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Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells
www.elsevier.com/locate/issn/10434666 Cytokine 39 (2007) 184–191
Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells Paul W. Thompson a
a,*
, Andrew I. Bayliffe b, Andrew P. Warren c, Jonathan R. Lamb
a
Translational Medicine and Genetics, GlaxoSmithKline, ACCI, Addenbrooke’s Hospital, Cambridge CB2 2GG, UK b GlaxoSmithKline, Upper Merion, PA, USA c GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, UK Received 1 May 2007; received in revised form 4 July 2007; accepted 26 July 2007
Abstract Activators of peroxisome proliferator-activated receptor (PPAR)-c are anti-inflammatory and have been proposed as therapeutic agents for the treatment of Th1-type inflammatory diseases. We report that nanomolar concentrations of rosiglitazone enhance the production of IL-10 from activated human mature monocyte-derived dendritic cells. Also, rosiglitazone specifically induces the production of IL-10 from TCR-activated human CD4+ T cells and that this effect is PPAR-c-dependent. We also demonstrate for the first time the presence of a functional PPAR response element (PPRE) in the human IL-10 promoter region. Finally we show that rosiglitazone can induce IL-10 in combination with 1,25 a-dihydroxyvitamin D3 to a greater extent than each treatment alone. In summary our findings demonstrate that IL-10 is upregulated by nanomolar TZDs in immune cells, and this may, in part, be responsible for the potential antiinflammatory effects of PPAR-c in humans. 2007 Elsevier Ltd. All rights reserved. Keywords: IL-10; PPAR agonists; T Lymphocytes; Nuclear receptors; Dendritic cells
1. Introduction The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear–hormone-receptor superfamily. PPARs control the transcription of many genes, especially those associated with lipid metabolism, through ligand-dependent transcriptional activation and repression [1]. Three different members of the PPAR family have been identified, designated as PPAR-a, PPAR-bd, and PPAR-c. The classical method of PPAR-induced transcriptional activation requires the presence of conserved PPAR response element (PPRE) sequences in the gene promoter. Typically PPARs form heterodimers with members of the retinoic acid receptor (RXR) family, and subsequently bind PPRE sequences. Specifically, PPAR-c ligands include
*
Corresponding author. Fax: +44 1223 296063. E-mail address: [email protected] (P.W. Thompson).
1043-4666/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.07.191
endogenous fatty acids such as 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2), and the synthetic thiazolidinedione (TZD) class of insulin sensitizers, including rosiglitazone, which is currently being used for the treatment of type II diabetes [2]. Evidence suggests that PPAR ligands, especially those of PPAR-a and -c, are anti-inflammatory [3]. In vitro, TZDs and 15dPGJ2 inhibit the production of many pro-inflammatory cytokines and chemokines by macrophages and dendritic cells [4,5]. In contrast, the effects of PPAR-c ligands upon T cells have been less well characterised. However, both TZDs and endogenous ligands have been reported to decrease IFN-c and IL-2 release from murine and human T cells, with modest effects on proliferation [6,7], although the majority of these effects require high micromolar concentrations, which in the case of TZDs may not be therapeutically relevant. The propensity for PPAR-c ligands to downregulate Th1 responses has been demonstrated in many a range of in vivo experimental mod-
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els of disease [8–10]. Furthermore, PPAR-c-deficient heterozygous mice elicit augmented Th1 responses and develop experimental allergic encephalitis (EAE; [11]). In addition to down-regulating pro-inflammatory cytokines, it is possible that PPAR-c ligands may also mediate their anti-inflammatory effects by promoting the production of anti-inflammatory cytokines. A possible target of PPAR-c activity is the inhibitory cytokine IL-10, which inhibits the activation and effector function of T cells, monocytes and macrophages [12]. Indeed, TZD treatment of a mouse model of acute colitis was accompanied by a marked increase in IL-10 mRNA levels, concomitant with decreased IFN-c and TNF-a mRNA levels [13]. Most recently, IL-10 has been implicated in the anti-inflammatory effect of TZDs in airway inflammation [14]. However, the target cells of drug action and the mechanism by which IL-10 is elevated were not investigated. Here, we report that nanomolar concentrations of the thiazolidinedione rosiglitazone increase production of IL10 from mature dendritic cells and activated CD4+ T cells in a dose-dependent and PPAR-c-dependent manner. We also identify a new PPRE in the human IL-10 promoter, which is conserved in murine IL-10 and demonstrate that it is functionally active. Our findings confirm that IL-10 is upregulated by nM concentrations of rosiglitazone in cells of the immune system, and that this may, at least in part, explain the anti-inflammatory actions of PPAR-c agonists in humans. 