Macrophages Inflammatory Response in Human Trichothecene Mycotoxins Activate

Trichothecene Mycotoxins Activate Inflammatory Response in Human Macrophages

This information is current as Päivi Kankkunen, Johanna Rintahaka, Annika Aalto, Marina of September 28, 2021. Leino, Marja-Leena Majuri, Harri Alenius, Henrik Wolff and Sampsa Matikainen J Immunol 2009; 182:6418-6425; ; doi: 10.4049/jimmunol.0803309

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Trichothecene Mycotoxins Activate Inﬂammatory Response in Human Macrophages1

Pa¨ivi Kankkunen, Johanna Rintahaka, Annika Aalto, Marina Leino, Marja-Leena Majuri, Harri Alenius, Henrik Wolff, and Sampsa Matikainen2

Damp building-related illnesses have caused concern for years in many countries. Although the problem is extensive, the knowledge of the immunological reactions behind damp building-related illnesses is still quite limited. Trichothecene mycotoxins form one major group of toxins, which possibly contribute to the illnesses. Stachybotrys chartarum is a well-known, but also controversial damp building mold and many strains of this mold are capable of producing trichothecenes. In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages. As a result, satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1␤ and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these Downloaded from mycotoxins also strongly enhanced LPS-dependent secretion of IL-1␤ and IL-18. The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3. In conclusion, our results indicate that human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1␤ and IL-18 from the LPS-primed cells. The Journal of Immunology, 2009, 182: 6418–6425. http://www.jimmunol.org/ ycotoxins, which are produced by several damp build- like receptors, detect pathogen-associated molecular patterns, and ing-associated mold species, are an important subject signal the presence of infection to the host by activating antimi- M of concern in water-damaged buildings (1). However, crobial host defense (6, 7). Peptidoglycans of bacterial origin trig- knowledge of the molecular immunology behind them, and espe- ger cytoplasmic NLRs, such as NOD 1 and NOD 2, which lead to cially knowledge of the significance of mycotoxins in damp build- the activation of NF-␬B and MAPK signaling pathways resulting ing-related illnesses (DBRIs)3 is limited and even controversial. in the transcriptional activation of IL-1␤, IL-6, and TNF-␣ genes Inflammasome activation triggered by different danger signals is (8). NLR proteins are also now known to detect different danger- the major recently described immunological finding (2). The action associated molecular patterns (DAMPs), which further reflect the by guest on September 28, 2021 of these inflammasome complexes may also relate to the recogni- innate immune response on a posttranscriptional level (2, 9). tion of mycotoxins by the cells of innate immunity. In general, Caspases are important proteases that are capable of specific sensing and defeating microbial infections is a challenging task for protein cleavages and by that way they participate in posttranscrip- the metazoan species, because of the heterogeneity and rapid evo- tional regulation of protein function (10, 11). Eleven caspases are lution of microbes. In addition to general variation among mi- presently known in humans, which are divided into proinflamma- crobes, many of them can express multiple virulence factors, for tory caspases (caspases 1, 4, and 5) and apoptotic caspases examples endotoxins, which can be involved in the uptake of the (caspases 2, 3, and 6–10) (12, 13). An important task of caspase- microbe and subsequent immune escape. Infecting microbes are 1-mediated proteolytic processing is to produce the proinflamma- recognized by the pattern recognition receptors (PRRs) of the in- tory cytokines IL-1␤ and IL-18 in their biologically active forms nate immune system, which also play a crucial role in the activa- (2). IL-1␤ is known as a major endogenous pyrogen, which is kept tion of mammalian host defense (3–5). PRRs, which include TLRs, strictly silent in normal circumstances (14, 15). Instead, IL-18, NOD-like receptors (NLRs), C-type lectin receptors, and RIG-I which is also a member of IL-1 family, is constitutively expressed in macrophages (15). The IL-18 was first described as an IFN-␥- Unit of Excellence for Immunotoxicology, Finnish Institute of Occupational Health, inducing factor, but since then it has also been connected to other Topeliuksenkatu 41 a A, Helsinki, Finland functions, including the induction of the synthesis of other acute Received for publication October 2, 2008. Accepted for publication March 11, 2009. phase cytokines, for example IL-1␤ and TNF-␣ (16). The process- The costs of publication of this article were defrayed in part by the payment of page ing of inactive IL-1␤ and IL-18 occurs in cytoplasmic multiprotein charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. complexes called inflammasomes, which belong to the NLR-family (2). Recent studies have shown that the NLR family, pyrin 1 This work was supported by grants from the Medical Society of Finland (Finska la¨karesa¨llskapet), the Nummela Foundation, Research Council for Biosciences and domain, containing 3 (NLRP3, also known as NALP3/Cryopyrin/ Environment of the Academy of Finland, and the Sigrid Juselius Foundation. CIAS1), an adapter molecule apoptosis-associated speck-like pro- 2 Address correspondence and reprint requests to Dr. Sampsa Matikainen, Unit of tein containing a CARD, and caspase-1, are all central components Excellence for Immunotoxicology, Finnish Institute of Occupational Health, Topeli- uksenkatu 41 a A, Helsinki, Finland. E-mail address: sampsa.matikainen@ttl.fi of NLRP3-inflammasomes (2). For example, monosodium urate and aluminum adjuvant are recently described as molecules that 3 Abbreviations used in this paper: DBRI, damp building related illness; PRR, pattern recognition receptor; NLR, NOD-like receptor; DAMP, danger-associated molecular activate NLRP3-inflammasome (17–19). Inflammasomes are also pattern; NLRP3, NLR family, pyrin domain, containing 3; NTC, no template control; reported to be important for recognizing danger signals caused by C , cycle threshold value. T bacterial toxins: nigericin and valinomycin have been found to Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 trigger the activation of NLRP3-inflammasome associated www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803309 The Journal of Immunology 6419 caspase-1 (20). Once pathogen-associated molecular patterns and satratoxin production (only minor amounts of stachybotrylactam and DAMP-induced innate immunity cascades through PRRs are ini- stachybotrytlactone are produced, Ͻ1 ng per 105 spores), whereas strain 72 tiated, high amounts of cytokines, which are essential for the ac- produces satratoxin G and H, stachybotrylactone, and stachybotrylactam in the respective amounts of 4 ng, 10 ng, 8 ␮g, and 2 ␮g per 105 spores. tivation of antimicrobial and inflammatory response via innate and Previous analysis verified that strain 29 did not produce detectable amounts adaptive immunity, are produced. of either satratoxin G or H, and that in contrast, strain 72 produced 28 ng Water-damaged buildings harbor and provide substrates for sev- of satratoxin G and 6 ng of satratoxin H per 1 ϫ 105 spores (37). Strain 29 eral mold species, which increases the risk of accumulation of was used as the nonsatratoxin producing strain (satratoxin negative S. chartarum), and strain 72 as the satratoxin-producing strain (satratoxin positive harmful fungi. Stachybotrys chartarum is a notorious, but contro- S. chartarum). The spores were suspended directly from the agar plates versial damp building