In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune and destructive joint disease affecting 1% of the population. RA is characterized by abnormal accumulation of immune cells in joints. These immune cells together with endothelial cells, fibroblasts, and chondrocytes express and export a complex mixture of proinflammatory mediators, such as cytokines (TNF-α, IL-1, IL-6, etc.), chemokines (MCP-1, MCP-4, etc.), lipids, growth factors, transcription factors (e.g., NF-κB, STATs, activator protein-1), and destructive enzymes (e.g., matrix metalloproteinases), which are critically involved in local tissue destruction and fibrotic processes (McInnes and Schett, 2007).
Glucocorticoids (GC) at high doses are used to control acute RA disease attacks (Kirwan and Power, 2007) and are prescribed at low concentrations as long-term medication. The anti-inflammatory effects of GCs are mediated by their ability to modulate expression of proinflammatory genes such as IL-1β or TNF-α, as well as T-cell proliferation (Chikanza, 2002). Although GCs are very potent anti-inflammatory drugs, their long-term usage is limited by a number of severe side effects, such as osteoporosis, insulin resistance, skin thinning, hypertension, obesity, and inhibition of wound repair. Moreover, approximately 30% of patients with RA fail to respond to steroid therapy (Barnes and Adcock, 2009). Therefore, the development of new anti-inflammatory drugs with the therapeutic potency of GCs but without their adverse effects is essential.
In our previous work, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as a potent inhibitor of proinflammatory gene expression in human alveolar epithelial A549/8 cells. SC reduced the expression of the human inducible nitric-oxide synthase (iNOS), an enzyme critically involved in proinflammatory immune processes, by inhibition of the cytokine-induced promoter activity. Studies on the mode of action revealed that SC inhibited the phosphorylation and thereby the activation of the tyrosine kinase (Janus kinase) JAK2 and, consecutively, of the transcription factor signal transducer and activator of transcription-1 (STAT-1) resulting in the inhibition of STAT-1-dependent gene expression (Yao et al., 2003). Inhibition of STAT-1-mediated signal transduction seems to be an attractive concept in RA therapy because it has been demonstrated that genes transcriptionally regulated by the JAK/STAT-1 pathway were up-regulated in RA patients with severe joint inflammation (van der Pouw Kraan et al., 2003).
Chondrocytes play an important role in the cartilage degradation observed in human joint diseases (Otero and Goldring, 2007). Under stress conditions, they produce a variety of cytokines, such as TNF-α or IL-1β, metalloproteases, NO, prostaglandins, and other mediators all associated with the inflammatory process. We formerly demonstrated that human C28/I2 chondrocytes did not respond to GC treatment because of the lack of glucocorticoid receptor α (GRα) expression. In contrast to GC, treatment of C28/I2 cells with SC inhibited cytokine induced iNOS expression (Schmidt et al., 2010). These findings suggest that SC may be useful as an anti-inflammatory agent to treat glucocorticoid-resistant inflammatory diseases.
In the current study, we used the mouse model of collagen-induced arthritis (CIA), which represents a wide accepted animal model of rheumatoid arthritis. In this model, immunization with heterologous collagen-type-II (CII) induces severe synovitis with redness and swelling of the joints. CII seems to be also a relevant autoantigen in humans, and there is evidence that in both human and mice, the disease is driven by CII-specific T and B cells and their secreted cytokines (Kim et al., 2000).
In this chronic inflammation model, SC treatment decreased the expression of important proinflammatory cytokines and chemokines, involved in the onset and progression of RA, in a similar manner as the glucocorticoid dexamethasone (Dex). In microarray analyses, we identified several immunomodulatory genes regulated during the course of CIA that are significantly affected by the therapeutic intervention with SC. Furthermore, SC reduced the expression of proinflammatory genes in human C28/I2 chondrocytes and human MonoMac6 monocytes at the mRNA and protein level. In conclusion, we demonstrate that SC possesses a similar therapeutic power as Dex in regulating proinflammatory gene expression in cell culture as well as in in vivo models.