2. Methods 2.1. Dendritic cell culture, activation and cytokine measurement Monocytes were isolated from human PBMCs by negative selection using MACS and seeded at 1 · 106 cells/ml in RPMI supplemented with 10% FCS. Differentiation was initiated by addition of rhGM-CSF (GSK; 30 ng/ml) and rhIL-4 (Becton–Dickinson; 10 ng/ml), and this was repeated every 2 days until day 6. At this point cells were checked using antibodies for the expression of immature monocyte-derived dendritic cell (MDDC) markers such as CD1a, CD83 and HLA-DR (Becton–Dickinson). Maturation of immature DCs1 was carried out by addition of either 10 ng/ml rhTNF-a (Becton–Dickinson) + 0.5 lg/ml rhCD40L (Alexis Biochemicals) or 10 ng/ml rhTNFa + 100 ng/ml LPS (Sigma), in the presence of different concentrations of rosiglitazone (5 lM, 500 nM or 10 nM). After 24 h, cells were pelleted and supernatants were removed for analysis using the BioPlex (Bio-Rad) fluid array system and the Bio-Rad custom 7-plex kit which 1
Abbreviations used: DC, dendritic cell; EMSA, electrophoretic mobility shift assay; IBD, inflammatory bowel disease; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; RXR, retinoic acid receptor; TZD, thiazolidinedione; VDR, vitamin D receptor; vitD3, 1,25 a-dihydroxyvitamin D3.
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measures rhTNF-a, IL-1b, IL-6, IL-8, IL-10, IL-12(p70) and GM-CSF. 2.2. CD4+ T cell purification and stimulation Human CD4+ T cells were isolated from peripheral blood of healthy volunteers by gradient centrifugation (Histopaque 1077; Sigma) by negative selection using MACS (Miltenyi Biotech). CD4+ T cell purities were always >96% as assessed by flow cytometry. Cells were resuspended at 1 · 106 cells/ml in RPMI-1640 (Gibco) supplemented with 10% FCS (Gibco) and plated into 12-well plates, uncoated or pre-coated with anti-CD3 antibody (1 lg/ml; R&D Systems). To these cultures soluble antiCD28 antibody (1 lg/ml; R&D Systems) was added together with vehicle or different concentrations (0.1 nM to 10 lM) of the PPAR-c agonist rosiglitazone and pioglitazone. In some experiments the PPAR-c antagonist GW9662 (500 nM) or 1,25 a-dihydroxyvitamin D3 (10 nM; Sigma) were also added to the cells at this point. Supernatants were collected at 48 h and the levels of IL10, TNF-a, IFN-c and IL-2 measured by ELISA (R&D Systems). Student’s t-tests were performed on all data to determine significance values for treatments compared to the vehicle controls, unless specified in figure legends. 2.3. EMSA, plasmids and luciferase assays The following oligonucleotides (ProOligo) were used for EMSAs; 10F 5 0 -TTGTCCACGTCACTGTGACCTAGGA ACAC-3 0 , 10R 5 0 -GTGTTCCTAGGTCACAGTGACGTG GACAA-3 0 ; 10Fshift 5 0 -ACGTCACTGTGACCTAGGA ACACGCGAATG-3 0 ; 10Rshift 5 0 -CATTCGCGTGTTC CTAGGTCACAGTGACGT-3 0 ; 102F 5 0 -GAAGGGAAGG TGAAGGCTCAATCAAAGG-3 0 , 102R 5 0 -CCT TTGAT TGAGCCTTCACCTTCCCCTTC-3 0 ; 103F 5 0 -TTTCAGG GAGCTCAAAGCTGATTCGGCAG-3 0 , 103R 3 0 -CTGC CGAATCAGCTTTGAGCTCCCTGAAA-3 0 . These oligonucleotides correspond to sequences within the human IL10 promoter (GenBank AF295024) with 5 0 end of forward primer positioned at 409 (10Fshift), 416 (10F), 1329 (102F), and 2267 (103F) nucleotides from the start of transcription. EMSAs were carried out as previously described [15]. For supershift assays, 1 ll of PPAR-c TransCruz GelShift reagent was used (Santa Cruz). Fragments of the human IL-10 promoter were cloned from human genomic DNA (Clontech) by PCR using the following primers; 384F 5 0 -GGGGTACCGAATGAGA ACCCACAGCTG-3 0 , 432F 5 0 -GGGGTACCGGCAATT TGTCCACGTCAC-3 0 , and 120R 5 0 -CCGCTCGAGGG CAGGTTGCCTGGGAAG-3 0 , yielding 2 fragments spanning from 384 to +120 and 432 to +120 of the promoter sequence. After fragments were subcloned into pCR2.1, sequences were checked and fragments were then ligated into the pGL3-Prom (Promega) vector to produce pGL3-P384 and pGL3-P432.
