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Microbes and Infection xx (2014) 1e10 www.elsevier.com/locate/micinf

Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome Caiqin Guo a,b,1, Mingkuan Chen b,1, Zhenzong Fa b,c, Ailing Lu b, Wei Fang c, Bing Sun b, Changbin Chen b, Wanqing Liao c,**, Guangxun Meng b,*

Q3

b

a College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200235, China Unit of Innate Immunity, Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China c Shanghai Key Laboratory of Molecular Medical Mycology, Changzheng Hospital, Shanghai 200003, China

Received 1 March 2014; accepted 25 August 2014

Abstract Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. Although cell surface receptors for recognition of C. neoformans have been studies intensively, cytoplasmic recognition of this pathogen remains unclear. As an important detector of pathogen infection, inflammasome can sense and get activated by infection of various pathogens, including pathogenic fungi such as Candida albicans and Aspergillus fumigatus. Our present study showed that acapsular C. neoformans (cap59D) activated the NLRP3-, but not AIM2-nor NLRC4- inflammasome. During this process, viability of the fungus was required. Moreover, our in vivo results showed that during the pulmonary infection of cap59D, immune cell infiltration into the lung and effective clearance of the fungus were both dependent on the presence of NLRP3 inflammasome. In summary, our data suggest that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome and indicate that manipulation of inflammasome activity maybe a novel approach to control C. neoformans infection. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: Cryptococcus neoformans; Capsule; Glucuronoxylomannan; NLRP3; Inflammasome

1. Introduction Fungi are a special group of microbes, some of which are pathogenic and infect immunocompromised hosts and often cause high mortality. Cryptococcus neoformans (C. neoformans) is such an opportunistic pathogenic fungus that mainly causes diseases in AIDS patients, people receiving corticosteroid treatment, and organ transplant recipients [1]. Among a number of pathogenic fungi, C. neoformans is unique by

* Corresponding author. Tel.: þ86 21 54923100. ** Corresponding author. Tel.: þ86 21 81885493. E-mail addresses: [email protected] (W. Liao), [email protected], [email protected] (G. Meng). 1 Both authors have equally contributed to the work and share the first authorship of this article.

carrying capsule outside of the cell wall. The capsule of C. neoformans is composed of glucuronoxylomannan (GXM, 95%), galactoxylomannans (GalXM, 5%) and other mannoproteins. Moreover, the capsule is a major virulent factor for C. neoformans to avoid being recognized and phagocytosed by host phagocytes [2]. In contrast, acapsular mutants of C. neoformans can be effectively recognized and induce proinflammatory cytokine secretion from macrophages [3]. However, the detailed mechanisms for the interaction of acapsular mutants of C. neoformans with host cells are not well understood. Host innate immune system takes the first line of defense against pathogen invasion. Pattern recognition receptors including membrane bound Toll-like receptors (TLRs) and Ctype lectin like receptors (CLRs), as well as cytosolic RIG-I like receptors (RLRs) and NOD like receptors (NLRs) are