mold. This fungus is a potent toxin pro- into PBS. S. chartarum suspensions were gamma-irradiated with 10 kGy ducer, and many of its toxins are thought to cause DBRI (1). One before use. major group of fungal secondary metabolites produced by a num- Cell stimulation ber of fungi, including Fusarium sp., Stachybotrys sp. and My- rothecium sp., is trichothecene mycotoxins (21–23). The trichoth- The functional concentrations of LPS and mycotoxins were pretitrated, and ecenes (type A–D trichothecenes) are roughly classified as we used the final amounts used in experiments for LPS (Escherichia coli 0111:B4) and roridin A, verrucarin A, and T-2 toxin (Sigma-Aldrich), nonmacrocyclic trichothecenes (types A–C) or macrocyclic tricho- which was 100 ng/ml. Satratoxin-positive S. chartarum (s. 72) and satra- thecenes (type D) (23). In general, trichothecenes are reported to toxin-negative S. chartarum (s. 29) were used at 3 ϫ 103 or 1 ϫ 105 cause many unfavorable effects on eukaryotic cells, including in- spores/ml. Macrophages were treated with LPS, roridin A, verrucarin A, hibition of RNA, DNA, and protein synthesis, as well programmed T-2 toxin, or S. chartarum spores in Macrophage SFM medium. RPMI 1640 medium supplemented with 2 mM L-glutamate, 1 mM HEPES, and cell death, apoptosis (24). Type A, B, and D trichothecenes appear Downloaded from antibiotic was used when cell culture supernatants were prepared for con- to be the most significant from the public health perspective. The centration. After each stimulation period, the cells and cell culture super- T-2 toxin, which belongs to the type A group, is possibly the most natant were harvested, and samples prepared for the RT-PCR, Western studied nonmacrocyclic trichothecene. It is mainly produced by the blotting, and ELISA. Fusarium species, and is reportedly the most potent naturally oc- Quantitative real-time RT-PCR assay curring nonmacrocyclic trichothecene among diacetoxyscirpenol We extracted total cellular RNA using TRIzol Reagent (Life Technologies) (another type A toxin) (24–27). The macrocyclic trichothecenes, http://www.jimmunol.org/ according to the manufacturer’s instructions. Absorbance at A260 and A280 including satratoxins, roridins, and verrucarins, which are pro- nm was measured to determine RNA concentration and purity in a spec- duced by several mold species e.g., many strains of S. chartarum, trophotometer (GeneQuant Pro RNA/DNA calculator, Amersham Bio- reportedly mediate toxicity by interfering with ribosome function sciences). Isolated RNA was temporarily stored at Ϫ70°C in nuclease-free and activating stress responses (28, 29). These toxins may also water. First, 0.5 ␮g of RNA was reverse transcribed into cDNA by the have a DNA-damaging capacity (30, 31). We have previously High Capacity cDNA Reverse Transcription kit (Applied Biosystems) in a 25-␮l reaction mixture containing optimized reverse transcription buffer, shown in a mouse model of asthma that exposure to S. chartarum random primers, deoxyNTP mixture, and MultiScribe reverse transcriptase. induces lung inflammation characterized by the infiltration of im- The conditions for cDNA synthesis were as follows: annealing at 25°C for mune cells and the formation of granulomas and giant cells (32). 20 min and synthesis at 37°C for 120 min. The reverse transcription reac- However, the specific immunological mechanisms and pathways tion was performed in an Applied Biosystems’ Thermal cycler version by guest on September 28, 2021 2720. The quantitative Real Time-PCR and TaqMan analysis was per- underlying S. chartarum-induced or trichothecene-induced apo- formed in a 96-well optical reaction plate in ABI Prism 7500 Fast Se- ptosis and inflammation have not yet been clearly defined. In our quence Detector (Applied Biosystems). The cDNA was amplified in 11 ␮l current study, we have focused on resolving the capability of tri- of 1ϫ TaqMan Fast universal PCR master mix with Predeveloped TaqMan chothecenes, satratoxins, roridin A, verrucarin A, and T-2 toxins to assay primers and probes (IL-1␤ Hs0017497_m1, IL-18 Hs00155517_m1, activate inflammasome-associated innate immunity reactions in Applied Biosystems). The primer and probe sets were designed and optimized according to Applied Biosystems guidelines. For each sample, PCR human primary macrophages. amplification of the endogenous 18S rRNA was determined to control the amount of cDNA added according to the manufacturer’s instruction (Ap- Materials and Methods plied Biosystems) and to allow normalization between the samples. Targets Differentiation of macrophages from human peripheral and controls were run on separate wells in optical 96-well plates. The thermocycling conditions consisted of an initial step of 95°C for 20 s, 40 blood-derived monocytes cycles of denaturation at 95°C for 3 s, and annealing and extension steps Monocytes from healthy blood donors (Finnish Red Cross Blood Trans- at 60°C for 30 s. Real time PCR was performed at least twice for each fusion Service) were isolated and purified as previously described (33, 34). cDNA product. No template control (NTC), in which molecular grade wa- In brief, we isolated PBMCs from normal human peripheral blood by low ter was used instead of template, was included in each assay. speed density gradient centrifugation over Ficoll-Paque Plus (Amersham Real-time PCR data analysis Biosciences). The cell layer containing lymphocytes, platelets, and monocytes was recovered from the interface, and further washed to remove The real-time PCR data was developed by using Sequence detector system platelets, excess plasma, and Ficoll-Paque Plus. After three sequential version 1.4 software (Applied Biosystems). We selected the cycle thresh- ϫ 6 washes with PBS, 7.5–10 10 mononuclear cells, which contained 0.75– old value (CT) of a sample according to the manufacturer’s guidelines. ϫ 6 1.5 10 monocytes, were transferred to Multiwell six-well culture dishes Relative units were calculated by a comparative CT method. First, we sub- L (Falcon) in 1 ml of RPMI 1640 supplemented with 2 mM -glutamate and tracted the CT of 18S rRNA sample from the corresponding target cytokine ␮ ␮ ⌬ antibiotics: 0.6 g/ml penicillin and 60 g/ml streptomycin (all from Life CT to get the CT. Next, we subtracted the average of CTs of 18S rRNA Technologies). The monocytes were allowed to attach to cell culture plates samples from the calibrator CT value of 40, which was obtained from NTC, ⌬ ⌬ ⌬ at 37°C and 5% CO2 for 1 h. The attached cells were washed three times to get the calibrator CT. The calibrator CT was subtracted from the CT ⌬⌬ with PBS and differentiated into macrophages by maintenance in Macro- of each experimental sample to get CT. Finally, we calculated the phage-SFM medium (Life Technologies) supplemented with 10 ng/ml amount of target normalized to an endogenous control, which was relative GM-CSF (Biosource) and antibiotics. The following day, fresh medium to the NTC calibrator, by the equation 2Ϫ⌬CT. was changed, and replaced every 2 days thereafter. After 7 days of culture, the resulting macrophages were used in experiments. Western blot analysis Microbe preparations Western blot analyses were performed either by using whole cell extract or by using concentrated cell supernatants. First, cells were lysed in protein Stachybotrys chartarum strains 29 and 72 (NRRL 6084) have been char- lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, and 25% acterized. They were grown on rice flour agar as previously described (35, ethylene glycol supplemented with complete mini protease inhibitor mix- 36). When grown on rice agar, S. chartarum strain 29 is not capable of ture (Roche Diagnostics) and homogenized with ultrasound sonicator 6420 MYCOTOXINS ACTIVATE INFLAMMATORY RESPONSE

