In these studies we utilized a chemically synthesized Hz to prevent contamination that could result from native Hz purification; the synthetic Hz is morphologically and chemically similar to native Plasmodium-isolated Hz (Fig. S1). Previously, we reported that both synthetic and native Hz induce similar expression profiles of chemokines and pro-inflammatory cytokines [7]. In addition, the synthetic Hz was subjected to elemental analysis to assess its purity. Theoretical calculated values of the molecular formula of Hz (C₆₈H₆₂N₈O₈Fe₂) give 66.35% of carbon (C), 5.08% of hydrogen (H) and 9.10% of nitrogen (N). We have obtained elemental values from our synthetic Hz preparation very close with the theoretical one (C: 66.5%; H: 5.3%; N: 8.9%). To further show the purity of Hz, we performed an agarose gel with 200 µg of Hz and we did not detect any trace DNA or RNA contamination (Fig. S2A) and treatment with DNase or RNase did not interfere with Hz-induced IL-1β production (Fig. S2B). These data indicate that our synthetic Hz preparation is high purity and free of contaminant.

To evaluate whether Hz activates the inflammasome, we measured IL-1β secretion by PMA-differentiated human monocytic cell line (THP-1) stimulated with increasing concentrations of Hz or MSU. Hz- and MSU-induced IL-1β production was found to be comparable (Fig. 1A). In accordance with previous studies showing that HSP-90 stability [11] modulates inflammasome assembly, we found that Hz-induced IL-1β secretion was reduced in the presence of the HSP-90 inhibitor geldanamycin D (Fig. 1B). Inhibition of caspase-1 activity using a specific competitor (Y-VAD-FMK) [12] or a broad caspase inhibitor (Z-VAD-CHO) also blocked Hz-induced IL-1β (Fig. 1C). To confirm the activation of caspase-1 we used the bone-marrow-derived macrophages (BMDM), since detection of the active form of caspase-1 in THP-1 cells is difficult as reported by others [13],[14]. Here, we show that Hz induced cleavage of caspase-1 to its enzymatically active (p10 subunit) form. BMDM were pre-stimulated with LPS in order to prime the induction of pro-IL-1β. As shown in Figure 1D, Hz and MSU, but not the pre-treatment with LPS, induced cleavage of caspase-1 and mature IL-1β production, which was completely abolished in BMDM from caspase-1 deficient mice.

These results suggest a role for the inflammasome in Hz-induced IL-1β production. To further establish which intracellular receptors and/or adaptor proteins are activated by Hz, we used BMDM from mice deficient in NLRP3, ASC or another NLR, NLRC4 (NLR containing CARD domain, also known as IPAF). We found that Hz- and MSU-induced caspase-1 activation and IL-1β maturation were dependent on NLRP3 and ASC but not NLRC4 (Fig. 2A). On the other hand, macrophages from NLRC4 mice failed to respond to Salmonella typhimurium infection (Fig. S3). To evaluate whether activation of the NLRP3 inflammasome is involved in Hz-induced inflammatory responses in vivo, mice were injected intraperitoneally with Hz and then neutrophil recruitment to the site of injection was examined. Hz induced significant recruitment of neutrophils to the peritoneal cavity in wild type, but not in ASC-deficient (Fig. 2B) or in NLRP3-deficient mice (Fig. 2C). As expected, NLRC4 was not involved in the inflammatory response induced by Hz (Fig. 2C). We further investigated whether IL-1β directly contributed to the recruitment of neutrophils. As expected, IL-1β deficient mice showed a significant decrease in the number of neutrophils elicited by Hz stimulation (Fig. 2D). However, we did not observe a complete abrogation of neutrophil influx as previously seen with IL-1 receptor-deficient mice stimulated with other inflammasome ligands [15]. These results suggest that a portion of the Hz-induced inflammatory response in vivo may results from other ligands of the IL-1 receptors and/or other cytokines and chemokines known to be induced by Hz [3],[7],[8].