http://dx.doi.org/10.1016/j.micinf.2014.08.013 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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responsible for recognition of pathogen associated molecular patterns derived from invading microbes and initiate inflammation [4]. Early studies reveal that mannose and beta-glucan receptors bind acapsular mutants of C. neoformans [5]. TLR2, TLR4 and CD14 also participate in the host response to GXM from C. neoformans [6]. Moreover, C. neoformans mannoprotein can be secreted and recognized by mannose receptor CD206 and CD209 [7]. However, cytoplasmic recognition of C. neoformans is still unclear. Recent studies reveal that cytoplasmic pattern recognition receptors such as NACHT, LRR and PYD domains-containing protein 1 (NLRP1), NOD-like receptor family, pyrin domaincontaining 3 (NLRP3), NLR family CARD domain-containing protein 4 (NLRC4) and absent in melanoma 2 (AIM2) can each form a large protein complex called inflammasome, which mediates activation of caspase-1, leading to the cleavage and secretion of interleukin-1b (IL-1b) and interleukin-18 (IL-18) upon cellular challenge with pathogen associated molecular patterns or danger-associated molecular patterns [8]. Moreover, IL-1b production via the NLRP3 inflammasome has been proved to be essential in host defense against fungal pathogens including Candida albicans and Aspergillus fumigatus from recent studies [9,10]. Research from our lab also reveal that biofilm of C. neoformans activates the NLRP3 inflammasome in human myeloid cells [11]. Infection with C. neoformans occurs following inhalation of acapsular or poorly encapsulated yeast cells from the environment [12]. In vitro study show that acapsular strain induce significantly higher production of IL-1b from human monocytes and mouse bone marrow derived dendritic cells (BMDCs) compared with encapsulated strains [13,14]. IL-1b production was also detected from acapsular or encapsulated C. neoformans infected mice [15,16]. However, the mechanism of IL-1b secretion induced by C. neoformans is not clear. Since the biofilm of C. neoformans is able to activate the NLRP3 inflammasome [11], and capsule shedding off is one important mechanism for the formation of fungal biofilm [17,18], we hypothesized that the capsule of C. neoformans is an inhibitory factor for host recognition of this fungus followed with inflammasome activation. Indeed, in the present study we found that an acapsular mutant of C. neoformans activated the NLRP3 inflammasome, and the process was dependent on potassium efflux. Interestingly, transferring of GXM from encapsulated C. neoformans to cap59D mutant inhibited the inflammasome activation by the modified mutant, which indicated that the capsule of C. neoformans interfered recognition of the fungus by host NLRP3 inflammasome. 2. Materials and methods 2.1. Mice and reagents Apoptosis-associated speck-like protein containing a CARD (Asc), Nlrc4, Nlrp3 and Aim2 deficient mice had been described before [19e21]. Caspase-1-deficient mice were from the Jackson Laboratory and crossed onto the C57BL/6 genetic background for 10 generations, these mice are also

deficient for a functional caspase-11 [22]. C57BL/6 wild type (WT) mice were bred in our specific pathogen free (SPF) animal facility. Animal care, use and experimental procedures complied with national guidelines and were approved by the Animal Care and Use Committee at Institute Pasteur of Shanghai. All chemical reagents were from Sigma unless stated otherwise. 2.2. Culture of C. neoformans C. neoformans reference strain H99 (serotype A) and acapsular mutant cap59D (based on H99) were gifts from Dr. John Perfect lab [23]. They were recovered from 80  C on yeast extract tryptone dextrose (YTD) agar plate. One clone was selected and inoculated into 2 ml YTD broth at 30  C on a shaker for 48 h, centrifuged at 2000 g for 5 min, washed 3 times in sterile Phosphate Buffer Solution (PBS) and counted using a hemocytometer prior to infection experiment. For inactivation of C. neoformans, the fungus was incubated at 60  C for 1 h (heat inactivation). No growth was observed from the inoculation of heat inactivated C. neoformans during a 2 days period on YTD agar plates. 2.3. GXM isolation and recapsule of acapsular mutants GXM isolation has been described before [24,25]. Briefly, C. neoformans H99 strain was grown for 5 days at 30  C. The culture supernatant was mixed with 95% EtOH followed with incubation at 4  C overnight. Then the mixture was centrifuged for 1 h at 15,000 g and the pellet was resuspended in 0.2 M NaCl and sonicated. CTAB (cetyltrimethylammonium bromide) was then added for precipitation, which was followed by washing in 10% EtOH and centrifugation at 18,000 g for 20 min. The pellet was resuspended in 1 M NaCl, sonicated and dialyzed versus distilled water for 5 days at 4  C. The supernatant was then lyophilized, weighted and resolved in PBS. For recapsulation of acapsular mutant, PBS washed cap59D yeast cells were counted and resuspended in 100 ml PBS at a concentration of 1  109 cells/ml. Then GXM was added to the yeast suspension to 10 mg/ml and incubated for 1 h at room temperature with rotating as reported before [26]. 2.4. Cell culture and in vitro C. neoformans challenge THP-1 cells were maintained in RPMI 1640 media with necessary supplements, including 10% FBS, 100 IU/ml penicillin, 1 mg/ml streptomycin, and 50 mM betamercaptoethanol. THP-1 cell derived macrophages were generated by treating THP-1 cells with PMA in a concentration of 100 ng/ml for 3 h, the cells were then rested for 48 h before use. Mouse BMDCs were prepared as described before [27]. For fungal challenging experiments, 1  105 BMDCs were pooled in plates with C. neoformans H99 or cap59D at multiplicity of infection (MOI) ¼ 10 (10 yeasts to 1 macrophage) unless stated otherwise, 6 h later cytokines were assayed via ELISA. In some cases, supernatants and cell extracts were collected for immunoblotting. In the indicated

Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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experiments, host cells were treated with the following chemicals for 30 min before infection with C. neoformans: AC-YVAD-CHO, Glyburide, Diphenyleneiodonium (DPI), Nacetyl-L-cysteine (NAC), butylated hydroxyanisole (BHA), potassium chloride (KCl) with indicated concentrations in respective figures. LPS (100 ng/ml) stimulation for 6 h with or without ATP (5 mM) pulse for 30 min served as controls.

with C. neoformans cap59D mutant for 6 h, then cells were pelleted and washed with PBS and cross-linked with disuccinimidyl suberate. The cross-linked pellets were fractionated on 12% SDS-PAGE, followed with immunoblotting with anti-human or mouse ASC antibodies, respectively.

2.5. Gene silencing in THP-1 cells Short hairpin RNA (shRNA) vectors against NLRP3, caspase-1, ASC, and their scramble vectors were from Dr. Jurg Tschopp lab [28]. Packaging plasmids pMD2-VSVG and pCMV-R8.91 were used for lenti virus production. Generation of SYK silencing THP-1 cells and related protocols had been described before [11,27]. The targeting sequences are: NLRP3, CAGGTTTGACTATCTGTTCT; CASPASE-1, GTGAAGAGATCCTTCTGTA; ASC, GATGCGGAAGCTCTTCAGTTTCA; SYK, CCTCATCAGGGAATATGTG.

Mice were challenged by intranasal infection of 1  106 cap59D mutant or H99 in 25 ml PBS or vehicle alone under avertin anesthesia. 4 or 12 h post infection, bronchial alveolar lavage fluid (BALF) was collected and cell number in the BALF was determined via counting. The lung was homogenated in 300 ml PBS, diluted and plated on YTD agar plates for colony determination after 48 h of incubation. Colony forming unit (CFU) was employed to indicate the fungus load in vivo. For tissue ELISA, the lungs were weighted and homogenated in 300 ml PBS, and were centrifuged at 10,000 g for 10 min. Supernatants were collected for ELISA.

2.6. Real-time PCR

2.10. Statistical analysis

Total RNA was extracted from the THP-1 cells using TRIzol reagent (Invitrogen). cDNA was synthesized with TaqMan Reverse Transcription Reagents (Applied Biosystems). Quantitative PCR was performed on 7900HT fast real-time PCR system using the SYBR Green qPCR Master Mix (TOYOBO). Relative quantification of genes was normalized against b-actin as relative unit (RU) via formula [2-⊿Ct(target geneb-actin)]. PCR primers used were:

Data were analyzed for statistical significance by two-tailed student's t test in Excel. Differences with P values 0.05 were considered statistically significant. *means P < 0.05, **means P < 0.01 in all figures if not stated additionally.

 IL-1b, 50 -CACGATGCACCTGTACGATCA-(forward) 30 ,  50 -GTTGCTCCATATCCTGTCCCT-(reverse) 30 ;  b-actin, 50 -AGTGTGACGTGGACATCCGCAAAG-(forward) 30 ,  50 -ATCCACATCTGCTGGAAGGTGGAC-(reverse) 30 .

2.7. Cytokine ELISA and immunoblotting Supernatants were analyzed for cytokine secretion by ELISA (eBiosciences). Antibodies for immunoblotting include: rabbit anti-human mature and pro-IL-1b (sc-1250, sc23459, Santa-Cruz), rabbit anti-mouse mature and pro-IL-1b (sc-7884, Santa Cruz), mouse anti-human or mouse NLRP3 (ALX-804-881, Enzo Life Sciences), rabbit anti-human or mouse ASC (sc-22514-R, Santa Cruz), rabbit anti-human caspase-1 (sc-515, Santa Cruz), rabbit anti-mouse caspase-1 (sc-514, Santa Cruz),mouse anti-human or mouse b-actin (KM9001, Tianjin Sungene Biotech Co., Ltd). Appropriate HRP-conjugated secondary antibodies were used for signal detection via ECL reagent (PerkinElmer). 2.8. ASC oligomerization detection ASC oligomerization detection was conducted as described before [29]. Briefly, BMDCs or THP-1 cells were infected