Materials and Methods
DBA/1 mice expressing a transgenic TCR β-chain (Vβ12), obtained from a CII-specific T-cell clone (Mori et al., 1992), were used. Mice were housed under specified pathogen-free conditions in accordance with standard animal care requirements and maintained on a 12-h light/dark cycle. Water and food were given ad libitum. The animal studies were approved by the ethics board, and they were performed with the current regulation on animal handling.
Induction and Treatment of CIA.
CIA induction was performed as described previously (Hess et al., 1996), with little modifications. CII (Sigma-Aldrich, Deisenhofen, Germany) was dissolved in 0.01 M acetic acid at 4 mg/ml. Animals were given intradermal injections of 200 μg of CII emulsified in complete Freund's adjuvant (CFA) (Becton Dickinson, Heidelberg, Germany) in both ears (50 μg each) and the base of the tail (100 μg). Two booster injections of 100 μg of CII in CFA were given intradermally 11 and 21 days later. Control animals were given intradermal injections of PBS. From day 21 to 37, CII-immunized mice were given intraperitoneal injections every 2nd day with 10 mg/kg (S)-curvularin, 5 mg/kg dexamethasone, or PBS/ethanol (EtOH) as solvent control. At days 21, 25, 29, 33, and 37, eight mice of each treatment group were euthanized by inhalation of CO2 for subsequent analyses.
Assessment of Arthritis.
Mice were visually checked for the appearance of arthritis in peripheral joints every 2 days, and grades of an established scoring system (Brand et al., 2007) ranging from 0 to 4 were allotted to each limb. A maximum score of 16 could be achieved for each mouse. The arthritis score was checked by a minimum of two people but not in a fully blinded manner.
(S)-Curvularin has been obtained by fermentation of the producer strain Penicillium sp. IBWF3-93 and isolation from the culture fluid by chromatographic methods was performed as described previously (Yao et al., 2003). The purity of S-curvularin as estimated by high-performance liquid chromatography equipped with diode-array detection and mass spectrometry analysis was greater than 99% (Supplemental Fig. 1).
Total RNA of paws was prepared by homogenizing the sample in GIT buffer (Chomczynski and Sacchi, 1987), and RNA was isolated as described previously (Rodriguez-Pascual et al., 2000). Total RNA from C28/I2 and MonoMac6 cells was prepared using the RNeasy Mini kit (QIAGEN, Hilden, Germany).
For microarray analysis, total RNA was prepared from all paws of each animal with the RNeasy Mini kit (QIAGEN), and then the RNA of all paws of one mouse was pooled. RNA quality analysis, generation of fluorescent-labeled cDNAs, microarray hybridization, and microarray data analysis were performed as described using mouse total genome OpArrays (Eurofins MWG Operon, Ebersberg, Germany) (Pautz et al., 2009). The study was conducted according to standards developed by the Microarray Gene Expression Data Society (MGED), and data complying with the “minimum information about microarray experiments” (MIAME) have been deposited in the EMBL-EBI ArrayExpress repository (http://www.ebi.ac.uk/arrayexpress; experiment name: HK_CIA_experiment).
Real-Time Reverse Transcription Polymerase Chain Reaction Analysis.
Gene expression in mouse paws was quantified in a two-step real-time RT-PCR as described previously (Schmidt et al., 2010). In the RT step, cDNA was reverse transcribed from 0.5 to 1 μg of total RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's recommendations. Real-time PCR analysis was performed in a total volume of 25 μl in a 96-well spectrofluorometric thermal cycler (iCycler; Bio-Rad Laboratories, München, Germany). For real-time PCR (40 cycles of 15 s at 94°C, 60 s at 60°C), the following oligonucleotides served as sense and antisense primers and TaqMan hybridization probe: MMP3, sense TGGAGATGCTCACTTTGACG, antisense ATGGAAACGGGACAAGTCTG, probe CACTCAGCCAAGGCTGAAGCTCTGA; RANKL, sense GTTCCTGTACTTTCGAGCGC, antisense TGTGTTGCAGTTCCTTCTGC, probe CATCGGGTTCCCATAAAGTCACTCTGTCCT; Pol2A, sense ACCACGTCCAATGATATTGTGGAG, antisense ATGTCATAGTGTCACACAGGAGCG, probe CTGGGCATTGAGGCTGTGCGGAA; and Notch2, sense TACCTGTCACATGCTCAGCC, antisense TCACACTTCTGCCCTGTGAG, probe TGCCTGTCTATCTCATCCCTGCGAA.
