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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.
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 . The mortality rate associated with invasive candidiasis has been reported to be as high as 40–50% . Candida species are the fourth most common pathogens isolated from nosocomial bloodstream infections in the USA and Europe . 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 . Particularly, the secretion of aspartic proteases (Saps), which are encoded by a gene family with ten members, has long been recognized as a virulence-associated trait of this pathogenic yeast .
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, Sap-induced cytokine production was independent of the proteolytic activity and of the optimal pH for the individual Sap activities . 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) . NLRP3 responds to multiple stimuli and forms an intracellular multiprotein complex, called NLRP3 inflammasome, consisting of the apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1, which triggers the secretion of IL-1β . A recent report by Kayagaki et al.  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 . 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 .
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].
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 . 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 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 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 , while nystatin is an inhibitor of the lipid raft-caveolae endocytosis pathway . 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 , CPZ must have caused the decrease in cytokine production following LPS-ATP stimulation (Fig. 2B). A CPZ concentration of 45 μM, which is commonly used to inhibit phagocytosis , 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  and many reports point to the involvement of the endolysosomal compartment in unconventional secretion , 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.
Table 1. Effect of chlorpromazine on IL-1β production induced by TLR agonists
Caspase-1 processes the inactive IL-1β and IL-18 precursors into their mature active forms, which are subsequently secreted by the cell . 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 LPS-ATP 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 , 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.
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 , 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 . 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 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.
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. . To investigate whether these mechanisms are involved during Sap-induced responses, we used recognized inhibitors such as glibenclamide, which inhibits K+ efflux  and butylated hydroxyanisole (BHA), a potent inhibitor of ROS production . 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 production is generally counteracted by upregulation of antioxidant defenses to avoid oxidative stress , 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 . Monocytes were treated for 2 h with 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 . In our experimental system, IC-B inhibited the production of IL-1β by 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 . 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).
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 , strongly induced upregulation of IL-1β, TNF-α, and IL-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 iso-enzyme family, which is one of the best known virulence determinants of C. albicans , 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 . Furthermore, it has been shown that mutants lacking functional SAP genes have a reduced potential to induce cytokine production in oral epithelial tissue models . While this process is possibly associated with reduced damage of SAP mutants, our study presented here suggests that single Sap proteins can directly activate the inflammasome.
This assertion is supported by evidence of direct activation of NLRP3 and caspase-1 by Sap2 and Sap6. In our experimental setting Sap2, Sap6, and the positive control LPS-ATP induce IL-1β and IL-18 to a similar extent. In previous studies, the amount of IL-1β secreted by human monocytes upon LPS plus ATP stimulation was much higher than the amount of IL-18 produced [36, 37]. However, the experimental conditions in these studies were different from ours. For example, Perregaux et al.  reported the production of IL-1β and IL-18 in plasma from LPS plus ATP-activated blood cells, while Piccini and collaborators  used a different concentration of cells and a lower concentration of ATP.
Silencing of NLRP3 mRNA provided direct evidence that Sap2 and Sap6 can activate the inflammasome. The kinetics of caspase-1 activation showed that Sap6-induced activation of caspase-1 occurred over longer periods (still evident 4 h poststimulation) than Sap2-induced activation. This may be due to the different epitopes of the Saps that could stimulate caspase-1 differently. Although Sap2 and Sap6 are both aspartic proteases and members of the Sap family, they belong to different subgroups. Sap2 belongs to the subfamily Sap1–3, while Sap6 belongs to the Sap4–6 subfamily . However, the kinetics of bioactive IL-1β expression was similar for both stimulating proteases. The expression was at a maximum 4 h after stimulation with Saps and the amount of secreted bioactive IL-1β was similar. This suggests that the extended activation of caspase-1 does not result in a more consistent cleavage of pro-IL-1β.
Using specific inhibitors of K+ efflux and ROS generation, we observed that Sap-induced inflammasome activation seems to be associated with physiological changes such as transient opening of ion channels and the production of ROS, as observed for NLRP3-induced inflammasome . Interestingly, when ROS generation was inhibited by BHA, which led to ROS levels similar to those of nonstimulated monocytes, cytokine production was only partially inhibited, but the reduction was significant. This suggests that inflammasome activation and cytokine production induced by Saps does not rely on just one signaling pattern but is likely driven by concurrent intracellular mechanisms. In agreement with this hypothesis, TNF-α is also strongly induced by Saps without any involvement of the NLRP3 inflammasome cascade. Coherently, cytokine production was also strongly affected, but not completely blocked, by an inhibitor of caspase-1, which, as expected, caused an accumulation of biologically inactive pro-IL-1β. All these observations suggest that Sap-mediated cytokine production is mediated via multiple pathways and confirm that Saps, well-known virulence factors of C. albicans, have a role as modulators of anticandidal immunity.