(Sanyo Electronics). We determined the total protein concentrations with Bio-Rad Dc Protein Assay (Bio-Rad) according to the manufacturer’s instructions. In contrast 5 ml of stimulated cell supernatants were concentrated by Amicon UItra-15 tubes, (Millipore) also according to the manufacturer’s instructions. Then, 10 ␮g of proteins from whole lysed cell extracts or 2.5 ␮l from 250 ␮l of concentrated cell supernatants were separated on 15% SDS-PAGE at 200 V and transferred onto Immobilon-P Transfer Membranes (Millipore) by the Isophor electro transfer apparatus PowerPac Basic (Bio-Rad) at ϩ4°C and 100 V for 2 h. The membranes were blocked in PBS containing 5% nonfat milk for 45 min after which they were incubated at ϩ4°C overnight. After this, membranes were incubated at room temperature for 1 h with appropriate HRP-conjugated secondary Abs (Dako A/S). Finally, proteins were visualized by the ECL system (PerkinElmer). To conﬁrm equal loading and transfer of the protein samples, membranes were stripped and stained with ready-to-use SYPRO Ruby Protein Blot Stain according to the manufacturers’ instructions (Sigma-Aldrich). The major protein band detected and basally expressed is shown as a loading control in each Western blot analysis. Anti-IL-1␤ and anti-IL-18 Abs have previously been described (34, 38). Anti-caspase-1 and anti-caspase-3 Abs were purchased from Sigma-Aldrich and Cell Sig- naling Technology, respectively. Downloaded from ELISA