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For luciferase assays, COS7 cells were seeded into 24well plates at 50% confluence. Cells were then transfected with either pGL3-P384 or pGL3-P432 (1 lg/well each) and phRL-TK (200 ng/well) alone or in conjunction with either pSG5-PPAR-c (100 ng) alone or pSG5-PPAR-c and pSG5-RXR-c (100 ng) together. Cells were left to express exogenous proteins overnight before treatment with vehicle or rosiglitazone (10 lM or 100 nM) for 24 h. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) using a luminometer and results were normalized against the Renilla luciferase control. A student’s t-test was performed to determine significance values for treatments compared to the vehicle controls. 3. Results 3.1. Rosiglitazone induces IL-10 production by activated mature MDDCs With the possible exception of regulatory T cells, cells of the myeloid lineage are the most potent producers of IL-10 in vivo, in particular the monocyte/macrophage and dendritic cells [12]. Therefore we tested whether rosiglitazone was capable of inducing IL-10 production from activated
mature monocyte-derived dendritic cells (MDDCs). MDDCs were generated by monocyte differentiation in the presence of GM-CSF and IL-4. Cells were characterised by morphology and the expression of cell-surface antigens typical of MDDCs (i.e., CD83, CD1a and HLA-DR; data not shown). MDDCs were activated with either LPS (100 ng/ml) or soluble rhCD40L (0.5 lg/ml) and supernatants tested for a panel of cytokines using the BioPlex system. Low nM concentrations of rosiglitazone specifically induced IL-10 production by approximately 75% compared to control treatments (Fig. 1a and b). These results were repeated using either CD40L or LPS as the primary stimulus (Fig. 1a: CD40L DMSO vs. 10 nM rosiglitazone, 360.6 ± 41.1 pg/ml vs. 633.0 + 68.4 pg/ml (n = 5, p < 0.05): Fig. 1b: LPS DMSO vs. 10 nM rosiglitazone, 599.2 ± 89.1 pg/ml vs. 1053.4 ± 146.6 pg/ml (n = 5, p < 0.01)). IL-12, a known target for PPAR-c-induced suppression, was dose dependently inhibited by rosiglitazone (Fig. 1c and d), once again proving the selectivity of the IL-10 response. No significant changes were observed in other cytokines measured by the BioPlex system (data not shown). Consistent with previously published data [5], rosiglitazone did alter the phenotype of mature MDDC with respect to expression of CD80 and CD86 (data not shown). Thus IL-10 is a target of PPAR-c activation in activated mature MDD.
Fig. 1. Rosiglitazone increases IL-10 production from both LPS- and CD40L-activated MMDDCs. MMDDCs were activated by either rhCD40L (a and c; 0.5 lg/ml) or LPS (b and d; 100 ng/ml) in the presence of different concentrations of rosiglitazone for 24 h. Supernatants were tested for the presence of IL-10 (a and b) and IL-12 (c and d) using the BioPlex system (n = 5). *p < 0.05, **p < 0.01, ***p < 0.005 relative to control IL-10 or IL-12 secretion.
