophages via Fas signaling. We because NK cells are the main source of IFN-g production upon therefore investigated whether cell–cell interaction between FasL- infection with L. monocytogenes in whole PECs (31). We further expressing cells and macrophages is required for cytokine pro- investigated whether cell–cell interaction between nonadherent duction via Fas signaling, and we found that the levels of IL-18/IL- cells including NK cells and macrophages infected with L. mono- 1b production were lower in the presence of Transwells than in cytogenes is required for cytokine production via Fas signaling. their absence (Fig. 3C). In addition, Cytochalasin D and Bafilo- Transwells (3.0 mM; Costar) were placed onto wells seeded with mycin A1, inhibitors for phagocytosis and vacuolar type H+ by guest on October 1, 2021 adherent PECs infected with L. monocytogenes; then nonadherent ATPase, respectively, did not inhibit cytokine production via Fas cells were added to the upper chamber of the Transwells. After signaling (Fig. 3D). These results indicate that cell–cell interaction incubation for 18 h, the cytokine levels in the supernatants were between FasL-expressing cells and Fas-expressing macrophages, analyzed using ELISA, and we found that the level of IL-18/IL-1b but not phagocytosis or acidification of phagosomes of macro- production was lower in the presence of the Transwells than in phages, is required for the production of IL-18/IL-1b via Fas sig- their absence (Fig. 2E). These results indicate that infection with naling pathway. L. monocytogenes induced the expression of FasL on NK cells, b which stimulated Fas expressed on infected macrophages by their Asc and ROS are required for IL-18/IL-1 production direct interactions, leading to the production of IL-18/IL-1b. It is well known that inflammasome formation and caspase-1 ac- tivation are required for production of mature IL-18/IL-1b.We b Cell–cell interaction is required for the production of IL-18/IL-1 have already shown that IL-18 production via Fas signaling is via Fas signaling induced by a caspase-1–independent mechanism (4) (Fig. 4A); To elucidate the molecular mechanisms of IL-18/IL-1b production however, the involvement of other molecules related to inflam- via Fas signaling, we used in vitro experiments using a murine masome activation is not clear. Therefore, we next investigated the FasL-expressing cell line (FasL/L5178Y) and its parental control involvement of molecules related to inflammasome formation for cell (L5178Y) (4). We first treated adherent PECs from normal the production of IL-18/IL-1b via Fas signaling. PECs or BMDMs mice with 0.1 mg/ml LPS for 6 h. Consistent with the published from normal mice or from mice deficient in genes involved in results, LPS-stimulated macrophages induced the gene expression inflammasome activation were stimulated with FasL; then cyto- of Fas (Supplemental Fig. 2). After washing cells with medium, kine levels in the cultured supernatants were measured using FasL cells or control cells were added to the cell culture to activate ELISA. We found that cytokine production was not influenced by Fas. After cultivation for 18 h, culture supernatants were collected the deletion of caspase-1, caspase-11, Nlrp3, and Nlrc4 (Fig. 4A, and the levels of cytokines were determined by ELISA. As shown in 4B). However, Asc2/2 macrophages produced low levels of IL-18/ Fig. 3A, FasL-stimulated macrophages produced higher levels of IL-1b compared with those seen in normal macrophages upon IL-18/IL-1b than did those treated with control cells. We also found FasL stimulation (Fig. 4A). Asc is shown to act as a regulator of that BMDMs produced IL-18/IL-1b after stimulation with FasL gene expression via the modulation of NF-kB activation in human (Supplemental Fig. 3). In the presence of anti-FasL neutralizing Ab, cells (33), so we checked the gene expression levels of Il18, Il1b, the production of IL-18/IL-1b was abolished (Fig. 3B). Further- and Fas in normal and Asc2/2 macrophages. We did not find any more, macrophages from Fas2/2 mice did not produce IL-18/IL-1b reduction in the gene expression levels of Il18, Il1b, and Fas in after stimulation with FasL (Fig. 3B, Supplemental Fig. 3). These Asc2/2 macrophages compared with those seen in normal mac- results indicate that this experimental system reflects the produc- rophages (Supplemental Fig. 2), indicating that Asc contributes to The Journal of Immunology 5 Downloaded from