The mRNA expression in human C28/I2 chondrocytes and MonoMac6 cells was analyzed by two-step real-time RT-PCR as described previously (Jung et al., 2009; Schmidt et al., 2010). cDNA was prepared from 0.5 to 1 μg of total cellular RNA. The reverse transcription was performed with the RevertAidTM H Minus First Strand cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. PCR products were synthesized from cDNA (100 or 300 ng) using functionally validated gene expression assays as the ABsolute qPCR SYBR Green fluorescein mix obtained from ABgene (Epsom, Surrey, UK). A two-step amplification protocol was chosen: initial denaturation step at 95°C for 10 min followed by 45 cycles with 15-s denaturation at 94°C, 30-s annealing at 56°C, and 30-s extension at 72°C. To analyze the mRNA expression of investigated genes, qRT-PCR was carried out using the following gene-specific primers: MCP-1, sense ATCAATGCCCCAGTCACC, antisense AGTCTTCGGAGTTTGGG; COX2, sense TTCAAATGAGATTGTGGGAAAATTGCT, antisense AGATCATCTCTGCCTGAGTATCTT; GAPDH, sense CCTCCGGGAAACTGTGG, antisense AGTGGGGACACGGAAG; IL-8, sense TGCCAAGGAGTGCTAAAG, antisense CTCCACAACCCTCTGCAC; TNF-α, sense TCTTCTGCCTGCTGCACTTTGG, antisense ATCTCTCAGCTCCACGCCATTG; and S100A8, sense CCATGCCGTCTACAGGGATG, antisense TATCCAACTCTTTGAACCAGACGTC. To calculate the relative mRNA expression, the 2[−ΔΔC(T)] method (Livak and Schmittgen, 2001) was used.
Western Blot Experiments.
To study protein-expression in mouse tissue, 50 to 100 μg of protein was separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes by semidry electroblotting. Total cell extracts of MonoMac6 cells or mouse tissue were prepared using RIPA detergent buffer [137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4·2 H2O, 1.5 mM KH2PO4, pH 7.4, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1.0 mM Na3VO4, and 1:50 complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)]. All further steps were performed as described previously (Rodriguez-Pascual et al., 2000). For murine protein detection, a monoclonal anti-S100A8 antibody (Santa Cruz Biotechnology, Heidelberg, Germany); polyclonal anti-PpiB and anti-MPO antibodies (both from R&D Systems, Wiesbaden, Germany); and anti-p38 MAPK antibodies (NEB, Frankfurt a.M., Germany) in a 1:1000 dilution were used. Human proteins were detected with anti-S100A8 and anti-GAPDH antibodies (1:1000 dilution; both from Santa Cruz Biotechnology).
Cell Culture and Stimulation Protocols.
Human immortalized C28/I2 chondrocyte cells (Goldring et al., 1994) were cultured as a superconfluent monolayer as described previously (Schmidt et al., 2010). For qRT-PCR and proteome profiling, cells were starved for 16 h in DMEM with 0.5% fetal calf serum. Cells were pretreated afterward with SC in serum-reduced medium for 1 h. Stimulation with a cytokine mixture (CM) consisting of 10 ng/ml TNF-α, 5 ng/ml IL-1β, and 10 ng/ml IFN-γ was performed for 6 (qRT-PCR) and 16 h (proteome profiling), respectively. Human immortalized MonoMac6 monocyte cells were cultured as described previously (Erkel et al., 2007). For qRT-PCR and Western blot analysis, cells were starved for 16 h in RPMI 1640 medium with 0.5% fetal calf serum, pretreated with SC in serum-reduced medium for 1 h, and then stimulated with a mixture of 1 μg/ml LPS, 10 ng/ml IFN-γ, 10 ng/ml TNF-α, and 5 ng/ml IL-1β for 24 h.