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3.2. Rosiglitazone increases IL-10 production in human CD4+ T cells in a PPAR-c-dependent manner To investigate the potential of the PPAR-c ligand rosiglitazone to modulate the function of TCR-activated CD4+ T cells from human donors, the compound was added over a broad range of concentrations (0.1 nM–10 lM—to ensure that the lowest concentration did not affect baseline cytokine levels)and cytokine secretion was determined. In agreement with the data from mature dendritic cells, the addition of rosiglitazone moderately, but significantly, enhanced IL-10 production (Fig. 2a). Individual donor data is shown in Fig. 2b. A bell-shaped dose–response curve was observed, with a concentration of 1 nM rosiglitazone inducing a 39% (501.3 ± 77.9 pg/ml; n = 5, p < 0.005) increase in IL-10 production compared to control levels (366.8 ± 63.6 pg/ml). A significant increase in IL-10 production also occurred in response to 10 nM rosiglitazone (465.5 ± 69.92 pg/ml; 29%, p < 0.01). The data fits to the quadratic model with an adjusted R2 value of 0.84 (data analysed using repeated mixed model ANOVA test). In contrast lM concentrations of rosiglitazone had no effect on IL-10 production. Bell-shaped dose–response curves for rosiglitazone on target genes have previously been shown [16]. We also noted that at high lM concentrations, rosiglitazone showed an anti-proliferative activity (data not shown), which may contribute to the shape of the response curve. Next, we used the specific PPAR-c antagonist GW9662 [15] to confirm that the enhancement of IL-10 production by rosiglitazone was specific to activation of PPAR-c. Treatment with 500 nM GW9662 abolished the effects of 10 nM rosiglitazone upon IL-10 production (Fig. 2c), thereby confirming that rosiglitazone-induced IL-10 production from CD4+ T cells is PPAR-c-specific. Treatment with GW9662 alone had no effect upon IL-10 production (Fig. 2c). In parallel, the effect of rosiglitazone on the production of Th1-type pro-inflammatory cytokines was also measured. Production of IFN-c by activated CD4+ T cells remained unchanged in the presence of 5 nM rosiglitazone concentrations which increased IL-10 secretion (Fig. 2d), thereby emphasising the selectivity of the IL-10 increase. IL-10 produced by CD4+ T cells in response to rosiglitazone in the absence of TCR-activation was also investigated. Although a similar dose–response curve for IL-10 secretion was noted with nM concentrations of compound, the absolute IL-10 concentrations produced were only between 3 and 15 pg/ml (data not shown). Rosiglitazone had no significant effect on the secretion of IL-2 or T cell proliferation at all doses tested (data not shown). IL-10 production by TCR-activated CD4+ T cells was increased by 35% after treatment with 100 nM pioglitazone (data not shown). High concentrations (10 lM) of pioglitazone markedly decreased IL-10 production, although this was accompanied by some cell toxicity (data not shown). The difference in optimal concentrations of rosiglitazone and pioglitazone required for the maximal IL-10 effect is reflected in the
Fig. 2. PPAR-c regulates IL-10 production by human CD4+ T cells. CD4+ T cells were stimulated with CD3/CD28 antibodies in the presence of different rosiglitazone concentrations, or vehicle control, for 48 h. (a) Supernatants were tested for IL-10 levels using ELISAs (n = 5; error bars = SEM). *p < 0.05, **p < 0.01, ***p < 0.005 relative to control IL-10 secretion. (b) Separate donor results for part (a). (c) CD4+ T cells were stimulated with CD3/CD28 with 10 nM rosiglitazone with or without GW9662 (500 nM), and supernatants were analysed for IL-10 levels (n = 5). (d) Supernatants of stimulated cells treated with rosiglitazone in the same experiments were analysed for both IL-10 and IFN-c levels (n = 3).
