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Trichothecene mycotoxins activate NLRP3 inflammasome through a P2X7 receptor and Src tyrosine kinase dependent pathway
Human Immunology 75 (2014) 134–140
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Trichothecene mycotoxins activate NLRP3 inflammasome through a P2X7 receptor and Src tyrosine kinase dependent pathway Päivi Kankkunen a,⇑, Elina Välimäki a,b, Johanna Rintahaka a,1, Jaana Palomäki a, Tuula Nyman a,b, Harri Alenius a, Henrik Wolff a, Sampsa Matikainen a a b
Finnish Institute of Occupational Health (FIOH), Topeliuksenkatu 41A, 00250 Helsinki, Finland Institute of Biotechnology, University of Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 18 April 2013 Accepted 11 November 2013 Available online 20 November 2013
a b s t r a c t Inflammasome is an intracellular molecular platform of the innate immunity that is a key mediator of inflammation. The inflammasome complex detects pathogens and different danger signals, and triggers cysteine protease caspase-1-dependent processing of pro-inflammatory cytokines IL-1b, and IL-18 in dendritic cells and macrophages. Previously, we have shown that water-damaged building associated trichothecene mycotoxins, including roridin A, trigger IL-1b and IL-18 secretion in human macrophages. However, the molecular basis as well as mechanism behind this trichothecene-induced cytokine secretion has remained uncharacterized. Here, we show that the trichothecene-induced IL-1b secretion is dependent on NLRP3 inflammasome in human primary macrophages. Pharmacological inhibition and small interfering RNA approach showed that the trichothecene-induced NLRP3 inflammasome activation is mediated through ATP-gated P2X7 receptor. Moreover, we show that trichothecene-triggered NLRP3 inflammasome activation is dependent on Src tyrosine kinase activity. In addition, gene silencing of cCbl, a negative autophagy-related regulator of c-Src, resulted in enhanced secretion of IL-1b and IL-18 in response to trichothecene mycotoxin stimulation in human macrophages. In conclusion, our results suggest that roridin A, a fungal trichothecene mycotoxin, acts as microbial danger signals that trigger activation of NLRP3 inflammasome through P2X7R and Src tyrosine kinase signaling dependent pathway in human primary macrophages. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The immune system recognizes and responds to infection, tissue damage, and/or cellular stress by pattern recognition receptors (PRRs) of the innate immune system. Interleukin (IL)-1b, IL-18, and tumor necrosis factor (TNF) are important pro-inflammatory cytokines which are produced by macrophages in response to PRR activation [1]. Production of TNF is mainly regulated at transcriptional level. In contrast, secretion of IL-1b and IL-18 is strictly regulated by post-translational mechanisms: both IL-1b and IL-18 are synthesized as immature forms: pro-IL-1b and pro-IL-18, respectively. Cysteine protease caspase-1 cleaves these pro-forms to biologically active equivalents which are then secreted via unconventional Abbreviations: AG126, tyrphostin AG 126; AZ, AZ11645373; DAMP, danger associated molecular pattern; NLR, NOD-like nucleotide-binding oligomerization domain; NLRP, NLR family, Pyrin domain containing; PP2, 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, RLR, RIG-I-like receptors; RU, relative unit; siRNA, small ribonucleic acid interference. ⇑ Corresponding author. E-mail address: paivi.kankkunen@ttl.fi (P. Kankkunen). 1 Present address: University of Helsinki, Finland.
protein secretion pathway [2,3]. Caspase-1 in turn is activated in a protein complex called the inflammasome. At present, several caspase-1 activating NOD-like receptor (NLR) structures have been characterized that include NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 inflammasomes [4–9]. Of these NLRP3 is the most extensively studied because of its function in antimicrobial defence, adjuvanticity, and autoinflammatory diseases [4,7]. In addition to NLRs, absent in melanoma 2 (AIM2)-like receptors are capable of forming a caspase-1 activating inflammasome complexes [10]. Fungi are heterotrophic eukaryotes which interact with humans in multiple ways establishing symbiotic, commensal, latent, and pathogenic relationships. Most fungi are ubiquitous in environment, and humans are exposed to inhaling fungi spores, and their metabolites including cell wall components and mycotoxins. Trichothecene mycotoxins are produced by several fungi including Fusarium, Trichoderma, Myrothecium, and Stachybotrys species. These fungal toxic metabolites were originally isolated as possible antimicrobial agents but they are also harmful and toxic to animals and humans. Trichothecene producing Stachybotrys chartarum is frequently linked to water-damaged buildings and its connection
0198-8859/$36.00 - see front matter Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2013.11.010
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to damp-building associated illnesses has also been proposed [11]. We have previously shown that trichothecene mycotoxins, including roridin A, verrucarin A and T-2 toxin, activate caspase-1 resulting in IL-1b and IL-18 secretion in LPS-primed human macrophages [12]. Here, we have further characterized the mechanism behind this trichothecene-induced cytokine release. 2. Materials and methods 2.1. Cells Human peripheral blood mononuclear cells (PBMCs) from healthy blood donors (Finnish Red Cross Blood Transfusion Service) were isolated and allowed to differentiated into macrophages according the protocol that has been previously described [12]. After seven days of culture, the resulting macrophages were used in the experiments.
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150 mM NaCl, 1% NP-40, and 1 mM EDTA supplemented with phosphatase inhibitor cocktails 2 and 3 and protease inhibitor cocktail (Sigma–Aldrich). After incubation on ice for 1 h the cell extract was centrifuged (12,000g for 30 min) and supernatants were collected and used for Western blot analysis. The proteins were visualized by ImageQuant LAS 4000 mini luminescent image analyzer according to manufacturer’s instruction (GE Healthcare BioScience Ab, Uppsala, Sweden). Anti-P2X7 Abs (H-265) and antiCbl (A-9) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-phospho-Src (Tyr416) from Cell Signalling Technology (Beverly, MA). To confirm equal protein loading and transfer during SDS–PAGE and immunoblotting, respectively, membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain according to the manufacturer’s instructions (Sigma–Aldrich). The major basally expressed protein band is shown as a loading control.
2.5. Statistical analyses 2.2. Reagents AZ11645373 (AZ; 1 lM), ATP (3 mM), LPS (Escherichia coli 0111:B4) (1 lg/ml), diphenyleneiodonium chloride (DPI; 1 lM), N-acetyl-L-cysteine (NAc; 5 mM), PP2 (5 lM), potassium chloride (KCl; +1 mM supplementation), and tyrphostin AG 126 (AG126; 50 lM), roridin A (10 ng/ml), verrucarin A (10 ng/ml), and poly(deoxyadenylic-thymidylic) acid sodium salt (poly(dA:dT); 2 lg/ml) were purchased from Sigma–Aldrich (St. Luis, MO). Cathepsin B inhibitor, CA-074-Me (10 lM), was purchased from Calbiochem (San Diego, CA). Optimal concentrations for the reagents were titrated in preliminary experiments.