2.9. In vivo C. neoformans infection

3. Results 3.1. Acapsular mutant of C. neoformans activates inflammasome Early studies have shown that acapsular strain of C. neoformans induced IL-1b secretion from human and mouse myeloid cells [13,14]. Since the maturation and secretion of IL-1b is largely dependent on the function of inflammasome, we set out to study whether C. neoformans was able to activate inflammasome in human monocytic cell derived macrophages. Interestingly, we found that an acapular mutant of C. neoformans, namely cap59D strain, but not the encapsulated wild type strain H99, induced robust IL-1b secretion from THP1 cell derived macrophages in a dose and time dependent manner (Fig. 1A and B). Moreover, cap59D strain also induced IL-1b and IL-18 secretion from mouse BMDCs (Fig. 1C and D). For most inflammasome assembly, the adapter molecule ASC acts as a bridge between caspase-1 and certain NOD-like receptor (NLR) by offering the CARD domain for CARDeCARD interactions and PYD domain for PYDePYD interactions. We found that the cap59D strain but not the encapsulated H99 strain induced ASC oligomerization, which is a key indicator for the activation of inflammasome (Fig. 1E). In addition, we also found cap59D strain activates caspase-1 maturation in human THP-1 cell derived macrophages (Fig. 1E). Therefore, C. neoformans acapsular mutant but not the encapsulated wild type strain induced IL-1b secretion and activated inflammasome from both human and mouse myeloid

Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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Fig. 1. Acapsular mutant of C. neoformans but not the encapsulated wild type strain activates inflammasome. (A and B) THP-1 cell derived macrophages were treated with C. neoformans reference strain H99 and Cap59 deletion mutant derived from H99 (cap59D) at indicated MOIs for 6 h (A), or for indicated time duration at MOI ¼ 10 (B), with PBS or LPS (100 ng/ml) stimulation for 6 h as controls. Cell culture supernatants were harvested for human IL-1b ELISA. (C and D) Mouse BMDCs were treated with cap59D mutant strain at indicated MOIs for 6 h for IL-1b detection or 10 h for IL-18 detection, with PBS or LPS (100 ng/ml) priming for 6 h and ATP (5 mM) pulse for 30 min as controls. The supernatants were then harvested for murine IL-1b and IL-18 ELISA. (E) ASC oligomerization, mature form of caspase-1 in cell culture supernatant (sup), and other indicated proteins from cell lysate (Lys) of THP-1 cell derived macrophages infected with H99 or cap59D mutant of C. neoformans were analyzed via immunoblotting, PBS or LPS served as controls. (F and G) Wild type female mice were infected through intranasal route with 1  106 cap59D mutant or H99 in 25 ml PBS. 4 h post infection, mice were sacrificed and the bronchial alveolar lavage fluid (BALF) was collected. The lung was weighed and homogenated in PBS and centrifuged to collect supernatants for IL-1b ELISA (F). Leukocyte number in the BALF was counted (G). *, P < 0.05; **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments.

cells. Meanwhile, during the early phase of infection, cap59D strain induced much stronger IL-1b production and immune cell infiltration in the lung of mice than H99 strain or PBS control (Fig. 1F and G), indicating the inflammasome activation by cap59D in vivo. 3.2. Acapsular mutant of C. neoformans activates the NLRP3 inflammasome in human macrophages As we have found that infection of myeloid cells with the acapsular mutant of C. neoformans cap59D resulted in IL1b secretion, we tried to determine whether caspase-1 activation was required for this process. To this end, we pre-treated the human THP-1 cell derived macrophages with a specific caspase-1 inhibitor AC-YVAD before challenging with cap59D strain. As shown in Fig. 2A, ACYVAD strongly inhibited IL-1b induction by cap59D mutant in a dose dependent manner, while the IL-8 secretion, which is independent from caspase-1 and/or inflammasome activity, was not affected (Fig. 2A and B). As NLRP3 inflammasome is involved in response to various fungi infection, we tested whether NLRP3 was required in cap59D mutant induced IL-1b secretion. When the NLRP3 inhibitor glyburide was applied in our experiment [30], we found that this drug significantly inhibited IL-1b secretion