approximate 10-fold difference in affinity for PPAR-c between these two TZDs [17] Taken together this data strongly suggests that rosiglitazone and pioglitazone can
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selectively increase IL-10 production by CD4+ T cells in a dose-dependent PPAR-c-dependent manner. 3.3. A functional PPRE is present in the IL-10 promoter The classical mechanism for PPAR-driven gene transcription requires the presence of a consensus DNA sequence, specific for PPARs, the PPRE. A PPRE was predicted to be present in the human IL-10 promoter (GenBank U63015), situated 407 bp from the start site of transcription (Fig. 3a). The IL-10 PPRE contains a high degree of sequence identity to the consensus PPRE (Fig. 3a), and is also conserved at the same position in the mouse IL-10 promoter. EMSAs revealed that the human IL-10 PPRE could bind PPAR-c/RXR-c heterodimers in vitro (Fig. 3b; arrow). Binding of the IL-10 PPRE to PPAR-c/RXR-c heterodimers was blocked by co-incubation with unlabelled IL-10 PPRE oligonucleotide. Furthermore, the IL-10 PPRE/PPAR complex was further retarded by co-incubation with an anti-PPAR-c antibody, proving the specificity of the interaction (Fig. 3b; arrowhead). Three other putative PPRE sequences were tested
for PPAR interaction by EMSAs (10shift, 102, and 103). However binding of PPAR-c/RXR-c heterodimers was not detectable for these sequences (data not shown). In order to test whether the IL-10 PPRE was functional in cells, 2 human IL-10 promoter luciferase-reporter gene constructs were synthesized, the first containing the IL-10 PPRE (pGL3-P432) and the second lacking the PPRE (pGL3-P384). After transfection of the IL-10 promoter constructs into COS7 cells, together with constructs encoding PPAR-c and RXR-c, the ability of rosiglitazone to increase luciferase transcription was assessed. After 24 h of treatment with rosiglitazone (10 lM), only transcription from pGL3-P432 was enhanced, increasing by 41% (p < 0.01), whereas transcription from pGL3-P384 remained unaffected (Fig. 3c). Specificity for the increased transcription is shown by the removal of RXR-c from the assay, which resulted in abrogation of the rosiglitazone-induced transcription increase. It is important to note that a concentration of rosiglitazone in the nanomolar range (100 nM) also induced a 25% increase in transcriptional activity that showed a trend towards significance (p = 0.09). In summary, we have identified a functional PPRE in the IL-10 gene promoter, to our knowledge this is the first demonstration of the presence of a functional PPRE in a cytokine gene promoter which is capable of transcriptional activation. 3.4. Rosiglitazone co-operates with vitamin D3 for enhanced IL-10 production
Fig. 3. A functional PPRE is present in the human IL-10 gene promoter. (a) The putative PPRE sequence in the human IL-10 gene is shown, aligned to the consensus PPRE motif, incorporating essential nucleotides at the 5 0 end of the sequence. (b) 33P-labelled IL-10 PPRE oligonucleotides were incubated with PPAR-c/RXR-c alone (-), PPAR-c/RXR-c plus unlabelled IL-10 PPRE oligonucleotides (Cold), or PPAR-c/RXR-c plus anti-PPAR-c antibody (PPAR-c Ab). Mixtures were analysed for interaction using gel-electrophoresis. Arrow indicates IL-10 PPRE/transcription factor complex, arrowhead indicates super-shifted complex. (c) Two human IL-10 promoter constructs, pGL3-P384 and pGL3-P432, were synthesized and transfected into COS7 cells alone, or together with PPAR-c/RXR-c or PPAR-c alone, and left to express overnight. After stimulation with vehicle control or rosiglitazone (10 lM or 100 nM) for 24 h, luciferase activity was measured. *p < 0.05, **p < 0.01, relative to control treatments.
Analysis of the human IL-10 promoter revealed the presence of a potential vitamin D receptor (VDR) element (data not shown). The vitamin D receptor [18] belongs to the same nuclear receptor superfamily as the PPARs, and also heterodimerizes with the retinoic acid receptor RXR, suggesting that there could be co-operation between the PPARs and VDRs during IL-10 transcription. Therefore we investigated the effects of vitamin D3 on CD4+ T cell IL-10 production. Levels of TNF-a were also monitored to ensure that responses were selective for IL-10. Treatment with 1,25 a-dihydroxyvitamin D3 (vitD3; 10 nM) significantly increased IL-10 by 52% (Fig. 4a, n = 7; p < 0.05), but did not significantly affect the production of TNF-a (Fig. 4b). The combination of vitD3 and rosiglitazone strongly increased IL-10 levels by 103% compared to control levels (Fig. 4a, n = 7; p < 0.01). The proliferation of cells in response to vitD3 and rosiglitazone remained unchanged from vitD3 alone (Fig. 4c). 4. Discussion Ligands of PPAR-c have been demonstrated to be antiinflammatory in many in vitro cell systems and in vivo disease models. Although the mechanism for PPAR-mediated down-regulation of pro-inflammatory cytokine production from immune cells is unclear, much evidence supports a transrepressive mechanism in which transcription factors
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Fig. 4. Combination of rosiglitazone and Vitamin D3 further enhance IL-10 production. CD4+ T cells were stimulated as previously in the presence of either vehicle, 10 nM 1,25 a-dihydroxyvitamin D3, or 1 nM rosiglitazone and 1,25 a-dihydroxyvitamin D3. IL-10 (a) and TNF-a (b) levels were measured by ELISA (n = 7). **p < 0.01, of combination relative to vitamin D3 alone. (c) Re-stimulation assays for combination treatments were performed as for rosiglitazone treatments in Fig. 3 (n = 4). *p < 0.05, relative to control treatments.