FIGURE 4. Requirement of Asc and intracellular ROS for the produc- tion of IL-18/IL-1b via Fas signaling. (A) Adherent PECs were prepared http://www.jimmunol.org/ from mice lacking genes related to the activation of the inflammasome. Cells were stimulated with LPS and FasL (ratio 1:0.5); then cytokine production was analyzed by ELISA. (B) BMDMs were prepared from normal or caspase-112/2 bone marrow, then stimulated with LPS and FasL (ratio 1:0.5). After 18 h of incubation, cytokine levels were analyzed by ELISA. (C) Adherent PECs from normal mice were treated with FasL (ratio 1:0.5) in the presence or absence of Nac (25 mM) for 18 h. Cytokine production was analyzed by ELISA. (D) Whole PECs from normal mice were infected with L. monocytogenes at an MOI of 5 in the presence or

absence of Nac (25 mM). After incubation for 18 h, cytokine production was by guest on October 1, 2021 analyzed by ELISA. Results shown are representative of three experiments. , FIGURE 3. Cell–cell interaction between FasL- and Fas-expressing *p 0.05; error bars indicate SD. cells was required for IL-18/IL-1b production via Fas signaling. (A) Peritoneal macrophages were treated with LPS for 6 h. After washing cells Caspase-8 activation via Fas signal and requirement for ROS with medium, cells were stimulated with FasL-expressing cells (FasL) or the parental control cells (Ctrl) in the described ratio of L5178Y cells We have already shown that a pan-caspase inhibitor (z-VAD-fmk) against macrophages. After incubation for 18 h, cytokine levels in the inhibited the Fas-mediated production of IL-18/IL-1b (4), indi- supernatants were determined by ELISA. (B) Contribution of Fas for IL- cating that the production of IL-18/IL-1b via Fas signaling is 18/IL-1b production was confirmed using anti-FasL neutralizing Ab or dependent on caspases except for caspase-1 (Fig. 4A). In addition, 2 2 macrophages derived from Fas / mice. Macrophages and FasL cells were recent reports have shown that caspase-8 and -11 are involved in cultured in a 1:0.5 ratio. (C) FasL cells were added into culture wells the production of IL-18/IL-1b (37–40). In this study, we investi- containing adherent PECs (ratio 1:0.5) in the presence or absence of gated the involvement of the caspases in Fas-mediated cytokine Transwells. After incubation for 18 h, cytokine levels in the supernatants production, and found that caspase-11 was not involved in the were determined by ELISA. (D) Adherent PECs were stimulated with production of IL-18/IL-1b via Fas signaling (Fig. 4B). In contrast, FasL (ratio 1:0.5) in the presence or absence of Cytochalasin D (CyD; 20 mM) or Bafilomycin A1 (BA; 200 nM). Cytokine levels were deter- it is well known that caspase-8 is critically important for the in- mined by ELISA. Results shown are representative of three experiments. duction of apoptosis via Fas signaling. In addition, a recent report *p , 0.05; error bars indicate SD. revealed that activated caspase-8 cleaves pro–IL-1b to yield the mature form via stimulation through TLR3, TLR4, and Fas sig- naling (40, 41). Based on this information, it is possible to spec- the production of active IL-18/IL-1b, but not their gene expres- ulate that caspase-8 is activated and involved in IL-18/IL-1b sion. production via Fas signaling in L. monocytogenes infection. We It was recently shown that intracellular ROS is required for IL- therefore analyzed the activation of caspase-8 in response to FasL 1b production (34–36). We therefore examined whether ROS stimulation or L. monocytogenes infection by immunoblot analy- signaling contributes to the production of IL-18/IL-1b via Fas sis. The active form of caspase-8 was detected in cell lysates from signaling. To elucidate this, we analyzed cytokine production in normal PECs stimulated with FasL, and this was abolished in FasL-stimulated (Fig. 4C) or L. monocytogenes–infected (Fig. 4D) Fas2/2 cells, indicating that caspase-8 activation was induced via cells in the presence or absence of Nac. We found that in the Fas signaling pathway (Fig. 5A). It is well known that active presence of Nac, IL-18/IL-1b production was inhibited (Fig. 4C, caspase-1 is released into the culture supernatant in response to 4D). These results indicate that intracellular ROS play important stimuli for inflammasome activation (28); we therefore analyzed roles in the production of IL-18/IL-1b via Fas signaling. whether activated caspase-8 was also released into the culture 6 IL-18 PRODUCTION VIA Fas SIGNALING IN LISTERIAL INFECTION Downloaded from