Each human macrophage sample represents a pool of separately stimulated cells from three different blood donors. ELISA results are combined through at least three different stimulations and data were expressed as means (±SD) unless otherwise mentioned. Quantitative RT-PCR and Western blot results are representative of at least two independent, but similarly performed experiments. In
2.3. Cell stimulations and small interfering RNA (siRNA) experiments Human macrophages were pretreated with LPS for 2 h prior to roridin A and verrucarin A stimulation for 4 h unless otherwise mentioned. When inhibitory reagents were used they were added to the cells 30 min prior to roridin A and verrucarin A stimulation. In ATP-stimulation experiments, the cells were treated with LPS for 6 h prior to ATP stimulation with 30 min. AZ were added to the cells 4.5 h prior to ATP addition. When Poly(dA:dT) was used, it was transfected into cell cytoplasm by lipofectamine 2000 (Life Technologies, Paisley, UK) according to the manufacturer’s instruction. For siRNA experiments, the cells were transfected with nontargeting control siRNA (AllStars Negative Control siRNA, Qiagen) or with siRNAs against NLRP3, P2X7, Cbl small interference RNA (siRNA) (Hs_CIAS1_6, Hs_CIAS1_9, Hs_CIAS1_10, Hs_CIAS1_11, and Hs_P2RX7_1, Hs_P2RX7_2, and Hs_CBL_7, Hs_CBL_8, respectively; Qiagen) treatments were performed as previously described [13,14]. 2.4. Quantitative RT-PCR, enzyme-linked immunosorbent (ELISA), and Western blot analysis Quantitative real-time RT-PCR analysis of expression of IL-1b, IL-18, and NLRP3 mRNA and ELISA analysis of secreted IL-1b level from human cells or cell culture supernatants were performed as previously described [13]. Pre-developed TaqManÒ assay primers and probes (IL-1b Hs_0017497_m1, IL-18 Hs_00155517_m1, NLRP3 Hs_00918082_m1 Applied Biosystems) were used according to manufacturer’s instructions. The ELISA analysis of human TNF was performed according to manufacturer’s instruction (eBioscience, San Diego, CA). For protein analysis, total cell lysates were prepared and Western blot analysis was performed as previously described [12]. For phospho-protein analysis, the cells were suspended in a lysis buffer containing 50 mM Hepes (pH 7.4),
Fig. 1. Roridin A activates NLRP3 inflammasome in human macrophages. (A and B) Human macrophages were treated with LPS (1 lg/ml) for 18 h or left untreated after which the cells were stimulated with roridin A (10 ng/ml; RoA) for the times indicated. After this the cell culture supernatants were collected and (A) IL-1b and (B) IL-18 secretion was analyzed by ELISA. Values are means ± SD from at least three independent analyses. A unpaired t-test was used to compare the differences between the samples. ⁄⁄⁄p < 0.001, ⁄p < 0.05. (C and D) Human macrophages were transfected with non-targeting siRNA control (NT-i) or NLRP3 specific siRNA molecules (NLRP3-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. (C) The cells were harvested, total cellular RNA was isolated, and RT-PCR analysis of NLRP3, IL-1b, and IL-18 mRNA expression was performed. For data expression, LPS and roridin A co-stimulation induced NLPR3, IL-1b, and IL-18 mRNA expression was set to 100%. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. Values are means ± SD from duplicate analyses from a single experiment. (D) Cell culture supernatants were collected and IL-1b IL18, and TNF secretion were analyzed with ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b, IL-18, or TNF secretion were set to 100%. Values are means ± SD from two independent analyses including 3 blood donors for each. ⁄p < 0.05.
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Fig. 2. Trichothecene induced IL-1b and IL-18 secretion is ROS, K+ fluxes, and cathepsin B release dependent process in LPS-primed human macrophages. Macrophages were left un-stimulated or stimulated with LPS (1 lg/ml) for 2 h, after which the cells were stimulated with roridin A (10 ng/ml; RoA) for 4 h in the absence and presence of DPI (1 lM), NAc (5 lM), K+ (+1 mM supplementation), or CA-074-Me (10 lM). The cell culture supernatants were collected and (A and E) IL-1b and (B and F) IL-18 secretion was analyzed with ELISAs. Values are means ± SD from three independent analyses. ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄p < 0.05. (C, D, G, and H) The cells were harvested, total cellular RNA was isolated, and RT-PCR analysis of IL-1b and IL-18 mRNA expression was performed. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. Results are expressed as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment.
RT-PCR figures, the RU values are means ± SD from duplicate analyses from a single experiment unless otherwise mentioned. All data were analyzed by using the GraphPad Prism 4 Software (GraphPad Software Inc., San Diego, CA). A one-tailed t-test was used to compare the differences between the samples unless otherwise mentioned. A p-value of <0.05 was considered to be statistically significant. 3. Results 3.1. Roridin A activates canonical caspase-1 containing NLRP3 inflammasome in human macrophages To study the secretion kinetics of IL-1b and IL-18 in response to trichothecene mycotoxins, macrophages were left untreated or treated with LPS for 18 h after which the cells were treated with roridin A for different time periods. Roridin A strongly activated IL-1b and IL-18 secretion at 4 h post-stimulation in LPS-primed macrophages (Fig. 1A and B). Roridin A alone was able to induce IL-18 secretion, but not that of IL-1b, at 8 h post-stimulation (Fig. 1A and B). IL-1b secretion activating molecular platforms include many different inflammasome structures [4–8]. To study whether the inflammasome structure activated by trichothecene mycotoxins is NLRP3, we performed gene silencing experiments. Macrophages were transfected with non-targeting or NLRP3 specific siRNA molecules as described in Section 2. After this macrophages were primed with LPS for 2 h and subsequently the cells were
stimulated with roridin A for 4 h. The cells were collected and total-RNA was isolated for RT-PCR analysis. Similarly, cell culture supernatants were collected and used for ELISA analysis. RT-PCR analysis verified the silencing of NLRP3 mRNA expression (Fig. 1C). In contrast, siRNA treatment had no effect on IL-1b mRNA expression (Fig. 1D). However, the NLRP3 gene-silencing decreased IL-1b and IL-18 secretion in LPS-primed and roridin A-stimulated macrophages (Fig. 1E). However, the treatment did not affect TNF secretion level (Fig. 1E). The results suggest that roridin A activates NLRP3 inflammasome resulting in IL-1b and IL-18 secretion in LPSprimed human macrophages. 3.2. ROS formation, potassium efflux, and cathepsin B activity are needed for roridin A-induced IL-1b and IL-18 secretion Microbial toxins and crystalloid substances trigger ROS formation, which has been suggested to be a key event in NLRP3 inflammasome activation [15]. Therefore, we analyzed whether ROS formation is required for roridin A-induced IL-1b and IL-18 release in human macrophages. Both ROS inhibitors, NAc and DPI, decreased roridin A-induced IL-1b and IL-18 release in LPS-primed macrophages indicating that ROS formation is essential for roridin A-triggered IL-1b and IL-18 release in human macrophages. ROS inhibitors had only little effect on IL-1b and IL-18 mRNA expression (Fig. 2C). In addition to ROS, potassium efflux has been shown to facilitate NLRP3 inflammasome activation in response to a variety of danger signals [16]. To study the role of potassium efflux in roridin
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Fig. 3. Roridin A activates NLRP3 inflammasome via a P2X7R-dependent pathway. (A and B) Macrophages were untreated or treated with LPS (1 lg/ml) for 2 h prior to roridin A (10 ng/ml; RoA) stimulation for 4 h in the presence or absence of P2X7 receptor inhibitor AZ11645373 (1 lM; AZ). In parallel, after LPS (1 lg/ml) priming for 6 h, the cells were exposed to ATP (3 mM) for 30 min in the presence or absence of AZ11645373 (1 lM; AZ). (A) The cells culture supernatants were collected and IL-1b secretion was analyzed by ELISA. Values are means ± SD from five independent analyses. ⁄⁄⁄p < 0.001. (B) Cells were harvested and used for IL-1b mRNA expression analysis. Results are expressed as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment. (C–E) Macrophages were transfected with non-targeting siRNA control (NT-i) or P2X7R siRNA molecules (P2X7-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. Cell culture supernatants were collected and the cells were harvested and cell lysates were prepared. (C) Western blot analysis of cell lysates was done with anti-P2X7R Ab. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. To confirm equal loading and transfer of the protein samples in Western blot assays, the membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain. The major protein band detected and basally expressed is shown as a loading control in Western Blot analysis (indicated as control). (D) IL-1b and (E) TNF secretion was analyzed from cell culture supernatants by ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b or TNF secretion levels without silencing were set to 100%. Values are means ± SD from two independent analyses.