such as NF-KB, NF-AT, Sp1 and AP-1 are inhibited by binding of activated PPAR [19–21]. Indeed, an additional consequence of these transrepressive mechanisms is that cell proliferation is blunted and autocrine growth factor production is inhibited (i.e., IL-2 for T cells in ref 20). We did not observe anti-proliferative effects at our highest concentration of rosiglitazone reported here, although we have observed anti-proliferative effects using concentrations greater than 25 lM. It is likely that either different concentrations used, different TZDs or PPAR-c agonist used, or the nature of cell stimulus employed may account for contrasting data produced by different research groups on PPAR-c ligands. Transrepression may well occur in vivo and explain observed reductions in TNF-a, IFN-c, and IL1b upon PPAR-c ligand treatment of mouse inflammatory disease models. However, in this study we have described a PPRE-dependent mechanism for PPAR-c-mediated upregulation of the anti-inflammatory cytokine IL-10 in both mature MDDCs and human CD4+ T cells. Increased production of IL-10 was only observed at low nM concentrations of rosiglitazone and slightly higher concentrations of pioglitazone and showed different magnitudes in the 2 different cell types. Very recently, rosiglitazone has been shown to increase IL-10 production in a mouse model of asthma [14]. Moreover, treatment with a soluble IL-10 receptor partially blocks the inhibitory effect of PPAR-c agonists on airway inflammation. A further report also showed a similar rosiglitazone-induced bell-shaped dose– response curve for IL-10 production from LPS-stimulated murine macrophages, although with higher concentrations
of rosiglitazone [22]. On the other hand, a previous report has shown that IL-10 production was dramatically reduced from dendritic cells cultured with high lM concentrations of rosiglitazone and other PPAR-c ligands [23], although these ligands were supplied at the beginning of monocyteto-DC differentiation (i.e., day 1). An earlier study reported no effect of 10 lM rosiglitazone on immature DC IL-10 production [5], which our data is in agreement with. Both of these PPAR-c/dendritic cell reports do not fully explore the relationship of IL-10 expression and concentration of the PPAR-c agonists. Therefore, a key issue with the majority of in vitro data generated with PPAR-c agonists is that of the concentration used. Concentrations of TZDs greater than 1 lM are not achieved systemically after daily 8 mg treatment of diabetic patients with rosiglitazone [24]. Moreover, with a half-life of 3–4 h, the concentration of systemic rosiglitazone falls below 20 nM approx 12 h after dosing with 2 mg rosiglitazone, with no accumulation of rosiglitazone occurring after repeat dosing. Thus, whilst in vitro data using lM concentrations may be more dynamic, translation of this data to the in vivo situation is less likely to be relevant. As previously mentioned, the ability of activated PPARs to transrepress various transcription factors as well as activate transcription through PPRE binding may result in complexed dose–response relationships, such as those observed in our study (Fig. 1). Furthermore, the magnitude of the PPAR response is likely to be determined by the strength and nature of the accompanying stimulus used, especially when considering PPRE-dependent responses
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in which PPARs would play a direct role in the formation of the multimeric transcriptional activation complex. Our data would suggest that the identified IL-10 PPRE is comparatively weak when compared to PPREs that are present in lipid metabolism gene promoters [25], and that this would impose a limit on the possible magnitude of IL-10 response to PPAR-c. The maximal upregulation of IL-10 observed in CD40L-stimulated MMDDCs and TCR-activated CD4+ T cells correlates well with the IL-10 PPRE driven transcription observed in the luciferase assays. Critically, the nature of the IL-10 upregulation in terms of concentration of rosiglitazone required