FIGURE 5. Immunoblot analysis of active caspase-8. (A) Activated caspase-8 in supernatant (SUP) and cell lysate (CL) from PECs stimulated with FasL (left, ratio 1:0.5) or infected with L. monocytogenes (right, MOI of 5) was analyzed. Asterisks indicate nonspecific bands; arrows indicate the active caspase-8 (p43/41). (B) Caspase-8 activation was analyzed in cells stimulated with FasL (left, ratio 1:0.5) or infected with L. mono- http://www.jimmunol.org/ cytogenes (right, MOI of 5) in the presence or absence of Nac (25 mM). Results shown are representative of three experiments. supernatant in response to FasL stimulation. Fig. 5A shows that the active caspase-8 fragment was also detected in culture super- natants, indicating the presence of secretion mechanisms of ac- tive caspase-8 in stimulated macrophages, as well as in the caspase-1

inflammasome. Upon infection with L. monocytogenes,wealso by guest on October 1, 2021 found that caspase-8 was activated via the Fas signaling pathway (Fig. 5A), because caspase-8 activation was abolished in Fas2/2 cells. This activation was dependent on intracellular ROS, because FIGURE 6. Endogenous type-I IFNs contribute to the production of IL- caspase-8 activation was inhibited in the presence of Nac (Fig. A b 2/2 18/IL-1b.( ) Gene expression of Ifn 1 in L. monocytogenes–infected 5B). However, Asc macrophages induced caspase-8 activation (MOI of 5) PECs was analyzed by quantitative real-time RT-PCR. (B) in response to FasL stimulation comparable with that of normal Whole PECs from normal or Ifnar12/2 mice were infected with L. mono- cells (Supplemental Fig. 4). These results indicate the possibility cytogenes (upper) or stimulated with FasL (lower) in the indicated ratio. that caspase-8 activation is induced in response to L. mono- After incubation for 18 h, cytokine levels in supernatant were analyzed by cytogenes infection via Fas and the intracellular ROS signaling ELISA. (C) PECs from normal mice were stimulated with LPS and FasL pathway, leading to mature IL-18/IL-1b production. (ratio 1:0.5) in the presence or absence of anti–IFN-aR1 neutralizing Ab for 18 h. Cytokine levels in supernatants were analyzed by ELISA. Results Endogenous type-I IFNs are required for Il18 gene expression shown are representative of three experiments. *p , 0.05; error bars in- in L. monocytogenes infection dicate SD. The production of IL-18/IL-1b has been shown to be regulated by type-I IFNs (39, 42). In addition, it is well known that L. mono- In contrast, surprisingly, IL-1b production was higher in deficient cytogenes infection induces the expression of type-I IFNs such as macrophages than in normal macrophages (Fig. 6B). These results IFN-b; we also detected gene expression of Ifnb1 postinfection were confirmed using anti–IFN-aR1 neutralizing Ab (Fig. 6C). with L. monocytogenes (Fig. 6A). Based on these results, we These results indicate that type-I IFNs contribute to the production speculate that type-I IFNs contribute to the production of IL-18/ of IL-18 via Fas signaling through positive regulation; in contrast, IL-1b via the Fas signaling pathway. To confirm this, we used IL-1b production via Fas signaling was negatively regulated by PECs from IFN-aR1–deficient mice. IL-18 production was abol- type-I IFNs. It is known that the gene expression of Il18 is reg- ished in L. monocytogenes–infected PECs from Ifnar12/2 mice, ulated by IFN consensus sequence binding protein (ICSBP), and we found low levels of IL-1b production in deficient cells which is activated by type-I IFNs by regulating the promoter re- at a low multiplicity