Thus far, we have shown that Hz-induced IL-1β production is dependent on the NLRP3 inflammasome, in addition, it is known that IL-1β is involved in malarial fever [4]. To evaluate the role of IL-1β and the NLRP3 inflammasome during malarial disease we infected IL-1β- and NLRP3-deficient mice with Plasmodium chabaudi adami DS, which is a mouse virulent strain. Of interest, both IL-1β- and NLRP3 mice presented a slight but significant lower body temperature (Fig. 3A and 3B) and parasitemia (Fig. 3C and 3D) in the early phase of infection. These knockout mice also showed a significantly prolonged survival compared with wild type mice, but ultimately succumbed to the infection (Fig. 3E and 3F). Finally, in the late phase of infection, the level of IL-1β was significantly lower in NLRP3-deficient mouse in comparison with wild type mice (Fig. 3G) and was not detectable in IL-1β-deficient mouse (data not shown). These results indicate that IL-1β is an important factor in the pathophysiology during malaria infection.

Hz is rapidly engulfed by phagocytes, both in infectious and experimental conditions [2]. Therefore, to test the importance of phagocytosis on Hz-induced IL-1β production, cells were treated with cytochalasin D - a powerful actin polymerization inhibitor - prior to the addition of the crystals. Consistent with other crystals that induce inflammasome activation [15],[16],[17], we found that Hz-induced IL-1β seems to be dependent on its internalization (Fig. 4A). Furthermore, under certain conditions phagocytosis requires cholesterol-rich lipid domains [18] and as expected, cholesterol depletion by MβCD inhibited HZ-induced IL-1β (Fig. S5A), which was due to the disruption of lipid rafts (Fig. S5C). Further characterization of Hz phagocytosis by confocal immunofluorescence microscopy revealed that Hz was internalized in a vacuole that acquired lysosomal features, as shown by the presence of Lamp-1 surrounding the engulfed Hz phagosomes (Fig. 4B). Phagocytosis is generally accompanied by the generation of reactive oxygen species (ROS), which modulates inflammasome activation by crystals such as silica [19], MSU [15] and asbestos [20]. Since Hz induces ROS production [7] its requirement in Hz-induced IL-1β production was evaluated. The ROS scavenger, N-acetylcysteine (NAC) inhibited both Hz- and MSU-induced IL-1β production (Fig. 4C), which suggests a potential upstream role for ROS in inflammasome activation by Hz. Cellular potassium efflux is another critical step in inflammasome activation induced by all known NLRP3 activators [21],[22]. As shown in the Figure 4D, inhibition of potassium efflux by high concentrations of extracellular potassium decreased IL-1β production induced by Hz. The above results suggest that Hz shares a common mechanistic pathway in the activation of the NRLP3 inflammasome with classical triggers such as ATP and others insoluble crystals [21],[23].

Recently, lysosomal destabilization has been proposed as one mechanism whereby inorganic materials such as silica and aluminum hydroxide activate the inflammasome [17]. To assess lysosomal morphology in the context of Hz stimulation, we performed a confocal analysis of PMA-matured THP-1 cells loaded with a self-quenched conjugate of ovalbumin (DQ-OVA) that fluoresces only upon proteolytic degradation. We found that Hz did not affect the shape of lysosomes in comparison to untreated cells. In contrast, silica-treated cells contained swollen lysosomes (Fig. 4E), suggesting that Hz may activate the inflammasome through distinct, but related pathway. Indeed, inhibition of the lysosomal cysteine protease (cathepsin B) by the specific inhibitor CA-074 abrogated IL-1β induced by Hz and silica (Fig. 4F) [17]. However, it is still unclear how this enzyme is involved in inflammasome activation and indeed, many of the proximal signaling events in NLRP3 and NLR activation remain unknown.

