Internalized Cryptococcus neoformans Activates the Canonical -1 and the Noncanonical Caspase-8

This information is current as Mingkuan Chen, Yue Xing, Ailing Lu, Wei Fang, Bing Sun, of September 25, 2021. Changbin Chen, Wanqing Liao and Guangxun Meng J Immunol 2015; 195:4962-4972; Prepublished online 14 October 2015; doi: 10.4049/jimmunol.1500865

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes

Mingkuan Chen,* Yue Xing,* Ailing Lu,* Wei Fang,† Bing Sun,* Changbin Chen,* Wanqing Liao,† and Guangxun Meng*

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococccosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3)

inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also Downloaded from activated the canonical NLRP3–apoptosis-associated speck-like protein containing a CARD (ASC)–caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3–ASC–caspase-8 inflammasome as well, which resulted in robust IL-1b secretion and from caspase-1–deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans– induced activation of either the canonical NLRP3–ASC–caspase-1 inflammasome or the noncanonical NLRP3–ASC–caspase-8 http://www.jimmunol.org/ inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3–ASC–caspase-8 inflam- masome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge. The Journal of Immunology, 2015, 195: 4962–4972.

s the first line of defense against pathogen invasion, host invading microbes and initiate innate immune responses, including innate immune system recognizes microbial pathogens inflammation (1). A such as viruses, bacteria, and fungi for elimination. This A number of NLRs and DNA sensors among these PRRs can by guest on September 25, 2021 recognition relies on a group of evolutionarily conserved pattern assemble into a protein complex called inflammasome, which recognition receptors (PRRs), including membrane-bound TLRs contains PRRs such as NLR family, pyrin domain-containing 3 and C-type lectin receptors, as well as cytoplasmic NOD-like re- (NLRP3), NLR family CARD domain-containing 4 (NLRC4), ceptors (NLRs), RIG-I like receptors, and cytosolic DNA sensors. or absent in melanoma 2 (AIM2), adaptor protein apoptosis- These PRRs recognize conserved molecular patterns expressed by associated speck-like protein containing a CARD (ASC), and procaspase-1. Assembly of inflammasome leads to the auto- cleavage and activation of caspase-1 (2). The activated caspase-1 *Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shang- hai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, triggers the processing and release of proinflammatory Shanghai 200031, China; and †Shanghai Key Laboratory of Molecular Medical My- IL-1b and IL-18, both of which are important for the shaping of cology, Changzheng Hospital, Shanghai 200003, China adaptive immune responses and elimination of invading pathogens Received for publication April 13, 2015. Accepted for publication September 18, (1). Furthermore, activation of caspase-1 triggers a form of cell 2015. death termed , which protects the host by eliminating This work was supported by Natural Science Foundation of China Grants 31170868, 31370892, 31300712, and 91429307; National Key Basic Research Programs Grants the replication niche of invading intracellular pathogens (3). 2014CB541905 and 2015CB554302; National Major Projects for Science and Tech- Among a large plethora of microbial pathogens, fungi are nology Grants 2012ZX10002007-003 and 2014ZX0801011B-001; Sanofi Aventis- attracting the attention of more and more scientists. That is because, Shanghai Institutes for Biological Sciences Scholarship Program, as well as the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs In- with the increasing number of immunocompromised hosts such as ternational Partnership Program for Creative Research Teams. AIDS patients and organ transplant recipients, mycoses have been Address correspondence and reprint requests to Dr. Guangxun Meng, Institut Pasteur emerging as life-threatening infectious diseases over the last three of Shanghai, Chinese Academy of Sciences, 320 Yueyang Road, Life Science Re- search Building, Shanghai 200031, China. E-mail address: [email protected] decades (4). Opportunistic fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans The online version of this article contains supplemental material. pose the biggest challenge in immunocompromised hosts. Of Abbreviations used in this article: AIM2, absent in melanoma 2; ASC, apoptosis- associated speck-like protein containing a CARD; BMDC, bone marrow–derived particular note, C. neoformans is a pathogenic fungus that causes dendritic cell; CPZ, chloropromazine; GXM, glucuronoxylomannan; LDH, lactate cryptococcosis, including life-threatening meningo encephalitis, dehydrogenase; NLR, NOD-like receptor; NLRC4, NLR family, CARD domain- . containing 4; NLRP3, NLR family, pyrin domain-containing 3; PAMP, pathogen- which affects about 1 million AIDS patients and causes 600,000 associated molecular pattern; PRR, pattern recognition receptor; ROS, reactive deaths worldwide annually (5, 6). C. neoformans is unique among oxygen species; shRNA, short hairpin RNA; WT, wild-type; YTD, yeast extract the pathogenic fungi by carrying a capsule outside of the cell wall, trypton dextrose. which is mainly composed of the antiphagocytic glucuronox- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 ylomannan (GXM) (7). However, the detailed mechanisms for the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500865 The Journal of Immunology 4963 interaction of C. neoformans with host cells are still poorly purity of CD11c+ cells higher than 80% tested by flow cytometry (data not studied. shown), and were used for fungal challenge experiments. For mouse serum Recent studies showed that the NLRP3 inflammasome was in- collection, WT C57BL/6 mice were anesthetized by injection of 2.4% avertin, and blood was collected by cardiac puncture. The whole blood was volved in host immune responses against a number of fungal left at room temperature for 2 h, and mouse serum was collected through pathogens, including C. albicans, A. fumigatus, and Microsporum centrifuging for 15 min at 3000 rpm at 4˚C. canis (8–11). In addition, research from the Meng Laboratory also In vitro C. neoformans challenge revealed that the biofilm from C. neoformans as well as the acapsular mutant of C. neoformans both activated the NLRP3 For fungal-challenging experiments, mouse BMDCs were pooled in plates inflammasome (12, 13). However, whether encapsulated C. neo- with C. neoformans H99 or Cap59 mutant at multiplicity of infection = 10 (10 yeasts to 1 macrophage) with or without 30 mg/ml anti-GXM Ab or formans activates inflammasome under certain conditions is not 10% mouse serum opsonization, unless stated otherwise. In some experi- known yet. Besides the canonical inflammasome activation men- ments, host cells were treated with the following chemicals for 30 min tioned above, C. albicans also induces noncanonical caspase-8 before infection with C. neoformans: Ac-YVAD-CHO (caspase-1 inhibitor), inflammasome activation in human dendritic cells (14). More- Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), b glyburide (NLRP3 inhibitor), CA-074Me, potassium chloride (KCl), Mito- over, caspase-8 is also involved in promoting -glucan–induced TEMPO (mitochondria-targeted antioxidant), chloropromazine (CPZ), with cell death and NLRP3 inflammasome-dependent IL-1b maturation indicated concentrations in figure legends. In indicated experiment, cells during C. albicans infection in mouse dendritic cells (15). How- were stimulated with LPS (100 ng/ml) for 6 h with ATP (5 mM) pulse for 30 ever, whether and how caspase-8 is activated during C. neofor- min. The supernatants were harvested for ELISA. In some cases, mans infection are still unknown. supernatants and cell extracts were collected for immunoblotting analysis.