of infection (MOI) of L. monocytogenes in- gion of Il18. We therefore analyzed gene expression in L. mono- fection (Fig. 6B). To determine the contribution of type-I IFNs in cytogenes–infected or LPS-stimulated macrophages from normal 2 2 the production of IL-18/IL-1b via Fas signaling, we stimulated or Ifnar1 / mice using quantitative real-time RT-PCR. We found peritoneal macrophages from normal or Ifnar12/2 mice with that in each stimulus condition, type-I IFNs were required for the FasL; then cytokine production was analyzed using ELISA. induction of Il18 gene expression (Fig. 7). We did not observe a Ifnar12/2 macrophages produced low levels of IL-18 upon FasL significant reduction in the expression of the Il1b and Fas genes stimulation compared with that of normal macrophages (Fig. 6B). in response to LPS stimulation or L. monocytogenes infection The Journal of Immunology 7

FIGURE 7. Gene expressions in Ifnar12/2 macrophages infected with FIGURE 8. Scheme of mechanism suggested by this study. Infected Downloaded from L. monocytogenes or stimulated with LPS. Adherent PECs from normal or macrophages induce the expression of type-I IFNs, which stimulate re- Ifnar12/2 mice were infected with L. monocytogenes (MOI of 5) or treated ceptors for type-I IFNs on neighboring cells; this induces Il18 gene ex- with LPS for 6 h; then the gene expression levels of Il18, Il1b, and Fas pression. FasL expressed on NK cells induces caspase-8 activation via an were analyzed by quantitative real-time RT-PCR. Results shown are rep- intracellular ROS-dependent mechanism, and this may contribute to the resentative of three experiments. *p , 0.05; error bars indicate SD. production of mature IL-18/IL-1b with Asc. http://www.jimmunol.org/ (Fig. 7). These results indicate that type-I IFNs induced the gene pression of inflammatory cytokines, and Fas has other functions expression of Il18; in contrast, IL-1b is negatively regulated by independent of its role in cleaving pro–IL-18 in this model. type-I IFNs at the translational level of the pro–IL-1b protein or Nonetheless, Fas signaling is likely to be important for the overall cleavage of the proform via the Fas signaling pathway (Fig. 8). production of mature IL-18 in vivo and in vitro because Fas2/2 cells and mice abolished the production of mature IL-18 in re- Discussion sponse to L. monocytogenes infection (Fig. 1B, 1D). In vivo, IL-18 In this study, we have shown that Fas signaling induced the pro- produced via the Fas signaling pathway in response to L. mono- duction of IL-18/IL-1b in response to L. monocytogenes infection cytogenes infection possibly resulted in the production of IFN-g, by guest on October 1, 2021 from PECs via a ROS- and Asc-mediated mechanism independent leading to the activation of macrophages and eradication of of the caspase-1 inflammasome. However, it is well known that L. monocytogenes. L. monocytogenes infection induces the activation of caspase-1 It is shown that FasL is expressed on activated NK cells (32), inflammasome in macrophages and dendritic cells, leading to T lymphocytes (43, 44), and NKT cells (45), and plays important the production of IL-18/IL-1b. Hara et al. (17) showed that lis- roles in cytotoxic effects against target cells. In this study, we have teriolysin O (LLO), a major virulence factor of L. monocytogenes, shown that FasL was expressed on NK cells in response to L. is required for caspase-1 activation and the production of IL-18/ monocytogenes infection. We checked the presence of NKT cells IL-1b in infected macrophages. In addition, it has been shown that in whole PECs by FACS analysis using cell-surface markers Nlrp3 was required for the activation of the caspase-1 inflamma- (NK1.1 and CD3ε), and found that in our experiments, there were some and IL-1b production in L. monocytogenes–infected cells no NKT cells (data not shown). In addition, we found small (20, 21). Furthermore, AIM2 was shown to contribute to the amounts of T lymphocytes in whole PECs; however, there was no caspase-1 activation and production of IL-18/IL-1b in L. mono- expression of FasL on these cells in response to L. monocytogenes cytogenes–infected cells (22–24). In this study, we