from THP-1 cell derived macrophages upon cap59D infection, and the IL-8 induction was not affected (Fig. 2C and D). These results indicated that the NLRP3 inflammasome was likely involved in the recognition of cap59D infection. To further confirm this possibility, we applied shRNA mediated silencing of NLRP3, ASC, and caspase-1 in THP1 cells as mentioned in our early studies [27,31]. With these cells carrying respective silenced genes as indicated, we found that upon cap59D strain infection, IL-1b but not IL-8 secretion was strongly abolished in the absence of NLRP3, ASC or caspase-1 (Fig. 2E and F). Thus, the IL-1b secretion induced by cap59D mutant in human macrophages was dependent on the activity of the NLRP3 inflammasome. Moreover, we also found that the caspase-1 activation was abolished in the absence of NLRP3, ASC or caspase-1 in the indicated THP1 cell lines upon cap59D infection (Fig. 2G). Interestingly, similar with our previous findings [27], the ASC oligomerization was strongly decreased in NLRP3 or ASC silenced cells, but caspase-1 “knock-down” did not affect the oligomerization of ASC (Fig. 2H). The successful silencing of NLRP3, ASC or caspase-1 was evident from immunoblotting data in Fig. 2G and H. These data thus demonstrated that cap59D mutant activated the NLRP3 inflammasome in human macrophages.

Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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Fig. 2. Acapsular mutant of C. neoformans activates the NLRP3 inflammasome in human macrophages. (A to D) THP-1 cell derived macrophages were pretreated with caspase-1 specific inhibitor AC-YVAD (AeB), or NLRP3 specific inhibitor Glyburide (CeD) for 30 min, then challenged with cap59D mutant as described in Fig. 1, IL-1b and IL-8 from cell culture supernatant were monitored via ELISA. (E and F) THP-1 cell derived macrophages with shRNA silencing of indicated genes were infected with cap59D mutant and IL-1b or IL-8 was detected as in A and B. (G and H) Mature form (p10) of caspase-1 and mature IL-1b (p17) in cell culture supernatant and other indicated proteins in cell lysate (G), as well as ASC oligomerization (H) from indicated cells challenged with cap59D mutant were analyzed with immunoblotting. *, P < 0.05; **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments.

3.3. cap59D mutant strain induces IL-1b secretion via NLRP3-, but not AIM2-nor NLRC4- inflammasome from mouse dendritic cells Since the inflammasome activation in human THP-1 cell derived macrophages is quite sensitive, we set out to demonstrate whether the acapsular mutant of C. neoformans was able to induce robust IL-1b secretion from mouse cells. As shown in Fig. 3A, infection with cap59D mutant led to clear IL-1b secretion from BMDCs of wild type mice (Fig. 3A). As expected, BMDCs from Nlrp3, Asc and Caspase-1/11 deficient mice did not secret any IL-1b upon cap59D infection, while the TNF-a secretion from all different genotypes of cells were comparable (Fig. 3A and B). This experiment revealed that cap59D induced IL-1b secretion from BMDCs was dependent on the activity of the NLRP3 inflammasome. Meanwhile, IL18 production was also NLRP3 inflammasome dependent (Fig. 3C). Moreover, we also found that the caspase-1 cleavage was abolished in BMDCs from Nlrp3/, Asc/ and Caspase-1/11/ mice but not wild type mice (Fig. 3D), while ASC oligomerization induced by cap59D mutant was abolished in BMDCs from Nlrp3/ and ASC/ mice, but not in Caspase-1/11/ and wild type mice (Fig. 3E). We further tested whether other inflammasomes were also involved in IL-1b production upon infection with the acapsular mutant of C. neoformans. In this case, AIM2 and NLRC4 were considered. We found that cap59D mutant

induced comparable IL-1b secretion from Aim2 or Nlrc4 deficient BMDCs in comparison with that from wild type mice (Fig. 3F). As expected, the TNF-a secretion was comparable among BMDCs from these “knock-out” and wild type mice (Fig. 3G). Taken together, these data demonstrated that acapsular mutant of C. neoformans activated NLRP3-, but not AIM2-nor NLRC4- inflammasome in murine dendritic cells. 3.4. Activation of the NLRP3 inflammasome by cap59D strain requires potassium efflux and ROS inhibitors interfered with the priming step Next we investigated which signaling pathway was involved in the activation of NLRP3 inflammasome by cap59D strain. Early study shows that inhibitors of reactive oxygen species (ROS) block NLRP3-mediated caspase-1 activation by inhibiting the priming step in myeloid cells [32]. In our study, it was found that treating mouse BMDCs with ROS inhibitor DPI decreased the secretion of both IL-1b and TNF-a in a dose dependent manner (Fig. 4A and B). Moreover, when other ROS inhibitor or scavenger (BHA and NAC, respectively) were applied in our experiments, they also inhibited IL-1b and TNF-a secretion induced by cap59D strain from mouse BMDCs (Fig. 4C, D, E and F). These data indicated that inhibition of ROS was mainly affecting the priming signal, although this did not exclude the possibility

Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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Fig. 3. Acapsular mutant of C. neoformans activates the NLRP3 inflammasome in mouse BMDCs. (A, B and C) Mouse BMDCs from wild type, Nlrp3/, Asc/ and Caspase-1/11/ mice were infected with C. neoformans cap59D mutant, then IL-1b, TNF-a and IL-18 from cell culture supernatant were monitored via ELISA. (D and E) Mature forms of caspase-1 (p10) and IL-1b (p17) in cell culture supernatant and other indicated proteins in cell lysate (D), as well as ASC oligomerization (E) from indicated BMDCs infected with cap59D mutant were analyzed via immunoblotting. (F and G) Mouse BMDCs from wild type, Nlrp3/, Nlrc4/ and Aim2/ mice were infected with cap59D mutant, then IL-1b and TNF-a from cell culture supernatant were detected with ELISA. KO, “knock-out”. **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments.

that these inhibitors may also affect the activation of caspase-1 at high concentration [33]. In addition, potassium efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter [34]. We also found that inhibition of potassium efflux with extracellular KCl inhibited

IL-1b secretion but not TNF-a production in BMDCs challenged with cap59D strain (Fig. 4G and H), indicating that potassium efflux was required for NLRP3 inflammasome activation, but not for the priming signal upon infection with cap59D.

Fig. 4. NLRP3 inflammasome activation by C. neoformans acapsular mutant requires potassium efflux and ROS inhibitors interfered with the priming step. BMDCs were pretreated with indicated ROS inhibitors or scavenger (DPI, BHA, NAC, A-F), or KCl (GeH), then challenged with C. neoformans cap59D mutant and the supernatant of BMDCs was harvested for IL-1b and TNF-a ELISA. **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments. Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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3.5. cap59D activation of inflammasome requires fungal cell viability and direct cell wall contact with host cells Early studies have uncovered several molecular mechanisms about host cell recognition of C. neoformans acapsular mutant, which include the finding that mannose and betaglucan receptors bind acapsular mutant [5], primary dendritic cells phagocytose acapsular C. neoformans via mannose receptors and Fc gamma receptor II [35]. To further characterize host cell inflammasome activation upon C. neoformans acapsular mutant challenge, we inactivated the cap59D strain via heating. Interestingly, the heat-inactivated acapsular mutant induced much lower level of IL-1b but comparable IL8 secretion or pro-IL-1b transcription from human macrophages compared with live fungal cell (Fig. 5A, B and C). Similarly, heat-inactivated cap59D mutant induced much lower level of IL-1b secretion from mouse BMDCs (Fig. 5D). Moreover, immunoblotting data showed that inactivation of cap59D also reduced IL-1b secretion, caspase-1 activation as well as ASC oligomerization in mouse BMDCs (Fig. 5E and F). These data showed that fungal cell viability was required for inflammasome activation in human macrophages and mouse dendritic cells by cap59D. In addition, treatment of BMDCs with the specific SYK inhibitor R406 clearly reduced IL-1b and TNF-a secretion induced by cap59D strain (Fig. 5G). Furthermore, shRNA mediated silencing of SYK also strongly decreased IL-1b induction from THP-1 derived macrophages (Fig. 5H), the