In the current study, we found that internalization of the Gene silencing in THP-1 cells and mouse BMDCs Downloaded from encapsulated C. neoformans with anti-GXM Ab or serum Short hairpin RNA (shRNA) vectors against NLRP3, caspase-1, ASC, opsonization activated the canonical NLRP3–ASC–caspase-1 and their scramble vectors were gifts of the J. Tschopp Laboratory (21). inflammasome. More interestingly, from dendritic cells deficient Generation of specific gene silencing THP-1 cells had been described for caspase-1, C. neoformans still induced clear secretion of IL-1b before (10). For knockdown of caspase-8 in mouse BMDCs, shRNA as well as cell death, both of which turned out to be mediated by vectors against mouse caspase-8 were constructed in the Meng Laboratory. Lentiviral particles were generated in 293T cells by transfection with a the noncanonical NLRP3–ASC–caspase-8 inflammasome activa- modified PLKO-shCasp8, pMD2-VSVG, and pCMV-R8.91 using poly- http://www.jimmunol.org/ tion. Of special note was that the activation of caspase-8 was ethylenimine. The viral particles were harvested 48 h later and used for strongly elevated in the absence of caspase-1. Collectively, our infection of BMDCs. BMDCs were infected on day 0, and then changed to findings establish a novel link between NLRP3–ASC and the regular medium 1 d later; on day 4, culture medium was changed and puromycin was added for screening; after another 3 d, cells were collected key inflammatory upon fungal challenge and reveal for experiments. The targeting sequences for respective genes are as follows: that caspase-1 is inhibitory for caspase-8 in dendritic cells upon human NLRP3,59-CAGGTTTGACTATCTGTTCT-39;humanCaspase-1, C. neoformans infection. 59-GTGAAGAGATCCTTCTGTA-39; human ASC,59-GATGCGGAAGCT- CTTCAGTTTCA-39;mouseCaspase-8-1,59-TCATCTCACAAGAACTA- 9 9 9 Materials and Methods TATT-3 ;andmouseCaspase-8-2,5-TCATCTCACAAGAACTATATT-3 . Mice and reagents mCherry–ASC overexpression in THP-1 cells by guest on September 25, 2021 C57BL/6 wild-type (WT) mice were obtained from The Jackson Laboratory For generation of mCherry–ASC–overexpressing THP-1 cell line, a len- and bred in our specific pathogen-free facility. Nlrp3, Asc, Nlrc4, tiviral vector containing mCherry–ASC fusion gene was constructed. Aim2, and Caspase-1/11–deficient mice had been described before (16– Lentiviral particles were generated in 293T cells and harvested for infec- 19). Animal care, use, and experimental procedures complied with national tion of THP-1 monocytes. The mCherry–ASC–expressing THP-1 mono- guidelines and were approved by the Animal Care and Use Committee cytes were screened and maintained with Geneticin (G418) selection. k at Institut Pasteur of Shanghai. Mouse anti-GXM mAb (IgG1, )to Cytokine ELISA and immunoblotting C. neoformans was purchased from Meridian Life Science. Zymosan was from InvivoGen. All chemical reagents were purchased from Sigma- Supernatants were analyzed for cytokine secretion by ELISA, according to Aldrich, unless stated otherwise. the manufacturer’s instructions (eBioscience). Abs for immunoblotting include the following: rabbit anti-mouse ASC (SC-22514-R; Santa Cruz), Culture of C. neoformans rabbit anti-mouse caspase-1 (sc-514; Santa Cruz), rabbit anti-mouse ma- b Encapsulated C. neoformans reference strain H99 (serotype A), R265 ture and pro–IL-1 (sc-7884; Santa-Cruz), rabbit anti-mouse caspase-8 (serotype B), B3501 (serotype D), and acapsular mutant Cap59 (H99 (9429; Cell Signaling), rat anti-mouse caspase-8 (Enzo Life Science), and b background) were from the J. Perfect Laboratory (20). The fungus was mouse anti-mouse -actin (Sigma-Aldrich). Appropriate HRP-conjugated maintained in glycerol stocks in 280˚C and was grown on yeast extract secondary Abs were used for signal detection via ECL reagent (Perkin tryptone dextrose (YTD) agar plate at 30˚C. Liquid cultures were grown in Elmer). YTD medium at 30˚C for 20–24 h in a shaking incubator at 180 rpm. ASC oligomerization detection Fungal cells were centrifuged at 2000 3 g for 2 min, washed three times, and resuspended in sterile PBS. In some experiments, the fungus was heat ASC oligomerization detection was conducted, as described before (22). inactivated by incubating at 70˚C for 1 h, and no growth was observed Briefly, BMDCs were cocultured with C. neoformans H99 under anti- from the following inoculation of inactivated C. neoformans during a 2-d GXM Ab opsonization for 6 h, and cells were lysed, pelleted, washed period on YTD agar plates. with PBS, and cross-linked with disuccinimidyl suberate. The cross-linked pellets were resuspended in 30 ml SDS loading buffer for immunoblotting Mammalian cell culture and mouse serum collection detection of ASC. THP-1 cells were maintained in RPMI 1640 media containing 10% FBS, Coimmunoprecipitation 100 IU/ml penicillin, 1 mg/ml streptomycin, and 50 mM 2-ME at 37˚C with 5% CO2. For differentiation into macrophages, THP-1 cells were incubated BMDCs were plated into six-well plates and infected with C. neoformans with 100 ng/ml PMA for 3 h, then washed twice with PBS and rested for Cap59 mutant at multiplicity of infection = 10. After 6 h, cells were lysed 48 h before use. Mouse bone marrow–derived dendritic cells (BMDCs) in buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% were prepared, as described before (12). Briefly, bone marrow cells were Nonidet P-40, and Complete Inhibitor Cocktail (Roche), and collected, suspended in PBS by addition of RBC lysis buffer for depletion centrifuged at 3000 3 g for 5 min at 4˚C. Part of the supernatants was of erythrocytes, and then seeded in 7.5 3 105 cells/ml in the RPMI 1640 taken as input control; the left was incubated with protein A agarose beads media with rGM-CSF (20 ng/ml; Peprotech) in a humidified incubator with (Invitrogen) and anti–caspase-8 Ab (Enzo Life Sciences) at 4˚C overnight. 5% CO2 at 37˚C. The cells were fed once at the interval of 3 d with the The beads were washed with lysis buffer, resuspended in sample buffer, identical dose of rGM-CSF. The cells were harvested on day 6, with the and processed for immunoblotting. 4964 INTERNALIZED C. neoformans ACTIVATES CASPASE-1 AND CASPASE-8

Immunofluorescence cretion from BMDCs was increased with higher doses of serum for A total of 5 3 105 BMDCs was seeded on glass coverslips in 24-well plates opsonization (Supplemental Fig. 1C). overnight and then cocultured with C. neoformans H99 strain under anti- The Cryptococcus species complex includes at least two sub- GXM Ab opsonization or acapsular mutant Cap59 for 6 h. For mito- species: C. neoformans and C. grubii. They are further divided chondrial reactive oxygen species (ROS) staining, MitoSox (Invitrogen) into five serotypes, including the hybrid serotype AD (A, D for was added to cells in the last 10 min. Then cells were washed gently three C. neoformans, and B, C for C. gattii), with serotype A isolates times with warm buffer and mounted in warm buffer for imaging. For detecting phagolysosome membrane leakage, THP-1–derived macro- responsible for the vast majority of cryptococcal infections (27). phages expressing mCherry–ASC were incubated with 5 mg/ml FITC- With the opsonization of anti-GXM Ab or mouse serum, all dextran (Sigma-Aldrich; 40-kDa molecular mass) molecules for 2 h at cryptococcal strains from serotype A, D, and B induced IL-1b and 37˚C. The cells were washed with PBS, chased with culture medium for TNF-a secretion from mouse BMDCs (Fig. 1C, Supplemental 3 h [to allow colocalization with lysosome (23)], and then challenged with C. neoformans in the presence of anti-GXM Ab. At the designated time Fig. 1D). postinfection, THP-1 cells were washed with PBS and visualized using Interestingly, CPZ, a clathrin inhibitor, inhibited anti-GXM 3100 objective fluorescence microscopy. For caspase-8 staining, BMDCs Ab-opsonized C. neoformans or acapsular mutant Cap59-induced were treated with FITC-IETD-FMK (CaspGLOW Fluorescein Active secretion of IL-1b and TNF-a from BMDCs (Fig. 1D, Supplemental caspase-8 staining kit; eBioscience) 2 h post-C. neoformans infection. Fig. 1E), indicating that internalization of C. neoformans was Then cells were washed with PBS, fixed with 4% (w/v) paraformaldehyde solution, permeabilized with 0.1% Trixton-100, and blocked with 10% essential for optimal inflammasome activation from host cells. FBS in PBS. For ASC speck staining, cells were incubated with anti-ASC Indeed, with immunoblotting, it was found that anti-GXM Ab- Ab, washed, and incubated with Alexa Fluor 555 goat anti-rabbit IgG opsonized C. neoformans induced caspase-1 maturation, ASC

(Invitrogen). For C. neoformans staining, cells were incubated with mouse speck formation, as well as ASC oligomerization in dendritic Downloaded from anti-GXM Ab and Alexa Fluor 488 anti-mouse IgG (Invitrogen). At last, cells were stained with DAPI in mounting buffer. Images were acquired by cells (Fig. 1E, 1F, Supplemental Fig. 1F), clearly indicating an inverted Leica DMI3000B fluorescence microscope and a 3100 objective. an activation of inflammasome. Moreover, our further study showed that inhibition of internalization of C. neoformans by CPZ Flow cytometry for mitochondrial ROS detection inhibited synthesis of pro–IL-1b as well as activation of caspase-1 MitoSox (Invitrogen) was added to cells 10 min before harvesting, and then (Supplemental Fig. 1G).

cells were washed in FACS buffer (PBS + 0.5% BSA) three times and In addition, a specific caspase-1 inhibitor Ac-YVAD greatly http://www.jimmunol.org/ resuspended in FACS buffer. Data were collected on BD LSRFortessa at reduced Ab-opsonized C. neoformans–induced IL-1b secretion, forward light scatter, side light scatter, and PE channels. but did not affect the secretion of TNF-a nor IL-8 from mouse TUNEL assay and lactate dehydrogenase release assay BMDCs or human THP-1 macrophages, respectively (Fig. 1G, DNA fragmentation and lactate dehydrogenase (LDH) release were Supplemental Fig. 1H). Taken together, these data demonstrate assessed using DeadEnd Fluorometric TUNEL System (Promega) and that internalized C. neoformans activates the canonical caspase-1 CytoTox-ONE Homogeneous Membrane Integrity Assay kit (Promega), inflammasome in myeloid cells from both humans and mice. respectively, according to the manufacturers’ instructions. Internalized C. neoformans activates the canonical NLRP3 Statistical analysis

inflammasome by guest on September 25, 2021 Data are presented as mean 6 SD and analyzed for statistical significance by two-tailed Student t test in Prism (GraphPad) software. Differences with To date, a number of different inflammasomes have been identified, p values ,0.05 were considered statistically significant (*p , 0.05). including NLRP1, NLRP3, NLRP6, NLRC4, AIM2, and IFI16 inflammasomes, which are activated by different stimuli (2). Among them, the NLRP3 and NLRC4 inflammasomes have been Results found to be involved in antifungal immunity (28, 29). In this study, Internalized C. neoformans activates the canonical caspase-1 we tested which inflammasome was responsible for the canonical inflammasome activation of caspase-1 upon C. neoformans challenge in the C. neoformans is unique among pathogenic fungi for its capsule presence of opsonin. To this end, we found that shRNA-mediated outside of the cell wall that is antiphagocytic (24). In the absence silencing of NLRP3 or ASC in human THP-1 macrophage greatly of opsonization, the phagocytosis rate of C. neoformans by host reduced the secretion of IL-1b but not IL-8 induced by C. neo- cells is quite low. We have previously demonstrated that an forman in the presence of anti-GXM Ab (Fig. 2A, Supplemental acapsular mutant of C. neoformans induced IL-1b secretion Fig. 2A). Similarly, BMDCs from Nlrp3-orAsc-deficient mice did through NLRP3–ASC–caspase-1 inflammasome (13), which not secrete IL-1b upon infection with acapsular mutant or en- might be resulted from enhanced phagocytosis of C. neoformans. capsulated C. neoformans opsonized with Ab or serum, whereas In the current study, we first examined whether internalized en- the TNF-a secretion from all different genotypes of BMDCs was capsulated C. neoformans activates inflammasome. Indeed, the comparable (Fig. 2B, Supplemental Fig. 2B). Moreover, it was encapsulated C. neoformans induced robust IL-1b production also found that internalization of encapsulated C. neoformans from human THP-1 macrophages in the presence of anti-GXM Ab induced comparable IL-1b and TNF-a secretion from Aim2-or (Supplemental Fig. 1A). In mouse bone marrow–derived dendritic Nlrc4-deficient BMDCs as from WT cells (Fig. 2C, Supplemental cells (BMDCs), anti-GXM Ab-opsonized C. neoformans also induced Fig. 2C). In addition, ASC oligomerization was abolished in clear IL-1b secretion in a time- and dose-dependent manner (Fig. 1A, BMDCs deficient for Nlrp3 or Asc, and was enhanced in caspase- Supplemental Fig. 1B). These results were consistent with an early 1/11–deficient cells, as found before (18) (Fig. 2D). Thus, the study finding that a mAb binding to the capsule of C. neoformans canonical caspase-1 activation induced by internalization of enhanced IL-1b production from human monocytes (25). C. neoformans is dependent on the NLRP3 inflammasome, but not The complement in serum is an important opsonin for C. neoformans, NLRC4 or AIM2 inflammasome. and the deposition of complement in the capsule of C. neoformans b is necessary for its phagocytosis (26). In the current study, we found Caspase-8 is responsible for the caspase-1–independent IL-1 that opsonized C. neoformans by mouse serum also induced IL-1b secretion secretion in mouse BMDCs, whereas heat-inactivated serum failed Very interestingly, we noticed that knockdown of caspase-1 to do so (Fig. 1B). Moreover, C. neoformans–induced IL-1b se- in human macrophages or deficiency of caspase-1 from mouse The Journal of Immunology 4965 Downloaded from

FIGURE 1. Internalized C. neoformans activates the canonical caspase-1 inflammasome. (A and B) BMDCs from WT mice were cocultured with C. http://www.jimmunol.org/ neoformans H99 strain (multiplicity of infection = 10) with or without anti-GXM Ab for indicated time duration (A) or opsonized with 10% live or heat- inactivated (HI) mouse serum for 6 h (B). The supernatants were harvested for murine IL-1b ELISA. (C) IL-1b secretion from WT mouse BMDCs infected with C. neoformans H99, R265, or B3501 strains under opsonization [as in (A) and (B)] was monitored via ELISA. (D) BMDCs from WT mice were pretreated with clathrin inhibitor CPZ for 30 min, and then cocultured with C. neoformans H99 strain with Ab opsonization or C. neoformans acapsular mutant Cap59 strain as in (A) for 6 h. IL-1b in supernatant was assayed as in (A). (E) BMDCs were primed with 100 ng/ml LPS or PBS for 3 h and then infected with C. neoformans H99 strain under anti-GXM Ab opsonization, or with C. neoformans acapsular mutant Cap59 strain. Supernatant (SN) and cell lysates were collected for immunoblotting. (F) BMDCs from WT mice were infected with C. neoformans H99strainwithAbopsonizationfor6h. Cells were fixed, permeabilized, and stained. The arrowhead denotes C. neoformans, and triangle denotes ASC speck. (G)BMDCsfromWTmice

were pretreated with caspase-1–specific inhibitor Ac-YVAD for 30 min, then cocultured with C. neoformans H99 strain with Ab opsonization as in by guest on September 25, 2021 (A)for6h;IL-1b and TNF-a from cell culture supernatant were monitored via ELISA. Data are mean 6 SD from one of three independent ex- periments. *p , 0.05.

BMDCs did not completely abolish the secretion of IL-1b during C. neoformans activates the noncanonical caspase-8 C. neoformans infection as in cells deficient for ASC or NLRP3 inflammasome in the absence of caspase-1 (Fig. 2A, 2B). To explore the mechanism for this caspase-1–in- Recent studies showed that caspase-8 is involved in noncanonical b dependent IL-1 secretion, we first treated the BMDCs from inflammasome activation upon fungal or bacterial challenges (15, caspase-1/11–deficient mouse with pan-caspase inhibitor Z-VAD. 30, 31). In this study, we found that the acapsular Cap59 or anti- b a In this study, we found that the secretion of IL-1 but not TNF- GXM Ab-opsonized C. neoformans H99 strains induced moderate induced with Ab-opsonized C. neoformans (H99) or acapsular level of caspase-8 activation in WT BMDCs (Fig. 3A), which mutant (Cap59) was completely abolished (Fig. 2E, Supplemental was dependent on both NLRP3 and ASC, as Nlrp3-orAsc- Fig. 2D), indicating that other caspases were required for the deficient cells failed to mount any caspase-8 activation (Fig. 3B, b moderate IL-1 secretion in the absence of caspase-1. Along Supplemental Fig. 3A). Very interestingly, the activation of this line, our further study showed that the caspase-8 inhibitor caspase-8 was greatly enhanced in caspase-1/11–deficient IETD-FMK, but not the caspase-1 inhibitor Ac-YVAD, abol- BMDCs (Fig. 3B, Supplemental Fig. 3A), which correlated ished IL-1b secretion from caspase-1/11–deficient BMDCs in- pretty well with the key role of caspase-8 in mediating IL-1b duced by Ab-opsonized C. neoformans or acapsular mutant secretion from cells deficient for caspase-1/11,aswehavejust (Fig. 2E), which clearly indicated that caspase-8 was playing a described (Fig. 2). Moreover, the secretion of IL-1b but not critical role in this process. In addition, when caspase-8–specific TNF-a from caspase-1/11–deficient BMDCs induced by inter- shRNA was applied for gene silencing, IL-1b secretion induced nalized C. neoformans wasinhibitedbytheNLRP3inhibitor by Ab-opsonized H99 strain or acapsular mutant Cap59 from glyburide, indicating that the caspase-8 activation in the ab- caspase-1/11–deficient BMDCs was significantly attenuated sence of caspase-1 was also dependent on NLRP3 (Fig. 3C). (Fig. 2F, Supplemental Fig. 2E, 2F). Intriguingly, knockdown of Then we set out to demonstrate whether ASC was also involved caspase-8 in WT BMDCs did not reduce IL-1b secretion in- in this process. Similar with caspase-8 activation, the oligomeri- duced by C. neoformans (Supplemental Fig. 2G, 2H). These zation of ASC was also enhanced in the absence of caspase-1 data suggest that caspase-8 is required for IL-1b secretion in- (Figs. 2D, 3B). Furthermore, through coimmunoprecipitation ex- duced by internalized C. neoformans in the absence of caspase- periment, we found that the endogenous caspase-8 interacted 1, whereas caspase-1 plays a dominant role for this process in with ASC in caspase-1/11–deficient BMDCs during acapsular WT cells. C. neoformans infection (Fig. 3D), and this interaction did not occur 4966 INTERNALIZED C. neoformans ACTIVATES CASPASE-1 AND CASPASE-8 Downloaded from http://www.jimmunol.org/

FIGURE 2. Internalized C. neoformans activates the canonical NLRP3 inflammasome, and caspase-8 is responsible for the noncanonical caspase-1– independent IL-1b secretion. (A) THP-1 cell–derived macrophages with shRNA silencing of indicated genes were cocultured with C. neoformans H99 strain under anti-GXM Ab opsonization for 6 h, and IL-1b was detected via ELISA. (B and C) BMDCs from WT, Nlrp32/2, Asc2/2, Caspase-1/112/2 (B), Nlrc42/2, and Aim22/2 mice (C) were cocultured with C. neoformans acapsular mutant Cap59 strain, or with C. neoformans H99 strain opsonized with Ab or mouse serum for 6 h. The supernatants were harvested for murine IL-1b ELISA. (D) ASC oligomerization from indicated BMDCs stimulated with C. neoformans H99 strain with anti-GXM Ab opsonization was analyzed via immunoblotting. (E) BMDCs from WT mice were pretreated with Z-VAD, Ac- YVAD, or Z-IETD for 30 min, and then challenged with C. neoformans H99 strain with Ab opsonization or the Cap59 strain. IL-1b secretion was assayed. (F) BMDCs from Caspase-1/112/2 mice with shRNA silencing of caspase-8 gene were cocultured with C. neoformans H99 strain under Ab opsonization or the Cap59 strain for 6 h, and IL-1b was detected via ELISA. Data are mean 6 SD from one of three independent experiments. *p , 0.05. by guest on September 25, 2021 in the same cells without fungal challenge (Fig. 3D). Moreover, it TUNEL assay, it was also evident that C. neoformans–induced was also evident that the internalized C. neoformans induced cell death was clearly reduced in the Nlrp3-orAsc-deficient colocalization of caspase-8 with ASC speck in caspase-1/11–de- BMDCs compared with WT cells (Fig. 4D). What’s more, the ficient BMDCs (Supplemental Fig. 3B). Thus, these data indicate formation of ASC specks was correlated with DNA fragmentation that dendritic cells turn to NLRP3–ASC–caspase-8 activation (Supplemental Fig. 3C). These data demonstrate that internaliza- when caspase-1 is absent during C. neoformans infection, and this tion of C. neoformans induces host cell death, which is dependent noncanonical caspase-8 inflammasome activation is suppressed by on both ASC and NLRP3. caspase-1 in WT cells. Interestingly, from caspase-1/11–deficient BMDCs, C. neofor- mans infection still induced a high level of LDH release, and the Internalized C. neoformans induces NLRP3–ASC–caspase-8– TUNEL-positive cell number in these cells was comparable with dependent cell death that from WT cells (Fig. 4C, 4D), indicating that caspase-8 might The canonical caspase-1 inflammasome activation usually leads to also be responsible for this cell death phenotype. Indeed, caspase- a form of cell death called pyroptosis (2). As the key component 8 inhibitor or silencing of caspase-8 with shRNA both effectively for noncanonical inflammasome activation, caspase-8 is also in- reduced acapsular C. neoformans–induced LDH release from volved in different types of cell death (32, 33). In the current caspase-1/11–deficient BMDCs (Fig. 4E, 4F). Therefore, these study, we found that internalized C. neoformans induced a clear data reveal that caspase-8 is required for internalized C. neofor- release of pro–IL-1b from caspase-1/11–deficient BMDCs, indi- mans–induced cell death in the absence of caspase-1 from den- cating a possibility of cell death during C. neoformans infection dritic cells. Of note was that silencing of caspase-8 did not reduce (Fig. 4A). Moreover, a clear activation of caspase-8 also occurred acapsular C. neoformans–induced LDH release from WT BMDCs, during this process (Fig. 4A), indicating a potential connection of which indicates that caspase-8 plays only a minor role in inducing caspase-8 and cell death in these cells. Indeed, in our further cell death in the presence of caspase-1 (Supplemental Fig. 3D). experiment, it was found that opsonized C. neoformans or acap- Taken together, internalized C. neoformans induces host cell death, sular C. neoformans infection of mouse BMDCs induced strong which is a process dependent on the activity of noncanonical LDH release, which was a clear indication for the permeabiliza- NLRP3–ASC–caspase-8 inflammasome in the absence of caspase-1. tion of host cell membrane during cell death (Fig. 4B). In addition, the LDH release was significantly reduced from cells deficient for Mechanisms for the internalized C. neoformans induced Nlrp3 or Asc (Fig. 4C). Besides LDH release, DNA fragmentation canonical and noncanonical NLRP3 inflammasome activation is another indicator for cell death, which can be detected by The NLRP3 inflammasome is activated by a big variety of different TUNEL assay with incorporating BrDU-FITC into DNA. With stimuli, and the mechanisms for its activation are still not fully The Journal of Immunology 4967

FIGURE 3. C. neoformans activates the non- canonical caspase-8 inflammasome in the absence of caspase-1. (A) BMDCs from WT mice were in- fected with C. neoformans acapsular mutant Cap59 strain, or with C. neoformans H99 strain with or without anti-GXM Ab for 6 h. Cell lysates were assayed via immunoblotting for caspase-8 activa- tion (p41). (B) BMDCs from indicated mice were infected with Cap59 strain for 6 h, and then cas- pase-8 activation was detected with immunoblot- ting. (C) BMDCs from Caspase-1/112/2 mice were pretreated with glyburide for 30 min, then chal- Downloaded from lenged with C. neoformans acapsular mutant Cap59 strain for 6 h. IL-1b and TNF-a from cell culture supernatant were assayed via ELISA. (D) BMDCs from WT and Caspase-1/112/2 mice were infected with C. neoformans acapsular mutant Cap59 strain for 6 h, cell extracts were subjected to immuno- http://www.jimmunol.org/ precipitation (IP) by caspase-8 Ab, and cell lysates and IP complex were assayed via immunoblotting with ASC Ab. Data are mean 6 SD from one of three independent experiments. *p , 0.05. ns band, non-specific band. by guest on September 25, 2021

understood (2). To understand how C. neoformans activated WT BMDCs (Fig. 5E), indicating potassium efflux was also in- inflammasome, we first found that heat-inactivated acapsular volved in C. neoformans–induced inflammasome activation. More- C. neoformans or Ab-opsonized heat-inactivated C. neoformans over, in caspase-1/11–deficient BMDCs, Cap59-induced IL-1b did not induce a significant level of IL-1b compared with live secretion but not TNF-a production was also inhibited by C. neoformans in WT BMDCs, whereas the TNF-a production B inhibitor and high extracellular potassium (Supplemental Fig. 4B, was only slightly compromised (Fig. 5A). In caspase-1/11–deficient 4C), indicating that the noncanonical caspase-8 inflammasome ac- BMDCs, internalization of heat-inactivated C. neoformans also tivation also requires lysosome damage and potassium efflux during induced much lower level of IL-1b in comparison with live fun- C. neoformans infection. gus, with the TNF-a production less affected (Supplemental Fig. In addition, a recent study reported that mitochondrial ROS 4A). These data demonstrate that activation of the canonical induces lysosomal damage and inflammasome activation (36). In caspase-1 inflammasome or the noncanonical caspase-8 inflam- our current study, internalized C. neoformans also induced mito- masome both require the viability of C. neoformans. chondrial ROS production in WT BMDCs (Supplemental Fig. 4D, Early studies reported that live C. neoformans causes host cell 4E). However, although the mitochondrial antioxidant Mito- phagolysosome permeabilization when residing in the phago- TEMPO clearly inhibited IL-1b secretion induced by LPS plus lysosome (34, 35). In our study, it was found that phagolysosome ATP, as reported (data not shown) (36), it did not inhibit IL-1b membrane permeabilization was correlated with ASC speck for- secretion triggered by internalized C. neoformans in these cells mation in C. neoformans–infected THP-1 macrophages expressing (Fig. 5F). This indicated that the inflammasome activation in this mCherry–ASC (Fig. 5B, 5C). This indicated that internalized case was not mediated by mitochondrial ROS, rather through a C. neoformans caused release of phagolysosome components, which different mechanism, which deserves further analysis in the future. may have led to the activation of inflammasome. Indeed, cathepsin Taken together, internalization of C. neoformans causes phago- B inhibitor CA-074Me also reduced C. neoformans–induced IL- lysosome membrane permeabilization and potassium efflux, 1b secretion, but not TNF-a production from WT BMDCs leading to activation of the canonical NLRP3–ASC–caspase-1 (Fig. 5D). Moreover, inhibition of potassium efflux also reduced inflammasome and the noncanonical NLRP3–ASC–caspase-8 the secretion of IL-1b induced by Ab-opsonized C. neoformans in inflammasome. 4968 INTERNALIZED C. neoformans ACTIVATES CASPASE-1 AND CASPASE-8

FIGURE 4. Internalized C. neoformans induces cell death in a manner dependent on NLRP3–ASC– caspase-8 inflammasome. (A) BMDCs from Cas- pase-1/112/2 mice were primed with LPS for 3 h and then infected with C. neoformans H99 strain with or without anti-GXM Ab opsonization, or with C. neoformans acapsular mutant Cap59 strain for 6 h, and then caspase-8 and IL-1b were detected B

via immunoblotting. ( ) BMDCs from WT mice Downloaded from were infected with C. neoformans H99 strain under opsonization or C. neoformans acapsular mutant Cap59 strain for 6 h; LDH release in cell culture was assayed. (C) BMDCs from indicated mice were incubated with C. neoformans Cap59 mutant, or C. neoformans H99 strain under opsonization for

6 h; LDH release was assayed. (D) BMDCs from http://www.jimmunol.org/ indicated mice were incubated with C. neoformans acapsular mutant Cap59 strain for 6 h; percentage of TUNEL-positive cells was counted. (E) BMDCs from Caspase-1/112/2 mice were pretreated with Z-IETD for 30 min, followed with C. neoformans acapsular mutant Cap59 strain challenged for 6 h, and LDH release was assayed. (F) BMDCs from Caspase-1/112/2 mice with lentivirus-mediated shRNA knockdown of caspase-8 were challenged with C. neoformans acapsular mutant Cap59, and by guest on September 25, 2021 LDH release was assayed. Data are mean 6 SD from one of three independent experiments. *p , 0.05.

Zymosan also activates caspase-8 in caspase-1/11–deficient signaling event (Fig. 6A). The same as in the case of internal- cells ized C. neoformans, zymosan did not induce IL-1b production Cryptococcal cell wall contains glucan, chitin, and glycosylated from Nlrp3-orAsc-deficient BMDCs (Fig. 6A). As expected, the b protein, which maintain the cell shape and regulate fungal per- IL-1 secretion from caspase-1/11–deficient BMDCs challenged meability (7). Recent studies show pathogen-associated molecular with zymosan was also blocked by caspase-8 inhibitor in a dose- a patterns (PAMPs) from fungal cell wall were involved in inflam- dependent manner, whereas TNF- production was not affected masome activation (14, 15, 29, 37, 38). To further identify the role (Fig. 6B). Similarly, knockdown of caspase-8 in these caspase-1/ of caspase-1 in fungal PAMP-induced caspase-8 activation, we set 11–deficient BMDCs also reduced IL-1b secretion as well as cell out to demonstrate whether the fungal cell wall products also in- death induced by zymosan (Fig. 6C, 6D). In addition, zymosan duced elevated caspase-8 activation in caspase-1/11–deficient challenge also resulted in elevated activation of caspase-8 in cells. To this end, we found that the fungal product zymosan, a BMDCs deficient for caspase-1 compared with WT cells (Fig. 6E, b-glucan found on the cell wall of fungi, induced clear IL-1b Fig. 7). These data suggest that caspase-8 is also critical for the secretion from caspase-1/11–deficient BMDCs, whereas LPS plus noncanonical NLRP3–ASC–inflammasome activation in response ATP failed to do so, indicating that this was a fungal-specific to fungal PAMPs. The Journal of Immunology 4969 Downloaded from FIGURE 5. C. neoformans induce phagolysosome membrane permeabilization and K+ efflux to activate inflammasome. (A) WT BMDCs were infected with living or heat-inactivated (HI) C. neoformans acapsular mutant Cap59 strain or H99 strain in the presence of anti-GXM Ab for 6 h; IL-1b and TNF-a secretion were assayed. (B and C) FITC-dextran diffusion and ASC speck formation in THP-1 macrophages with overexpression of mCherry–ASC fusion protein infected with C. neoformans H99 strain under opsonization for 4 h were assayed by immunofluorescence microscopy. The arrowhead denotes C. neoformans, and triangle denotes ASC speck. Original magnification 3400 (B). Four hundred cells were randomly chosen to calculate ASC speck for- mation ratio among cells with FTIC-dextran punctate, punctate/diffuse, or diffuse (C). (D and E) WT BMDCs were pretreated with CA-074Me (D), or KCl (E), and then challenged with Ab-opsonized C. neoformans H99 strain, and the supernatants were harvested for ELISA. (F) WT BMDCs were pretreated http://www.jimmunol.org/ with Mito-TEMPO and then challenged with C. neoformans H99 strain with Ab opsonization; IL-1b secretion was assayed. Data are mean 6 SD from one of three independent experiments. *p , 0.05.

Discussion fungal cell is much larger, so the interaction between host cell and The inflammasome has emerged as an important cytoplasmic fungal cell is very different from other types of pathogens (40). platform responsible for proteolytic processing of inflammatory C. neoformans is an important fungal pathogen, whereas the host cytokines IL-1b and IL-18 as well as pyroptotic cell death (39). cell interaction with this fungus is not well understood. In previ-

Although many pathogens have been found to activate inflam- ous studies, we have found that the biofilm as well as the acapsular by guest on September 25, 2021 masomes, the fungal pathogens activating inflammasomes are not mutant of C. neoformans activate the NLRP3 inflammasome in widely studied (2). Different from viruses or bacteria, the size of myeloid cells (12, 13). However, a question remaining is whether

FIGURE 6. Zymosan also activates caspase-8 in caspase-1/11–deficient cells. (A) BMDCs from indicated mice were stimulated with 200 mg/ml zymosan for 12 h, or with LPS/ATP as control. Culture supernatants were harvested for IL-1b and TNF-a ELISA. (B) BMDCs from Caspase-1/112/2 mice were pretreated with Z-IETD for 30 min, followed with zymosan challenged for 12 h; IL-1b and TNF-a secretion were assayed. (C and D) BMDCs from Caspase-1/112/2 mice with shRNA knockdown of caspase-8 were challenged with zymosan; IL-1b and TNF-a secretion (C) or LDH release (D) was assayed. (E) BMDCs from WT and Caspase-1/112/2 mice were challenged with zymosan; cell lysates were assayed by immunoblotting for caspase-8 activation. Data are mean 6 SD from one of three independent experiments. *p , 0.05. 4970 INTERNALIZED C. neoformans ACTIVATES CASPASE-1 AND CASPASE-8 the naturally existing encapsulated C. neoformans activates in- The function of caspase-8 in fungal pathogen-induced inflam- flammasome under certain circumstances. In the current study, we masome activation has also been explored. C. albicans induces found that internalization of encapsulated C. neoformans with caspase-8 activation in human and mouse phagocytic cells and opsonin activated inflammasome in a NLRP3–ASC–dependent leads to IL-1b secretion (14, 15, 30). Another fungal pathogen, manner. Importantly, C. neoformans activated both the canonical A. fumigatus, induces AIM2 and NLRP3 activation, which initiates caspase-1 inflammasome and the noncanonical caspase-8 inflam- assembly of inflammasome composed of ASC along with caspase- masome, although the canonical inflammasome activation was 1 and caspase-8, leading to the maturation of IL-1b and IL-18 dominant in WT cells. Our findings thus uncover a previously (51). In our study, internalized C. neoformans induced moderate unknown regulation of caspase-1 on caspase-8 in dendritic cells level of caspase-8 activation in WT BMDCs, but resulted in much upon fungal pathogen challenge (Fig. 7). stronger activation of caspase-8 in caspase-1/11–deficient cells. As an initiating and apical activating caspase, caspase-8 is well This indicated that the canonical caspase-1 inflammasome inhibits known for its important role in apoptosis (41). Interestingly, re- the noncanonical caspase-8 inflammasome activation during cent studies have found that caspase-8 is involved in modulating C. neoformans infection in WT cells. Probably because the canonical IL-1b maturation (42–47). Caspase-8 is also involved in priming caspase-1 function is dominant, we did not see any inhibition of inflammasome for activation during Salmonella infection (31). caspase-1 activation by caspase-8. When caspase-8 was silenced, Moreover, it has been found that caspase-8 is an apical mediator the IL-1b secretion from WT cells did not change (Supplemental for the canonical and noncanonical NLRP3 inflammasome acti- Fig. 2G). Because both caspase-1 and caspase-8 activations re- vation induced by LPS or Citrobacter rodentium (48). In addition, quire NLRP3 and ASC, most likely they compete with each other caspase-8 promotes NLRP1/NLRP3 inflammasome activation and for binding with NLRP3–ASC to form different complexes upon Downloaded from TLR4-mediated IL-1b production in acute glaucoma (49). On the fungal infection (Fig. 7). contrary, there is also a study that found that caspase-8 blocks Many pathogens cause host cell death during infection. RIPK3-mediated activation of the NLRP3 inflammasome (50). Inflammasomes are important mediators for the rapid lytic cell These studies indicate a very complex role of caspase-8 in several death termed pyroptosis (2). And recent studies show that caspase- important signaling pathways. 8 participates in cell death resulted from inflammasome activation.

For example, ubiquitination of NLRC4 by Sug1 formed cyto- http://www.jimmunol.org/ plasmic aggregates containing caspase-8, leading to its activation and cell death (52). Upon transfection of DNA, AIM2 and NLRP3 inflammasomes activate caspase-8 and caspase-1, leading to both apoptotic and pyroptotic cell death (53). Caspase-8 is also re- quired for pathogenic Yersinia YopJ protein or Yersinia pestis– induced cell death (54, 55). Francisella also triggers AIM2/ASC/ caspase-8–dependent apoptosis in caspase-1–deficient macro- phages (32). Moreover, fungal pathogen C. albicans infection also causes host cell death via caspase-8 (15). In this study, we found by guest on September 25, 2021 C. neoformans induced host cell death in NLRP3/ASC-dependent manner. In caspase-1/11–deficient BMDCs, activation of caspase- 8 by internalized C. neoformans induced host cell death as well as caspase-3 and caspase-7 activation (data not shown). It is thus very interesting to explore the relationship between caspase-1 and caspase-8 upon different pathogen challenge in terms of cell death. Although dendritic cells play a key role in immune response against C. neoformans, recognition of C. neoformans by dendritic cells is less studied (56). We found that Ab- or serum-opsonized C. neoformans induced NF- kB and MAPK activation as well as enhanced expression of NLRP3 in mouse BMDCs (data not shown). After opsonic phagocytosis by immune cells, C. neofor- mans can survive and replicate in the phagosome or phago- FIGURE 7. Canonical and noncanonical NLRP3 inflammasome acti- lysosome, where the fungus secrete virulent factors, including vation upon fungal challenge in dendritic cells. Opsonization of C. neo- formans with Ab or complement triggers phagocytosis of the pathogen into phospholipase B, which may cause phagolysosome membrane phagosome, subsequently forming phagolysosome. Meanwhile, phagocy- permeabilization (57, 58). It has been found that particulate tosis of the pathogen activates MAPK and NF-kB signaling pathways, stimuli, including silica and alum, activate the NLRP3 inflam- which induces expression of pro–IL-1b and NLRP3. C. neoformans re- masome through phagosomal destabilization (59). In this study, siding in phagolysosome may secret virulent factors, causing destabiliza- we found that phagolysosome membrane destabilization also tion of phagolysosomal membrane. The leakage of phagolysosome leads to contributed to C. neoformans activation of the NLRP3 inflam- + the release of components such as and causes K efflux. masome. Akin to previous finding, K+ efflux was also necessary Subsequent activated canonical NLRP3–ASC–Caspase-1 inflammasome for NLRP3 inflammasome activation induced by C. neoformans b results in the processing of pro–IL-1 as well as host cell death. In the (60). Therefore, C. neoformans activates NF-kBandMAPK absence of caspase-1, internalized C. neoformans induces noncanonical b pathways for the priming signal, and then induces phagolysosome NLRP3–ASC–Caspase-8 inflammasome activation, which leads to IL-1 + secretion and host cell death as well. Zymosan can also induce enhanced membrane destabilization and K efflux to activate the NLRP3 noncanonical NLRP3–ASC–Caspase-8 inflammasome activation in the inflammasome (Fig. 7). absence of caspase-1. Thus, canonical caspase-1 inflammasome and non- Different types of cells exhibit difference in the activation of canonical caspase-8 inflammasome coordinate to mount innate immune inflammasome. For example, the threshold for inflammasome responses against fungal pathogens. activation in mouse macrophages is much higher than in human The Journal of Immunology 4971 macrophages (61). As a facultative intracellular fungal pathogen, production in response to b-glucans and the fungal pathogen, Candida albicans. J. Immunol. 193: 2519–2530. C. neoformans can replicate in host phagocytic cells (62, 63). 16. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee, However, different phagocytic cells respond differently to M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of C. neoformans; thus, the intracellular recognition of this pathogen the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218. 17. Rathinam, V. A., Z. Jiang, S. N. Waggoner, S. Sharma, L. E. Cole, L. Waggoner, by different host cells may also be variable (57). In contrast to S. K. Vanaja, B. G. Monks, S. Ganesan, E. Latz, et al. 2010. The AIM2 the data we showed in this study from dendritic cells, phagocytosis inflammasome is essential for host defense against cytosolic bacteria and DNA of C. neoformans by mouse macrophages did not induce clear IL-1b viruses. Nat. Immunol. 11: 395–402. 18. Mao, K., S. Chen, M. Chen, Y. Ma, Y. Wang, B. Huang, Z. He, Y. Zeng, Y. Hu, production (data not shown). This indicates that C. neoformans S. Sun, et al. 2013. Nitric oxide suppresses NLRP3 inflammasome activation and applies specific mechanisms to avoid inflammasome activation protects against LPS-induced septic shock. Cell Res. 23: 201–212. in macrophages, which are invalid in dendritic cells. As the most 19. Kayagaki, N., S. Warming, M. Lamkanfi, L. Vande Walle, S. Louie, J. Dong, K. Newton, Y. Qu, J. Liu, S. Heldens, et al. 2011. Non-canonical inflammasome important APCs, dendritic cells are much more sensitive than activation targets caspase-11. Nature 479: 117–121. other cells in sensing invading pathogens. Probably that is also the 20. Fang, W., M. S. Price, D. L. Toffaletti, J. Tenor, M. Betancourt-Quiroz, reason that the threshold for inflammasome activation is much J. L. Price, W. H. Pan, W. Q. Liao, and J. R. Perfect. 2012. Pleiotropic effects of deubiquitinating Ubp5 on growth and pathogenesis of Cryptococcus lower in these cells (64). To understand the molecular mechanisms neoformans. PLoS One 7: e38326. accounting for the difference between dendritic cells and macro- 21. Pe´trilli, V., S. Papin, C. Dostert, A. Mayor, F. Martinon, and J. Tschopp. 2007. Activation of the NALP3 inflammasome is triggered by low intracellular po- phages upon C. neoformans infection is an interesting topic for tassium concentration. Cell Death Differ. 14: 1583–1589. future investigation. 22. Fernandes-Alnemri, T., and E. S. Alnemri. 2008. Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol. 442: 251–270. 23. Bewley, M. A., M. Naughton, J. Preston, A. Mitchell, A. 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