The pathway that leads to Sap induced NLRP3 inflammasome activation is not fully understood. However, our data may contribute to unraveling important aspects of the induction process. First, it appears that inflammasome activation and cytokine production occur through Sap internalization via a clathrin-dependent endocytic process. This process is strongly affected by chlorpromazine, and left unaffected by nystatin and cytochalasin D, which are inhibitors of other mechanisms of intracellular entry . Second, one of the sensing events could be an alternative cellular system involved in the recognition of microbial molecules, that is, the NLR pathway [40, 41]. Indeed, unlike several PRR, the NLR protein family senses microbial molecules intracellularly in the cytosol [42-46]. This mechanism has been described for some viral and bacterial proteins [47-49] and for some adjuvants such as aluminum salts . Since the inhibition of Sap internalization via the clathrin-dependent pathway results in a consistent inhibition of IL-1β and IL-18, it can be concluded that Saps must reach the cytoplasmic compartment to induce inflammasome activation. Proteases could be internalized in endosomes and then released in the cytosol as suggested by the lysosomal damage observed. Our results show that the inflammasome activation and cytokine production induced by Saps in monocytes are different from those induced by LPS-ATP treatment. In fact, unlike LPS-ATP, Sap2 and Sap6 inflammasome induction results in lysosome damage.
Since our experiments were generally performed in primary human monocytes, which have been reported to display constitutive inflammasome activity , we performed some additional experiments with other cell types (MDMs and DCs), which do not display constitutive inflammasome activation. In these cells, activation of the inflammasome usually occurs in two stages. In the first stage, often called priming, upregulation of NLRP3 and pro-IL-1β, caused by exposure to microbial ligands, is necessary. In the second stage, NLRP3 needs to be directly activated. Indeed Sap2 and Sap6 provide the second signal causing direct NLRP3 activation, as demonstrated by their capacity of inducing caspase-1-dependent IL-1β production. These data suggest that Saps can genuinely activate the inflammasome, rather than spuriously affecting inflammasome auto-activation.
There is overwhelming experimental, and a certain amount of clinical evidence that Saps are important factors of C. albicans virulence [5, 15, 51-54]. The demonstration that proinflammatory cytokine production is induced by Saps through inflammasome activation implies a new role for these virulence factors related to their structural features. In addition, the observation that selected Saps are capable of inducing the inflammasome directly, regardless of C. albicans, suggests that inflammasome activation triggered during the course of infection may be independent of dectin-1 recognition and Syk activation pathway, previously described for C. albicans .
Inflammasome activation may contribute to severe inflammation and recruitment of neutrophils, a scenario characteristic in the pathology of vaginal candidiasis . Expression of Sap proteases may contribute to the pathology of these superficial infections via activation of the inflammasome. This hypothesis is supported by the fact that expression of SAP genes and Sap antigens, including Sap2 and Sap6, is frequently detected during vaginal infection [15, 52], and that antibodies against Sap2 have been shown to be protective against C. albicans challenges in a rat vaginal infection model [56, 57]. Differences in the magnitude and mechanisms of induction are also likely, as in part suggested by some differences shown here between Sap2 and Sap6 in the kinetics of caspase-1 activation. In conclusion, while these aspects remain to be investigated, our data suggest that Sap production may contribute to the excessive inflammatory response observed during C. albicans infections, which may be the hallmark of at least some Candida pathologies.
Materials and methods
Aspartic protease production
As previously described by Borg-von Zepelin et al.  recombinant C. albicans aspartic proteases Sap1, Sap2, Sap3, and Sap6 were expressed as recombinant proteins using Pichia pastoris clones. After purification, Saps tested negative for endotoxin contamination in a Limulus assay (E-toxate, Sigma) with a sensitivity of 10 pg/mL of Escherichia coli LPS. Nevertheless, selected experiments were carried out at least once in the presence of 10 μg/mL of polymyxin B (Sigma) to neutralize any undetected contamination with bacterial LPS.
PBMCs were separated by density gradient centrifugation over Ficoll-Hypaque Plus (Pharmacia Biotech), recovered, washed twice, and suspended in RPMI 1640 supplemented with 10% FCS, 100 U penicillin/mL, and 100 μg streptomycin/mL, plated in cell culture flasks (Corning), and incubated for 1 h at a density of 2–3 × 106/mL. Adherent cells recovered were >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 . 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), 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 serum-free buffer with 150 mM KCl (10 mM Hepes, 5 mM NaH2PO4, 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 KH2PO4, 1 mM MgCl2, 1 mM CaCl2, 1% BSA, pH 7.4) . After incubation, supernatant fluids were recovered, then the presence of human IL-1β, IL-18, and TNF-α was measured by ELISA kit (Biosciences).
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
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.
Sap2 and Sap6 uptake
The uptake of Sap2 and Sap6 by human monocytes was analyzed by flow cytometry as previously described . 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 FITC-labeled 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.
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.
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 pro-IL-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).
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.
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.
The production of superoxide was analyzed using a membrane permeable probe: 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, Sigma), which is oxidized by H2O2 to dichlorofluorescein. Monocytes (2.5 × 105) were pretreated with 10 μM of H2DCF-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 H2DCF-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 H2DCFDA; 355 nm excitation and 485 nm emission for MCB).
Determination of lysosomal damage
To measure lysosomal membrane integrity, healthy, or apoptotic cells were stained with acridine orange (Sigma) as previously described . 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.
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.
The study was supported by the EU Marie-Curie project N° FP7-214004-2, FINSysB. We thank Catherine Macpherson for editorial assistance.
Conflict of interests
The authors declare no financial or commercial conflict of interest.