The cell culture cytokine concentrations were determined by ELISA ac- ␤ cording to the manufacturer’s instructions. Human IL-1␤ Eli-pair and hu- FIGURE 1. S. chartarum activates IL-1 and IL-18 mRNA expression man IL-18 ELISA were purchased from Diaclone and Medical and Bio- in human macrophages. Human macrophages were stimulated with S. logical Laboratories, respectively. chartarum or activated with LPS for the times indicated, after which total RNA was extracted and cDNA was synthesized. A, IL-1␤ and B, IL-18

mRNA expression was analyzed by real-time PCR. The data represents http://www.jimmunol.org/ Statistical analyses relative units (RU), that is, fold change in gene expression, which is nor- Each macrophage sample represents a pool of separately stimulated cells malized to an endogenous reference gene (18S rRNA) and is relative to from three different blood donors. The RT-PCR and Western blot results no-template-control (NTC)-calibrator. Each macrophage sample represents are representative of three independent, but similarly performed experi- a pool of separately stimulated cells from three different blood donors and ments, and ELISA results are combined from three different stimulations. the results are representative of three independent, but similarly performed Data were analyzed with the GraphPad Prism 4 Software (GraphPad Soft- experiments. Values are means Ϯ SD from three independent analyses. ware). An unpaired t test or Mann-Whitney U test was used to compare the .p Ͻ 0.001 ,ءءء ;p Ͻ 0.05 ,ء differences between the groups. A p value Ͻ0.05 was considered to be statistically significant. In RT-PCR and ELISA figures, data were expressed as means (ϮSD). by guest on September 28, 2021 Satratoxin-positive S. chartarum and LPS synergistically activate Results caspase-1 and caspase-3-mediated inflammatory and apoptotic Regulation of IL-1␤ and IL-18 mRNA expression in human cascades in human macrophages macrophages in response to Satratoxin positive S. chartarum To analyze whether costimulation of macrophages by satratoxin- and LPS positive S. chartarum and LPS results in the secretion of mature ␤ To compare S. chartarum- and LPS-induced proinflammatory cy- IL-1 and IL-18, we stimulated the cells with S. chartarum spores ␤ tokine response in vitro, human primary macrophages were stim- and LPS for 12 h. IL-1 and IL-18 were determined by ELISA ulated for different time periods with satratoxin-positive S. char- from the stimulated cell culture supernatants. Results showed that ␤ tarum and LPS, and the expression of IL-1␤ and IL-18 genes was LPS and S. chartarum alone elicited very little secretion of IL-1 studied by RT-PCR. As shown in Fig. 1, LPS strongly activated and IL-18. However, the enhancement in the release of the cyto- IL-1␤ mRNA expression, and its maximal induction was seen at kines was seen after the costimulation: together S. chartarum and ␤ 3 h after stimulation. In contrast to LPS, S. chartarum induced only LPS caused increased secretion of IL-1 and IL-18 in human mac- a modest up-regulation of IL-1␤ gene expression, and the highest rophages (Fig. 3, A and B). ␤ level was detected at 3 to 6 h after S. chartarum exposure. IL-18 IL-1 and IL-18 are synthesized as inactive 31 kDa (p31) and mRNA was expressed at high basal level in macrophages: LPS and 24 kDa (p24) precursors, respectively, which remain in the cyto- S. chartarum spores had little effect on IL-18 mRNA expression. plasm. Their biological activity is dependent on caspase-1, which cleavages them into smaller subunits, to the conformation in which these cytokines can be secreted (15). To study the effect of satra- Satratoxin-positive S. chartarum and LPS synergistically activate toxin-positive S. chartarum and LPS on IL-1␤ and IL-18 protein ␤ IL-1 and IL-18 gene expression processing, we performed Western blot analysis with anti-IL-1␤ In damp buildings, S. chartarum grows together with other molds and anti-IL-18 specific Abs. As shown in Fig. 3C, LPS induced and bacteria, and this coexposure of fungi and bacteria may have synthesis of pro-IL-1␤, which was not seen in the cells stimulated harmful effects on human health. Therefore, we studied the pos- by satratoxin-positive S. chartarum. Pro-IL-1␤ was also absent in sible synergistic effects of satratoxin-positive S. chartarum and macrophages that were cotreated with LPS and S. chartarum LPS on the expression of IL-1␤ and IL-18 genes in human mac- spores. S. chartarum activated the formation of IL-18 p18, which rophages. The cells were stimulated for 6 h with three different is the caspase-1 cleavage product and the biologically active form amounts of satratoxin-positive S. chartarum spores both with and of IL-18. In addition, we observed the caspase-3 processed cleav- without LPS treatment. We found that satratoxin-positive S. char- age products of IL-18, IL-18 p15/p16, in macrophages, which were tarum and LPS had a strong synergistic effect on IL-1␤ and IL-18 activated by S. chartarum. In agreement with our previous results, mRNA expression (Fig. 2). LPS did not induce proteolytic processing of pro-IL-18. However, The Journal of Immunology 6421