Whereas we obtained clear evidence that Hz can induce IL-1β production in an inflammasome-dependent manner that required active cathepsin B, we did not find evidence of Hz-induced lysosomal rupture as previously reported with silica [17]. Release of cathepsin B without lysosomal rupture has been observed in monocytes treated with the potassium ionophore nigericin [24]. In addition, the widely expressed Spleen Tyrosine Kinase (Syk) was shown to be required for cathepsin B release into the cytosol in a model of B cell receptor-mediated apoptosis [25]. We therefore screened Hz-activated macrophages for changes in their tyrosine phosphorylation profiles. Consistent with the possible involvement of Syk, we observed a band with an apparent molecular weight of 72 kDa that was phosphorylated in response to Hz, but not MSU (Fig. 5A). We then carried out anti-Syk immunoprecipitation, followed by anti phospho-tyrosine analysis and found that Syk was phosphorylated in response to Hz, but not MSU stimulation (Fig. 5B). Even by extending the time-course of stimulation, MSU did not induce Syk phosphorylation (Fig S4A).

Syk is typically activated via receptors or adaptor proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs) or ITAM-like domains phosphorylated by Scr family kinases following receptor clustering [26],[27]. The Src kinase inhibitor PP2 decreased the Hz-induced Syk phosphorylation in a dose dependent manner (Fig. 5C). Syk activation can be mediated by the Scr family kinase member Lyn [28]. Lyn is typically found in lipid raft signaling platforms and disruption of these rafts by MβCD (Fig. S5C) indeed blocked, in dose-dependent manner, Syk phosphorylation in Hz-stimulated monocytes (Fig. S5B). Using BMDM from Lyn-deficient mice, we found that Hz-induced Syk phosphorylation required Lyn, and further confirmed that MSU does not utilize this signaling pathway in either murine or human macrophages (Fig. 5).

Next we evaluated the role of Lyn and Syk in Hz-induced IL-1β production. IL-1β secretion stimulated by Hz was inhibited in macrophages treated with the Syk inhibitor piceatannol (Fig. 6A), the Scr kinase inhibitor PP2 (Fig. 6B), and more specifically using Lyn-deficient BMDM (Fig. 6C). Importantly, in this last experiment, Hz-induced IL-1β production was only partially inhibited, which suggest that another member of the Src kinase family could play the same role of Lyn, since these kinases are known to be functionally redundant [28]. Of note, MSU-induced IL-1β production was not affected in Lyn-deficient BMDM pre-treated with LPS. To evaluate the relative roles of LPS and Hz in the induction of this signaling pathway, we treated BMDM with LPS and we observed that LPS by itself did not induce phospho-Syk, and indeed pre-treatment with LPS reduced Hz-induced Syk phosphorylation (Fig. S4B). Furthermore, Hz-induced Syk activation is not affected by the absence of the MyD88 adaptor protein (Fig. S4C). However, MyD88-deficient cells show a delay in the phosphorylation of c-jun N-terminal kinase (JNK) stimulated by LPS (Fig. S4C), similar as previously reported [29]. These results rule out a possible effect of LPS on Syk phosphorylation. Consistent with the involvement of this kinase in a pathway upstream of the inflammasome, NLRP3-, ASC- and NLRC4-deficient macrophages exhibited normal Syk phosphorylation upon Hz stimulation (Fig. 6D).

Syk activates various downstream signaling pathways, including phosphoinositide 3-kinase (PI3K) [30] and extracellular signal-regulated kinase (ERK). To test whether the PI3K pathway is required for propagation of the Syk signaling pathway following Hz exposure, the PI3K inhibitor wortmannin was used prior to Hz stimulation. Inhibition of PI3K indeed abrogated IL-1β maturation (Fig. 7A). We have previously identified MAPK activation upon Hz stimulation of macrophages [31]. We therefore attempted to isolate which pathways might be required for Hz-induced IL-1β production using known p38 and ERK kinase inhibitors. Whereas p38 phosphorylation can be observed following Hz stimulation, inhibition of p38 with SB203580 failed to block Hz-induced IL-1β production (Fig. 7B–D). On the other hand, inhibition of ERK with Apigenin abrogated Hz-induced IL-1β secretion (Fig. 7E). Altogether, these results reveal that Lyn/Syk activation following Hz exposure initiates the PI3K and ERK signaling pathways and these pathways appear to regulate the production of mature IL-1β.