The cytotoxicity of (S)-curvularin against C28/I2 cells was determined in a Giemsa staining assay according to Mirabelli et al. (1985). Absorbances were read by a Lambda Spectral 340 Microplate reader (MWG Biotech, Ebersberg, Germany) at a wavelength of 600 nm and a reference wavelength of 405 nm. Data were displayed as percentage of the control absorbances (relative to untreated cells).
To analyze proinflammatory protein expression in murine samples or in culture supernatants of C28/I2 cells, we used the proteome profiler Mouse Cytokine Array Panel A and the Human Cytokine Array Panel A according to the protocol of the manufacturer (R&D Systems). The QuantityOne software (Bio-Rad Laboratories) was used for quantification.
Data represent means + S.E.M. Statistical differences were determined by factorial analysis of variance followed by Fisher's protected least-significant-difference test for comparison of multiple means.
The Expression of Different Proinflammatory Mediators Was Decreased by SC.
Our previous analyses showed that SC inhibits the JAK/STAT pathway (STAT-1α-phosphorylation) and thereby proinflammatory gene expression in different human and murine cell systems (Yao et al., 2003; Elzner et al., 2008; Schmidt et al., 2010; and data not shown). In addition, SC was able to reduce proinflammatory gene expression in LPS-treated mice in vivo (data not shown). Therefore, we evaluated the anti-inflammatory properties of SC in the CIA mouse model. CIA was induced in transgenic Vβ12 DBA/1 mice as described under Materials and Methods. In comparison to PBS-injected control mice, mice immunized with CII displayed severe symptoms of an inflammation, visible as redness and swelling of the digits or other parts of the paws. First signs of inflammation were seen between days 21 and 25 after the first immunization with collagen type II, and the maximum was reached around day 33, with an average arthritis score of 8.2 ± 2.22 (data not shown).
To investigate the effect of (S)-curvularin in the setting of a chronic inflammation in the CIA model, the CII-immunized mice were given intraperitoneal injections every 2nd day from day 21 on with (S)-curvularin dissolved in PBS/10% EtOH (CII+SC) (10 mg/kg), dexamethasone (CII+Dex) (5 mg/kg) dissolved in PBS/10% EtOH, or PBS/10% EtOH (CII) as a solvent control. To analyze the effects of SC and Dex on the expression of different CIA-related cytokines and chemokines, we performed a proteome profile analysis with a mouse cytokine/chemokine array using cell lysates isolated from paws on day 33 after immunization with CII (Fig. 1A). CII treatment (CII) significantly elevated the expression (between 1.6- and 32-fold), which is presented as log 2 ratios, of chemokines such as component 5a (C5a), MCP-1, CCL17, Mip-1α, MIP-1β, MIP-2, or CXCL12. These chemokines are responsible for the recruitment of immune cells to the inflamed tissue (Fig. 1B). The largest effect of Dex and SC was detected on CCL17 expression, which was nearly reduced to the level of control animals by both substances. CII induced protein expression of MCP-1, and Mip-1α was reduced to 30 and 60% by Dex, respectively, and to 40 and 80% by SC, respectively. Thus, in almost all cases, the SC effects were similar to those of Dex. The expression of different proinflammatory RA-relevant cytokines, such as IFN-γ, IL-1β, IL-12p70, IL-6, or TNF-α, was also increased (1.8–10-fold) upon CII treatment (Fig. 1C). Dex and SC markedly inhibited CII-induced IL12p70 expression (reduction to 18 and 22%, respectively). The effect of both substances on TNF-α- or IL-10 production was less pronounced. Dex treatment resulted in a 70% inhibition of CII-induced TNF-α and 50% inhibition of IL-10 production, whereas SC therapy led to a 60% decrease of TNF-α- and a 40% inhibition of IL-10 production. It is surprising that Dex and SC had opposite effects on IFN-γ and IL-1β expression. Only SC treatment led to a significant reduction of IL-1β production (reduction to 30%), whereas Dex further increased CII-induced IL-1β expression (2-fold). Similar results were obtained for IFN-γ. SC did not affect IFN-γ levels, whereas Dex enhanced IFN-γ production (6-fold) in the CIA setting. In summary, SC treatment of CIA was nearly as effective as the therapy with Dex. SC significantly reduced the expression of many proinflammatory genes critically involved in the pathogenesis of CIA and RA.