FIGURE 2. LPS activates significant IL-1␤ (A) and IL-18 (B) mRNA expression in synergy with satratoxin positive S. chartarum spores. Mac- Downloaded from rophages were stimulated with different concentrations of S. chartarum in the absence or presence of LPS for 6 h after which total cellular RNA was prepared, cDNA synthetized, and the TaqMan analysis was performed. .p Ͻ 0.01 ,ءء .Values are means Ϯ SD from three independent analyses http://www.jimmunol.org/ LPS enhanced the S. chartarum-induced formation of both IL-18 p18 and IL-18 p15/p16 (Fig. 3C). Caspase-1 is expressed as an inactive proenzyme (p45), which must be activated by proteolytic cleavage. The functional caspase-1 comprises of p10 and p20 subunits, which assemble into a heterotetramer (38). Like caspase-1, caspase-3 is a latent zymogen, which is processed upon activation into smaller polypeptide chains: p17 and p19, which in turn form the bioactive enzyme (10, 39, 40). To study the effects of LPS and S. chartarum on caspase-1 by guest on September 28, 2021 and caspase-3 activation, we performed Western blot analysis with anti-caspase-1 p20 and anti-caspase-3 p17/p19-specific Abs. As shown in Fig. 3C, satratoxin-positive S. chartarum activated the formation of caspase-1 p20 as well as caspase-3 p17/p19. Further- more, S. chartarum and LPS together enhanced the formation of both caspase-1 p20 and caspase-3 p17/19. LPS was unable to induce the formation of either caspase-1 p20 or caspase-3 p17/p19. Instead, it induced the caspase-1 p22 subunit (Fig. 3C). Thus, the satratoxin-positive S. chartarum activates both caspase-1 and caspase-3, a process which was enhanced by LPS costimulation. FIGURE 3. A and B, S. chartarum and LPS synergistically activate secretion of IL-1␤ and IL-18. Macrophages were activated with S. chartarum Trichothecene mycotoxins activate caspase-1 and caspase-3 in and LPS or their combination for 24 h. IL-1␤ and IL-18 secretion was human macrophages analyzed using ELISA. Each macrophage sample represents a pool of separately stimulated cells from three different blood donors. The mean of two p Ͻ 0.001. C, LPS ,ءءء ;p Ͻ 0.05 ,ء .Trichothecene mycotoxins such as satratoxins, roridins, and ver- separate experiments (ϮSD) is shown rucarins are secondary metabolites of several strains of S. charta- enhances S. chartarum-dependent caspase-1 and caspase-3 activation. rum (41–44). Trichothecenes have been linked to many harmful Macrophages were left untreated or activated with different amounts of S. health effects (1). To study the potential role of satratoxin in S. chartarum (satratoxin-producing strain s.72) in both the absence and pres- chartarum-induced caspase-1 and caspase-3 activation, macro- ence of LPS or their combination for 12 h. After stimulation, total cellular phages were exposed to satratoxin-positive (s. 72) and satratoxin- protein lysates were prepared, separated on 15% SDS-PAGE, and transferred on membranes. The proteins were visualized by ECL with anti-IL- negative (s. 29) S. chartarum strains and we analyzed caspase-1 1␤, anti-IL-18, anti-caspase-1 p20, and anti-caspase-3 p17/19 specific Abs. and caspase-3 activation with Western blotting. In accordance with Equal loading of protein samples was controlled by using SYPRO Ruby the results shown in Fig. 4A, satratoxin-positive S. chartarum ac- protein stain. The results are representative of three separate experiments. tivated the formation of caspase-1 p20 and caspase-3 p17/p19. Activation of caspase-1 and caspase-3 were seen 6 to 10 h after fungal exposure. In accordance with these results, processing of the trichothecenes, satratoxin G and H, produced by S. chartarum pro-IL-18 to smaller IL-18 p18 and IL-18 p15/16 pieces were seen, strain 72, are the probable reason for the activation of caspase-1 which further confirms activations of caspase-1 and caspase-3, re- and caspase-3 in human macrophages. spectively. In contrast, satratoxin-negative S. chartarum was not To further study the role of trichothecenes in the activation of able to activate caspase-1 or caspase-3, nor the processing of IL-18 caspase-1 and caspase-3, macrophages were stimulated with the at any of the time-points studied (Fig. 4A). Our results suggest that purified trichothecenes, roridin A, verrucarin A, and T-2 toxin for 6422 MYCOTOXINS ACTIVATE INFLAMMATORY RESPONSE

