Eur. J. Immunol. 2013. 43: 1–14
DOI: 10.1002/eji.201242691
Immunity to infection
Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome Donatella Pietrella∗1 , Neelam Pandey∗1 , Elena Gabrielli1 , Eva Pericolini1 , Stefano Perito1 , Lydia Kasper2 , Francesco Bistoni1 , Antonio Cassone1 , Bernhard Hube2,3,4 and Anna Vecchiarelli1 1
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy 2 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany 3 Friedrich Schiller University, Jena, Germany 4 Center for Sepsis Control and Care (CSCC), Jena, Germany In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL-1β, TNF-α, and IL-6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL-1β and IL-18 production through inflammasome activation. This occurs via NLRP3 and caspase-1 activation, which cleaves pro-IL-1β into secreted bioactive IL-1β, a cytokine that was induced by Saps in monocytes, in monocyte-derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL-1β. Inflammasome activation required Sap internalization via a clathrin-dependent mechanism, intracellular induction of K+ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS-ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.
Keywords: Aspartic proteases
r
C. albicans
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IL-1β
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Inflammasome r Virulence factor
Introduction Candida albicans is a commensal fungus that colonizes human mucosal surfaces such as the vaginal and gastrointestinal tracts without causing harm. However, under conditions of primary or secondary immunodeficiency, this yeast can cause opportunistic infections such as mucosal inflammation and systemic sepsis [1]. The mortality rate associated with invasive candidiasis
Correspondence: Dr. Anna Vecchiarelli e-mail:
[email protected]
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has been reported to be as high as 40–50% [2]. Candida species are the fourth most common pathogens isolated from nosocomial bloodstream infections in the USA and Europe [3]. Although the immune status of the host plays a key role in the prevention or pathogenesis of C. albicans infections, a number of virulence attributes of C. albicans, such as factors that mediate adhesion, enzyme secretion, or hyphal formation, contribute to the disease process [4]. Particularly, the secretion of aspartic proteases (Saps),
∗
These authors contributed equally to this work.
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which are encoded by a gene family with ten members, has long been recognized as a virulence-associated trait of this pathogenic yeast [5]. We recently reported that various members of the Sap family, including Sap1, Sap2, Sap3, and Sap6, have different abilities to induce secretion of pro-inflammatory cytokines by human monocytes via Akt/NF-κB activation. Sap1, Sap2, and Sap6 potently induced IL-1β, TNF-α, and IL-6 production. Importantly, Sapinduced cytokine production was independent of the proteolytic activity and of the optimal pH for the individual Sap activities [6]. These data suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity. Immune cells of the innate system recognize pathogens through PRR that include membrane-bound TLRs and intracellular proteins such as NOD-like receptors (NLRs) [7]. NLRP3 responds to multiple stimuli and forms an intracellular multiprotein complex, called NLRP3 inflammasome, consisting of the apoptosisassociated speck-like protein containing a caspase recruitment domain, and caspase-1, which triggers the secretion of IL-1β [8]. A recent report by Kayagaki et al. [9] demonstrated that caspase-11 is also involved in a noncanonical inflammasome activation. NLRP3 inflammasome is activated in response to a range of bacterial, viral, and fungal pathogens, including Aspergillus fumigatus and C. albicans [10–12]. IL-1β production via the inflammasome generally requires two signals: an NF-κB-dependent signal that induces the synthesis of pro-IL-1β, and a second signal that triggers proteolytic pro-IL-1β processing to produce mature IL-1β. Spleen tyrosine kinase (Syk) coupled to fungal pattern recognition receptors, such as Dectin-1, controls both pro-IL-1β synthesis and the activation of the inflammasome after cell stimulation with C. albicans [13]. IL-1β and IL-18, which are both induced by activation of the inflammasome, are involved in the initiation of the adaptive Th1 and Th17 cellular responses to C. albicans [14]. To investigate whether the inflammasome activation is triggered by Saps, we selected Sap2 and Sap6 as members of two Sap subfamilies, Sap1–3 and Sap4–6 respectively, which differ in their biological properties and potential roles in different types of C. albicans infections [5, 15].