The Expression of Markers for Synovial Joint Destruction Was Decreased by SC.
As markers for destruction of cartilage and bone loss in the synovial joint, we measured the mRNA levels of the matrix metalloproteinase 3 (MMP3) and the receptor activator of nuclear factor-κB ligand (RANKL) by qRT-PCR. On day 33 after the first immunization with CII (CII), we detected a 20-fold increase in MMP3 mRNA levels and a 6-fold enhanced RANKL mRNA expression. In CII-immunized mice treated with SC (CII+SC) or Dex (CII+Dex), the elevated mRNA levels of MMP3 and RANKL were reduced to the level of PBS-treated control mice (PBS) (Fig. 2, A and B).
Microarray Analyses to Identify CIA- and SC-Regulated Genes.
To identify genes that are regulated by Dex and SC during CIA development, we performed microarray experiments using whole-genome mouse arrays, allowing analysis of the expression of 25,000 genes in parallel. RNA was isolated from the paws of PBS-, CII-, CII+Dex-, or CII+SC-treated mice on day 33 and used for labeled cDNA probe preparation.
Immunization with CII significantly altered the expression of 2228 genes at least 2-fold (P value threshold ≤0.05) compared with control PBS mice. The analysis of the microarray data obtained after Dex and SC treatment revealed that 100 genes were at least 2-fold up- or down-regulated (P value threshold ≤0.05) by the treatment with Dex and SC compared with CII-immunized mice (data not shown; the array data have been deposited in the EMBL-EBI ArrayExpress repository, http://www.ebi.ac.uk/arrayexpress; experiment name: HK_CIA_experiment). We next focused our microarray data analysis on genes differentially regulated by SC treatment in CII-immunized mice. In silico biological process analysis by PANTHER database (Thomas et al., 2003) revealed that 20 genes (Fig. 3, A and B) involved in signal transduction and immune defense mechanisms were significantly up- or down-regulated (log2 scale) by SC treatment. Among others, the mRNA expression of cathelicidin antimicrobial peptide (CAMP), peptidylprolyl isomerase B (Pbip), defensin beta (Defb6), myeloperoxidase (Mpo), and the calcium-binding protein S100A8 (S100A8) was significantly increased (induction 10–100-fold) in the paws of the animals upon CII immunization. In mice injected with SC, we detected an approximately 50% reduced expression of those genes, whereas Dex treatment seems to be less effective (Fig. 3A). Furthermore, administration of SC reversed the down-regulation of genes such as the notch homolog 2 (Notch2), the forkhead box 1 (Foxc1) transcription factor, or the autoimmune regulator inhibitor (AIRE) during arthritis development (Fig. 3B).
To confirm the data obtained from our microarray analyses, Western blot experiments for the protein expression of selected genes were performed. We detected a marked increase in the protein expression of MPO, Pbip, and S100A8 in the paws of CII-treated mice (CII), which was significantly reduced by the administration of SC (CII+SC) or Dex (CII+Dex) (Fig. 4A). In qRT-PCR experiments, we measured a 4-fold up-regulation of Notch2 mRNA-levels in SC-treated CII mice. These results confirmed our microarray data. On the other hand, treatment of CII-mice with Dex not only showed no reversion of the reduction of Notch2 mRNA levels but also resulted in an additional repression of this gene (Fig. 4B).
SC Decreased Proinflammatory Gene Expression in C28/I2 Chondrocytes.
To extend our studies to the human system, we tested the effect of SC on the mRNA expression of relevant proinflammatory genes in human C28/I2 chondrocytes by qRT-PCR. The cells were stimulated with a mixture of proinflammatory cytokines (CM) and treated with 30 μg/ml SC (CM + 30 μg/ml SC) as described. Compared with control cells, the mRNA expression of IL-8, MCP-1, TNF-α, and COX-2 was significantly enhanced (minimal 5-fold; maximal 100-fold) upon cytokine stimulation and reduced by SC treatment (reduction to 30–60%; Fig. 5A).