FIGURE 5. Roridin A dose-dependently activates secretion of IL-1␤ in LPS-primed human macrophages. The cells were first stimulated with LPS for 6 h after which the cells were treated with different concentrations of roridin A for 18 h. Secretion of IL-1␤ from cell culture supernatants was studied by ELISA. Each macrophage sample represents a pool of separately stimulated cells from three different blood donors. The mean of three .p Ͻ 0.01 ,ءء .separate experiments (ϮSD) is shown Downloaded from Costimulation with roridin A, verrucarin A, or T-2 toxin and LPS enhance secretion of IL-1␤ and IL-18 FIGURE 4. Mycotoxins are involved in caspase-1 and caspase-3 activation. A, Human macrophages were left untreated or stimulated with both To determine the effect of the cooperation between the trichoth- with satratoxin-producing strain (s.72) and the nonproducing strain (s.29) ecenes and LPS on cytokine production, macrophages were first of S. chartarum for the times indicated. After this, total cellular protein stimulated by LPS for 6 h. After this, the cells were further stim- lysates were prepared, separated on 15% SDS-PAGE, and transferred onto ulated with the trichothecene mycotoxins, roridin A, verrucarin A, http://www.jimmunol.org/ membranes. The proteins were visualized by ECL with anti-IL-18, anti- and T-2 toxin, for 18 h. We then collected the cell culture super- caspase-1 p20, and anti-caspase-3-specific Abs. B, Macrophages were left natants, and the secretion of IL-1␤ and IL-18 was studied using untreated or activated with roridin A, verrucarin A, or T-2 toxin for 24 h. ELISA. As a result, all the trichothecenes, together with LPS, elic- Protein lysates were prepared and analyzed by Western blotting. The pro- ited a high secretion of both IL-1␤ and IL-18 (Fig. 6). The syn- teins were visualized with anti-caspase-1 p20 and anti-caspase-3 p17/19 specific Abs. Equal loading of protein samples was controlled by using ergistic effect of mycotoxins and LPS on IL-18 production was not SYPRO Ruby protein stain. The results are representative of three separate experiments. by guest on September 28, 2021

18 h, after which caspase-1 activation was studied by Western blotting. Results presented in Fig. 4B show that caspase-1 was activated in response to all trichothecenes used. The trichothecenes also induced further degradation of caspase-1 p20 to a smaller fragment (the lowest arrow in caspase-1 picture in Fig. 4B). In addition, caspase-3 was clearly activated in response to toxins (Fig. 4B).