Results IL-1β and IL-18 production by monocytes in response to Saps We first analyzed whether Sap2 and Sap6 from C. albicans were able to induce the production of IL-18 and IL-1β by human monocytes [16]. Figure 1 shows that Sap2 and Sap6 induce IL-1β and IL-18 to a similar extent. The cytokine production levels were generally comparable with those observed after stimulation with a positive control stimulant (LPS-ATP). In contrast, Sap3 did not induce significant upregulation of IL-18, even though a modest increase of this cytokine was observed. To rule out any LPS involvement, we added polymyxin B to select basic assays. As shown in Figure 1, addition of polymyxin B did not cause any C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2013. 43: 1–14
Figure 1. IL-1β and IL-18 production by monocytes in response to Saps. Monocytes were treated for 4 h with Sap2, Sap3, and Sap6 (20 μg/mL) or LPS (1 μg/mL) plus 5 mM ATP. Selected experiments were carried out in the presence of 10 μg/mL of polymyxin B. After incubation, supernatants were recovered and tested for the presence of IL-1β or IL-18. Data are expressed as means ± SEM for six samples pooled from three independent experiments. *p < 0.05, **p < 0.01, (Sap or LPS-ATP-treated cells versus untreated cells), ##p < 0.01, (polymyxin B plus Sap or LPSATP-treated cells versus Sap or LPS-ATP-treated cells). Differences were analyzed by ANOVA test.
significant decrease in the level of Sap-induced cytokine production, but it strongly reduced IL-1β and IL-18 production after LPS-ATP stimulation of monocytes (see below for additional evidence of the polymyxin B effect). To investigate whether the production of Sap-induced IL-1β requires proteolytic activity of Saps, monocytes were treated with Sap2 and Sap6 in combination with the aspartic protease inhibitor pepstatin A (15 μM). Pepstatin A did not affect the Sap2- or Sap6-induced production of IL-1β (data not shown), confirming that the activation of the inflammasome by Saps is independent from their enzymatic activity. To further confirm that the recombinant Sap proteins were not contaminated with other molecules that may be responsible for the observed activation, experiments were repeated with heat-inactivated Saps and with Proteinase K. Heat inactivation and addition of Proteinase K abrogated the IL-1β production by
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Eur. J. Immunol. 2013. 43: 1–14
monocytes stimulated with Sap2 or Sap6 indicating that native Sap proteins were required for inflammasome activation.
Inhibition of Sap internalization and regulation of IL-1β and IL-18 production To test whether Saps must be internalized for cytokine induction, monocytes were treated with known inhibitors of endocytic activity such as nystatin, chlorpromazine (CPZ), and cytochalasin D. CPZ is known to inhibit clathrin-mediated endocytosis [17], while nystatin is an inhibitor of the lipid raft-caveolae endocytosis pathway [18]. As shown in Figure 2A, the treatment with nystatin or cytochalasin D did not cause any decrease of Sap2- or Sap6-induced IL-1β production. Conversely, treatment with CPZ markedly inhibited IL-1β secretion in response to both Sap2 and Sap6. Since it is known that LPS-ATP recognition by monocytes also depends on clathrin-mediated endocytosis [19], CPZ must have caused the decrease in cytokine production following LPSATP stimulation (Fig. 2B). A CPZ concentration of 45 μM, which is commonly used to inhibit phagocytosis [20], was effective in producing inhibitory effects. In parallel experiments, we also demonstrated that CPZ treatment also downregulated IL-18 production after induction by Sap2 and Sap6 (Fig. 2C). Because TNF-α production is induced through regulatory pathways that differ from those leading to IL-1β and IL-18 production, we tested whether CPZ was able to affect TNF-α production induced by Saps. As shown in Figure 2D, treatment with CPZ did not modify Sap induced TNF-α production. Next, we tested the uptake of FITC-conjugated Sap2 and Sap6 by monocytes with and without CPZ treatment. Figure 2E and F shows a significant decrease in Sap2 and Sap6 uptake by CPZ-treated monocytes. When FITC-conjugated LPS was used, differences in LPS uptake after CPZ treatment were also observed (Fig. 2G). This is consistent with previous data showing that internalization of LPS occurs via a clathrin-dependent mechanism [21, 22]. FITC-conjugated CTLA-4 F(ab)2 was used as a negative control for protein uptake (Figure 2H). Since recent data indicate that autophagosome formation modulates IL-1β secretion [23] and many reports point to the involvement of the endolysosomal compartment in unconventional secretion [24], it is possible that CPZ affects not only endocytosis but also intracellular trafficking of endolysosomes or autophagosomes. To settle this point, we carried out experiments with two TLR agonists that do not require clathrin-mediated endocytosis, FSL-1 (TLR2 agonist), and flagellin (TLR5 agonist). Both agonists were able to induce IL-1β secretion by monocytes (Table 1). Pretreatment of monocytes with 90, 45, 15 μM of CPZ did not significantly affect the IL-1β production induced by either TLR agonist.