To study the effect of SC on proinflammatory protein expression in C28/I2 cells, we used a human proteome profiler. We detected a 15-fold up-regulation of MCP-1 upon cytokine stimulation that was reversed by SC to a level of 10% of CM-incubated C28/I2 cells. The cytokine-induced expression of IL-8 (2-fold) and CXCL10 (7-fold) was slightly reduced by SC treatment (reduction to 90%), whereas the effect on cytokine-induced IL-28 (2-fold) and CXCL11 (6-fold) expression was more pronounced. Here we detected a 40% reduction compared with cytokine-stimulated control cells (Fig. 5B). The data are presented as log2 ratios in comparison with untreated control cells or CM-treated cells to evaluate the SC effects. In comparison with the data obtained in the CIA mouse model, C5a expression was also significantly reduced upon SC treatment in CM-stimulated C28/I2 cells. Giemsa staining of C28/I2 cells demonstrated that SC had no pronounced cytotoxic effects in the concentrations used in our experiments (Fig. 5C). These data showed that SC reduced the expression of different proinflammatory cytokines and chemokines also in human C28/I2 chondrocytes.
SC Decreased the Expression of S100A8 in Human MonoMac6 Cells.
Monocytes and macrophages are critically involved in the pathogenesis of RA and are the major source of the proinflammatory marker S100A8. In the human monocyte cell line MonoMac6, pretreatment with 20 μg/ml SC (CM+SC) prevented the 3-fold LPS/CM-induced increase of S100A8 (CM) mRNA and protein expression (Fig. 6, A–C). These data highlighted that SC effects detected in the mouse in vivo model were reproducible in the human system. In summary, our data demonstrate that SC is a potent inhibitor of proinflammatory gene expression in a murine in vivo model of RA as well as in different human cells important for RA disease pathogenesis.
We previously demonstrated that the fungal macrocyclic lactone SC reduced the expression of the proinflammatory enzyme iNOS by inhibition of the JAK/STAT pathway (Yao et al., 2003). In addition, we observed inhibition of NF-κB-dependent promoter activity by SC in human A549/8 cells (data not shown). Because the JAK/STAT and NF-κB signal pathways play a pivotal role in the initiation and progression of inflammation and recruitment of immune cells, we tested the effect of SC in the murine CIA model.
Chemokines promote CIA and RA by recruitment of immune cells to the synovial tissue. For example, the complement C5a attracts neutrophils and macrophages to the synovium (Wang et al., 1995; Grant et al., 2002). Beside C5a, the expression of other important chemokines such as MCP-1, Mip-1α, Mip-1β, Mip-2, CXCL12, or CCL17 was inhibited by SC treatment. These effects were similar to those observed in the Dex-treated control group (Fig. 1B). These data imply that SC may improve CIA or RA by diminishing the recruitment of immune cells to the inflamed joint.
The imbalance of pro- and anti-inflammatory cytokines promotes chronic inflammation and destruction of the joints in the CIA model and in RA. In consistence, we detected a significant increase of proinflammatory cytokines upon immunization with CII. Our data demonstrated that SC decreased the expression of these cytokines (Fig. 1C). Beside IFN-γ and IL-1β, the effects observed with SC were similar to the Dex-mediated effects. It is surprising that Dex intervention elevated the protein amount of IFN-γ and IL-1β in the paws. Increase of IL-1β expression after treatment with high doses of glucocorticoids has also been described in human peripheral blood monocytes (Markova et al., 2007).
TNF-α is a key mediator of inflammation in the joints by stimulating the production of additional proinflammatory cytokines such as IL-1, IL-8, IL-6, or GM-CSF or different chemokines. The significant down-regulation of TNF-α expression by SC might account for the reduction of the expression of a number of cytokines and chemokines seen in our experiments (Fig. 1C). The development of drugs that block the activity of TNF-α, IL-1, and IL-6 has been a major clinical advance in the treatment of RA, indicating the benefit of therapeutic strategies to modulate proinflammatory signal pathways. (Brennan and McInnes, 2008). An important hallmark of RA is the destruction of cartilage and bone. SC inhibited the expression of RANKL, which regulates osteoclast differentiation, maturation, and induction of resorptive activity. The expression of RANKL is regulated by inflammatory cytokines and mediators such as TNF-α (Karmakar et al., 2010). In addition, we found that SC inhibited the expression of MMP9 (data not shown) and MMP 3, enzymes that degrade cartilage and noncollagen matrix components of the joint (Fig. 2, A and B). These results indicate that SC may improve the disease by reducing the expression of proteins involved in degenerative processes in the joint.