A-induced IL-1b and IL-18 secretion, human LPS-primed macrophages were stimulated with roridin A in the presence and absence of additional potassium chloride as described in Section 2. The enhanced potassium chloride concentration completely blocked roridin A-induced IL-1b and IL-18 release (Fig. 2 A and B). However, the enhanced potassium chloride concentration did not significantly affect IL-1b nor IL-18 gene expression (Fig. 2C). Lysosomal destabilization and the release of cathepsin B from the lysomal compartment is involved in the activation of NLRP3 inflammasome [4]. To study the role of cathepsin B in trichothecene induced NLRP3 inflammasome activation we used cathepsin B specific inhibitor, CA-074-Me. As shown in Fig. 2E and F, both IL-1b and IL-18 secretion in response to roridin A stimulation was reduced in LPS-primed macrophages in the presence of CA074-Me (Fig. 2E and F). In contrast, the inhibitor did not affect IL1b or IL-18 mRNA expression level (Fig. 2G and H). 3.3. Roridin A induced NLRP3 inflammasome activation is mediated through P2X7R Recently, it has been suggested that NLRP3 inflammasome activation in response to monosodium urate, silica, and alum is dependent on purinergic P2X7 receptor (P2X7R) [17]. We used pharmacological inhibitor of P2X7R and siRNA approach to study the role of this receptor in roridin A-triggered NLRP3 inflammasome activation. Macrophages were untreated or treated with LPS and roridin A in the absence and presence of P2X7R antagonist AZ11645373 (AZ). AZ-treatment decreased IL-1b secretion from LPS-primed human macrophages in response to roridin A stimulation (Fig. 3A). The treatment had no effect on IL-1b mRNA expression (Fig. 3B). In parallel, the AZ treatment blocked ATP-induced IL-1b secretion in LPS-primed human macrophages (Fig. 3A). Next, we performed siRNA studies with P2X7R specific siRNAs in human
primary macrophages. In line with the inhibitor data, the P2X7R specific siRNA treatment of macrophages resulted in decreased IL-1b secretion in LPS-primed and roridin A-stimulated macrophages (Fig. 3C and D). However, gene silencing had no effect on TNF secretion (Fig. 3E). The efficacy of the siRNA-treatment was verified with Western blotting (Fig. 3F). 3.4. Src kinase activity is essential for roridin A-induced NLRP3 inflammasome activation in human macrophages It has been shown that ATP-induced IL-1b secretion is inhibited by a synthetic tyrosine kinase inhibitor, tyrphostin AG126 (AG126) [18,19]. To study the role of tyrosine kinase signaling in roridin Ainduced NLRP3 inflammasome activation, LPS-primed macrophages were stimulated with roridin A in the presence and absence of AG126. The inhibitor abolished roridin A-induced IL-1b and IL18 secretion in LPS-primed cells indicating that tyrosine kinase signaling is needed for roridin A-induced inflammasome activation (Fig. 4A and B). The inhibitor had no effect on IL-1b or IL-18 mRNA expression levels (Fig. 4C and D). Src family of tyrosine kinases has an important role in the activation of innate immune response in TLR and RLR signaling pathways [20,21]. To study whether this pathway is important for NLRP3 inflammasome activation caused by roridin A, we used the Src tyrosine kinase inhibitor PP2. Macrophages were untreated or treated with LPS for 2 h prior to roridin A stimulation for 4 h in the presence and absence PP2. The inhibitor abrogated secretion of IL-1b and IL-18 in LPS-primed and roridin A-stimulated macrophages (Fig. 4E and F). In contrast, PP2-treatment had no effect on IL-1b or IL-18 mRNA expression in LPS-primed and roridin A co-stimulated cells (Fig 4G and H). In addition, PP2 inhibited IL-1b secretion induced by verrucarin A (Fig. 4I), which is also a caspase-1 activating trichothecene mycotoxin [12]. In line with
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Fig. 4. Src tyrosine kinase signaling is essential for trichothecene engaged NLRP3 inflammasome activation. Human macrophages were primed with LPS (1 lg/ml) for 2 h after which they were exposed to (A–H) roridin A (10 ng/ml; RoA) for 4 h hour in the absence or presence of (A–D) AG126 (50 lM) or (E–H) PP2 (5 lM). (A and E) IL-1b and (B and F) IL-18 secretion was measured by ELISA from the collected cell culture supernatants. The means at least three separate experiments (±SD) are shown. ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄ p < 0.05. (C, D, G, and H) The cells were collected, total RNA was isolated. After this (C and G) IL-1b (D and H) IL-18 mRNA expression was analyzed by RT-PCR. The results are representative of three independent, but similarly performed experiments. The analyzed results are shown as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment. (I and J) Macrophages were left untreated or primed with LPS (1 lg/ml) for 2 h and stimulated with verrucarin A (10 ng/ml; VerA) or (J) transfected with Poly(dA:dT) (2 lg/ml) for 4 h in absence and presence of PP2 (5 lM). IL-1b secretion was analyzed from cell culture supernatants with ELISA. The means of two separate experiments (±SD) are shown. (K) Macrophages were stimulated with roridin A (10 ng/ml; RoA) for the times indicated, cell lysates were prepared and Western blot analysis was performed with anti-phopho-Src Abs. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. To confirm equal loading and transfer of the protein samples, membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain. The major protein band detected and basally expressed is shown as a loading control in Western Blot analysis (indicated as control).
the PP2 data, also Src inhibitor I reduced IL-1b and IL-18 secretion triggered by roridin A (Supplementary Fig. 1). The inhibitor did not significantly influence on IL-1b and IL-18 mRNA transcription level (Supplementary Fig. 1). Next we studied the role of Src tyrosine kinases in AIM2 inflammasome activation. Macrophages were primed with LPS for 2 h and after that transfected with synthetic double-stranded DNA, poly(dA:dT), for 4 h in the presence and absence of PP2. After this cell culture supernatants were harvested and secretion of IL-1b was analyzed. PP2 treatment clearly inhibited IL-1b secretion triggered by poly(dA:dT) transfection in LPSprimed human macrophages (Fig. 4J). After this we wanted to verify that Src family of tyrosine kinases are activated in macrophages in response to roridin A stimulation. Src activity is positively regulated by tyrosine phosphorylation at Tyr416 in the activation loop of the kinase domain [22]. Macrophages were exposed to roridin A for different time periods after which protein lysates were prepared and Src tyrosine kinase activity was analyzed by Western blotting with anti-phospho-Src family (Tyr416) Abs. As shown in Fig. 4K, roridin A activated tyrosine phosphorylation of Src (60 kDa) at 1 h post-stimulation. 3.5. Autophagy-related c-Cbl negatively regulates NLRP3 inflammasome activation in human macrophages Recently, it was shown that E3 ubiquitin ligase c-Cbl acts as an autophagy cargo receptor for Src to mediate Src degradation [23]. To study whether c-Cbl regulates Src function and NLRP3
inflammasome activation in human macrophages, we silenced cCbl expression with siRNA approach. Efficiency of the siRNA treatments were verified with Western blotting (Fig. 5A). As shown in Fig. 5B and C, silencing of c-Cbl enhanced IL-1b or IL-18 secretion in LPS-primed and roridin A-stimulated human macrophages (Fig. 5B and C). In contrast, there were no changes in TNF expression level (Fig. 5D). 4. Discussion The inflammasome is a cytoplasmic multiprotein complex that controls secretion of pro-inflammatory cytokine IL-1b through activation of cysteine protease caspase-1 [24]. NLRP3, a member of the NLR family of PRRs is the most studied inflammasome structure. NLRP3 together with adaptor protein ASC, drives caspase-1 activation in response to diverse stimuli including those of endogenous and microbial origin, as well as environmental crystalline particles [4,7]. These endogenous and exogenous stimuli are chemically and structurally heterogeneous suggesting that they are not directly recognized by the NLRP3 inflammasome. It is more likely that they activate inflammasome indirectly by inducing changes in endogenous molecules and/or disturbances in intracellular ionic concentrations. Here we provide evidence that purinergic P2X7R is involved in NLRP3 inflammasome activation in response to fungal trichothecene mycotoxins in human macrophages: both pharmacological inhibition and gene silencing of P2X7R reduced inflammasome activation in response to roridin A (Fig. 3). However, gene
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Fig. 5. Trichothecene induced IL-1b and IL-18 secretion is regulated through c-Cbl dependent pathway in LPS-primed human macrophages. Human macrophages were transfected with non-targeting siRNA control (NT-i) or c-Cbl specific siRNA molecules (CBL-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. (A) Western blot analysis of c-Cbl expression from cell lysates was done as described in Section 2. (B–D) Cell culture supernatants were collected and (B) IL-1b, (C) IL-18, and (D) TNF secretion were analyzed with ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b, IL-18 or TNF secretion was set to 100%. Values are means ± SD from three independent analyses.