Intracellular signaling induced by Saps Caspase-1 processes the inactive IL-1β and IL-18 precursors into their mature active forms, which are subsequently secreted by the
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Immunity to infection
cell [25]. We next examined the intracellular production of the inactive precursor of IL-1β (pro-IL-1β) after addition of Saps by cytofluorimetric analysis. The results documented in Figure 3A show that both Sap2 and Sap6 induce abundant production of the IL-1β precursor. The addition of the specific caspase-1 inhibitor (Ac-YVAD 25 μM, referred to as IC-1) resulted in an increase of the inactive precursor of IL-1β (Fig. 3A), as expected when cytokine maturation is inhibited (see also Fig. 4C). Immunoblot analysis was also performed to unravel the dynamics of IL-1β secretion in response to Saps. As shown in Figure 3B, a pro-IL-1β band was observed 2 h after stimulation with Sap2 and Sap6 only in the presence of the inhibitor, while pro-IL-1β and IL-1β active bands were manifested after 2 h of stimulation with LPS-ATP independently of the inhibitor presence. However, pro-IL-1β and active IL-1β bands were detected in response to 4 h of stimulation with Sap2, Sap6, and LPS-ATP. Two different doses of IC-1 (25 and 100 μM) were used to inhibit the activation of caspase-1. IC-1 treatment clearly downregulated the formation of the active IL-1β at 4 h at the concentration of 25 μM, while the higher dose completely abolished the IL-1β maturation (Fig. 3C). The kinetics of caspase-1 activation was analyzed by western blot (Fig. 4A and quantification in B). The results showed that stimulation with Sap2 or Sap6 led to different kinetics of caspase-1 expression. Sap2 was able to induce the activation of caspase-1 after 1 h of stimulation in a pattern similar to that manifested after LPSATP stimulation. In contrast, Sap6 caused a weak activation of caspase-1 after 1 h of incubation, while a strong expression was observed after 2 h (Fig. 4A and B). The inhibition of caspase-1 by IC-1 resulted in downregulation of caspase-1 activation in all experiments, independently of whether Sap2, Sap6, or LPS-ATP were used as stimulants (Fig. 4A and B). The secretion of both IL-1β (Fig. 4C) and IL-18 (Fig. 4F) significantly decreased in the presence of the IC-1. However, the inhibitor did not affect TNF-α production (Fig. 4G). Since monocytes, macrophages and dendritic cells (DCs) have different requirements for inflammasome activation and the processing and release of IL-1β [26], we tested Sap ability to stimulate human monocyte-derived macrophages (MDMs) and human DCs to produce IL-1β. As shown in Figure 4D and E, IL-1β was also produced by both MDMs and DCs, following stimulation with Sap2 and Sap6. Similarly, IC-1 caused a significant decrease in IL-1β production. The activation of the NLRP3 inflammasome requires, in most cell types, two different stimuli. One, often called priming, is necessary for the upregulation of NLRP3 and pro-IL-1β and is usually caused by microbial ligands. The second stimulus is the real activator of the NLRP3 inflammasome (e.g. ATP). It has been demonstrated that human monocytes need only one stimulus, because they constitutively express active caspase-1 or because they can secrete ATP in response to stimulation with microbial ligands. To clarify whether Saps act exclusively like other microbial ligands or whether they are direct activators of the NLRP3 inflammasome, macrophages primed with LPS (signal 1) were then stimulated with Saps. The results shown in Figure 4H demonstrate that Saps are able to directly activate the production of IL-1β.
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Figure 2. Inhibition of Sap internalization and regulation of IL-1β and IL-18 production by monocytes. (A–D) Monocytes were pretreated with nystatin (5 μg/mL), chlorpromazine (45 μM), cytochalasin D (2 μM) for 30 min and then stimulated with Sap2, Sap6, or LPS-ATP. After incubation, supernatants were tested for the presence of (A, B) IL-1β, (C) IL-18, and (D) TNF-α. (E–H) To inhibit Sap internalization, monocytes were treated with chlorpromazine (45 μM) and then incubated for 10, 30, and 120 min with 20 μg/mL of FITC-labeled (E) Sap2, (F) Sap6, (G) LPS, or (H) CTLA4 (Fab)2 . Fluorescence was analyzed by flow cytometry and it is expressed as mean intensity fluorescence. Data are expressed as means ± SEM of six samples pooled from three independent experiments carried out with monocytes from three different donors. *p < 0.05, **p < 0.01 (chlorpromazine plus Saps or LPSATP-treated cells versus Saps or LPS-ATP-treated cells). Differences were analyzed by ANOVA test.