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successful silencing of SYK gene had been shown in our previous study [27]. Thus, SYK signaling pathway was required for C. neoformans acapsular mutant induced inflammasome activation. Interestingly, when the C. neoformans acapsular mutant was recapsulated with GXM purified from the capsule of encapsulated H99 strain, its ability to induce IL-1b secretion was strongly reduced (Fig. 5I). This indicated that a direct interaction of cap59D cell wall component with host cells was essential for the activation of NLRP3 inflammasome. Taken together, cell wall component from acapsular mutant of C. neoformans directly interacted with host cells and triggered SYK signaling pathway for inflammasome activation, which was inhibited in the encapsulated strain due to the presence of capsule. 3.6. In vivo function of the NLRP3 against acapsular C. neoformans infection Most clinical isolates of C. neoformans are encapsulated, but there are also poorly encapsulated clinical isolates from AIDS patients reported [36]. Importantly, the well accepted concept is that infectious propagule of C. neoformans should be small desiccated yeast cells or spores produced by sexual reproduction, which have no capsule or very thin capsule outside of the fungal cell wall. Although the acapsular mutant is generally avirulent in experimental animal models of infection, the study of cap59D is still instructive for

Fig. 5. The activation of inflammasome by C. neoformans cap59D mutant requires fungal cell viability, SYK signaling, and is inhibited by GXM. (AeB) THP1 cell derived macrophages were infected with viable or inactivated (heating) cap59D mutant for 6 h, and IL-1b as well as IL-8 in cell culture supernatant were detected via ELISA. (C) THP-1 cell derived macrophages were infected with viable or inactivated (heating) cap59D mutant for 2 h, pro-IL-1b mRNA expression level was assayed with real-time PCR. (DeF) BMDCs were infected with viable or heat-killed (HK) cap59D mutant for 6 h and IL-1b in cell culture supernatant was detected with ELISA (D) or immunoblotting (EeF) as described in Fig. 3(G) BMDCs were pretreated with SYK inhibitor R406 for 30 min, then challenged with cap59D mutant, the cell culture supernatant was harvested for IL-1b ELISA, with Candida albicans (MOI ¼ 10) infection as control. (H) THP-1 cell derived macrophages with silencing of SYK or scramble were infected with cap59D mutant for 6 h, and IL-1b in cell culture supernatant was assayed via ELISA. (I) THP1 cell derived macrophages were infected with cap59D mutant or cap59D strain recapsulated with GXM for 6 h, and IL-1b in cell culture supernatant was assayed with ELISA. *, P < 0.05; **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments. Please cite this article in press as: Guo C, et al., Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.08.013

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understanding the interaction of C. neoformans with the host. As the cap59D strain was able to activate the NLRP3 inflammasome in vitro, we asked whether this mutant could also activate the inflammasome in vivo, and whether the inflammasome played a critical role in controlling acapsular C. neoformans infection. With a mouse model of pulmonary C. neoformans infection, we found that shortly (4 h) after cap59D infection, immune cells were quickly recruited to the lung of wild type mice, but this was clearly compromised in the Nlrp3 deficient animal (Fig. 6A), although the total fungal load did not show a clear difference between these mice (Fig. 6B), probably due to that the time point to harvest tissues was too early. Indeed, 12 h after cap59D infection, leukocyte infiltration into the lung was again high in the wild type animal, and the infiltrated cell number in Nlrp3/ mice was significantly decreased (Fig. 6C). Importantly, in this case the fungal load in Nlrp3/ mice was significantly higher than that in the wild type control animals (Fig. 6D). These data indicated that the activity of NLRP3 was rather important for control of C. neoformans infection in vivo.

Fig. 6. In vivo function of the NLRP3 in the control of C. neoformans infection. Indicated genotypes of female mice were infected through intranasal route with 1  106 cap59D mutant in 25 ml PBS. 4 h (AeB) or 12 h (CeD) post infection, mice were sacrificed and the bronchial alveolar lavage fluid (BALF) was collected and the lung was homogenated in PBS. Leukocyte number in the BALF was counted (A and C), and samples from the lung homogenate was plated for CFU counting to monitor the fungal load of C. neoformans (B and D). KO, “knock-out”. *, P < 0.05; **, P < 0.01. Data are mean ± standard deviations from one out of three independent experiments.