Roridin A dose-dependently activates secretion of IL-1␤ in LPS-primed cells It is commonly known that LPS is able to activate the expression of pro-IL-1␤, but it is unable to induce processing of pro-IL-1␤ to its biological active form. LPS-treated cells need second stimulus, such as ATP, for caspase-1 activation before pro-IL-1␤ is processed and mature IL-1␤ is secreted (45, 46). Therefore, we studied the capability of roridin A to activate secretion of IL-1␤ in LPS-primed cells. Macrophages were ﬁrst stimulated with LPS for 6 h, and the cells were further incubated with roridin A concentration at 100, 10, or 1 ng/ml for 18 h. As ELISA results show in Fig. 5, the stimulation of LPS-primed cells with roridin A concentration at 100 and 10 ng/ml induced the highest secretion of IL-1␤ into the cell culture supernatants. Also 1 ng/ml of roridin A in- FIGURE 6. LPS and roridin A, verrucarin A, or T-2 toxin synergistically activate IL-1␤ (A) and IL-18 (B) secretion. Macrophages were stim- duced minor IL-1␤ secretion levels in human macrophages. Rori- ␤ ulated with LPS for 6 h after which the cells were further stimulated with din A had a synergistic effect on IL-1 secretion also with lower roridin A, verrucarin A, and T-2 toxins for 18 h. After this, the cell culture LPS concentrations (10 ng/ml; data not shown). In further studies, supernatants were collected and IL-1␤ and IL-18 secretion was studied ,ء .the trichothecene mycotoxins and LPS were used at a concentra- using ELISA. The mean of three separate experiments (ϮSD) is shown .p Ͻ 0.01 ,ءء ,tion 100 ng/ml. p Ͻ 0.05 The Journal of Immunology 6423

macrophages with LPS and trichothecenes is required for the ef- ﬁcient secretion of biologically active forms of IL-1␤ and IL-18.

Discussion Multicellular organisms have evolved to live in the presence of microbes. During evolution, the host cells have developed many mechanisms to keep the interaction in balance. However, for some reason this harmony is easily broken, resulting in different clinical symptoms, which are mainly caused by the reaction of our own immune systems. Stachybotrys chartarum is a notorious but also controversial damp building mold. This toxic mold has often been associated to the DBRI (1). Mycotoxins, especially the trichothecene mycotoxins it produces, are probably a reason behind the symptoms (1, 47, 48). Trichothecenes have immunoregulatory and cytotoxic effects on immune cells (28, 36, 49–52). In addition, it has been concluded that these mycotoxins cause MAPK-mediated ribotoxic stress and apoptosis (53–55). We have previously shown in a mouse model of asthma that exposure to toxic S. chartarum

FIGURE 7. Costimulation of macrophages with LPS and the trichoth- induces lung inflammation characterized by the infiltration of im- Downloaded from ␤ ecenes activate the processing and secretion of IL-1 and IL-18. Macro- mune cells, as well as the formation of granulomas and giant cells phages were stimulated with LPS for 6 h and further for 18 h with the (32). However, the exact mechanism for S. chartarum-induced toxins. The cells were collected and lysed or cell supernatants were concentrated. A, The total cell lysates and B, the concentrated cell culture lung inflammation has not been determined. A potential explana- supernatants were used for Western blot analysis. The proteins were visu- tion for the inflammation may found in the ability of the trichoth- alized by ECL with anti-IL-1␤, and anti-IL-18 specific Abs. Equal loading ecene mycotoxins to cause these reactions. In the present work, we of protein samples was controlled by using SYPRO Ruby protein stain. The have studied the effect of S. chartarum and purified trichothecene http://www.jimmunol.org/ results are representative of three similarly performed but independent mycotoxins on proinflammatory cytokine response in human pri- experiments. mary macrophages. Activated macrophages are the primary source of proinflammatory cytokines, including IL-1␤ and IL-18. It is well characterized statistically significant due to high interindividual variation of that TLR ligands, including LPS, induces transcription of IL-1␤ IL-18 secretion levels. However, in each separate experiment, a and expression of pro-IL-1␤ protein (15). A second stimulus is synergistic effect of mycotoxins and LPS on IL-18 secretion was needed to engage inflammasome-associated caspase-1 activation, clearly seen (data not shown). Hence, IL-1␤ and IL-18 release, in which leads to pro-IL-1␤ processing and secretion of mature IL-1␤ LPS-primed cells, indicated that the trichothecenes activate the protein (56). Caspase-1 also processes pro-IL-18 for its biologi- by guest on September 28, 2021 inflammasome associated caspase-1 mediated inflammatory re- cally active form and enables the secretion of IL-18 (2). In our sponse in human macrophages (Fig. 4, 5, and 6). experiment, the satratoxin-positive S. chartarum activated gene expression of IL-1␤ and IL-18 (Fig. 1). Furthermore, satratoxin- Trichothecenes, roridin A, verrucarin A, and T-2 toxin, active positive S. chartarum and LPS synergistically activated IL-1␤ and ␤ proteolytic processing and secretion of IL-1 and IL-18 in IL-18 gene expression (Fig. 2). This enhancement was also seen in LPS-primed human macrophages the secretion level of the cytokines promoted by costimulation, To further characterize the proteolytic processing of pro-IL-1␤ and indicating activation of inflammasome-associated caspase-1 (Fig. pro-IL-18 proteins, we performed Western blot analysis from cell 3). These results also suggest that S. chartarum spores include lysates, and from concentrated cell culture supernatants. The sam- some substance(s), which is sensed as a danger signal by human ples were prepared from macrophages, which were left either un- macrophages. stimulated or stimulated with LPS and/or the purified trichoth- Many bacterial toxins are now known to be involved in the ecene mycotoxins. Western blot samples performed from the cell activation of innate immune response. However, because toxins lysates show that, unlike LPS, the mycotoxins were not able to are structurally a diverse group of molecules, they cannot be rec- activate the synthesis of pro-IL-1␤ protein (Fig. 7A). Instead, the ognized directly by specific PRRs. In contrast, toxins may, for costimulation of macrophages with the toxins and LPS caused both example, trigger changes in cellular ion composition, which the the production and the processing of pro-IL-1␤, and this cotreat- cells of the innate immunity system sense as a danger signal (20). ment also triggered the appearance of the biologically active 17- Complexes formed by the NLR-family components, adapter mol- kDa form of IL-1␤ (Fig. 7A). In addition, results from the cell ecules, and caspases are important in the sensing of different lysates showed that stimulation of macrophages with roridin A, DAMP signals, including ATP release of the host cells (2). The verrucarin A and T-2 toxin activates the processing of pro-IL-18 to recently described NLRP3-inflammasome complex is activated by IL-18 p15/p16 isoforms, which are the caspase-3 cleavage prod- a potassium (Kϩ) efflux, triggered by pore-forming bacterial toxins ucts of IL-18 (Fig. 7A). The costimulation of macrophages with (57). This inflammasome-induced caspase-1 activation enables the LPS and the mycotoxins also resulted in the appearance of the secretion of IL-1␤ and IL-18 (2). In our experiments, the satra- biologically active 18-kDa form of IL-18 in whole cell lysates toxin-positive S. chartarum induced the formation of the active (Fig. 7A). Further, the secretion of the biologically active form of caspase-1 p20 subunit, but the satratoxin negative strain of S. char- IL-1␤ and IL-18, as well as the pro-forms of these cytokines, was tarum did not (Fig. 4A). LPS also failed to activate the formation clearly seen in concentrated cell culture supernatants, which were of caspase-1 p20. Instead, the biologically inactive caspase-1 p22 prepared from macrophages that were costimulated with LPS and was generated, which is not capable of processing pro-IL-1␤ or the mycotoxins (Fig. 7B). These results are in line with the ELISA pro-IL-18 into the biologically active forms of the cytokines (Fig. results shown in Fig. 6, and further confirm that costimulation of 3C). Our result also shows that the purified trichothecenes, roridin 6424 MYCOTOXINS ACTIVATE INFLAMMATORY RESPONSE

A, verrucarin A, and T-2 toxin activate caspase-1 and they also novel role for the trichothecenes as a danger signal, which acti- seem to have a potency for further caspase-1 degradation (Fig. 4B). vates inflammasome-associated caspase-1 as a part of the myco- The role of degraded caspase-1 is still unclear. The costimulation toxin-triggered innate immunity response. with LPS and the trichothecene-induced enhancement in the secretion of IL-1␤ and IL-18, which further verifies the role of the Acknowledgments trichothecenes as DAMPs, triggering the activation of inflamma- We thank Dr. Maria Andersson for preparing the fungal culture extract, Dr. some associated caspase-1 (Figs. 5–7). Further studies are needed Ilkka Julkunen for providing anti-IL-18 Abs, Tuula Stjernvall for technical for the detailed characterization of the mechanisms for inflamma- assistance, Prof. Emer. Bruce Jarvis for his valuable advice and Eeva-Liisa some activation triggered by trichothecenes. Hintikka and all the laboratory staff for useful discussions. In addition to inflammation, exposure of mouse alveolar cells to S. chartarum toxins has also been associated with cell death (53). Disclosures Cell death may be due to necrosis or apoptosis. Most of the mor- The authors have no financial conflict of interest. phological changes associated with cell death are caused by a set of cysteine proteases, which are specifically activated in apoptotic References cells (10). These death proteases are functionally and structurally 1. Pestka, J. J., I. Yike, D. G. Dearborn, M. D. Ward, and J. R. Harkema. 2008. homologous to each other, and belong to a large caspase protein Stachybotrys chartarum, trichothecene mycotoxins, and damp building-related family (12). Caspase-3 is a central executioner of the apoptotic illness: new insights into a public health enigma. Toxicol. Sci. 104: 4–26. 2. Petrilli, V., C. Dostert, D. A. Muruve, and J. Tschopp. 2007. The inflammasome: pathway. 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