Because the tyrosine kinase Syk is involved in the control of both pro-IL-1β synthesis and the activation of the inflammasome induced by Candida [10], we carried out experiments to assess whether Syk is also involved in the activation of the NLRP3 inflammasome induced by Saps. The phosphorylation of Tyr525/526 is essential for Syk function [27]. No activation of Syk was observed after 4 h of stimulation with Sap2, Sap6, or LPS-ATP (data not shown). The direct involvement of NLRP3 in the induction of IL-1β by Saps was investigated. By using Western blot analysis, a significant
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increase in NLRP3 expression upon stimulation with Sap2, Sap6, or LPS-ATP was observed (Fig. 5A and B). Then, the involvement of NLRP3 was also analyzed by using small interfering RNA (siRNA). An RT-PCR control experiment (Fig. 5C) showed that electroporation of NLRP3 siRNA had a strong silencing efficiency (about 80%). As shown in Figure 5D, IL-1β production in silenced cells stimulated with Sap2, Sap6, or LPS-ATP was markedly attenuated compared with that of control cells. Conversely, TNF-α secretion was barely affected, indicating that Sap-induced IL-1β production is regulated via NLRP3.
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Immunity to infection
Table 1. Effect of chlorpromazine on IL-1β production induced by TLR agonists.
Chlorpromazinea)
Untreated cells
Flagellin (Bacillus subtilis) 100 ng/mL
FSL-1 12.5 μg/mL
— 15 μM 45 μM 90 μM
9.4 ± 2.2 5.0 ± 2.1 5.6 ± 2.7 6.2 ± 1.4
31.0 ± 8.7b),c) 42.8 ± 22.9 33.1 ± 8.5 22.4 ± 2.0
21.4 ± 2.4c) 21.2 ± 4.0 14.3 ± 0.8 17.5 ± 1.3
a)
Monocytes were pretreated with chlorpromazine for 30 min and then stimulated with flagellin (TLR5 agonist) and FSL-1 (TLR2 agonist). b) IL-1β is expressed as pg/mL. c) p < 0.05, Flagellin or FSL-1-treated cells versus untreated cells. Differences were analyzed by ANOVA test.
Inhibition of potassium efflux, superoxide production, and induction of lysosomal damage by Saps Activation of the NLRP3 inflammasome can be mediated by different, often concurrent, mechanisms, including stimulation of K+ efflux, induction of lysosomal damage, and production of ROS, as reviewed by Cassel et al. [28]. To investigate whether these mechanisms are involved during Sap-induced responses, we used recognized inhibitors such as glibenclamide, which inhibits K+ efflux
[29] and butylated hydroxyanisole (BHA), a potent inhibitor of ROS production [29]. Preliminary tests ruled out that the concentrations of these inhibitors used in our assays were toxic for monocytes, both in the presence and in the absence of Sap stimulation (data not shown). Glibenclamide was able to inhibit production of IL-1β upon stimulation with Saps, particularly with Sap6, in a dose-dependent fashion (data not shown). Additional experiments using a buffer rich in K+ (150 mM) were carried out. A reduction of IL-1β production by monocytes stimulated with Sap2 and Sap6 was observed (Fig. 6A), confirming that K+ efflux is involved in Sap-induced inflammasome activation. In a second series of experiments, BHA was used to inhibit ROS or cytokine production. Figure 6B shows that an increase of superoxide anions was observed upon addition of Saps or LPS-ATP. In the presence of BHA the production of ROS was reversed to the levels of unstimulated monocytes. As shown in Figure 6C and D, BHA also caused a statistically significant reduction of IL-1β and IL-18 upon stimulation, although cytokine production was not reversed to the level of unstimulated cells, suggesting that additional pathways that are not inhibited by BHA are involved in cytokine production after Sap induction. In further experiments, we evaluated the kinetics of ROS production. Stimulation of monocytes with Saps induced generation of intracellular ROS that peaked at 30 min and decreased thereafter (Fig. 6E). Because ROS
Figure 3. Expression of immature pro-IL-1β and active IL-1β induced by Saps. Monocytes were stimulated with Sap2 or Sap6 (20 μg/mL) or LPS-ATP for 4 h. (A) After incubation, cells were first fixed, permeabilized, and incubated with anti-human pro-IL-1β. To inhibit the activation of caspase-1 monocytes were pretreated with IC-1 (25 and 100 μM), for 30 min. Fluorescence was analyzed by flow cytometer and expressed as mean intensity fluorescence. Data are expressed as means ± SEM of three samples pooled from three independent experiments. *p < 0.05 (Sap-treated cells versus untreated cells), § p < 0.05 (Sap plus IC-1-treated cells versus Sap-treated cells). Differences were analyzed by ANOVA test. (B) For western blotting analysis, after incubation, cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with antibodies against pro and active IL-1β. (C) An antibody to β-actin was used for normalization. The Western blots and normalization data are from one experiment representative of four independent experiments with similar results.