In total genome microarray experiments, we detected considerable differences in the effects of SC and Dex on CII-modulated gene expression, indicating that SC- and Dex-induced effects are mediated by different pathways. A more detailed analysis revealed that SC treatment primarily affected the expression of genes involved in signal and immune processes; therefore, we focused on those genes whose expression was regulated by CII and SC in an opposite manner (Fig. 3, A and B).
Peptidylprolyl cis-trans-isomerase B (Ppib, cyclophilin B) was up-regulated by CII in Vβ12 mice and down-regulated by SC treatment (Figs. 3A and 4A). In the articular cartilage, Ppib is secreted by chondrocytes, and its release is mediated by matrix metalloproteinases (De Ceuninck et al., 2003). Ppib has been shown to induce efficiently chemotaxis of human neutrophils and T cells (Pakula et al., 2007), and down-regulation of Ppib expression by SC could ameliorate CIA or RA symptoms by inhibiting the recruitment of immune cells.
MPO has proinflammatory and pro-oxidative properties and is expressed in different immune cells. In inflamed tissues, the enzyme can be released to the extracellular space resulting in enhanced production of reactive oxygen species and increased oxidative stress. Reactive oxygen species, as a second messenger, can activate NF-κB and thus promote the onset and progression of arthritis (Miesel et al., 1996). In chronic inflammatory joint diseases, elevated levels of MPO were detected (Mäki-Petäjä et al., 2008; Feijóo et al., 2009). In the study of Mäki-Petäjä et al. (2008), MPO levels correlated positively with the amount of the inflammation marker C-reactive protein and iNOS activity and negatively with the endothelial function. These data indicate that the inflammatory and oxidative processes in RA trigger the development of endothelial dysfunction and thereby may promote the cardiovascular risk of RA patients. Our data showed that SC and Dex treatment significantly reduced MPO mRNA and protein expression in the CIA model (Figs. 3A and 4A). Thus, SC-mediated decrease of MPO expression may result in reduced oxidative stress during RA.
The highly conserved Notch signaling pathway plays a critical role in cell proliferation, differentiation, and apoptosis (Baron, 2003). Notch2 expression in the synovium of RA patients has been demonstrated, but the role of this protein in the context of RA is poorly understood (Ishii et al., 2001). One report linked Notch2 signaling with the induction of apoptosis in chondrocytes (Hattori et al., 2005). Moreover, an altered expression of Notch2 was detected in T-helper cells of RA patients with an active disease (Jiao et al., 2010). Thus, modification of Notch2 expression by SC may have beneficial effects in RA therapy. It is unexpected that, in case of dexamethasone, the qRT-PCR analyses do not reflect the microarray data on Notch2 mRNA expression (Figs. 3B and 4B).
The S100 calcium-binding proteins (S100A8, S100A9, and S100A12) are primarily expressed by neutrophils, monocytes, and activated macrophages and mediate proinflammatory and chemotactic effects partly by amplifying NF-κB-dependent gene expression (Ryckman et al., 2003; Sunahori et al., 2006). S100A8 and S100A9 form heterodimers, and elevated levels of these complexes were detected in the synovial fluid and serum of RA patients (Frosch et al., 2000; Bovin et al., 2004). In addition, a positive correlation of S100A8 expression with the severity of arthritis symptoms was described in RA patients as well as in the mouse model of antigen-induced arthritis (Sunahori et al., 2006; van Lent et al., 2008). Deletion of the gene coding for S100A9 also abrogates the expression of S100A8. In S100A9−/− mice, a significant reduction of the swelling of the paws as well as an inhibition of MMP-mediated degradation of articular cartilage was detected (van Lent et al., 2008). As S100A8 seems to be important for the pathogenesis of CIA and RA, down-regulation of the protein by SC treatment (Fig. 4A) in combination with reduced expression of other mediators (see above) may have a significant effect on the course of the disease.