silencing of P2X7R did not completely abolish roridin A-induced IL1b release suggesting that also other purinergic receptors may participate in NLRP3 inflammasome activation in human macrophages during exposure to fungal toxins. It may be that roridin A directly interacts with purinergic receptors which results in NLRP3 inflammasome activation. Alternatively, it is more likely that roridin A indirectly activates purinergic receptors by inducing changes in intracellular ionic concentrations. In addition to trichothecene mycotoxins, it has been recently shown that NLRP3 inflammasome activation by crystalline substances is dependent purinergic receptors [17]. The common feature of NLRP3 inflammasome activators is their ability to induce ROS production [25]. In addition, it is well documented that ROS scavengers attenuate NLRP3 activation. However, the cellular source of ROS that activates NLRP3 is unknown. Zhou and co-workers recently provided evidence that ROS of mitochondrial origin activates the NLRP3 inflammasome [26]. However, the mechanism by which NLRP3 senses ROS is still unclear, albeit the production of ROS has been suggested to be a prerequisite for the inflammasome activation [25]. It has been shown that DPI, a flavoprotein inhibitor of NADPH oxidases, and NAc, a general scavenger of ROS, both prevent up-regulation of NLRP3 expression in response to TLR4 activation [27] which has been suggested to be a prerequisite for the inflammasome activation [4,28]. We have previously shown that roridin A alone can activate the secretion of IL-18 in human macrophages [12]. Roridin A stimulation of human macrophages has no effect on NLRP3 mRNA expression (data not shown). This implies that NLRP3 inflammasome activation can proceed also in un-primed human macrophages without upregulation of NLRP3 expression. Our current results also show that both DPI and NAc abolish roridin A-induced IL-1b and IL-18 secretion in LPS-primed macrophages (Fig. 2A and B). This suggests a
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direct role for ROS in NLRP3 inflammasome activation in response to trichothecene mycotoxins. Stimulation of P2X7R is known to trigger ROS production [29]. Therefore, it is likely that P2X7R-induced ROS is involved in roridin A-induced NLRP3 inflammasome activation. Cathepsins are lysosomal cysteine and aspartic specific proteases that are intimately involved in the regulation of innate immune response. They have key roles in antigen processing and presentation, cytokine regulation, apoptosis, and TLR signalling [30]. In addition to their function in lysosomes, recent studies have shown that cathepsins have also important roles in the cytoplasm and extracellular milieu. It has been suggested that the release of cathepsin B from the lysosomal compartment to cytosol activates NLRP3 inflammasome by a yet uncharacterized mechanism [31]. We have seen that cathepsin B specific inhibitor, CA-074-Me, blocks inflammasome activation in response to b-glucans [13] and trichothecene mycotoxins (Fig. 2E and F). Interestingly, it was recently shown that ATP is able to induce the release of cathepsins, including cathepsin B, through a P2X7R-dependent pathway in macrophages [32]. Furthermore, secreted cathepsin B was biologically active and was able to degrade collagen extracellular matrix [32] demonstrating that cathepsin B can function in neutral pH. Moreover, it has been previously shown that proteolytic processing of TLR9 by cathepsins in endolysomes is essential for TLR9 activity [33,34]. Therefore, it is tempting to speculate that NLRP3 could be a direct target of cathepsin B. However, future studies are needed to elucidate the cytosolic targets of cathepsin B that are involved in NLRP3 inflammasome activation. Members of the Src tyrosine kinases have important roles both in innate and adaptive immune response [35]. For example, malarial hemozoin activates the NLRP3 inflammasome through Lyn, which is a member of Src tyrosine kinases [36]. Similarly, we have shown that Src kinase activity is required for NLRP3 inflammasome activation during influenza a virus infection [37]. It is also known that tyrosine kinase activity is essential for P2X7R function. More specifically, Src tyrosine kinase signaling has been linked in P2X7R function through its C-terminal region that is known to bind Src tyrosine kinase [38]. Our present results demonstrate that roridin A specifically induce tyrosine phosphorylation of Src at Tyr416. Furthermore, Src tyrosine kinases were essential for NLRP3 inflammasome activation caused by trichothecene mycotoxins (Fig. 4). In parallel with NLRP3 inflammasome, the activation of AIM2 inflammasomes was dependent on Src tyrosine kinase activity (Fig. 4J). These results emphasize the role of Src tyrosine kinases in the activation inflammatory response. It was recently shown that the activity of Src kinase is negatively regulated by the E3 ubiquitin ligase c-Cbl: c-Cbl targeted active form of c-Src to autophagy for degradation [23]. In our experiments gene silencing of c-Cbl enhanced secretion of both IL-1b and IL-18 in response to roridin A stimulation. These results suggest that c-Cbl is involved in pathway that leads to NLRP3 inflammasome activation. Further studies are needed to elucidate whether c-Cbl regulates the inflammasome activity by directly targeting c-Src for degradation. In conclusion, our results demonstrate that fungal trichothecene mycotoxins are important microbial danger signals that activate NLRP3 inflammasome through purinergic receptor and Src tyrosine kinase-dependent pathway in human primary macrophages. Acknowledgments This work was supported by grants from the Finnish Work Environment Fund, the Research Council for Biosciences and Environment of the Academy of Finland, the Sigrid Juselius Foundation, SYTYKE Graduate School in Environmental Health, and the Nummela foundation. The authors have no conflicting financial interests.
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[19] Mehta VB, Hart J, Wewers MD. ATP-stimulated release of interleukin (IL)-1beta and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage. J Biol Chem 2001;276:3820. [20] Johnsen IB, Nguyen TT, Bergstroem B, Fitzgerald KA, Anthonsen MW. The tyrosine kinase c-Src enhances RIG-I (retinoic acid-inducible gene I)-elicited antiviral signaling. J Biol Chem 2009;284:19122. [21] Johnsen IB, Nguyen TT, Ringdal M, Tryggestad AM, Bakke O, Lien E, et al. Tolllike receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling. EMBO J 2006;25:3335. [22] Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 1997;13:513. [23] Sandilands E, Serrels B, McEwan DG, Morton JP, Macagno JP, McLeod K, et al. Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling. Nat Cell Biol 2011;14:51. [24] Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 2002;10:417. [25] Tschopp J. Mitochondria: Sovereign of inflammation? Eur J Immunol 2011;41:1196. [26] Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature 2010;469:221. [27] Bauernfeind F, Bartok E, Rieger A, Franchi L, Nunez G, Hornung V. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J Immunol 2011;187:613. [28] Franchi L, Eigenbrod T, Nunez G. Cutting edge: TNF-alpha mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation. J Immunol 2009;183:792. [29] Lenertz LY, Gavala ML, Hill LM, Bertics PJ. Cell signaling via the P2X(7) nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking. Purinergic Signal 2009;5:175. [30] Colbert JD, Matthews SP, Miller G, Watts C. Diverse regulatory roles for lysosomal proteases in the immune response. Eur J Immunol 2009;39:2955. [31] Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008;9:847. [32] Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. J Immunol 2010;185:2611. [33] Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA, et al. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 2008;456:658. [34] Park B, Brinkmann MM, Spooner E, Lee CC, Kim YM, Ploegh HL. Proteolytic cleavage in an endolysosomal compartment is required for activation of Tolllike receptor 9. Nat Immunol 2008;9:1407. [35] Bradshaw JM. The Src, Syk, and Tec family kinases: distinct types of molecular switches. Cell Signal 2010;22:1175. [36] Shio MT, Eisenbarth SC, Savaria M, Vinet AF, Bellemare MJ, Harder KW, et al. Malarial hemozoin activates the NLRP3 inflammasome through Lyn and Syk kinases. PLoS Pathog 2009;5:e1000559. [37] Rintahaka J, Lietzen N, Ohman T, Nyman TA, Matikainen S. Recognition of cytoplasmic RNA results in cathepsin-dependent inflammasome activation and apoptosis in human macrophages. J Immunol 2011;186:3085. [38] Costa-Junior HM, Sarmento Vieira F, Coutinho-Silva R. C terminus of the P2X7 receptor: treasure hunting. Purinergic Signal 2011;7:7.