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Figure 4. Caspase-1 activation induced by Saps. Monocytes, MDMs, or DCs were stimulated with Sap2 or Sap6 (20 μg/mL). (A) After incubation cells were lysed and analyzed by western blotting to examine caspase-1 activation. (A–H) To inhibit the activation of caspase-1, cells were pretreated with IC-1 (25 μM). (B) Normalization of caspase-1 expression against β-actin was carried out. (C–G) Supernatants were recovered and tested for the presence of (C–E) IL-1β, (F) IL-18, and (G) TNF-α. (H) MDMs were pretreated with LPS (1 μg/mL) and then stimulated with Saps for 2 h before lysis and western blotting analysis. Data are expressed as means + SEM of three samples pooled from three independent experiments. *p < 0.05, **p < 0.01 ((A) Sap-treated cells versus untreated cells (C–H) Sap plus IC-1-treated cells versus Sap-treated cells). Differences were analyzed by ANOVA test. The western blots and normalization data are from one out experiment representative of two performed with similar results.
production is generally counteracted by upregulation of antioxidant defenses to avoid oxidative stress [30], we analyzed the modulation of intracellular glutathione (GSH). GSH was absent after 30 min of stimulation and reached a maximum at 1 h (Fig. 6F). These data suggest that ROS have a priming role in the induction of IL-1β production, which is found only after 4 h of stimulation. In addition, we examined whether Sap stimulation could provoke lysosome rupture thereby inducing inflammasome activation by cathepsin B release [31]. Monocytes were treated for 2 h with
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Saps and then analyzed for lysosome damage, assessed by the decrement of acridine orange fluorescence intensity. The results shown in Figure 7A show that there is a consistent decrease of fluorescence intensity in cells treated with Sap2 or Sap6, but not with LPS-ATP, suggesting that Saps are able to induce cathepsin B release by lysosomes. It has been reported that the inhibition of the lysosomal cysteine protease (cathepsin B) by its specific inhibitor CA-074 (IC-B) abrogated IL-1β production [32]. In our experimental system, IC-B inhibited the production of IL-1β by
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Immunity to infection
Figure 5. The role of NLRP3 in the production and release of IL-1β. (A, B) Expression of NLRP3 in monocytes was evaluated by western blotting after stimulation for 4 h with Sap2 or Sap6. (C) Monocytes were transfected with siRNA NLRP3 or nonsilencing control (siC). RT-PCR of lysates after overnight incubation was performed for NLRP3 expression normalized against that of GAPDH. (D–E) After 24 h cells were primed for 4 h with Sap2, Sap6, or LPS plus ATP. (D) IL-1β or (E) TNF-α were assessed in the supernatants. The western blots and normalization data are from one experiment representative of three independent experiments with similar results. Cytokine production data are expressed as means + SEM of six samples pooled from three independent experiments. *p < 0.05, **p < 0.01 (siRNA NLRP3-treated cells versus siC-treated cells). Differences were analyzed by ANOVA test.
monocytes stimulated with Sap2 or Sap6 (Fig. 7B). However, we cannot completely exclude that this effect is due to nonspecific effects of IC-B as previously observed [10]. Moreover, to confirm that lysosomal damage is not secondary to the induction of pyroptosis, experiments were carried out in the presence of the caspase-1 inhibitor IC-1. IC-1 did not significantly affect the lysosome damage, assessed by the decrement of acridine orange fluorescence intensity in cells treated with Sap2, Sap6, or LPS-ATP (data not shown).