found that infection (data not shown). These results indicate that FasL on NK heat-killed L. monocytogenes abolished the production of IL-18/ cells, but not NKT cells or T lymphocytes, plays a pivotal role in IL-1b from infected macrophages. Furthermore, caspase-12/2 the stimulation of Fas in L. monocytogenes–infected PECs macrophages exhibited no IL-18/IL-1b production in response in vitro. In this study, we could not address the mechanism of the to L. monocytogenes infection (data not shown). These results expression of FasL on NK cells in response to L. monocytogenes indicate that L. monocytogenes infection induces IL-18/IL-1b infection; however, it is possible that cytokines such as IL-12 or production with two phases, via Fas-independent (caspase-1 IL-18 activated NK cells and induced FasL expression (32). inflammasome-dependent) and Fas-dependent mechanisms (as Our data demonstrate the contribution of Asc and intracellular shown in this study). Caspase-1 activation upon infection with ROS to the production of IL-18/IL-1b via the Fas signaling L. monocytogenes is regarded as the primary mechanism for the pathway. Asc is known as an important molecule for the activation production of IL-18/IL-1b, and we concluded that Fas-dependent of the caspase-1 inflammasome (18, 19). In addition, recent re- IL-18/IL-1b production constitutes to the amplification phase ports have shown that the contribution of Asc to the regulation of inflammatory cytokines in response to L. monocytogenes in- of inflammatory responses is independent of caspase-1 inflam- fection. masome activation. Hasegawa et al. (33) showed that caspase-8 We showed that the expression levels of Il18/Il1b genes in Fas2/2 associated with Asc via the pyrin domain, leading to NF-kB ac- cells were higher than those in normal cells in L. monocytogenes tivation and IL-8 production in response to muramyl dipeptide infection in vitro (Fig. 1C). These results indicate the possibility stimulation. In addition, Gringhuis et al. (38) showed that Asc that Fas signaling contributes to the downregulation of gene ex- is required for IL-1b processing via “non-canonical caspase-8 8 IL-18 PRODUCTION VIA Fas SIGNALING IN LISTERIAL INFECTION inflammasome” formation. The authors indicate the possibility pression in L. monocytogenes infection. These observations allow that Asc plays a supporting role in pro–IL-1b processing in the us to speculate that type-I IFNs contribute to the protection against inflammasome, but not in caspase-8 activation (38). In this study, L. monocytogenes infection via inflammatory cytokine production we have also shown that Asc is required for IL-18/IL-1b pro- in vivo. However, previous reports demonstrated that defective duction without any effect on gene expression and caspase-8 ac- type-I IFNs result in the protective effects in regulation of L. tivation (Supplemental Figs. 2, 4). In contrast, Bossaller et al. (40) monocytogenes infection in vivo (55). Consistent with this, we also showed that FasL-stimulated cells induced the production of IL- confirmed that Ifnar12/2 mice were more resistant to L. mono- 1b in an Asc-independent manner. The inconsistent results for the cytogenes infection than normal mice (data not shown). Rayamajhi contribution of Asc to Fas-mediated cytokine production are et al. (56) showed that IFN-a/b released from L. monocytogenes– possibly caused by differences in the cells used, the conditions of infected cells downregulated the cell-surface receptor for IFN-g priming activation using a TLR agonist, or the FasL used in the in macrophages, thereby preventing the activation of macrophages experiments. In this study, we could not address the precise mo- by IFN-g. In addition, it was shown that type-I IFNs sensitized lecular mechanisms underlying the contribution of Asc to Fas- infected macrophages and lymphocytes to L. monocytogenes–in- mediated cytokine production. Pierini et al. (46) showed that ac- duced cell death, resulting in the increase of susceptibility to L. tivated caspase-8 specifically colocalizes with the AIM2/Asc monocytogenes infection in vivo (57, 58). On the basis of these specks in response to Francisella infection. In addition, Hase- results, we