4. Discussion In the present study, we found that the acapsular C. neoformans activated the NLRP3-, but not the AIM2-nor the NLRC4- inflammasome. Importantly, recapsulation of the acapsular mutant cap59D with GXM abolished the ability of cap59D to activate the NLRP3 inflammasome. This indicated that the capsule of C. neoformans prevents the otherwise recognition of the fungus by NLRP3 directly or indirectly. Moreover, our in vivo data showed that during the pulmonary infection with cap59D, leukocyte infiltration into the lung, as well as the clearance of the fungus were both dependent on the NLRP3 inflammasome. As C. neoformans is a facultative intracellular pathogen [37], so the intracellular recognition of this pathogen is important for the clearance. It's known that the encapsulated C. neoformans can be phagocytosed by host cells and reside in the phagocytes when opsonized with complement or antibody. In this case, C. neoformans can replicate in the phagosomes and/or phagolysosomes and maintain the host cell in a silent state [38,39]. Meanwhile, the replication of C. neoformans in host phagocytes produce large amounts of polysaccharidecontaining capsule, which can disrupt host cell function mechanically or chemically [40]. Intracellular C. neoformans can also activate NF-kB signaling pathway and result in apoptosis [41]. However, the death of macrophages induced by C. neoformans infection is different from apoptosis [41e44], which indicates that inflammasome mediated pyroptosis maybe involved in the cell death process upon C. neoformans infection. This would be an interesting and important topic to study in the future. As key cytokines controlled by inflammasome activation, IL1b and IL-18 are both important for host defense against various pathogens [45]. Quite some studies have revealed the importance of IL-18 for control of C. neoformans infection [46]. It is thought that IL-18 promotion of the Th1 cell differentiation and IFN-g production are mechanisms for its protective role. Although IL1b production was detected from acapsular or encapsulated C. neoformans infected mice, there are data showing that IL-18 signal but not IL-1b signal plays a protective role in pulmonary infection with a virulent strain of C. neoformans (H99) [15]. In that study, although both the IL-18R-deficient mice and TLR9deficient mice exhibited higher death rate than wild type control animal upon C. neoformans infection, IL-1b production in the infected lung homogenates from IL-18R-deficient mice was clearly decreased, while in the lung of TLR9-deficient mice the level of IL-1b was totally comparable with that from the wild type control [15]. So the protective role of the NLRP3 inflammasome from our current study (Fig. 6) and the previous one may be mediated by IL-18 [11]. The innate immune system of the host is very critical in combating with the infection from fungal pathogens such as C. albicans, A. fumigatus and C. neoformans [47]. All these fungi were recently found activating the NLRP3 inflammasome [9e11,48]. It was found that fungi cell wall components, including b-glucans, chitin and mannans are the major stimulants triggering host innate immune responses. Although we

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have not identified the exact component from the acapsular strain cap59D that was activating the NLRP3 inflammasome, our study revealed that viability of the fungus was essential for its activation of inflammasome. Since heating of cap59D abolished the induction of IL-1b and IL-8, it is likely that certain thermo labile protein was the fungal component in cap59D strain causing cell activation, leading to NF-kB activation as well as inflammasome activation. Further studies are required to identify the exact component that engages the NLRP3 inflammasome activation upon cap59D strain infection. Up to now, the control of cryptococcosis remains challenging, especially in the immunocompromised patients. Although some animal studies have found that the Th1- and Th17- cell responses were protective while the Th2 response was deleterious [49,50], the balance between different T helper cell responses and inflammatory responses have to be well controlled. On the one hand, these immune responses are important for clearance of Cryptococcus infection, on the other hand, overwhelming inflammation can also cause deleterious effect. Our current study implied that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome, thus indicating the importance of NLRP3 in recognition and control of C. neoformans infection, although the activation of inflammasome may also cause deleterious results such as tissue damage. Fine tuning the function of NLRP3 inflammasome maybe a novel approach to control cryptococcosis. Q1

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 wish to thank Dr. John Perfect for providing C. neoformans strains (H99 and cap59D mutant) as well as Dr. Jiangye Chen for providing Candida albicans for our experiments. This work was supported by grants from Natural Science Foundation of China (31370892, 31300712, 31170868), National Key Basic Research Programs (2013CB531600, 2014CB541900), National Major Projects for Science and Technology (2012ZX10002007-003, 2014ZX0801011B-001), the opening project of Shanghai Key Laboratory of Molecular Medical Mycology (10dz2220100), SA-SIBS Scholarship Program, as well as the CAS/SAFEA International Partnership Program for Creative Research Teams. References [1] Casadevall A. Cryptococci at the brain gate: break and enter or use a Trojan horse? J Clin Investigation 2010;120:1389e92. [2] Kozel TR, Gotschlich EC. The capsule of cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages. J Immunol 1982;129:1675e80.

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Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome.

Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. A...
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