Discussion In a recent paper, we reported the capacity of secreted aspartic proteases of C. albicans to induce secretion of proinflammatory cytokines by human monocytes. In particular, Sap2 and Sap6, which are members of two Sap subfamilies (Sap1–3 and Sap4–6) with distinct roles in C. albicans pathogenicity and disease spectrum [5], strongly induced upregulation of IL-1β, TNF-α, and IL-6 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6] independently from their proteolytic activity, from the optimal pH of each individual Sap activity, and from activation of protease-activated receptors. In this study, we propose that IL-1β and IL-18 production caused by Sap2 or Sap6 is the result of NLRP3 inflammasome and caspase-1 activation. We demonstrate that Sap2 and Sap6 induction of proinflammatory cytokines was due to a cascade of events causing upstream inflammasome activation and downstream caspase-1 mediated cytokine synthesis by procytokines as proposed in Figure 8. To our knowledge, there has been no prior demonstration that any single member of the Sap isoenzyme family, which is one of the best known virulence determinants of C. albicans [33], nor any protein of C. albicans or any other fungus, can induce inflammasome activation. However, although the biological relevance remains unclear, it has been reported that selected proteinases of C. albicans can activate pro-IL-1β proteolytically to IL-1β [34]. Furthermore, it has been shown that mutants lacking functional SAP genes have a reduced potential to induce cytokine production in oral
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Figure 6. Inhibition of potassium efflux and superoxide production induced by Saps. Monocytes were stimulated with Saps for 4 h in a serum-free buffer with 150 mM KCl. In parallel, a buffer with 150 mM NaCl was used. (A) After incubation, supernatants were tested for the presence of IL-1β. To analyze superoxide production, cells were pretreated with 10 μM of H2 DCFDA. (B) To inhibit superoxide production, monocytes were pretreated with BHA (50 μM) for 30 min. After incubation, cells were fixed and underwent cytofluorimetric analysis. (C, D) Supernatant fluids were recovered to test for the presence of (C) IL-1β and (D) IL-18. (E, F) To study the kinetics of ROS production, monocytes were stimulated with 20 μg/mL of Sap2 or Sap6 for 30 min, 1, 2, and 4 h. (E) A 10 μM of H2 DCFDA to test intracellular ROS or (F) 100 μM of MCB to assess intracellular GSH were added to cultures 30 min before the end of the incubation. Results are expressed as relative fluorescence units (RFU). Data are expressed as means ± SEM of six samples pooled from three independent experiments. *p < 0.05, **p 95% CD14+ , evaluated by flow cytometry (FACScan, Becton Dickinson).
In vitro generation of DCs and MDMs The generation of DCs and MDMs from monocytes was performed as previously described, with minor modifications [58]. Immature DCs were obtained by treating monocytes with 50 ng/mL of human recombinant GM-CSF (Biosource) and 30 ng/mL of human recombinant IL-4 (Biosource) for 5–6 days. MDMs were obtained by treatment with human recombinant M-CSF (50 ng/mL) for 5 days.
Determination of proinflammatory cytokine production Monocytes, MDMs, or DCs (2.5 × 105 ) were incubated at 37◦ C for 4 h with Sap2, Sap6 (20 μg/mL). As a positive control LPS (1 μg/mL) plus ATP (5 mM) was used for a further 15 min. In selected experiments monocytes were stimulated with 12.5 μg/mL of diacylated lipoprotein FSL-1 (Pam2CGDPKHPKSF, InvivoGen) or with flagellin from Bacillus subtilis (100 ng/mL, InvivoGen). In selected experiments cells were pretreated with nystatin (5 μg/mL, Sigma), chlorpromazine (90, 45, 15 μM),
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cytochalasin D (2 μM, Sigma), caspase-1 inhibitor Ac-YVAD-cmk (25 μM) (IC-1), glibenclamide (10, 25, 50, 75, and 100 μM), BHA (50 μM) for 30 min at 37◦ C, or treated with CA074me (10 μM, Peptanova), an inhibitor of cathepsin B (IC-B). To inhibit the K+ efflux monocytes were stimulated with Saps for 4 h in a serumfree buffer with 150 mM KCl (10 mM Hepes, 5 mM NaH2 PO4 , 150 mM KCl, 1 mM MgCl2 , 1 mM CaCl2 , 1% BSA, pH 7.4). In parallel, a buffer with 150 mM NaCl was used (10 mM Hepes, 150 mM NaCl, 5 mM KH2 PO4, 1 mM MgCl2, 1 mM CaCl2 , 1% BSA, pH 7.4) [59]. After incubation, supernatant fluids were recovered, then the presence of human IL-1β, IL-18, and TNF-α was measured by ELISA kit (Biosciences).
Cytotoxicity assay Monocytes (2.5 × 105 ) were treated for 4 and 18 h at 37◦ C with 7.5, 15, 30, 45, and 90 μM of chlorpromazine (Sigma); 10, 25, 50, 75, and 100 μM of glibenclamide (Sigma); 10, 25, and 50 μM of Ac-YVAD-cmk (Bachem AG). After incubation cell viability was evaluated by the use of an ATP bioluminescence kit (Via Light kit; Cambrex).
Protease FITC labeling
Immunity to infection
Immunoblotting For immunoblotting, 106 cells were stimulated with 20 μg/mL of Sap2, Sap6, or LPS+ATP for the indicated times. In selected experiments, MDMs were pretreated with LPS for 1 h and then stimulated with Sap2 and Sap6 (20 μg/mL). After stimulation cells were lysed and 30 μg of protein of each sample were separated by SDS-PAGE and transferred to nitrocellulose membrane (Pierce). The membrane was blocked with 5% w/v milk powder in PBS and incubated overnight at 4◦ C with goat anti human proIL-1β or goat anti-human-activated caspase-1, rabbit anti-human Syk (Santa Cruz Biotechnology), rabbit anti-human phospho-Syk (Abcam) or rabbit anti-human NLRP3 (Imgenex) in TBST. After overnight incubation, the blots were washed with TBST and incubated with HRP-conjugated goat anti-rabbit antibody or mouse anti goat for 1 h at room temperature and incubated with the substrate ChemiLucent Trial Kit (Chemicon Int.). Immunoreactive bands were visualized and quantified by Chemidoc Instrument (Bio-Rad).