We could translate our data from the CIA mouse model to human immortalized C28/I2 chondrocytes where SC treatment efficiently reduced the cytokine-stimulated expression of various proinflammatory mediators. This demonstrates the anti-inflammatory efficacy of SC also in a RA-relevant human cell model (Fig. 5A). We found that SC inhibited the synthesis of the chemotactic factors C5a and MCP-1 in CM-induced C28/I2 cells (Fig. 5B) corroborating the results of the murine proteome profile analyses. These data indicate that SC treatment in the human disease may also efficiently inhibit the recruitment of immune cells to the inflamed tissue. We previously described that C28/I2 cells are resistant to glucocorticoid treatment because of a lack of GRα expression (Schmidt et al., 2010). In contrast, SC markedly reduced proinflammatory gene expression in these cells, and this clearly indicates that SC-mediated effects are not mediated via glucocorticoid signal transduction pathways.
Recent studies have shown that S100A8 expression is strongly enhanced in the synovial fluid of RA patients and could serve as an early biomarker for the onset of the disease (Liao et al., 2004; Baillet et al., 2010). It has been described that glucocorticoids increases S100A8 expression in human macrophages (Hsu et al., 2005). In contrast, SC inhibited the expression of this important marker in the human monocyte cell line MonoMac6 (Fig. 6, A–C).
Furthermore, we have no evidence for major adverse effects of SC. Mice treated with SC neither show a loss of weight nor abnormalities in cytochrome P450 expression (data not shown). In addition, Giemsa staining of C28/I2 cells demonstrated that the reduced cytokine and chemokine expression was not attributed to cytotoxic effects of SC (Fig. 5C). According to data in the literature, myelosuppressive side effects could be expected through inhibition of the JAK2/STAT1 signal transduction pathway (Purandare et al., 2012). Whether this occurs in SC-treated mice must be investigated in further experiments.
In summary, our study demonstrated that in the CIA mouse model the fungal compound SC reduced the expression of proinflammatory genes nearly as effectively as the established glucocorticoid Dex. Moreover, we provide evidence that SC is also effective in a glucocorticoid-resistant human cellular model. Altogether, SC may serve as a promising lead structure for the development of new therapeutics for the treatment of chronic inflammatory diseases, such as RA or inflammatory bowel disease. However, it will be important to describe efficacy of SC using a therapeutic dosing regime in future experiments.
Participated in research design: Erkel, Kleinert, and Pautz.
Conducted experiments: Schmidt, Art, Forsch, Werner, and Jung.
Contributed new reagents or analytic tools: Erkel.
Performed data analysis: Schmidt, Pautz, Kleinert, and Erkel.
Wrote or contributed to the writing of the manuscript: Pautz, Kleinert, Erkel, and Horke.
The Vβ12 transgenic DBA/1 mice (Mori et al., 1992) were a generous gift from Dr. Mori (Experimental Immunology, Department of Biomedicine University Hospital, University of Basel, Basel, Switzerland). We thank Dr. E. Schmitt and S. Gerecht (Department of Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany) for their support. The human C28/I2 chondrocytes were a generous gift of Dr. M. B. Goldring (Laboratory for Cartilage Biology, Hospital for Special Surgery, New York, NY).
This work was supported by the Innovation Foundation of the State of Rhineland-Palatinate [Grants 1512-366261/758K, 961-386261/917K]; the Collaborative Research Center SFB 553 [Project A7] (to H.K.); and the Deutsche Forschungsgemeinschaft [Grant LI 1759/1-1] (to H.K.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- rheumatoid arthritis
- autoimmune regulator
- collagen-induced arthritis
- chicken collagen II
- complement component 5a
- chemokine (C-C motive) ligand
- glucocorticoid receptor
- cytokine mixture
- Janus kinase
- monocyte chemotactic protein
- macrophage inflammatory protein
- matrix metalloprotease
- nuclear factor κB
- inducible NO synthase
- peptidylprolyl cis-trans-isomerase B
- quantitative real-time reverse transcription polymerase chain reaction
- receptor activator of nuclear factor-κB ligand
- signal transducer and activator of transcription-1α
- T-cell receptor
- tumor necrosis factor-α
- complete Freund's adjuvant
- Received January 18, 2012.
- Accepted July 3, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics