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
J Immunol November 15, 2015, 195 (10) 4962-4972; DOI: https://doi.org/10.4049/jimmunol.1500865

Abstract

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 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-1β secretion and cell death 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 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3–ASC–caspase-8 inflammasome 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.

Introduction

As the first line of defense against pathogen invasion, host innate immune system recognizes microbial pathogens such as viruses, bacteria, and fungi for elimination. This recognition relies on a group of evolutionarily conserved pattern recognition receptors (PRRs), including membrane-bound TLRs and C-type lectin receptors, as well as cytoplasmic NOD-like receptors (NLRs), RIG-I like receptors, and cytosolic DNA sensors. These PRRs recognize conserved molecular patterns expressed by invading microbes and initiate innate immune responses, including inflammation (1).

A number of NLRs and DNA sensors among these PRRs can assemble into a protein complex called inflammasome, which contains PRRs such as NLR family, pyrin domain-containing 3 (NLRP3), NLR family CARD domain-containing 4 (NLRC4), or absent in melanoma 2 (AIM2), adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and procaspase-1. Assembly of inflammasome leads to the autocleavage and activation of caspase-1 (2). The activated caspase-1 triggers the processing and release of proinflammatory cytokines IL-1β and IL-18, both of which are important for the shaping of adaptive immune responses and elimination of invading pathogens (1). Furthermore, activation of caspase-1 triggers a form of cell death termed pyroptosis, which protects the host by eliminating the replication niche of invading intracellular pathogens (3).

Among a large plethora of microbial pathogens, fungi are attracting the attention of more and more scientists. That is because, with the increasing number of immunocompromised hosts such as AIDS patients and organ transplant recipients, mycoses have been emerging as life-threatening infectious diseases over the last three decades (4). Opportunistic fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans pose the biggest challenge in immunocompromised hosts. Of particular note, C. neoformans is a pathogenic fungus that causes cryptococcosis, including life-threatening meningo encephalitis, which affects about 1 million AIDS patients and causes >600,000 deaths worldwide annually (5, 6). C. neoformans is unique among the pathogenic fungi by carrying a capsule outside of the cell wall, which is mainly composed of the antiphagocytic glucuronoxylomannan (GXM) (7). However, the detailed mechanisms for the interaction of C. neoformans with host cells are still poorly studied.

Recent studies showed that the NLRP3 inflammasome was involved in host immune responses against a number of fungal pathogens, including C. albicans, A. fumigatus, and Microsporum canis (8–11). In addition, research from the Meng Laboratory also revealed that the biofilm from C. neoformans as well as the acapsular mutant of C. neoformans both activated the NLRP3 inflammasome (12, 13). However, whether encapsulated C. neoformans activates inflammasome under certain conditions is not known yet. Besides the canonical inflammasome activation mentioned above, C. albicans also induces noncanonical caspase-8 inflammasome activation in human dendritic cells (14). Moreover, caspase-8 is also involved in promoting β-glucan–induced cell death and NLRP3 inflammasome-dependent IL-1β maturation during C. albicans infection in mouse dendritic cells (15). However, whether and how caspase-8 is activated during C. neoformans infection are still unknown.

In the current study, we found that internalization of the encapsulated C. neoformans with anti-GXM Ab or serum opsonization activated the canonical NLRP3–ASC–caspase-1 inflammasome. More interestingly, from dendritic cells deficient for caspase-1, C. neoformans still induced clear secretion of IL-1β as well as cell death, both of which turned out to be mediated by the noncanonical NLRP3–ASC–caspase-8 inflammasome activation. Of special note was that the activation of caspase-8 was strongly elevated in the absence of caspase-1. Collectively, our findings establish a novel link between NLRP3–ASC and the key inflammatory caspases upon fungal challenge and reveal that caspase-1 is inhibitory for caspase-8 in dendritic cells upon C. neoformans infection.

Materials and Methods

Mice and reagents

C57BL/6 wild-type (WT) mice were obtained from The Jackson Laboratory and bred in our specific pathogen-free animal facility. Nlrp3, Asc, Nlrc4, Aim2, and Caspase-1/11–deficient mice had been described before (16–19). Animal care, use, and experimental procedures complied with national guidelines and were approved by the Animal Care and Use Committee at Institut Pasteur of Shanghai. Mouse anti-GXM mAb (IgG1, κ) to C. neoformans was purchased from Meridian Life Science. Zymosan was from InvivoGen. All chemical reagents were purchased from Sigma-Aldrich, unless stated otherwise.

Culture of C. neoformans

Encapsulated C. neoformans reference strain H99 (serotype A), R265 (serotype B), B3501 (serotype D), and acapsular mutant Cap59 (H99 background) were from the J. Perfect Laboratory (20). The fungus was maintained in glycerol stocks in −80°C and was grown on yeast extract tryptone dextrose (YTD) agar plate at 30°C. Liquid cultures were grown in YTD medium at 30°C for 20–24 h in a shaking incubator at 180 rpm. Fungal cells were centrifuged at 2000 × g for 2 min, washed three times, and resuspended in sterile PBS. In some experiments, the fungus was heat inactivated by incubating at 70°C for 1 h, and no growth was observed from the following inoculation of inactivated C. neoformans during a 2-d period on YTD agar plates.

Mammalian cell culture and mouse serum collection

THP-1 cells were maintained in RPMI 1640 media containing 10% FBS, 100 IU/ml penicillin, 1 mg/ml streptomycin, and 50 μM 2-ME at 37°C with 5% CO2. For differentiation into macrophages, THP-1 cells were incubated with 100 ng/ml PMA for 3 h, then washed twice with PBS and rested for 48 h before use. Mouse bone marrow–derived dendritic cells (BMDCs) were prepared, as described before (12). Briefly, bone marrow cells were collected, suspended in PBS by addition of RBC lysis buffer for depletion of erythrocytes, and then seeded in 7.5 × 105 cells/ml in the RPMI 1640 media with rGM-CSF (20 ng/ml; Peprotech) in a humidified incubator with 5% CO2 at 37°C. The cells were fed once at the interval of 3 d with the identical dose of rGM-CSF. The cells were harvested on day 6, with the purity of CD11c+ cells higher than 80% tested by flow cytometry (data not shown), and were used for fungal challenge experiments. For mouse serum collection, WT C57BL/6 mice were anesthetized by injection of 2.4% avertin, and blood was collected by cardiac puncture. The whole blood was left at room temperature for 2 h, and mouse serum was collected through centrifuging for 15 min at 3000 rpm at 4°C.

In vitro C. neoformans challenge

For fungal-challenging experiments, mouse BMDCs were pooled in plates with C. neoformans H99 or Cap59 mutant at multiplicity of infection = 10 (10 yeasts to 1 macrophage) with or without 30 μg/ml anti-GXM Ab or 10% mouse serum opsonization, unless stated otherwise. In some experiments, host cells were treated with the following chemicals for 30 min before infection with C. neoformans: Ac-YVAD-CHO (caspase-1 inhibitor), Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), glyburide (NLRP3 inhibitor), CA-074Me, potassium chloride (KCl), Mito-TEMPO (mitochondria-targeted antioxidant), chloropromazine (CPZ), with indicated concentrations in figure legends. In indicated experiment, cells were stimulated with LPS (100 ng/ml) for 6 h with ATP (5 mM) pulse for 30 min. The supernatants were harvested for cytokine ELISA. In some cases, supernatants and cell extracts were collected for immunoblotting analysis.

Gene silencing in THP-1 cells and mouse BMDCs

Short hairpin RNA (shRNA) vectors against NLRP3, caspase-1, ASC, and their scramble vectors were gifts of the J. Tschopp Laboratory (21). Generation of specific gene silencing THP-1 cells had been described before (10). For knockdown of caspase-8 in mouse BMDCs, shRNA vectors against mouse caspase-8 were constructed in the Meng Laboratory. Lentiviral particles were generated in 293T cells by transfection with a modified PLKO-shCasp8, pMD2-VSVG, and pCMV-R8.91 using polyethylenimine. The viral particles were harvested 48 h later and used for infection of BMDCs. BMDCs were infected on day 0, and then changed to 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 for experiments. The targeting sequences for respective genes are as follows: human NLRP3, 5′-CAGGTTTGACTATCTGTTCT-3′; human Caspase-1, 5′-GTGAAGAGATCCTTCTGTA-3′; human ASC, 5′-GATGCGGAAGCTCTTCAGTTTCA-3′; mouse Caspase-8-1, 5′-TCATCTCACAAGAACTATATT-3′; and mouse Caspase-8-2, 5′-TCATCTCACAAGAACTATATT-3′.

mCherry–ASC overexpression in THP-1 cells

For generation of mCherry–ASC–overexpressing THP-1 cell line, a lentiviral vector containing mCherry–ASC fusion gene was constructed. Lentiviral particles were generated in 293T cells and harvested for infection of THP-1 monocytes. The mCherry–ASC–expressing THP-1 monocytes were screened and maintained with Geneticin (G418) selection.

Cytokine ELISA and immunoblotting

Supernatants were analyzed for cytokine secretion by ELISA, according to the manufacturer’s instructions (eBioscience). Abs for immunoblotting include the following: rabbit anti-mouse ASC (SC-22514-R; Santa Cruz), rabbit anti-mouse caspase-1 (sc-514; Santa Cruz), rabbit anti-mouse mature and pro–IL-1β (sc-7884; Santa-Cruz), rabbit anti-mouse caspase-8 (9429; Cell Signaling), rat anti-mouse caspase-8 (Enzo Life Science), and mouse anti-mouse β-actin (Sigma-Aldrich). Appropriate HRP-conjugated secondary Abs were used for signal detection via ECL reagent (Perkin Elmer).

ASC oligomerization detection

ASC oligomerization detection was conducted, as described before (22). Briefly, BMDCs were cocultured with C. neoformans H99 under anti-GXM Ab opsonization for 6 h, and cells were lysed, pelleted, washed with PBS, and cross-linked with disuccinimidyl suberate. The cross-linked pellets were resuspended in 30 μl SDS loading buffer for immunoblotting detection of ASC.

Coimmunoprecipitation

BMDCs were plated into six-well plates and infected with C. neoformans Cap59 mutant at multiplicity of infection = 10. After 6 h, cells were lysed in buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% Nonidet P-40, and Complete Protease Inhibitor Cocktail (Roche), and centrifuged at 3000 × g for 5 min at 4°C. Part of the supernatants was taken as input control; the left was incubated with protein A agarose beads (Invitrogen) and anti–caspase-8 Ab (Enzo Life Sciences) at 4°C overnight. The beads were washed with lysis buffer, resuspended in sample buffer, and processed for immunoblotting.

Immunofluorescence

A total of 5 × 105 BMDCs was seeded on glass coverslips in 24-well plates overnight and then cocultured with C. neoformans H99 strain under anti-GXM Ab opsonization or acapsular mutant Cap59 for 6 h. For mitochondrial reactive oxygen species (ROS) staining, MitoSox (Invitrogen) was added to cells in the last 10 min. Then cells were washed gently three times with warm buffer and mounted in warm buffer for imaging. For detecting phagolysosome membrane leakage, THP-1–derived macrophages expressing mCherry–ASC were incubated with 5 mg/ml FITC-dextran (Sigma-Aldrich; 40-kDa molecular mass) molecules for 2 h at 37°C. The cells were washed with PBS, chased with culture medium for 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 postinfection, THP-1 cells were washed with PBS and visualized using ×100 objective fluorescence microscopy. For caspase-8 staining, BMDCs were treated with FITC-IETD-FMK (CaspGLOW Fluorescein Active caspase-8 staining kit; eBioscience) 2 h post-C. neoformans infection. Then cells were washed with PBS, fixed with 4% (w/v) paraformaldehyde solution, permeabilized with 0.1% Trixton-100, and blocked with 10% FBS in PBS. For ASC speck staining, cells were incubated with anti-ASC Ab, washed, and incubated with Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen). For C. neoformans staining, cells were incubated with mouse 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 an inverted Leica DMI3000B fluorescence microscope and a ×100 objective.

Flow cytometry for mitochondrial ROS detection

MitoSox (Invitrogen) was added to cells 10 min before harvesting, and then cells were washed in FACS buffer (PBS + 0.5% BSA) three times and resuspended in FACS buffer. Data were collected on BD LSRFortessa at forward light scatter, side light scatter, and PE channels.

TUNEL assay and lactate dehydrogenase release assay

DNA fragmentation and lactate dehydrogenase (LDH) release were assessed using DeadEnd Fluorometric TUNEL System (Promega) and CytoTox-ONE Homogeneous Membrane Integrity Assay kit (Promega), respectively, according to the manufacturers’ instructions.

Statistical analysis

Data are presented as mean ± SD and analyzed for statistical significance by two-tailed Student t test in Prism (GraphPad) software. Differences with p values <0.05 were considered statistically significant (*p < 0.05).

Results

Internalized C. neoformans activates the canonical caspase-1 inflammasome

C. neoformans is unique among pathogenic fungi for its capsule outside of the cell wall that is antiphagocytic (24). In the absence of opsonization, the phagocytosis rate of C. neoformans by host cells is quite low. We have previously demonstrated that an acapsular mutant of C. neoformans induced IL-1β secretion through NLRP3–ASC–caspase-1 inflammasome (13), which might be resulted from enhanced phagocytosis of C. neoformans. In the current study, we first examined whether internalized encapsulated C. neoformans activates inflammasome. Indeed, the encapsulated C. neoformans induced robust IL-1β production from human THP-1 macrophages in the presence of anti-GXM Ab (Supplemental Fig. 1A). In mouse bone marrow–derived dendritic cells (BMDCs), anti-GXM Ab-opsonized C. neoformans also induced clear IL-1β secretion in a time- and dose-dependent manner (Fig. 1A, Supplemental Fig. 1B). These results were consistent with an early study finding that a mAb binding to the capsule of C. neoformans enhanced IL-1β production from human monocytes (25).

FIGURE 1.
FIGURE 1.

Internalized C. neoformans activates the canonical caspase-1 inflammasome. (A and B) BMDCs from WT mice were cocultured with C. 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-1β ELISA. (C) IL-1β 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-1β 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 H99 strain with Ab opsonization for 6 h. Cells were fixed, permeabilized, and stained. The arrowhead denotes C. neoformans, and triangle denotes ASC speck. (G) BMDCs from WT mice were pretreated with caspase-1–specific inhibitor Ac-YVAD for 30 min, then cocultured with C. neoformans H99 strain with Ab opsonization as in (A) for 6 h; IL-1β and TNF-α from cell culture supernatant were monitored via ELISA. Data are mean ± SD from one of three independent experiments. *p < 0.05.

The complement in serum is an important opsonin for C. neoformans, and the deposition of complement in the capsule of C. neoformans is necessary for its phagocytosis (26). In the current study, we found that opsonized C. neoformans by mouse serum also induced IL-1β secretion in mouse BMDCs, whereas heat-inactivated serum failed to do so (Fig. 1B). Moreover, C. neoformans–induced IL-1β secretion from BMDCs was increased with higher doses of serum for opsonization (Supplemental Fig. 1C).

The Cryptococcus species complex includes at least two subspecies: C. neoformans and C. grubii. They are further divided into five serotypes, including the hybrid serotype AD (A, D for C. neoformans, and B, C for C. gattii), with serotype A isolates responsible for the vast majority of cryptococcal infections (27). With the opsonization of anti-GXM Ab or mouse serum, all cryptococcal strains from serotype A, D, and B induced IL-1β and TNF-α secretion from mouse BMDCs (Fig. 1C, Supplemental Fig. 1D).

Interestingly, CPZ, a clathrin inhibitor, inhibited anti-GXM Ab-opsonized C. neoformans or acapsular mutant Cap59-induced secretion of IL-1β and TNF-α from BMDCs (Fig. 1D, Supplemental Fig. 1E), indicating that internalization of C. neoformans was essential for optimal inflammasome activation from host cells. Indeed, with immunoblotting, it was found that anti-GXM Ab-opsonized C. neoformans induced caspase-1 maturation, ASC speck formation, as well as ASC oligomerization in dendritic cells (Fig. 1E, 1F, Supplemental Fig. 1F), clearly indicating an activation of inflammasome. Moreover, our further study showed that inhibition of internalization of C. neoformans by CPZ inhibited synthesis of pro–IL-1β as well as activation of caspase-1 (Supplemental Fig. 1G).

In addition, a specific caspase-1 inhibitor Ac-YVAD greatly reduced Ab-opsonized C. neoformans–induced IL-1β secretion, but did not affect the secretion of TNF-α nor IL-8 from mouse BMDCs or human THP-1 macrophages, respectively (Fig. 1G, Supplemental Fig. 1H). Taken together, these data demonstrate that internalized C. neoformans activates the canonical caspase-1 inflammasome in myeloid cells from both humans and mice.

Internalized C. neoformans activates the canonical NLRP3 inflammasome

To date, a number of different inflammasomes have been identified, 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 found to be involved in antifungal immunity (28, 29). In this study, we tested which inflammasome was responsible for the canonical activation of caspase-1 upon C. neoformans challenge in the presence of opsonin. To this end, we found that shRNA-mediated silencing of NLRP3 or ASC in human THP-1 macrophage greatly reduced the secretion of IL-1β but not IL-8 induced by C. neoforman in the presence of anti-GXM Ab (Fig. 2A, Supplemental Fig. 2A). Similarly, BMDCs from Nlrp3- or Asc-deficient mice did not secrete IL-1β upon infection with acapsular mutant or encapsulated C. neoformans opsonized with Ab or serum, whereas the TNF-α secretion from all different genotypes of BMDCs was comparable (Fig. 2B, Supplemental Fig. 2B). Moreover, it was also found that internalization of encapsulated C. neoformans induced comparable IL-1β and TNF-α secretion from Aim2- or Nlrc4-deficient BMDCs as from WT cells (Fig. 2C, Supplemental Fig. 2C). In addition, ASC oligomerization was abolished in BMDCs deficient for Nlrp3 or Asc, and was enhanced in caspase-1/11–deficient cells, as found before (18) (Fig. 2D). Thus, the canonical caspase-1 activation induced by internalization of C. neoformans is dependent on the NLRP3 inflammasome, but not NLRC4 or AIM2 inflammasome.

FIGURE 2.
FIGURE 2.

Internalized C. neoformans activates the canonical NLRP3 inflammasome, and caspase-8 is responsible for the noncanonical caspase-1–independent IL-1β 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-1β was detected via ELISA. (B and C) BMDCs from WT, Nlrp3−/−, Asc−/−, Caspase-1/11−/− (B), Nlrc4−/−, and Aim2−/− 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-1β 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-1β secretion was assayed. (F) BMDCs from Caspase-1/11−/− 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-1β was detected via ELISA. Data are mean ± SD from one of three independent experiments. *p < 0.05.

Caspase-8 is responsible for the caspase-1–independent IL-1β secretion

Very interestingly, we noticed that knockdown of caspase-1 in human macrophages or deficiency of caspase-1 from mouse BMDCs did not completely abolish the secretion of IL-1β during C. neoformans infection as in cells deficient for ASC or NLRP3 (Fig. 2A, 2B). To explore the mechanism for this caspase-1–independent IL-1β secretion, we first treated the BMDCs from caspase-1/11–deficient mouse with pan-caspase inhibitor Z-VAD. In this study, we found that the secretion of IL-1β but not TNF-α induced with Ab-opsonized C. neoformans (H99) or acapsular mutant (Cap59) was completely abolished (Fig. 2E, Supplemental Fig. 2D), indicating that other caspases were required for the moderate IL-1β secretion in the absence of caspase-1. Along this line, our further study showed that the caspase-8 inhibitor IETD-FMK, but not the caspase-1 inhibitor Ac-YVAD, abolished IL-1β secretion from caspase-1/11–deficient BMDCs induced by Ab-opsonized C. neoformans or acapsular mutant (Fig. 2E), which clearly indicated that caspase-8 was playing a critical role in this process. In addition, when caspase-8–specific shRNA was applied for gene silencing, IL-1β secretion induced by Ab-opsonized H99 strain or acapsular mutant Cap59 from caspase-1/11–deficient BMDCs was significantly attenuated (Fig. 2F, Supplemental Fig. 2E, 2F). Intriguingly, knockdown of caspase-8 in WT BMDCs did not reduce IL-1β secretion induced by C. neoformans (Supplemental Fig. 2G, 2H). These data suggest that caspase-8 is required for IL-1β secretion induced by internalized C. neoformans in the absence of caspase-1, whereas caspase-1 plays a dominant role for this process in WT cells.

C. neoformans activates the noncanonical caspase-8 inflammasome in the absence of caspase-1

Recent studies showed that caspase-8 is involved in noncanonical inflammasome activation upon fungal or bacterial challenges (15, 30, 31). In this study, we found that the acapsular Cap59 or anti-GXM Ab-opsonized C. neoformans H99 strains induced moderate level of caspase-8 activation in WT BMDCs (Fig. 3A), which was dependent on both NLRP3 and ASC, as Nlrp3- or Asc-deficient cells failed to mount any caspase-8 activation (Fig. 3B, Supplemental Fig. 3A). Very interestingly, the activation of caspase-8 was greatly enhanced in caspase-1/11–deficient BMDCs (Fig. 3B, Supplemental Fig. 3A), which correlated pretty well with the key role of caspase-8 in mediating IL-1β secretion from cells deficient for caspase-1/11, as we have just described (Fig. 2). Moreover, the secretion of IL-1β but not TNF-α from caspase-1/11–deficient BMDCs induced by internalized C. neoformans was inhibited by the NLRP3 inhibitor glyburide, indicating that the caspase-8 activation in the absence of caspase-1 was also dependent on NLRP3 (Fig. 3C).

FIGURE 3.
FIGURE 3.

C. neoformans activates the noncanonical caspase-8 inflammasome in the absence of caspase-1. (A) BMDCs from WT mice were infected 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 activation (p41). (B) BMDCs from indicated mice were infected with Cap59 strain for 6 h, and then caspase-8 activation was detected with immunoblotting. (C) BMDCs from Caspase-1/11−/− mice were pretreated with glyburide for 30 min, then challenged with C. neoformans acapsular mutant Cap59 strain for 6 h. IL-1β and TNF-α from cell culture supernatant were assayed via ELISA. (D) BMDCs from WT and Caspase-1/11−/− mice were infected with C. neoformans acapsular mutant Cap59 strain for 6 h, cell extracts were subjected to immunoprecipitation (IP) by caspase-8 Ab, and cell lysates and IP complex were assayed via immunoblotting with ASC Ab. Data are mean ± SD from one of three independent experiments. *p < 0.05. ns band, non-specific band.

Then we set out to demonstrate whether ASC was also involved in this process. Similar with caspase-8 activation, the oligomerization of ASC was also enhanced in the absence of caspase-1 (Figs. 2D, 3B). Furthermore, through coimmunoprecipitation experiment, we found that the endogenous caspase-8 interacted with ASC in caspase-1/11–deficient BMDCs during acapsular C. neoformans infection (Fig. 3D), and this interaction did not occur in the same cells without fungal challenge (Fig. 3D). Moreover, it was also evident that the internalized C. neoformans induced colocalization of caspase-8 with ASC speck in caspase-1/11–deficient BMDCs (Supplemental Fig. 3B). Thus, these data indicate that dendritic cells turn to NLRP3–ASC–caspase-8 activation when caspase-1 is absent during C. neoformans infection, and this noncanonical caspase-8 inflammasome activation is suppressed by caspase-1 in WT cells.

Internalized C. neoformans induces NLRP3–ASC–caspase-8–dependent cell death

The canonical caspase-1 inflammasome activation usually leads to a form of cell death called pyroptosis (2). As the key component for noncanonical inflammasome activation, caspase-8 is also involved in different types of cell death (32, 33). In the current study, we found that internalized C. neoformans induced a clear release of pro–IL-1β from caspase-1/11–deficient BMDCs, indicating a possibility of cell death during C. neoformans infection (Fig. 4A). Moreover, a clear activation of caspase-8 also occurred during this process (Fig. 4A), indicating a potential connection of caspase-8 and cell death in these cells. Indeed, in our further experiment, it was found that opsonized C. neoformans or acapsular C. neoformans infection of mouse BMDCs induced strong LDH release, which was a clear indication for the permeabilization of host cell membrane during cell death (Fig. 4B). In addition, the LDH release was significantly reduced from cells deficient for Nlrp3 or Asc (Fig. 4C). Besides LDH release, DNA fragmentation is another indicator for cell death, which can be detected by TUNEL assay with incorporating BrDU-FITC into DNA. With TUNEL assay, it was also evident that C. neoformans–induced cell death was clearly reduced in the Nlrp3- or Asc-deficient BMDCs compared with WT cells (Fig. 4D). What’s more, the formation of ASC specks was correlated with DNA fragmentation (Supplemental Fig. 3C). These data demonstrate that internalization of C. neoformans induces host cell death, which is dependent on both ASC and NLRP3.

FIGURE 4.
FIGURE 4.

Internalized C. neoformans induces cell death in a manner dependent on NLRP3–ASC–caspase-8 inflammasome. (A) BMDCs from Caspase-1/11−/− 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-1β were detected via immunoblotting. (B) BMDCs from WT mice 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 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-1/11−/− 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-1/11−/− mice with lentivirus-mediated shRNA knockdown of caspase-8 were challenged with C. neoformans acapsular mutant Cap59, and LDH release was assayed. Data are mean ± SD from one of three independent experiments. *p < 0.05.

Interestingly, from caspase-1/11–deficient BMDCs, C. neoformans infection still induced a high level of LDH release, and the TUNEL-positive cell number in these cells was comparable with that from WT cells (Fig. 4C, 4D), indicating that caspase-8 might also be responsible for this cell death phenotype. Indeed, caspase-8 inhibitor or silencing of caspase-8 with shRNA both effectively reduced acapsular C. neoformans–induced LDH release from caspase-1/11–deficient BMDCs (Fig. 4E, 4F). Therefore, these data reveal that caspase-8 is required for internalized C. neoformans–induced cell death in the absence of caspase-1 from dendritic cells. Of note was that silencing of caspase-8 did not reduce acapsular C. neoformans–induced LDH release from WT BMDCs, which indicates that caspase-8 plays only a minor role in inducing cell death in the presence of caspase-1 (Supplemental Fig. 3D). Taken together, internalized C. neoformans induces host cell death, which is a process dependent on the activity of noncanonical NLRP3–ASC–caspase-8 inflammasome in the absence of caspase-1.

Mechanisms for the internalized C. neoformans induced canonical and noncanonical NLRP3 inflammasome activation

The NLRP3 inflammasome is activated by a big variety of different stimuli, and the mechanisms for its activation are still not fully understood (2). To understand how C. neoformans activated inflammasome, we first found that heat-inactivated acapsular C. neoformans or Ab-opsonized heat-inactivated C. neoformans did not induce a significant level of IL-1β compared with live C. neoformans in WT BMDCs, whereas the TNF-α production was only slightly compromised (Fig. 5A). In caspase-1/11–deficient BMDCs, internalization of heat-inactivated C. neoformans also induced much lower level of IL-1β in comparison with live fungus, with the TNF-α production less affected (Supplemental Fig. 4A). These data demonstrate that activation of the canonical caspase-1 inflammasome or the noncanonical caspase-8 inflammasome both require the viability of C. neoformans.

FIGURE 5.
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-1β and TNF-α 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 ×400 (B). Four hundred cells were randomly chosen to calculate ASC speck formation 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 with Mito-TEMPO and then challenged with C. neoformans H99 strain with Ab opsonization; IL-1β secretion was assayed. Data are mean ± SD from one of three independent experiments. *p < 0.05.

Early studies reported that live C. neoformans causes host cell phagolysosome permeabilization when residing in the phagolysosome (34, 35). In our study, it was found that phagolysosome membrane permeabilization was correlated with ASC speck formation in C. neoformans–infected THP-1 macrophages expressing mCherry–ASC (Fig. 5B, 5C). This indicated that internalized C. neoformans caused release of phagolysosome components, which may have led to the activation of inflammasome. Indeed, cathepsin B inhibitor CA-074Me also reduced C. neoformans–induced IL-1β secretion, but not TNF-α production from WT BMDCs (Fig. 5D). Moreover, inhibition of potassium efflux also reduced the secretion of IL-1β induced by Ab-opsonized C. neoformans in WT BMDCs (Fig. 5E), indicating potassium efflux was also involved in C. neoformans–induced inflammasome activation. Moreover, in caspase-1/11–deficient BMDCs, Cap59-induced IL-1β secretion but not TNF-α production was also inhibited by cathepsin B inhibitor and high extracellular potassium (Supplemental Fig. 4B, 4C), indicating that the noncanonical caspase-8 inflammasome activation also requires lysosome damage and potassium efflux during C. neoformans infection.