siRNA experiments
Proteases and CTLA-4 F(ab)2 (Ancell) were labeled with fluorescein using the Fluoro Tag FITC Conjugation kit (Sigma). Briefly, fresh FITC solution in carbonate-bicarbonate buffer was added to the Sap solutions and samples were incubated for 2 h at room temperature with gentle stirring. The labeled proteins were purified from unconjugated fluorescein by use of a Sephadex G-25M column.
For siRNA experiments, the primary monocyte protocol for electroporation in the Amaxa chamber (AmaxaBiosystems) was used, according to the instructions of the manufacturer. Specific sets of siRNA for NLRP3 as well as nonsilencing siRNA for the control were obtained from Ambion. Monocytes were resuspended in 100 μL of Human Monocyte Nucleofactor solution. Two microgram siRNA per 106 cells was added, electroporation was performed and monocytes were allowed to recover overnight and stimulated with Sap2 or Sap6 or LPS.
Sap2 and Sap6 uptake
RT-PCR
The uptake of Sap2 and Sap6 by human monocytes was analyzed by flow cytometry as previously described [58]. Monocytes (2.5 × 105 ) were treated with or without 45 μM of chlorpromazine for 30 min at 37◦ C, incubated with 20 μg/mL of FITClabeled Sap2, Sap6, LPS, or CTLA-4 F(ab)2 for 30, 60, and 120 min at 37◦ C in the presence of 5% CO2 . After incubation cell fluorescence was evaluated by using FACScan and expressed as the mean intensity fluorescence.
Control experiments to check the inhibition of NLRP3 expression were performed by RT-PCR. A total of 106 monocytes were treated with nonsilencing siRNA or with NLRP3 siRNA. After 18 h of incubation at 37◦ C, total RNA was extracted, reverse-transcribed into complementary DNA. PCR was performed using a Bio-Rad Thermal Cycler-200. PCR conditions were as follows: 2 min at 50◦ C and 10 min at 95◦ C, followed by 30 cycles of PCR reaction at 94◦ C for 45 s, 70◦ C for 2 min, and 59◦ C for 1 min. GAPDH was used as a reference gene. The PCR products were run on 1% agarose gels stained with ethidium bromide.
Determination of pro-IL-1β by flow cytometry Monocytes were stimulated with 20 μg/mL of Sap2 or Sap6 at 37◦ C for 4 h, fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin (Sigma) in PBS, and labeled with goat anti human pro-IL-1β (Santa Cruz Biotechnology). As a secondary antibody a FITC-conjugated anti-goat antibody was used. Cells expressing pro-IL-1β were analyzed using FACScan.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Superoxide assay The production of superoxide was analyzed using a membrane permeable probe: 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCF-DA, Sigma), which is oxidized by H2 O2 to dichlorofluorescein. Monocytes (2.5 × 105 ) were pretreated with 10 μM
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of H2 DCF-DA for 1 h at 37◦ C, then incubated for 2 h with 20 μg/mL of Sap2 or Sap6. Cells were then fixed in 4% paraformaldehyde and analyzed using a flow cytometer. In selected experiments, to inhibit the superoxide production, monocytes were pretreated with a broad superoxide scavenger, BHA (50 μM). To study the kinetics of ROS production, monocytes were stimulated with 20 μg/mL of Sap2 or Sap6 for 30 min, 1, 2, 4 h. A 10 μM of H2 DCF-DA to test intracellular ROS or 100 μM of monochlorobimane (MCB, Sigma) to assess intracellular GSH were added to cultures 30 min before the end of the incubation. Fluorescence was measured in cell lysates with a microplate fluorometer (480 nm excitation, 530 nm emission for H2 DCFDA; 355 nm excitation and 485 nm emission for MCB).