While a number of stimuli are known to activate the NLRP3 inflammasome, there is no evidence that NLRP3 directly recognizes these ligands. Therefore an indirect pathway of NLRP3 activation is likely, however the identity of the direct molecular switch of NLRP3 has not been identified. Our studies provide the first evidence for a role of tyrosine kinase signaling molecules in NLRP3 activation. To examine whether Syk can modulate the inflammasome by directly interacting with its components, we immunoprecipitated Syk and then immunoblotted for potential partners associated with Syk by silver staining and western blotting (Fig. 8A). Selected differential bands were analyzed by LC-tandem mass spectrometry. Interestingly, two to three different peptides covering 11–23% of the Pyrin domain (Pyd) [32] were identified. Pyrin domains are known to mediate protein-protein interactions and are crucial in many of the NLR inflammasome complexes, and in particular, mediate the NLRP3 and ASC interaction [6]. We therefore confirmed by western blotting whether NLRP3 or ASC can be co-immunoprecipitated (co-IP) with Syk. Whereas NLRP3 was shown to weakly interact with Syk, ASC was found to strongly associate with this kinase upon Hz stimulation (Fig. 8B). These findings suggest that Syk, and possibly other unidentified signaling kinases, can associated with the ASC/NRLP3 inflammasome.

Another possible mechanism is that Syk could be controlling the NLRP3 inflammasome by regulating cathepsin B activation. First, we tested if Hz can induce release of the active form of cathepsin B in the supernatant and as showed in the Figure 9A, Hz did not induce cathepsin B release into supernatant as has been observed with MSU and silica. However, using a cathepsin B substrate that emits red fluorescence upon cleavage we demonstrated that Hz induces rapid (30 min) and transient (maximum 1.5 h) intra-compartmental cathepsin B activation that was dependent on Syk activation (Fig. 9B). These results indicate that Syk not only can associate with the inflammasome component but it can also modulate cathepsin B activation.

With the subheading Ethics Statement, all protocols used in this study were approved by the Institutional Animal Care and Use Committees at the McGill University or Yale University. IL-1β- and Lyn-deficient mice were provided by Dr. G.Sébire and Dr. K. W. Harder (University of Sherbrooke, Quebec and University of British Columbia, Vancouver, Canada), respectively. The generation of IL-1β-, Lyn-, NLRP3-, ASC-, caspase-1-, and NLRC4-deficient mice has been described previously [47],[48],[49],[50],[51]. Caspase-1-, ASC-, and NLRP3-deficient mice were backcrossed onto the C57BL/6 genetic background for at least nine generations. NLRC4-deficient mice were backcrossed onto the C57BL/6 genetic background for at least six generations. Age- and sex-matched C57BL/6 mice purchased from the National Cancer Institute or Charles River were used as WT controls.

Hemin (>99% of purity) was purchased from Fluka; RPMI-1640 medium, Penicillin-Streptomycin-Glutamine (PSG) from Wisent, fetal bovine serum (FBS), Alpha MEM medium from Gibco; CV-Cathepsin B detection kit, PP2, piceatannol, geldanamycin, cytochalasin D, Y-VAD-FMK and Z-VAD-CHO from Biomol; MSU, anti-human NLRP3 and ASC from Alexis Biochemical; inhibitor protease cocktail from Roche; CHAPs from Fisher; A/G-coupled agarose beads, anti-human pro-IL-1β, anti-human or murine caspase-1 and anti-Syk from Santa Cruz; True Blot anti-rabbit Ig, anti-phosphoY/HRP from eBioscience; PVDF from Bio-rad; anti-LAMP-1 Ab from Developmental Studies Hybridoma Bank at the University of Iowa; anti-human mature IL-1β, anti-pp38 and anti-p38 from Cell signal; anti-pSyk and anti-pY (4G10) from Upstate; rat or goat anti-murine IL-1β and recombinant IL-1β from R&D system; DQ-OVA from Invitrogen; anti-rat AlexaFluor 568, cholera toxin B-AlexaFluor 568 from Molecular Probes; DRAQ5 from Biostatus; Fluoromount-G from Southern Biotechnology; all others unlisted or not indicated reagents were purchased from Sigma. L929 and THP-1 cell line from ATCC. MyD88 KO BMDM was generated from MyD88-deficient mice and kindly supplied by Dr. Danuta Radzioch (McGill University, Montreal, Canada).

Native and Synthetic Hz have been obtained as previously described [8],[31]. We have modified synthetic Hz preparation, using high purity chemical reagents (>99% of purity), as follows: 0.8 mmol Hemin was dissolved in degassed NaOH (0.1 M) for 30 minutes with mild stirring. pH 4.0 was adjusted adding drop-wise propionic acid. The mixture was allowed to anneal at 70°C for 18 hours. Then washed three times with NaHCO₃ (0.1 M) for three hours and the last wash with MeOH. All washes were alternated with distilled H₂O. Finally, the sample was then dried in a vacuum oven overnight over phosphorous pentoxyde. All synthetic hemozoin samples were analyzed by X-ray powder diffraction, field emission gun scanning electron microscopy, and infra-red spectroscopy to characterize the crystalline state of Hz. Hz purity was assessed by elemental analysis [52].

THP-1 cells (ATCC) were cultured with RPMI-1640 medium supplemented with 10% FBS, 1% PSG, 50 µM of 2-β-mercaptoetanol, Glucose 4.5 g/L and 1 mM sodium pyruvate. THP-1 differentiation: (1.5×10⁶ cells/mL) were incubated with 0.5 µM of PMA, after three hours cells were washed and plated at 0.75×10⁶ cells/mL or 0.2×10⁶ cell/0.5 mL in 12 well plates (IL-1β) or 24 well plates containing coverslips (confocal) and incubated for 20–24 hours. This treatment increases the phagocytic properties of the cells and induces a constitutive production of pro-IL-1β. Prior to stimulation, cells were washed and 500 µL of Alpha MEM medium without FBS was replaced. Cells were pre-treated with different drugs for 1 hour and stimulate with Hz, MSU or silica as indicated in figure legends.

Gender and age matched wild type (WT), NLRP3- or IL-1β-deficient mice were injected i.p. with 5×10⁴ Plasmodium chabaudi adami DS infected red blood cells obtained from syngeneic infected mice. Parasitemia was assessed at day 5, 7 and then every day by examination of Giemsa stained blood smears and was expressed as mean parasitemia. Body temperature was measured using an infrared thermometer (La Crosse Technology). Survival of mice was monitored and blood serum was collected when the temperature dropped down to 26°C. IL-1β was measured by ELISA with rat monoclonal and goat anti-mouse IL-1β. The detection limit was 6.25 pg/mL of IL-1β.

Bone marrow cells were obtained by flushing the femurs and tibias from mice. Cells were used from fresh or from frozen marrows. Erythrocytes were lysed with 2 mL of NH₄Cl (155 mM) in Tris/HCl (10 mM), pH 7.2 (9∶1 solution)/mouse. Bone marrow cells were adjusted to 7×10⁶ cells/10 mL and plated in 100 mm dishes with RPMI-1640 medium supplemented with 1% of PSG, 10% FBS and 30% (v/v) L929 cell culture supernatant. The supernatants of bone marrow cells were changed every two days in order to renew the cytokines and nutrients. After 7 days, the culture dishes were washed with PBS and replaced by ice cold PBS, incubated on ice for 15 min and cells were vigorously detached. BMDM were adjusted to 1.5×10⁶/2 mL or 0.2×10⁶ cells/0.5 mL in RPMI medium supplemented with 5% FBS (Gibco) and 1% of PSG and plated in 6 well plates (IL-1β) or 24 wells plate (confocal). The next day, cells were washed with warm PBS (37°C) and replaced by 500 µL of Alpha MEM medium without FBS. Cells were, as indicated in figure legends, stimulated with Hz, MSU or infected with Salmonella typhimurium as described by Franchi et al. [53].

Supernatant and cell extract analysis: After designated incubation time, supernatants were collected and protein was precipitated with trichloroacetic acid at 10% final concentration. Precipitates were then dissolved in Tris/HCl 0.1 mM pH 8.0 and Laemmli sample load buffer. Cell extracts were obtained by lysing cells with Igepal 1% (for signaling, in 1× PBS, 20% Glycerol, 1× inhibitor protease cocktail, 2 mM Na₃VO₄ and 1 mM NaF) or triton 1% (for caspase-1, in TNE buffer: 10 mM Tris/HCl pH 7.5, 150 mM NaCl, 5 mM EDTA and 1.5× inhibitor protease cocktail). Whole supernatant protein and equal amount of protein or cell lysate were subjected to SDS-PAGE and immunoblot analysis.

IP: Cells lysates were extracted with lysis buffer (1% CHAPs detergent in TNE buffer, 1× inhibitor cocktail, 2 mM Na₃VO₄ and 1 mM NaF). Cells lysates were pre-incubated for two hours at 4°C with protein A/G-coupled agarose beads and 1 µg of unspecific matched isotype control antibody (Ab). Equal amount of protein were immunoprecipitated with protein A/G-coupled agarose beads or True Blot anti-rabbit Ig and 2 µg of specific or unspecific matched isotype control Ab overnight. Beads were spun down 3 times with lysis buffer and proteins were denatured in Laemmli load buffer.

SDS-PAGE/Immunoblot: Samples from supernatants, cell extracts or IP were subjected to 10% (signaling) or 15% (IL-1β and caspase-1) acrylamide gel (all reagents from Laboratoire Mat. Inc., Montreal, Qc, Canada) or 4–12% NuPAGE® gel (for p10 caspase-1 and IP, Invitrogen). After transfer onto PVDF membranes, they were subjected to immunoblot analysis with the indicated Ab and matched secondary HRP-conjugated Ab. In some experiments, optical density was determined using AlphaDigiDoc 1000 v3.2 software (Alpha Innotech corporation).

OVA uptake: THP-1 cells (0.2×10⁶ cells/coverslip 12 mm from Fisher) were treated with 10 µg of DQ-OVA in the absence or presence of Hz (200 µg/mL) or Silica (400 µg/mL) for 30 min, washed and incubated up to three hours. Laser settings were adjusted on DQ-OVA fluorescence emission that is stronger than hemozoin or silica. Phagosome: BMDM were fixed, permeabilized using 0.1% Triton X-100, and non-specific surface Fcγ-receptor binding were blocked as described [54]. For immunofluorescence experiments, cells were labelled with the rat anti-LAMP-1 Ab and an anti-rat AlexaFluor 568. DRAQ5 was used to visualize DNA. Cathepsin B activity: THP-1 cells (0.2×10⁶ cells/coverslip 12 mm from Fisher) were pre-treated for 30 min with 5 µM of piceatannol and stimulated or not with Hz (200 µg/mL). A cathepsin B substract (Arg-Arg)₂ linked with cresyl violet were given 30 min before the end of incubation time and cleaved substract generated a red fluorescence. All coverslips (THP-1/OVA or BMDM) were mounted on slides with Fluoromount-G. Detailed analysis of protein localization on the phagosome was performed by using an oil immersion Nikon Plan Apo 100 (N.A. 1.4) objective mounted on a Nikon Eclipse E800 microscope equipped with a Bio-Rad Radiance 2000 confocal imaging system (Bio-Rad Laboratories, Hercules, CA).

WT, IL-1β-, NLRP3-, ASC-, caspase-1- and NLRC4-deficient mice were injected intraperitoneally with 800 µg of hemozoin in 1 ml of endotoxin-free PBS. Control groups were injected with 1 mL of PBS. After six hours, the mice were euthanized and the peritoneal cavity was washed with 10 mL of PBS. Cells recovered from the peritoneum were counted and the percentage of neutrophils was determined from an H&E stain (DiffQuick; Dade Behring, Inc.) of a cytospun sample.

Unpaired Student's t-test was used when comparing two groups and ANOVA/Bonferroni test when comparing more than two groups. The differences were considered significant when p<0.05. Survival curves for infected and control mice were compared using the Mantel-Haenszel test. Statistical analysis was performed using Prism 5.00 software (GraphPad, San Diego, Calif.).