In addition, a recent study reported that mitochondrial ROS induces lysosomal damage and inflammasome activation (36). In our current study, internalized C. neoformans also induced mitochondrial ROS production in WT BMDCs (Supplemental Fig. 4D, 4E). However, although the mitochondrial antioxidant Mito-TEMPO clearly inhibited IL-1β secretion induced by LPS plus ATP, as reported (data not shown) (36), it did not inhibit IL-1β secretion triggered by internalized C. neoformans in these cells (Fig. 5F). This indicated that the inflammasome activation in this case was not mediated by mitochondrial ROS, rather through a different mechanism, which deserves further analysis in the future. Taken together, internalization of C. neoformans causes phagolysosome membrane permeabilization and potassium efflux, leading to activation of the canonical NLRP3–ASC–caspase-1 inflammasome and the noncanonical NLRP3–ASC–caspase-8 inflammasome.

Zymosan also activates caspase-8 in caspase-1/11–deficient cells

Cryptococcal cell wall contains glucan, chitin, and glycosylated protein, which maintain the cell shape and regulate fungal permeability (7). Recent studies show pathogen-associated molecular patterns (PAMPs) from fungal cell wall were involved in inflammasome activation (14, 15, 29, 37, 38). To further identify the role of caspase-1 in fungal PAMP-induced caspase-8 activation, we set out to demonstrate whether the fungal cell wall products also induced elevated caspase-8 activation in caspase-1/11–deficient cells. To this end, we found that the fungal product zymosan, a β-glucan found on the cell wall of fungi, induced clear IL-1β secretion from caspase-1/11–deficient BMDCs, whereas LPS plus ATP failed to do so, indicating that this was a fungal-specific signaling event (Fig. 6A). The same as in the case of internalized C. neoformans, zymosan did not induce IL-1β production from Nlrp3- or Asc-deficient BMDCs (Fig. 6A). As expected, the IL-1β secretion from caspase-1/11–deficient BMDCs challenged with zymosan was also blocked by caspase-8 inhibitor in a dose-dependent manner, whereas TNF-α production was not affected (Fig. 6B). Similarly, knockdown of caspase-8 in these caspase-1/11–deficient BMDCs also reduced IL-1β secretion as well as cell death induced by zymosan (Fig. 6C, 6D). In addition, zymosan challenge also resulted in elevated activation of caspase-8 in BMDCs deficient for caspase-1 compared with WT cells (Fig. 6E, Fig. 7). These data suggest that caspase-8 is also critical for the noncanonical NLRP3–ASC–inflammasome activation in response to fungal PAMPs.

FIGURE 6.
FIGURE 6.

Zymosan also activates caspase-8 in caspase-1/11–deficient cells. (A) BMDCs from indicated mice were stimulated with 200 μg/ml zymosan for 12 h, or with LPS/ATP as control. Culture supernatants were harvested for IL-1β and TNF-α ELISA. (B) BMDCs from Caspase-1/11−/− mice were pretreated with Z-IETD for 30 min, followed with zymosan challenged for 12 h; IL-1β and TNF-α secretion were assayed. (C and D) BMDCs from Caspase-1/11−/− mice with shRNA knockdown of caspase-8 were challenged with zymosan; IL-1β and TNF-α secretion (C) or LDH release (D) was assayed. (E) BMDCs from WT and Caspase-1/11−/− mice were challenged with zymosan; cell lysates were assayed by immunoblotting for caspase-8 activation. Data are mean ± SD from one of three independent experiments. *p < 0.05.

FIGURE 7.
FIGURE 7.

Canonical and noncanonical NLRP3 inflammasome activation upon fungal challenge in dendritic cells. Opsonization of C. neoformans with Ab or complement triggers phagocytosis of the pathogen into phagosome, subsequently forming phagolysosome. Meanwhile, phagocytosis of the pathogen activates MAPK and NF-κB signaling pathways, which induces expression of pro–IL-1β and NLRP3. C. neoformans residing in phagolysosome may secret virulent factors, causing destabilization of phagolysosomal membrane. The leakage of phagolysosome leads to the release of components such as cathepsin B and causes K+ efflux. Subsequent activated canonical NLRP3–ASC–Caspase-1 inflammasome results in the processing of pro–IL-1β as well as host cell death. In the absence of caspase-1, internalized C. neoformans induces noncanonical NLRP3–ASC–Caspase-8 inflammasome activation, which leads to IL-1β secretion and host cell death as well. Zymosan can also induce enhanced noncanonical NLRP3–ASC–Caspase-8 inflammasome activation in the absence of caspase-1. Thus, canonical caspase-1 inflammasome and noncanonical caspase-8 inflammasome coordinate to mount innate immune responses against fungal pathogens.

Discussion

The inflammasome has emerged as an important cytoplasmic platform responsible for proteolytic processing of inflammatory cytokines IL-1β and IL-18 as well as pyroptotic cell death (39). Although many pathogens have been found to activate inflammasomes, the fungal pathogens activating inflammasomes are not widely studied (2). Different from viruses or bacteria, the size of fungal cell is much larger, so the interaction between host cell and fungal cell is very different from other types of pathogens (40). C. neoformans is an important fungal pathogen, whereas the host cell interaction with this fungus is not well understood. In previous studies, we have found that the biofilm as well as the acapsular mutant of C. neoformans activate the NLRP3 inflammasome in myeloid cells (12, 13). However, a question remaining is whether the naturally existing encapsulated C. neoformans activates inflammasome under certain circumstances. In the current study, we found that internalization of encapsulated C. neoformans with opsonin activated inflammasome in a NLRP3–ASC–dependent manner. Importantly, C. neoformans activated both the canonical caspase-1 inflammasome and the noncanonical caspase-8 inflammasome, although the canonical inflammasome activation was dominant in WT cells. Our findings thus uncover a previously unknown regulation of caspase-1 on caspase-8 in dendritic cells upon fungal pathogen challenge (Fig. 7).

As an initiating and apical activating caspase, caspase-8 is well known for its important role in apoptosis (41). Interestingly, recent studies have found that caspase-8 is involved in modulating IL-1β maturation (42–47). Caspase-8 is also involved in priming inflammasome for activation during Salmonella infection (31). Moreover, it has been found that caspase-8 is an apical mediator for the canonical and noncanonical NLRP3 inflammasome activation induced by LPS or Citrobacter rodentium (48). In addition, caspase-8 promotes NLRP1/NLRP3 inflammasome activation and TLR4-mediated IL-1β production in acute glaucoma (49). On the contrary, there is also a study that found that caspase-8 blocks RIPK3-mediated activation of the NLRP3 inflammasome (50). These studies indicate a very complex role of caspase-8 in several important signaling pathways.

The function of caspase-8 in fungal pathogen-induced inflammasome activation has also been explored. C. albicans induces caspase-8 activation in human and mouse phagocytic cells and leads to IL-1β secretion (14, 15, 30). Another fungal pathogen, A. fumigatus, induces AIM2 and NLRP3 activation, which initiates assembly of inflammasome composed of ASC along with caspase-1 and caspase-8, leading to the maturation of IL-1β and IL-18 (51). In our study, internalized C. neoformans induced moderate level of caspase-8 activation in WT BMDCs, but resulted in much stronger activation of caspase-8 in caspase-1/11–deficient cells. This indicated that the canonical caspase-1 inflammasome inhibits the noncanonical caspase-8 inflammasome activation during C. neoformans infection in WT cells. Probably because the canonical caspase-1 function is dominant, we did not see any inhibition of caspase-1 activation by caspase-8. When caspase-8 was silenced, the IL-1β secretion from WT cells did not change (Supplemental Fig. 2G). Because both caspase-1 and caspase-8 activations require NLRP3 and ASC, most likely they compete with each other for binding with NLRP3–ASC to form different complexes upon fungal infection (Fig. 7).