Eur. J. Immunol. 2013. 43: 1–14
3 Chen, L. Y., Kuo, S. C., Wu, H. S., Yang, S. P., Chan, Y. J., Chen, L. K. and Wang, F. D., Associated clinical characteristics of patients with candidemia among different Candida species. J. Microbiol. Immunol. Infect 2012. In press. doi: 10.1016/j.jmii.2012.08.001. 4 Calderone, R. A. and Fonzi, W. A., Virulence factors of Candida albicans. Trends Microbiol. 2001. 9: 327–335. 5 Naglik, J., Albrecht, A., Bader, O. and Hube, B., Candida albicans proteinases and host/pathogen interactions. Cell Microbiol. 2004. 6: 915–926. 6 Pietrella, D., Rachini, A., Pandey, N., Schild, L., Netea, M., Bistoni, F., Hube, B. et al., The Inflammatory response induced by aspartic proteases of Candida albicans is independent of proteolytic activity. Infect Immun. 2010. 78: 4754–4762. 7 Akira, S., Uematsu, S. and Takeuchi, O., Pathogen recognition and innate immunity. Cell 2006. 124: 783–801. 8 Franchi,
L.,
Eigenbrod,
T.,
Munoz-Planillo,
R.
and
Nunez,
G.,
The inflammasome: a caspase-1-activation platform that regulates
Determination of lysosomal damage
immune responses and disease pathogenesis. Nat. Immunol. 2009. 10: 241–247.
To measure lysosomal membrane integrity, healthy, or apoptotic cells were stained with acridine orange (Sigma) as previously described [60]. Briefly, monocytes after stimulation with 20 μg/mL Sap2 or Sap6 or LPS+ATP for 2 h, were collected, washed, and incubated with 10 μM of acridine orange (Sigma Aldrich) for 10 min. Monocytes were then analyzed using a FACScan flow cytometer. In selected experiments cells were pretreated with caspase-1 inhibitor.
9 Kayagaki, N., Warming, S., Lamkanfi, M., Vande Walle, L., Louie, S., Dong, J., Newton, K. et al., Non-canonical inflammasome activation targets caspase-11. Nature 2011. 479: 117–121. 10 Gross, O., Poeck, H., Bscheider, M., Dostert, C., Hannesschlager, N., Endres, S., Hartmann, G. et al., Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence. Nature 2009. 459: 433–436. 11 Joly, S. and Sutterwala, F. S., Fungal pathogen recognition by the NLRP3 inflammasome. Virulence 2010. 1: 276–280. 12 Said-Sadier, N., Padilla, E., Langsley, G. and Ojcius, D. M., Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway
Statistical analysis Statistical significance was determined using ANOVA test. Results are presented as means ± SEM from replicate samples of 2-6 experiments. Post hoc comparison were done with Bonferroni’s Test. A value of p < 0.05 was considered significant.
requiring ROS production and the Syk tyrosine kinase. PLoS One 2010. 5: e10008. 13 Cheng, S. C., van de Veerdonk, F. L., Lenardon, M., Stoffels, M., Plantinga, T., Smeekens, S., Rizzetto, L. et al., The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans. J. Leukoc. Biol. 2011. 90: 357–366. 14 van de Veerdonk, F. L., Joosten, L. A., Shaw, P. J., Smeekens, S. P., Malireddi, R. K., van der Meer, J. W., Kullberg, B. J. et al., The inflammasome drives protective Th1 and Th17 cellular responses in disseminated candidiasis. Eur. J. Immunol. 2011. 41: 2260–2268. 15 Naglik, J. R., Challacombe, S. J. and Hube, B., Candida albicans secreted
Acknowledgments: The study was supported by the EU MarieCurie project N◦ FP7-214004-2, FINSysB. We thank Catherine Macpherson for editorial assistance.
aspartyl proteinases in virulence and pathogenesis. Microbiol. Mol. Biol. Rev. 2003. 67: 400–428. 16 van de Veerdonk, F. L., Netea, M. G., Dinarello, C. A. and Joosten, L. A., Inflammasome activation and IL-1beta and IL-18 processing during infection. Trends Immunol. 2011. 32: 110–116.
Conflict of interests: The authors declare no financial or commercial conflict of interest.
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Abbreviations: BHA: butylated hydroxyanisole · CPZ: chlorpromazine ·
Bernardis, F., A highly immunogenic recombinant and truncated protein
IC-1: caspase-1 inhibitor · IC-B: cathepsin B inhibitor · MDM: monocyte-
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derived macrophage · NLR: NOD-like receptor · Sap: aspartic protease · Syk: spleen tyrosine kinase Full correspondence: Dr. Anna Vecchiarelli, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06126 Perugia, Italy Fax: +39-075-585-7407 e-mail:
[email protected]
4359–4367. 59 Cassel, S. L., Eisenbarth, S. C., Iyer, S. S., Sadler, J. J., Colegio, O. R., Tephly, L. A., Carter, A. B. et al., The Nalp3 inflammasome is essential for the development of silicosis. Proc. Natl. Acad. Sci. USA 2008. 105: 9035–9040.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 22/5/2012 Revised: 6/12/2012 Accepted: 20/12/2012 Accepted article online: 26/12/2012
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