speculated that type-I IFNs reduce the killing function gawa et al. (33) also showed that caspase-8 colocalized with of macrophages against L. monocytogenes infection in vivo, and speck-forming Asc. On the basis of these reports, our data may these phenomena overcome the Fas-mediated inflammatory re- imply that active caspase-8 contributes to the basal production of sponses and activation of infected macrophages. IL-18/IL-1b, and that speck-forming Asc might accelerate it by In contrast, Guarda et al. (42) showed that type-I IFNs inhibited Downloaded from unidentified mechanisms. the production of IL-1b in the process of pro–IL-1b production Recent research has revealed new aspects of intracellular ROS and the activation of the Nlrp3-dependent inflammasome. We also in the induction of inflammatory cytokines such as IL-1b. Saitoh found that IL-1b produced via Fas signaling was inhibited by et al. (36) showed that Atg16L1 deficiency resulted in augmented type-I IFNs without any reduction in Il1b gene expression in vitro, induction of IL-1b in response to LPS stimulation, accompanied showing that type-I IFNs negatively regulate the production of IL- by intracellular ROS production. The authors showed that intra- 1b in the process of Il1b transcriptional, cleavage of pro–IL-1b, http://www.jimmunol.org/ cellular ROS scavenging by Nac abolished the production of IL- or secretion of active IL-1b. The precise molecular mechanisms 1b. In addition, Gross et al. (35) have shown that fungal infection and the functional roles in regulation of L. monocytogenes in- induced IL-1b production in dendritic cells via protein kinase Syk fection by which IL-1b production is regulated by type-I IFNs are signaling coupled with Nlrp3 and the caspase-1 inflammasome, a not yet clear, and further study is required to elucidate these process that required intracellularROS.Furthermore,Dostertetal. points. (47) showed that macrophages stimulated with silica or mono- In summary, this study revealed a novel role of Fas in the in- sodium urate crystals induced IL-1b production via the formation duction of the inflammatory response against L. monocytogenes of the Nlrp3 inflammasome accompanied by ROS production, and infection. This system was regulated by type-I IFNs produced by guest on October 1, 2021 Nac inhibited IL-1b production. In this study, we also found that from macrophages infected with L. monocytogenes (Fig. 8). This intracellular ROS was required for IL-18/IL-1b production in kind of innate immune system might be common in other bacterial response to L. monocytogenes infection via the Fas signaling infections such as those involving intracellular bacteria; therefore, pathway, and that ROS contributed to the activation of caspase-8. this study might accelerate our understanding of novel mecha- The precise molecular mechanism of caspase-8 activation via the nisms of innate immunity against bacterial infection. ROS signaling pathway is under consideration; however, it is plausible that intracellular ROS plays an important role in the Fas Acknowledgments signaling pathway leading to caspase-8 activation in microbial We thank Dr. Vishva M. Dixit (Genentech) for providing the Nlrp32/2 and infection by the activation of the noncanonical caspase-8 inflam- Nlrc42/2 mice, Prof. Shigekazu Nagata (Kyoto University) for providing masome (48, 49). the Ifnar12/2 mice, and Dr. N. Kayagaki (Genentech) for providing L5178 It is well known that type-I IFNs play important roles in antiviral cells, anti-FasL neutralizing Ab, and caspase-112/2 bone marrow cells. and antitumor activities. In addition, recent studies have revealed We also thank N. Iwami and T. Mizobuchi (Hyogo College of Medicine) new roles of type-I IFNs in the regulation of bacterial infection for technical support, and Prof. K. Nakanishi (Hyogo College of Medicine) (50). In L. monocytogenes infection, IFN-b production is induced for enthusiastic discussion. in infected macrophages in an LLO-dependent manner (51), and the extent of IFN-b production in L. monocytogenes infection is Disclosures related to its virulence (52). In addition, Yamamoto et al. 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