Many pathogens cause host cell death during infection. Inflammasomes are important mediators for the rapid lytic cell death termed pyroptosis (2). And recent studies show that caspase-8 participates in cell death resulted from inflammasome activation. For example, ubiquitination of NLRC4 by Sug1 formed cytoplasmic 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 required 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 macrophages (32). Moreover, fungal pathogen C. albicans infection also causes host cell death via caspase-8 (15). In this study, we found 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-κB and MAPK activation as well as enhanced expression of NLRP3 in mouse BMDCs (data not shown). After opsonic phagocytosis by immune cells, C. neoformans can survive and replicate in the phagosome or phagolysosome, where the fungus secrete virulent factors, including phospholipase B, which may cause phagolysosome membrane permeabilization (57, 58). It has been found that particulate stimuli, including silica and alum, activate the NLRP3 inflammasome through phagosomal destabilization (59). In this study, we found that phagolysosome membrane destabilization also contributed to C. neoformans activation of the NLRP3 inflammasome. Akin to previous finding, K+ efflux was also necessary for NLRP3 inflammasome activation induced by C. neoformans (60). Therefore, C. neoformans activates NF-κB and MAPK pathways for the priming signal, and then induces phagolysosome membrane destabilization and K+ efflux to activate the NLRP3 inflammasome (Fig. 7).

Different types of cells exhibit difference in the activation of inflammasome. For example, the threshold for inflammasome activation in mouse macrophages is much higher than in human macrophages (61). As a facultative intracellular fungal pathogen, C. neoformans can replicate in host phagocytic cells (62, 63). However, different phagocytic cells respond differently to C. neoformans; thus, the intracellular recognition of this pathogen by different host cells may also be variable (57). In contrast to the data we showed in this study from dendritic cells, phagocytosis of C. neoformans by mouse macrophages did not induce clear IL-1β production (data not shown). This indicates that C. neoformans applies specific mechanisms to avoid inflammasome activation in macrophages, which are invalid in dendritic cells. As the most important APCs, dendritic cells are much more sensitive than other cells in sensing invading pathogens. Probably that is also the reason that the threshold for inflammasome activation is much lower in these cells (64). To understand the molecular mechanisms accounting for the difference between dendritic cells and macrophages upon C. neoformans infection is an interesting topic for future investigation.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Jurg Tschopp for providing shRNA constructs against human NLRP3, CASPASE-1, ASC, and scramble. We are grateful to Dr. Warren Strober for sharing Nlrp3-deficient mice, Dr. Vishva M. Dixit for providing Asc and Nlrc4-deficient mice, and Dr. Katherine A. Fitzgerald for providing Aim2-deficient mice. We thank Dr. John Perfect for providing C. neoformans strains (H99 and Cap59 mutant) for experiments. We also thank Dr. Hongbin Wang for critical reading of this paper.

Footnotes

  • This work was supported by Natural Science Foundation of China Grants 31170868, 31370892, 31300712, and 91429307; National Key Basic Research Programs Grants 2014CB541905 and 2015CB554302; National Major Projects for Science and Technology Grants 2012ZX10002007-003 and 2014ZX0801011B-001; Sanofi Aventis-Shanghai Institutes for Biological Sciences Scholarship Program, as well as the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs International Partnership Program for Creative Research Teams.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AIM2
    absent in melanoma 2
    ASC
    apoptosis-associated speck-like protein containing a CARD
    BMDC
    bone marrow–derived dendritic cell
    CPZ
    chloropromazine
    GXM
    glucuronoxylomannan
    LDH
    lactate dehydrogenase
    NLR
    NOD-like receptor
    NLRC4
    NLR family, CARD domain-containing 4
    NLRP3
    NLR family, pyrin domain-containing 3
    PAMP
    pathogen-associated molecular pattern
    PRR
    pattern recognition receptor
    ROS
    reactive oxygen species
    shRNA
    short hairpin RNA
    WT
    wild-type
    YTD
    yeast extract trypton dextrose.

  • Received April 13, 2015.
  • Accepted September 18, 2015.

References

  1. ↵
    1. Takeuchi O.,
    2. S. Akira
    . 2010. Pattern recognition receptors and inflammation. Cell 140: 805–820.
    OpenUrlCrossRefPubMed
  2. ↵
    1. de Zoete M. R.,
    2. N. W. Palm,
    3. S. Zhu,
    4. R. A. Flavell
    . 2014. Inflammasomes. Cold Spring Harb. Perspect. Biol. 6: a016287.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Bergsbaken T.,
    2. S. L. Fink,
    3. B. T. Cookson
    . 2009. Pyroptosis: host cell death and inflammation. Nat. Rev. Microbiol. 7: 99–109.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Ramirez-Ortiz Z. G.,
    2. T. K. Means
    . 2012. The role of dendritic cells in the innate recognition of pathogenic fungi (A. fumigatus, C. neoformans and C. albicans). Virulence 3: 635–646.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Del Poeta M.,
    2. A. Casadevall
    . 2012. Ten challenges on Cryptococcus and cryptococcosis. Mycopathologia 173: 303–310.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Park B. J.,
    2. K. A. Wannemuehler,
    3. B. J. Marston,
    4. N. Govender,
    5. P. G. Pappas,
    6. T. M. Chiller
    . 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23: 525–530.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Doering T. L.
    2009. How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans. Annu. Rev. Microbiol. 63: 223–247.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Saïd-Sadier N.,
    2. E. Padilla,
    3. G. Langsley,
    4. D. M. Ojcius
    . 2010. Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase. PLoS One 5: e10008.
    OpenUrlCrossRefPubMed
    1. Hise A. G.,
    2. J. Tomalka,
    3. S. Ganesan,
    4. K. Patel,
    5. B. A. Hall,
    6. G. D. Brown,
    7. K. A. Fitzgerald
    . 2009. An essential role for the NLRP3 inflammasome in host defense against the human fungal pathogen Candida albicans. Cell Host Microbe 5: 487–497.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Mao L.,
    2. L. Zhang,
    3. H. Li,
    4. W. Chen,
    5. H. Wang,
    6. S. Wu,
    7. C. Guo,
    8. A. Lu,
    9. G. Yang,
    10. L. An,
    11. et al
    . 2014. Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome. Infect. Immun. 82: 882–892.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Gross O.,
    2. H. Poeck,
    3. M. Bscheider,
    4. C. Dostert,
    5. N. Hannesschläger,
    6. S. Endres,
    7. G. Hartmann,
    8. A. Tardivel,
    9. E. Schweighoffer,
    10. V. Tybulewicz,
    11. et al
    . 2009. Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence. Nature 459: 433–436.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Lei G.,
    2. M. Chen,
    3. H. Li,
    4. J. L. Niu,
    5. S. Wu,
    6. L. Mao,
    7. A. Lu,
    8. H. Wang,
    9. W. Chen,
    10. B. Xu,
    11. et al
    . 2013. Biofilm from a clinical strain of Cryptococcus neoformans activates the NLRP3 inflammasome. Cell Res. 23: 965–968.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Guo C.,
    2. M. Chen,
    3. Z. Fa,
    4. A. Lu,
    5. W. Fang,
    6. B. Sun,
    7. C. Chen,
    8. W. Liao,
    9. G. Meng
    . 2014. Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome. Microbes Infect. 16: 845–854.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Gringhuis S. I.,
    2. T. M. Kaptein,
    3. B. A. Wevers,
    4. B. Theelen,
    5. M. van der Vlist,
    6. T. Boekhout,
    7. T. B. Geijtenbeek
    . 2012. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1β via a noncanonical caspase-8 inflammasome. Nat. Immunol. 13: 246–254.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Ganesan S.,
    2. V. A. Rathinam,
    3. L. Bossaller,
    4. K. Army,
    5. W. J. Kaiser,
    6. E. S. Mocarski,
    7. C. P. Dillon,
    8. D. R. Green,
    9. T. N. Mayadas,
    10. S. M. Levitz,
    11. et al
    . 2014. Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1β production in response to β-glucans and the fungal pathogen, Candida albicans. J. Immunol. 193: 2519–2530.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Mariathasan S.,
    2. K. Newton,
    3. D. M. Monack,
    4. D. Vucic,
    5. D. M. French,
    6. W. P. Lee,
    7. M. Roose-Girma,
    8. S. Erickson,
    9. V. M. Dixit
    . 2004. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218.
    OpenUrlCrossRefPubMed
    1. Rathinam V. A.,
    2. Z. Jiang,
    3. S. N. Waggoner,
    4. S. Sharma,
    5. L. E. Cole,
    6. L. Waggoner,
    7. S. K. Vanaja,
    8. B. G. Monks,
    9. S. Ganesan,
    10. E. Latz,
    11. et al
    . 2010. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat. Immunol. 11: 395–402.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Mao K.,
    2. S. Chen,
    3. M. Chen,
    4. Y. Ma,
    5. Y. Wang,
    6. B. Huang,
    7. Z. He,
    8. Y. Zeng,
    9. Y. Hu,
    10. S. Sun,
    11. et al
    . 2013. Nitric oxide suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock. Cell Res. 23: 201–212.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Kayagaki N.,
    2. S. Warming,
    3. M. Lamkanfi,
    4. L. Vande Walle,
    5. S. Louie,
    6. J. Dong,
    7. K. Newton,
    8. Y. Qu,
    9. J. Liu,
    10. S. Heldens,
    11. et al
    . 2011. Non-canonical inflammasome activation targets caspase-11. Nature 479: 117–121.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Fang W.,
    2. M. S. Price,
    3. D. L. Toffaletti,
    4. J. Tenor,
    5. M. Betancourt-Quiroz,
    6. J. L. Price,
    7. W. H. Pan,
    8. W. Q. Liao,
    9. J. R. Perfect
    . 2012. Pleiotropic effects of deubiquitinating enzyme Ubp5 on growth and pathogenesis of Cryptococcus neoformans. PLoS One 7: e38326.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Pétrilli V.,
    2. S. Papin,
    3. C. Dostert,
    4. A. Mayor,
    5. F. Martinon,
    6. J. Tschopp
    . 2007. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14: 1583–1589.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Fernandes-Alnemri T.,
    2. E. S. Alnemri
    . 2008. Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol. 442: 251–270.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Bewley M. A.,
    2. M. Naughton,
    3. J. Preston,
    4. A. Mitchell,
    5. A. Holmes,
    6. H. M. Marriott,
    7. R. C. Read,
    8. T. J. Mitchell,
    9. M. K. Whyte,
    10. D. H. Dockrell
    . 2014. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation. MBio 5: e01710–e01714.
    OpenUrlPubMed
  24. ↵
    1. Kozel T. R.,
    2. E. C. Gotschlich
    . 1982. The capsule of cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages. J. Immunol. 129: 1675–1680.
    OpenUrlAbstract
  25. ↵
    1. Vecchiarelli A.,
    2. C. Retini,
    3. C. Monari,
    4. A. Casadevall
    . 1998. Specific antibody to Cryptococcus neoformans alters human leukocyte cytokine synthesis and promotes T-cell proliferation. Infect. Immun. 66: 1244–1247.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Kozel T. R.,
    2. B. Highison,
    3. C. J. Stratton
    . 1984. Localization on encapsulated Cryptococcus neoformans of serum components opsonic for phagocytosis by macrophages and neutrophils. Infect. Immun. 43: 574–579.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Lin, X. 2009. Cryptococcus neoformans: morphogenesis, infection, and evolution. Infect. Genet. Evol. 9: 401-416.
  28. ↵
    1. Tomalka J.,
    2. S. Ganesan,
    3. E. Azodi,
    4. K. Patel,
    5. P. Majmudar,
    6. B. A. Hall,
    7. K. A. Fitzgerald,
    8. A. G. Hise
    . 2011. A novel role for the NLRC4 inflammasome in mucosal defenses against the fungal pathogen Candida albicans. PLoS Pathog. 7: e1002379.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Kumar H.,
    2. Y. Kumagai,
    3. T. Tsuchida,
    4. P. A. Koenig,
    5. T. Satoh,
    6. Z. Guo,
    7. M. H. Jang,
    8. T. Saitoh,
    9. S. Akira,
    10. T. Kawai
    . 2009. Involvement of the NLRP3 inflammasome in innate and humoral adaptive immune responses to fungal beta-glucan. J. Immunol. 183: 8061–8067.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Zwolanek F.,
    2. M. Riedelberger,
    3. V. Stolz,
    4. S. Jenull,
    5. F. Istel,
    6. A. D. Köprülü,
    7. W. Ellmeier,
    8. K. Kuchler
    . 2014. The non-receptor tyrosine kinase Tec controls assembly and activity of the noncanonical caspase-8 inflammasome. PLoS Pathog. 10: e1004525.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Man S. M.,
    2. P. Tourlomousis,
    3. L. Hopkins,
    4. T. P. Monie,
    5. K. A. Fitzgerald,
    6. C. E. Bryant
    . 2013. Salmonella infection induces recruitment of Caspase-8 to the inflammasome to modulate IL-1β production. J. Immunol. 191: 5239–5246.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Pierini R.,
    2. C. Juruj,
    3. M. Perret,
    4. C. L. Jones,
    5. P. Mangeot,
    6. D. S. Weiss,
    7. T. Henry
    . 2012. AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages. Cell Death Differ. 19: 1709–1721.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Maelfait J.,
    2. R. Beyaert
    . 2008. Non-apoptotic functions of caspase-8. Biochem. Pharmacol. 76: 1365–1373.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Davis M. J.,
    2. A. J. Eastman,
    3. Y. Qiu,
    4. B. Gregorka,
    5. T. R. Kozel,
    6. J. J. Osterholzer,
    7. J. L. Curtis,
    8. J. A. Swanson,
    9. M. A. Olszewski
    . 2015. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence. J. Immunol. 194: 2219–2231.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Tucker S. C.,
    2. A. Casadevall
    . 2002. Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad. Sci. USA 99: 3165–3170.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Heid M. E.,
    2. P. A. Keyel,
    3. C. Kamga,
    4. S. Shiva,
    5. S. C. Watkins,
    6. R. D. Salter
    . 2013. Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation. J. Immunol. 191: 5230–5238.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Kankkunen P.,
    2. L. Teirilä,
    3. J. Rintahaka,
    4. H. Alenius,
    5. H. Wolff,
    6. S. Matikainen
    . 2010. (1,3)-beta-glucans activate both dectin-1 and NLRP3 inflammasome in human macrophages. J. Immunol. 184: 6335–6342.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Lamkanfi M.,
    2. R. K. Malireddi,
    3. T. D. Kanneganti
    . 2009. Fungal zymosan and mannan activate the cryopyrin inflammasome. J. Biol. Chem. 284: 20574–20581.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Lamkanfi M.,
    2. V. M. Dixit
    . 2012. Inflammasomes and their roles in health and disease. Annu. Rev. Cell Dev. Biol. 28: 137–161.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Seider K.,
    2. A. Heyken,
    3. A. Lüttich,
    4. P. Miramón,
    5. B. Hube
    . 2010. Interaction of pathogenic yeasts with phagocytes: survival, persistence and escape. Curr. Opin. Microbiol. 13: 392–400.
    OpenUrlCrossRefPubMed
  41. ↵
    1. McIlwain D. R.,
    2. T. Berger,
    3. T. W. Mak
    . 2013. Caspase functions in cell death and disease. Cold Spring Harb. Perspect. Biol. 5: a008656.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Gurung P.,
    2. T. D. Kanneganti
    . 2015. Novel roles for caspase-8 in IL-1β and inflammasome regulation. Am. J. Pathol. 185: 17–25.
    OpenUrlCrossRefPubMed
    1. Vince J. E.,
    2. W. W. Wong,
    3. I. Gentle,
    4. K. E. Lawlor,
    5. R. Allam,
    6. L. O’Reilly,
    7. K. Mason,
    8. O. Gross,
    9. S. Ma,
    10. G. Guarda,
    11. et al
    . 2012. Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation. Immunity 36: 215–227.
    OpenUrlCrossRefPubMed
    1. Antonopoulos C.,
    2. C. El Sanadi,
    3. W. J. Kaiser,
    4. E. S. Mocarski,
    5. G. R. Dubyak
    . 2013. Proapoptotic chemotherapeutic drugs induce noncanonical processing and release of IL-1β via caspase-8 in dendritic cells. J. Immunol. 191: 4789–4803.
    OpenUrlAbstract/FREE Full Text
    1. Uchiyama R.,
    2. S. Yonehara,
    3. H. Tsutsui
    . 2013. Fas-mediated inflammatory response in Listeria monocytogenes infection. J. Immunol. 190: 4245–4254.
    OpenUrlAbstract/FREE Full Text
    1. Antonopoulos C.,
    2. G. R. Dubyak
    . 2014. Chemotherapy engages multiple pathways leading to IL-1β production by myeloid leukocytes. OncoImmunology 3: e27499.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Shenderov K.,
    2. N. Riteau,
    3. R. Yip,
    4. K. D. Mayer-Barber,
    5. S. Oland,
    6. S. Hieny,
    7. P. Fitzgerald,
    8. A. Oberst,
    9. C. P. Dillon,
    10. D. R. Green,
    11. et al
    . 2014. Cutting edge: Endoplasmic reticulum stress licenses macrophages to produce mature IL-1β in response to TLR4 stimulation through a caspase-8- and TRIF-dependent pathway. J. Immunol. 192: 2029–2033.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Gurung P.,
    2. P. K. Anand,
    3. R. K. Malireddi,
    4. L. Vande Walle,
    5. N. Van Opdenbosch,
    6. C. P. Dillon,
    7. R. Weinlich,
    8. D. R. Green,
    9. M. Lamkanfi,
    10. T. D. Kanneganti
    . 2014. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes. J. Immunol. 192: 1835–1846.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Chi W.,
    2. F. Li,
    3. H. Chen,
    4. Y. Wang,
    5. Y. Zhu,
    6. X. Yang,
    7. J. Zhu,
    8. F. Wu,
    9. H. Ouyang,
    10. J. Ge,
    11. et al
    . 2014. Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1β production in acute glaucoma. Proc. Natl. Acad. Sci. USA 111: 11181–11186.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Kang T. B.,
    2. S. H. Yang,
    3. B. Toth,
    4. A. Kovalenko,
    5. D. Wallach
    . 2013. Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome. Immunity 38: 27–40.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Karki R.,
    2. S. M. Man,
    3. R. K. Malireddi,
    4. P. Gurung,
    5. P. Vogel,
    6. M. Lamkanfi,
    7. T. D. Kanneganti
    . 2015. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17: 357–368.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Kumar Y.,
    2. V. Radha,
    3. G. Swarup
    . 2010. Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death. Biochem. J. 427: 91–104.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Sagulenko V.,
    2. S. J. Thygesen,
    3. D. P. Sester,
    4. A. Idris,
    5. J. A. Cridland,
    6. P. R. Vajjhala,
    7. T. L. Roberts,
    8. K. Schroder,
    9. J. E. Vince,
    10. J. M. Hill,
    11. et al
    . 2013. AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ. 20: 1149–1160.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Philip N. H.,
    2. C. P. Dillon,
    3. A. G. Snyder,
    4. P. Fitzgerald,
    5. M. A. Wynosky-Dolfi,
    6. E. E. Zwack,
    7. B. Hu,
    8. L. Fitzgerald,
    9. E. A. Mauldin,
    10. A. M. Copenhaver,
    11. et al
    . 2014. Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling. Proc. Natl. Acad. Sci. USA 111: 7385–7390.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Weng D.,
    2. R. Marty-Roix,
    3. S. Ganesan,
    4. M. K. Proulx,
    5. G. I. Vladimer,
    6. W. J. Kaiser,
    7. E. S. Mocarski,
    8. K. Pouliot,
    9. F. K. Chan,
    10. M. A. Kelliher,
    11. et al
    . 2014. Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. Proc. Natl. Acad. Sci. USA 111: 7391–7396.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Kelly R. M.,
    2. J. Chen,
    3. L. E. Yauch,
    4. S. M. Levitz
    . 2005. Opsonic requirements for dendritic cell-mediated responses to Cryptococcus neoformans. Infect. Immun. 73: 592–598.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Voelz K.,
    2. R. C. May
    . 2010. Cryptococcal interactions with the host immune system. Eukaryot. Cell 9: 835–846.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Johnston S. A.,
    2. R. C. May
    . 2013. Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen. Cell. Microbiol. 15: 403–411.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Hornung V.,
    2. F. Bauernfeind,
    3. A. Halle,
    4. E. O. Samstad,
    5. H. Kono,
    6. K. L. Rock,
    7. K. A. Fitzgerald,
    8. E. Latz
    . 2008. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: 847–856.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Muñoz-Planillo R.,
    2. P. Kuffa,
    3. G. Martínez-Colón,
    4. B. L. Smith,
    5. T. M. Rajendiran,
    6. G. Núñez
    . 2013. K⁺ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38: 1142–1153.
    OpenUrlCrossRefPubMed
  61. ↵
    1. Wang H.,
    2. L. Mao,
    3. G. Meng
    . 2013. The NLRP3 inflammasome activation in human or mouse cells, sensitivity causes puzzle. Protein Cell 4: 565–568.
    OpenUrlCrossRefPubMed
  62. ↵
    1. Feldmesser M.,
    2. Y. Kress,
    3. P. Novikoff,
    4. A. Casadevall
    . 2000. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 68: 4225–4237.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Coelho C.,
    2. A. L. Bocca,
    3. A. Casadevall
    . 2014. The intracellular life of Cryptococcus neoformans. Annu. Rev. Pathol. 9: 219–238.
    OpenUrlCrossRefPubMed
  64. ↵
    1. He Y.,
    2. L. Franchi,
    3. G. Núñez
    . 2013. TLR agonists stimulate Nlrp3-dependent IL-1β production independently of the purinergic P2X7 receptor in dendritic cells and in vivo. J. Immunol. 190: 334–339.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section