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Trichothecene mycotoxins activate NLRP3 inflammasome through a P2X7 receptor and Src tyrosine kinase dependent pathway Päivi Kankkunen a,⇑, Elina Välimäki a,b, Johanna Rintahaka a,1, Jaana Palomäki a, Tuula Nyman a,b, Harri Alenius a, Henrik Wolff a, Sampsa Matikainen a a b
Finnish Institute of Occupational Health (FIOH), Topeliuksenkatu 41A, 00250 Helsinki, Finland Institute of Biotechnology, University of Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 18 April 2013 Accepted 11 November 2013 Available online 20 November 2013
a b s t r a c t Inflammasome is an intracellular molecular platform of the innate immunity that is a key mediator of inflammation. The inflammasome complex detects pathogens and different danger signals, and triggers cysteine protease caspase-1-dependent processing of pro-inflammatory cytokines IL-1b, and IL-18 in dendritic cells and macrophages. Previously, we have shown that water-damaged building associated trichothecene mycotoxins, including roridin A, trigger IL-1b and IL-18 secretion in human macrophages. However, the molecular basis as well as mechanism behind this trichothecene-induced cytokine secretion has remained uncharacterized. Here, we show that the trichothecene-induced IL-1b secretion is dependent on NLRP3 inflammasome in human primary macrophages. Pharmacological inhibition and small interfering RNA approach showed that the trichothecene-induced NLRP3 inflammasome activation is mediated through ATP-gated P2X7 receptor. Moreover, we show that trichothecene-triggered NLRP3 inflammasome activation is dependent on Src tyrosine kinase activity. In addition, gene silencing of cCbl, a negative autophagy-related regulator of c-Src, resulted in enhanced secretion of IL-1b and IL-18 in response to trichothecene mycotoxin stimulation in human macrophages. In conclusion, our results suggest that roridin A, a fungal trichothecene mycotoxin, acts as microbial danger signals that trigger activation of NLRP3 inflammasome through P2X7R and Src tyrosine kinase signaling dependent pathway in human primary macrophages. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The immune system recognizes and responds to infection, tissue damage, and/or cellular stress by pattern recognition receptors (PRRs) of the innate immune system. Interleukin (IL)-1b, IL-18, and tumor necrosis factor (TNF) are important pro-inflammatory cytokines which are produced by macrophages in response to PRR activation [1]. Production of TNF is mainly regulated at transcriptional level. In contrast, secretion of IL-1b and IL-18 is strictly regulated by post-translational mechanisms: both IL-1b and IL-18 are synthesized as immature forms: pro-IL-1b and pro-IL-18, respectively. Cysteine protease caspase-1 cleaves these pro-forms to biologically active equivalents which are then secreted via unconventional Abbreviations: AG126, tyrphostin AG 126; AZ, AZ11645373; DAMP, danger associated molecular pattern; NLR, NOD-like nucleotide-binding oligomerization domain; NLRP, NLR family, Pyrin domain containing; PP2, 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, RLR, RIG-I-like receptors; RU, relative unit; siRNA, small ribonucleic acid interference. ⇑ Corresponding author. E-mail address: paivi.kankkunen@ttl.fi (P. Kankkunen). 1 Present address: University of Helsinki, Finland.
protein secretion pathway [2,3]. Caspase-1 in turn is activated in a protein complex called the inflammasome. At present, several caspase-1 activating NOD-like receptor (NLR) structures have been characterized that include NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 inflammasomes [4–9]. Of these NLRP3 is the most extensively studied because of its function in antimicrobial defence, adjuvanticity, and autoinflammatory diseases [4,7]. In addition to NLRs, absent in melanoma 2 (AIM2)-like receptors are capable of forming a caspase-1 activating inflammasome complexes [10]. Fungi are heterotrophic eukaryotes which interact with humans in multiple ways establishing symbiotic, commensal, latent, and pathogenic relationships. Most fungi are ubiquitous in environment, and humans are exposed to inhaling fungi spores, and their metabolites including cell wall components and mycotoxins. Trichothecene mycotoxins are produced by several fungi including Fusarium, Trichoderma, Myrothecium, and Stachybotrys species. These fungal toxic metabolites were originally isolated as possible antimicrobial agents but they are also harmful and toxic to animals and humans. Trichothecene producing Stachybotrys chartarum is frequently linked to water-damaged buildings and its connection
0198-8859/$36.00 - see front matter Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2013.11.010
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to damp-building associated illnesses has also been proposed [11]. We have previously shown that trichothecene mycotoxins, including roridin A, verrucarin A and T-2 toxin, activate caspase-1 resulting in IL-1b and IL-18 secretion in LPS-primed human macrophages [12]. Here, we have further characterized the mechanism behind this trichothecene-induced cytokine release. 2. Materials and methods 2.1. Cells Human peripheral blood mononuclear cells (PBMCs) from healthy blood donors (Finnish Red Cross Blood Transfusion Service) were isolated and allowed to differentiated into macrophages according the protocol that has been previously described [12]. After seven days of culture, the resulting macrophages were used in the experiments.
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150 mM NaCl, 1% NP-40, and 1 mM EDTA supplemented with phosphatase inhibitor cocktails 2 and 3 and protease inhibitor cocktail (Sigma–Aldrich). After incubation on ice for 1 h the cell extract was centrifuged (12,000g for 30 min) and supernatants were collected and used for Western blot analysis. The proteins were visualized by ImageQuant LAS 4000 mini luminescent image analyzer according to manufacturer’s instruction (GE Healthcare BioScience Ab, Uppsala, Sweden). Anti-P2X7 Abs (H-265) and antiCbl (A-9) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-phospho-Src (Tyr416) from Cell Signalling Technology (Beverly, MA). To confirm equal protein loading and transfer during SDS–PAGE and immunoblotting, respectively, membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain according to the manufacturer’s instructions (Sigma–Aldrich). The major basally expressed protein band is shown as a loading control.
2.5. Statistical analyses 2.2. Reagents AZ11645373 (AZ; 1 lM), ATP (3 mM), LPS (Escherichia coli 0111:B4) (1 lg/ml), diphenyleneiodonium chloride (DPI; 1 lM), N-acetyl-L-cysteine (NAc; 5 mM), PP2 (5 lM), potassium chloride (KCl; +1 mM supplementation), and tyrphostin AG 126 (AG126; 50 lM), roridin A (10 ng/ml), verrucarin A (10 ng/ml), and poly(deoxyadenylic-thymidylic) acid sodium salt (poly(dA:dT); 2 lg/ml) were purchased from Sigma–Aldrich (St. Luis, MO). Cathepsin B inhibitor, CA-074-Me (10 lM), was purchased from Calbiochem (San Diego, CA). Optimal concentrations for the reagents were titrated in preliminary experiments.
Each human macrophage sample represents a pool of separately stimulated cells from three different blood donors. ELISA results are combined through at least three different stimulations and data were expressed as means (±SD) unless otherwise mentioned. Quantitative RT-PCR and Western blot results are representative of at least two independent, but similarly performed experiments. In
2.3. Cell stimulations and small interfering RNA (siRNA) experiments Human macrophages were pretreated with LPS for 2 h prior to roridin A and verrucarin A stimulation for 4 h unless otherwise mentioned. When inhibitory reagents were used they were added to the cells 30 min prior to roridin A and verrucarin A stimulation. In ATP-stimulation experiments, the cells were treated with LPS for 6 h prior to ATP stimulation with 30 min. AZ were added to the cells 4.5 h prior to ATP addition. When Poly(dA:dT) was used, it was transfected into cell cytoplasm by lipofectamine 2000 (Life Technologies, Paisley, UK) according to the manufacturer’s instruction. For siRNA experiments, the cells were transfected with nontargeting control siRNA (AllStars Negative Control siRNA, Qiagen) or with siRNAs against NLRP3, P2X7, Cbl small interference RNA (siRNA) (Hs_CIAS1_6, Hs_CIAS1_9, Hs_CIAS1_10, Hs_CIAS1_11, and Hs_P2RX7_1, Hs_P2RX7_2, and Hs_CBL_7, Hs_CBL_8, respectively; Qiagen) treatments were performed as previously described [13,14]. 2.4. Quantitative RT-PCR, enzyme-linked immunosorbent (ELISA), and Western blot analysis Quantitative real-time RT-PCR analysis of expression of IL-1b, IL-18, and NLRP3 mRNA and ELISA analysis of secreted IL-1b level from human cells or cell culture supernatants were performed as previously described [13]. Pre-developed TaqManÒ assay primers and probes (IL-1b Hs_0017497_m1, IL-18 Hs_00155517_m1, NLRP3 Hs_00918082_m1 Applied Biosystems) were used according to manufacturer’s instructions. The ELISA analysis of human TNF was performed according to manufacturer’s instruction (eBioscience, San Diego, CA). For protein analysis, total cell lysates were prepared and Western blot analysis was performed as previously described [12]. For phospho-protein analysis, the cells were suspended in a lysis buffer containing 50 mM Hepes (pH 7.4),
Fig. 1. Roridin A activates NLRP3 inflammasome in human macrophages. (A and B) Human macrophages were treated with LPS (1 lg/ml) for 18 h or left untreated after which the cells were stimulated with roridin A (10 ng/ml; RoA) for the times indicated. After this the cell culture supernatants were collected and (A) IL-1b and (B) IL-18 secretion was analyzed by ELISA. Values are means ± SD from at least three independent analyses. A unpaired t-test was used to compare the differences between the samples. ⁄⁄⁄p < 0.001, ⁄p < 0.05. (C and D) Human macrophages were transfected with non-targeting siRNA control (NT-i) or NLRP3 specific siRNA molecules (NLRP3-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. (C) The cells were harvested, total cellular RNA was isolated, and RT-PCR analysis of NLRP3, IL-1b, and IL-18 mRNA expression was performed. For data expression, LPS and roridin A co-stimulation induced NLPR3, IL-1b, and IL-18 mRNA expression was set to 100%. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. Values are means ± SD from duplicate analyses from a single experiment. (D) Cell culture supernatants were collected and IL-1b IL18, and TNF secretion were analyzed with ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b, IL-18, or TNF secretion were set to 100%. Values are means ± SD from two independent analyses including 3 blood donors for each. ⁄p < 0.05.
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Fig. 2. Trichothecene induced IL-1b and IL-18 secretion is ROS, K+ fluxes, and cathepsin B release dependent process in LPS-primed human macrophages. Macrophages were left un-stimulated or stimulated with LPS (1 lg/ml) for 2 h, after which the cells were stimulated with roridin A (10 ng/ml; RoA) for 4 h in the absence and presence of DPI (1 lM), NAc (5 lM), K+ (+1 mM supplementation), or CA-074-Me (10 lM). The cell culture supernatants were collected and (A and E) IL-1b and (B and F) IL-18 secretion was analyzed with ELISAs. Values are means ± SD from three independent analyses. ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄p < 0.05. (C, D, G, and H) The cells were harvested, total cellular RNA was isolated, and RT-PCR analysis of IL-1b and IL-18 mRNA expression was performed. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. Results are expressed as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment.
RT-PCR figures, the RU values are means ± SD from duplicate analyses from a single experiment unless otherwise mentioned. All data were analyzed by using the GraphPad Prism 4 Software (GraphPad Software Inc., San Diego, CA). A one-tailed t-test was used to compare the differences between the samples unless otherwise mentioned. A p-value of <0.05 was considered to be statistically significant. 3. Results 3.1. Roridin A activates canonical caspase-1 containing NLRP3 inflammasome in human macrophages To study the secretion kinetics of IL-1b and IL-18 in response to trichothecene mycotoxins, macrophages were left untreated or treated with LPS for 18 h after which the cells were treated with roridin A for different time periods. Roridin A strongly activated IL-1b and IL-18 secretion at 4 h post-stimulation in LPS-primed macrophages (Fig. 1A and B). Roridin A alone was able to induce IL-18 secretion, but not that of IL-1b, at 8 h post-stimulation (Fig. 1A and B). IL-1b secretion activating molecular platforms include many different inflammasome structures [4–8]. To study whether the inflammasome structure activated by trichothecene mycotoxins is NLRP3, we performed gene silencing experiments. Macrophages were transfected with non-targeting or NLRP3 specific siRNA molecules as described in Section 2. After this macrophages were primed with LPS for 2 h and subsequently the cells were
stimulated with roridin A for 4 h. The cells were collected and total-RNA was isolated for RT-PCR analysis. Similarly, cell culture supernatants were collected and used for ELISA analysis. RT-PCR analysis verified the silencing of NLRP3 mRNA expression (Fig. 1C). In contrast, siRNA treatment had no effect on IL-1b mRNA expression (Fig. 1D). However, the NLRP3 gene-silencing decreased IL-1b and IL-18 secretion in LPS-primed and roridin A-stimulated macrophages (Fig. 1E). However, the treatment did not affect TNF secretion level (Fig. 1E). The results suggest that roridin A activates NLRP3 inflammasome resulting in IL-1b and IL-18 secretion in LPSprimed human macrophages. 3.2. ROS formation, potassium efflux, and cathepsin B activity are needed for roridin A-induced IL-1b and IL-18 secretion Microbial toxins and crystalloid substances trigger ROS formation, which has been suggested to be a key event in NLRP3 inflammasome activation [15]. Therefore, we analyzed whether ROS formation is required for roridin A-induced IL-1b and IL-18 release in human macrophages. Both ROS inhibitors, NAc and DPI, decreased roridin A-induced IL-1b and IL-18 release in LPS-primed macrophages indicating that ROS formation is essential for roridin A-triggered IL-1b and IL-18 release in human macrophages. ROS inhibitors had only little effect on IL-1b and IL-18 mRNA expression (Fig. 2C). In addition to ROS, potassium efflux has been shown to facilitate NLRP3 inflammasome activation in response to a variety of danger signals [16]. To study the role of potassium efflux in roridin
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Fig. 3. Roridin A activates NLRP3 inflammasome via a P2X7R-dependent pathway. (A and B) Macrophages were untreated or treated with LPS (1 lg/ml) for 2 h prior to roridin A (10 ng/ml; RoA) stimulation for 4 h in the presence or absence of P2X7 receptor inhibitor AZ11645373 (1 lM; AZ). In parallel, after LPS (1 lg/ml) priming for 6 h, the cells were exposed to ATP (3 mM) for 30 min in the presence or absence of AZ11645373 (1 lM; AZ). (A) The cells culture supernatants were collected and IL-1b secretion was analyzed by ELISA. Values are means ± SD from five independent analyses. ⁄⁄⁄p < 0.001. (B) Cells were harvested and used for IL-1b mRNA expression analysis. Results are expressed as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment. (C–E) Macrophages were transfected with non-targeting siRNA control (NT-i) or P2X7R siRNA molecules (P2X7-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. Cell culture supernatants were collected and the cells were harvested and cell lysates were prepared. (C) Western blot analysis of cell lysates was done with anti-P2X7R Ab. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. To confirm equal loading and transfer of the protein samples in Western blot assays, the membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain. The major protein band detected and basally expressed is shown as a loading control in Western Blot analysis (indicated as control). (D) IL-1b and (E) TNF secretion was analyzed from cell culture supernatants by ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b or TNF secretion levels without silencing were set to 100%. Values are means ± SD from two independent analyses.
A-induced IL-1b and IL-18 secretion, human LPS-primed macrophages were stimulated with roridin A in the presence and absence of additional potassium chloride as described in Section 2. The enhanced potassium chloride concentration completely blocked roridin A-induced IL-1b and IL-18 release (Fig. 2 A and B). However, the enhanced potassium chloride concentration did not significantly affect IL-1b nor IL-18 gene expression (Fig. 2C). Lysosomal destabilization and the release of cathepsin B from the lysomal compartment is involved in the activation of NLRP3 inflammasome [4]. To study the role of cathepsin B in trichothecene induced NLRP3 inflammasome activation we used cathepsin B specific inhibitor, CA-074-Me. As shown in Fig. 2E and F, both IL-1b and IL-18 secretion in response to roridin A stimulation was reduced in LPS-primed macrophages in the presence of CA074-Me (Fig. 2E and F). In contrast, the inhibitor did not affect IL1b or IL-18 mRNA expression level (Fig. 2G and H). 3.3. Roridin A induced NLRP3 inflammasome activation is mediated through P2X7R Recently, it has been suggested that NLRP3 inflammasome activation in response to monosodium urate, silica, and alum is dependent on purinergic P2X7 receptor (P2X7R) [17]. We used pharmacological inhibitor of P2X7R and siRNA approach to study the role of this receptor in roridin A-triggered NLRP3 inflammasome activation. Macrophages were untreated or treated with LPS and roridin A in the absence and presence of P2X7R antagonist AZ11645373 (AZ). AZ-treatment decreased IL-1b secretion from LPS-primed human macrophages in response to roridin A stimulation (Fig. 3A). The treatment had no effect on IL-1b mRNA expression (Fig. 3B). In parallel, the AZ treatment blocked ATP-induced IL-1b secretion in LPS-primed human macrophages (Fig. 3A). Next, we performed siRNA studies with P2X7R specific siRNAs in human
primary macrophages. In line with the inhibitor data, the P2X7R specific siRNA treatment of macrophages resulted in decreased IL-1b secretion in LPS-primed and roridin A-stimulated macrophages (Fig. 3C and D). However, gene silencing had no effect on TNF secretion (Fig. 3E). The efficacy of the siRNA-treatment was verified with Western blotting (Fig. 3F). 3.4. Src kinase activity is essential for roridin A-induced NLRP3 inflammasome activation in human macrophages It has been shown that ATP-induced IL-1b secretion is inhibited by a synthetic tyrosine kinase inhibitor, tyrphostin AG126 (AG126) [18,19]. To study the role of tyrosine kinase signaling in roridin Ainduced NLRP3 inflammasome activation, LPS-primed macrophages were stimulated with roridin A in the presence and absence of AG126. The inhibitor abolished roridin A-induced IL-1b and IL18 secretion in LPS-primed cells indicating that tyrosine kinase signaling is needed for roridin A-induced inflammasome activation (Fig. 4A and B). The inhibitor had no effect on IL-1b or IL-18 mRNA expression levels (Fig. 4C and D). Src family of tyrosine kinases has an important role in the activation of innate immune response in TLR and RLR signaling pathways [20,21]. To study whether this pathway is important for NLRP3 inflammasome activation caused by roridin A, we used the Src tyrosine kinase inhibitor PP2. Macrophages were untreated or treated with LPS for 2 h prior to roridin A stimulation for 4 h in the presence and absence PP2. The inhibitor abrogated secretion of IL-1b and IL-18 in LPS-primed and roridin A-stimulated macrophages (Fig. 4E and F). In contrast, PP2-treatment had no effect on IL-1b or IL-18 mRNA expression in LPS-primed and roridin A co-stimulated cells (Fig 4G and H). In addition, PP2 inhibited IL-1b secretion induced by verrucarin A (Fig. 4I), which is also a caspase-1 activating trichothecene mycotoxin [12]. In line with
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Fig. 4. Src tyrosine kinase signaling is essential for trichothecene engaged NLRP3 inflammasome activation. Human macrophages were primed with LPS (1 lg/ml) for 2 h after which they were exposed to (A–H) roridin A (10 ng/ml; RoA) for 4 h hour in the absence or presence of (A–D) AG126 (50 lM) or (E–H) PP2 (5 lM). (A and E) IL-1b and (B and F) IL-18 secretion was measured by ELISA from the collected cell culture supernatants. The means at least three separate experiments (±SD) are shown. ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄ p < 0.05. (C, D, G, and H) The cells were collected, total RNA was isolated. After this (C and G) IL-1b (D and H) IL-18 mRNA expression was analyzed by RT-PCR. The results are representative of three independent, but similarly performed experiments. The analyzed results are shown as relative unit (RU). Values are means ± SD from duplicate analyses from a single experiment. (I and J) Macrophages were left untreated or primed with LPS (1 lg/ml) for 2 h and stimulated with verrucarin A (10 ng/ml; VerA) or (J) transfected with Poly(dA:dT) (2 lg/ml) for 4 h in absence and presence of PP2 (5 lM). IL-1b secretion was analyzed from cell culture supernatants with ELISA. The means of two separate experiments (±SD) are shown. (K) Macrophages were stimulated with roridin A (10 ng/ml; RoA) for the times indicated, cell lysates were prepared and Western blot analysis was performed with anti-phopho-Src Abs. Each sample represents a pool of separately stimulated cells from three different blood donors and the results are representative of three independent, but similarly performed experiments. To confirm equal loading and transfer of the protein samples, membranes were stained with ready-to-use SYPRO Ruby Protein Blot Stain. The major protein band detected and basally expressed is shown as a loading control in Western Blot analysis (indicated as control).
the PP2 data, also Src inhibitor I reduced IL-1b and IL-18 secretion triggered by roridin A (Supplementary Fig. 1). The inhibitor did not significantly influence on IL-1b and IL-18 mRNA transcription level (Supplementary Fig. 1). Next we studied the role of Src tyrosine kinases in AIM2 inflammasome activation. Macrophages were primed with LPS for 2 h and after that transfected with synthetic double-stranded DNA, poly(dA:dT), for 4 h in the presence and absence of PP2. After this cell culture supernatants were harvested and secretion of IL-1b was analyzed. PP2 treatment clearly inhibited IL-1b secretion triggered by poly(dA:dT) transfection in LPSprimed human macrophages (Fig. 4J). After this we wanted to verify that Src family of tyrosine kinases are activated in macrophages in response to roridin A stimulation. Src activity is positively regulated by tyrosine phosphorylation at Tyr416 in the activation loop of the kinase domain [22]. Macrophages were exposed to roridin A for different time periods after which protein lysates were prepared and Src tyrosine kinase activity was analyzed by Western blotting with anti-phospho-Src family (Tyr416) Abs. As shown in Fig. 4K, roridin A activated tyrosine phosphorylation of Src (60 kDa) at 1 h post-stimulation. 3.5. Autophagy-related c-Cbl negatively regulates NLRP3 inflammasome activation in human macrophages Recently, it was shown that E3 ubiquitin ligase c-Cbl acts as an autophagy cargo receptor for Src to mediate Src degradation [23]. To study whether c-Cbl regulates Src function and NLRP3
inflammasome activation in human macrophages, we silenced cCbl expression with siRNA approach. Efficiency of the siRNA treatments were verified with Western blotting (Fig. 5A). As shown in Fig. 5B and C, silencing of c-Cbl enhanced IL-1b or IL-18 secretion in LPS-primed and roridin A-stimulated human macrophages (Fig. 5B and C). In contrast, there were no changes in TNF expression level (Fig. 5D). 4. Discussion The inflammasome is a cytoplasmic multiprotein complex that controls secretion of pro-inflammatory cytokine IL-1b through activation of cysteine protease caspase-1 [24]. NLRP3, a member of the NLR family of PRRs is the most studied inflammasome structure. NLRP3 together with adaptor protein ASC, drives caspase-1 activation in response to diverse stimuli including those of endogenous and microbial origin, as well as environmental crystalline particles [4,7]. These endogenous and exogenous stimuli are chemically and structurally heterogeneous suggesting that they are not directly recognized by the NLRP3 inflammasome. It is more likely that they activate inflammasome indirectly by inducing changes in endogenous molecules and/or disturbances in intracellular ionic concentrations. Here we provide evidence that purinergic P2X7R is involved in NLRP3 inflammasome activation in response to fungal trichothecene mycotoxins in human macrophages: both pharmacological inhibition and gene silencing of P2X7R reduced inflammasome activation in response to roridin A (Fig. 3). However, gene
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Fig. 5. Trichothecene induced IL-1b and IL-18 secretion is regulated through c-Cbl dependent pathway in LPS-primed human macrophages. Human macrophages were transfected with non-targeting siRNA control (NT-i) or c-Cbl specific siRNA molecules (CBL-i) as described in Section 2. After this the cells were primed with LPS (1 lg/ml) for 2 h followed by roridin A (10 ng/ml; RoA) stimulation for 4 h. (A) Western blot analysis of c-Cbl expression from cell lysates was done as described in Section 2. (B–D) Cell culture supernatants were collected and (B) IL-1b, (C) IL-18, and (D) TNF secretion were analyzed with ELISAs. For data expression, LPS and roridin A co-stimulation induced IL-1b, IL-18 or TNF secretion was set to 100%. Values are means ± SD from three independent analyses.
silencing of P2X7R did not completely abolish roridin A-induced IL1b release suggesting that also other purinergic receptors may participate in NLRP3 inflammasome activation in human macrophages during exposure to fungal toxins. It may be that roridin A directly interacts with purinergic receptors which results in NLRP3 inflammasome activation. Alternatively, it is more likely that roridin A indirectly activates purinergic receptors by inducing changes in intracellular ionic concentrations. In addition to trichothecene mycotoxins, it has been recently shown that NLRP3 inflammasome activation by crystalline substances is dependent purinergic receptors [17]. The common feature of NLRP3 inflammasome activators is their ability to induce ROS production [25]. In addition, it is well documented that ROS scavengers attenuate NLRP3 activation. However, the cellular source of ROS that activates NLRP3 is unknown. Zhou and co-workers recently provided evidence that ROS of mitochondrial origin activates the NLRP3 inflammasome [26]. However, the mechanism by which NLRP3 senses ROS is still unclear, albeit the production of ROS has been suggested to be a prerequisite for the inflammasome activation [25]. It has been shown that DPI, a flavoprotein inhibitor of NADPH oxidases, and NAc, a general scavenger of ROS, both prevent up-regulation of NLRP3 expression in response to TLR4 activation [27] which has been suggested to be a prerequisite for the inflammasome activation [4,28]. We have previously shown that roridin A alone can activate the secretion of IL-18 in human macrophages [12]. Roridin A stimulation of human macrophages has no effect on NLRP3 mRNA expression (data not shown). This implies that NLRP3 inflammasome activation can proceed also in un-primed human macrophages without upregulation of NLRP3 expression. Our current results also show that both DPI and NAc abolish roridin A-induced IL-1b and IL-18 secretion in LPS-primed macrophages (Fig. 2A and B). This suggests a
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direct role for ROS in NLRP3 inflammasome activation in response to trichothecene mycotoxins. Stimulation of P2X7R is known to trigger ROS production [29]. Therefore, it is likely that P2X7R-induced ROS is involved in roridin A-induced NLRP3 inflammasome activation. Cathepsins are lysosomal cysteine and aspartic specific proteases that are intimately involved in the regulation of innate immune response. They have key roles in antigen processing and presentation, cytokine regulation, apoptosis, and TLR signalling [30]. In addition to their function in lysosomes, recent studies have shown that cathepsins have also important roles in the cytoplasm and extracellular milieu. It has been suggested that the release of cathepsin B from the lysosomal compartment to cytosol activates NLRP3 inflammasome by a yet uncharacterized mechanism [31]. We have seen that cathepsin B specific inhibitor, CA-074-Me, blocks inflammasome activation in response to b-glucans [13] and trichothecene mycotoxins (Fig. 2E and F). Interestingly, it was recently shown that ATP is able to induce the release of cathepsins, including cathepsin B, through a P2X7R-dependent pathway in macrophages [32]. Furthermore, secreted cathepsin B was biologically active and was able to degrade collagen extracellular matrix [32] demonstrating that cathepsin B can function in neutral pH. Moreover, it has been previously shown that proteolytic processing of TLR9 by cathepsins in endolysomes is essential for TLR9 activity [33,34]. Therefore, it is tempting to speculate that NLRP3 could be a direct target of cathepsin B. However, future studies are needed to elucidate the cytosolic targets of cathepsin B that are involved in NLRP3 inflammasome activation. Members of the Src tyrosine kinases have important roles both in innate and adaptive immune response [35]. For example, malarial hemozoin activates the NLRP3 inflammasome through Lyn, which is a member of Src tyrosine kinases [36]. Similarly, we have shown that Src kinase activity is required for NLRP3 inflammasome activation during influenza a virus infection [37]. It is also known that tyrosine kinase activity is essential for P2X7R function. More specifically, Src tyrosine kinase signaling has been linked in P2X7R function through its C-terminal region that is known to bind Src tyrosine kinase [38]. Our present results demonstrate that roridin A specifically induce tyrosine phosphorylation of Src at Tyr416. Furthermore, Src tyrosine kinases were essential for NLRP3 inflammasome activation caused by trichothecene mycotoxins (Fig. 4). In parallel with NLRP3 inflammasome, the activation of AIM2 inflammasomes was dependent on Src tyrosine kinase activity (Fig. 4J). These results emphasize the role of Src tyrosine kinases in the activation inflammatory response. It was recently shown that the activity of Src kinase is negatively regulated by the E3 ubiquitin ligase c-Cbl: c-Cbl targeted active form of c-Src to autophagy for degradation [23]. In our experiments gene silencing of c-Cbl enhanced secretion of both IL-1b and IL-18 in response to roridin A stimulation. These results suggest that c-Cbl is involved in pathway that leads to NLRP3 inflammasome activation. Further studies are needed to elucidate whether c-Cbl regulates the inflammasome activity by directly targeting c-Src for degradation. In conclusion, our results demonstrate that fungal trichothecene mycotoxins are important microbial danger signals that activate NLRP3 inflammasome through purinergic receptor and Src tyrosine kinase-dependent pathway in human primary macrophages. Acknowledgments This work was supported by grants from the Finnish Work Environment Fund, the Research Council for Biosciences and Environment of the Academy of Finland, the Sigrid Juselius Foundation, SYTYKE Graduate School in Environmental Health, and the Nummela foundation. The authors have no conflicting financial interests.
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