and selectivity was repeated in different cell-types, stimulation protocols and measurement methods, thus emphasizing the importance and reproducibility of this finding. Our data clearly demonstrate that rosiglitazone (and probably pioglitazone) can selectively increase IL-10 production by mature MDDCs and CD4+ T cells in a bellshaped dose-dependent and PPAR-c-dependent manner. The ability of dendritic cells and activated T cells to synthesize increased amounts of IL-10 protein in response to PPAR-c ligands may be a potential explanation for their mechanism of action in inflammatory disease. Of further interest is the co-operation shown between rosiglitazone and 1,25 a-dihydroxyvitamin D3 towards IL-10 production from TCR-activated CD4+ T cells. Co-operation between vitamin D3 and other nuclear receptor activators (dexamethasone) towards IL-10 production has been previously described [26] In conclusion we have shown that IL-10 is significantly upregulated by nanomolar rosiglitazone in cells of the human immune system. This regulation may be a critical part of the mechanism of action of PPAR-c ligands in the potential resolution of inflammatory disease in humans. Acknowledgments We thank Filippo Volpe (Bioinformatics group, GlaxoSmithKline) for bioinformatics analysis of the IL-10 promoter, Liang Ye for technical assistance, Steve Smith for suggestions and advice, and Bill Davis for helpful discussions. References [1] Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 2002;53:409–35. [2] van Bilsen M, van der Vusse GJ, Gilde AJ, Lindhout M, van der Lee KA. Peroxisome proliferator-activated receptors: lipid binding proteins controling gene expression. Mol Cell Biochem 2002;239:131–8. [3] Daynes RA, Jones DC. Emerging roles of PPARs in inflammation and immunity. Nat. Rev Immunol 2002;2:748–59. [4] Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 1998;391:82–6. [5] Gosset P, Charbonnier AS, Delerive P, Fontaine J, Staels B, et al. Peroxisome proliferator-activated receptor gamma agonists affect the maturation of human monocyte-derived dendritic cells. Eur J Immunol 2001;31:2857–65.
[6] Clark RB, Bishop-Bailey D, Estrada-Hernandez T, Hla T, Puddington L, Padula SJ. The nuclear receptor PPAR gamma and immunoregulation: PPAR gamma mediates inhibition of helper T cell responses. J Immunol 2000;164:1364–71. [7] Marx N, Kehrle B, Kohlhammer K, Grub M, Koenig W, Hombach V, et al. PPAR activators as antiinflammatory mediators in human T lymphocytes: implications for atherosclerosis and transplantationassociated arteriosclerosis. Circ Res 2002;90:703–10. [8] Niino M, Iwabuchi K, Kikuchi S, Ato M, Morohashi T, Ogata A, et al. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferator-activated receptor-gamma. J Neuroimmunol 2001;116:40–8. [9] Takagi T, Naito Y, Tomatsuri N, Handa O, Ichikawa H, Yoshida N, et al. Pioglitazone, a PPAR-gamma ligand, provides protection from dextran sulfate sodium-induced colitis in mice in association with inhibition of the NF-kappaB-cytokine cascade. Redox Rep 2002;7:283–9. [10] Yuan Z, Liu Y, Liu Y, Zhang J, Kishimoto C, Wang Y, et al. Peroxisome proliferation-activated receptor-gamma ligands ameliorate experimental autoimmune myocarditis. Cardiovasc Res 2003;59:685–94. [11] Natarajan C, Muthian G, Barak Y, Evans RM, Bright JJ. Peroxisome proliferator-activated receptor-gamma-deficient heterozygous mice develop an exacerbated neural antigen-induced Th1 response and experimental allergic encephalomyelitis. J Immunol 2003;171:5743–50. [12] Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765. [13] Saubermann LJ, Nakajima A, Wada K, Zhao S, Terauchi Y, Kadowaki T, et al. Peroxisome proliferator-activated receptor gamma agonist ligands stimulate a Th2 cytokine response and prevent acute colitis. Inflamm Bowel Dis 2002;8:330–9. [14] Kim SR, Lee KS, Park HS, Park SJ, Min KH, Jin SM, et al. Involvement of IL-10 in PPAR{gamma}-mediated anti-inflammatory response in asthma. Mol Pharmacol 2005;68:1568–75. [15] Hong G, Lockhart A, Davis B, Rahmoune H, Baker S, Ye L, et al. PPARgamma activation enhances cell surface ENaCalpha via upregulation of SGK1 in human collecting duct cells. FASEB J 2003;17:1966–8. [16] Patel L, Buckels AC, Kinghorn IJ, Murdock PR, Holbrook JD, Plumpton C, et al. Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators. Biochem Biophys Res Comm 2003;300:472–6. [17] Henke BR, Blanchard SG, Brackeen MF, Brown KK, Cobb JE, Collins JL, et al. N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents. J Med Chem 1998;41:5020–36. [18] Nagpal S, Lu J, Boehm MF. Vitamin D analogs: mechanism of action and therapeutic applications. Curr Med Chem 2001;8:1661–79. [19] Wang P, Anderson PO, Chen S, Paulsson KM, Sjogren HO, Li S. Inhibition of the transcription factors AP-1 and NF-kappaB in CD4 T cells by peroxisome proliferator-activated receptor gamma ligands. Int Immunopharmacol 2001;1:803–12. [20] Yang XY, Wang LH, Chen T, Hodge DR, Resau JH, DaSilva L, et al. Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma co-association with transcription factor NFAT. J Biol Chem 2000;275:4541–4. [21] Sugawara A, Uruno A, Kudo M, Ikeda Y, Sato K, Taniyama Y, et al. Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-gamma via an interaction with Sp1 in vascular smooth muscle cells. J Biol Chem 2002;277:9676–83. [22] Alleva DG, Johnson EB, Lio FM, Boehme SA, Conlon PJ, Crowe PD. Regulation of murine macrophage proinflammatory and antiinflammatory cytokines by ligands for peroxisome proliferatoractivated receptor-c: counter-regulatory activity by IFN-c. J Leuk Biol 2002;71:677–85.
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[25] Juge-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA, et al. DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. J Biol Chem 1997;272:25252–9. [26] Barrat FJ, Cua DJ, Boonstra A, Richards DF, Crain C, Savelkoul HF, et al. In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J Exp Med 2002;195:603–16.
Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells Paul W. Thompson a
a,*
, Andrew I. Bayliffe b, Andrew P. Warren c, Jonathan R. Lamb
a
Translational Medicine and Genetics, GlaxoSmithKline, ACCI, Addenbrooke’s Hospital, Cambridge CB2 2GG, UK b GlaxoSmithKline, Upper Merion, PA, USA c GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, UK Received 1 May 2007; received in revised form 4 July 2007; accepted 26 July 2007
Abstract Activators of peroxisome proliferator-activated receptor (PPAR)-c are anti-inflammatory and have been proposed as therapeutic agents for the treatment of Th1-type inflammatory diseases. We report that nanomolar concentrations of rosiglitazone enhance the production of IL-10 from activated human mature monocyte-derived dendritic cells. Also, rosiglitazone specifically induces the production of IL-10 from TCR-activated human CD4+ T cells and that this effect is PPAR-c-dependent. We also demonstrate for the first time the presence of a functional PPAR response element (PPRE) in the human IL-10 promoter region. Finally we show that rosiglitazone can induce IL-10 in combination with 1,25 a-dihydroxyvitamin D3 to a greater extent than each treatment alone. In summary our findings demonstrate that IL-10 is upregulated by nanomolar TZDs in immune cells, and this may, in part, be responsible for the potential antiinflammatory effects of PPAR-c in humans. 2007 Elsevier Ltd. All rights reserved. Keywords: IL-10; PPAR agonists; T Lymphocytes; Nuclear receptors; Dendritic cells
1. Introduction The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear–hormone-receptor superfamily. PPARs control the transcription of many genes, especially those associated with lipid metabolism, through ligand-dependent transcriptional activation and repression [1]. Three different members of the PPAR family have been identified, designated as PPAR-a, PPAR-bd, and PPAR-c. The classical method of PPAR-induced transcriptional activation requires the presence of conserved PPAR response element (PPRE) sequences in the gene promoter. Typically PPARs form heterodimers with members of the retinoic acid receptor (RXR) family, and subsequently bind PPRE sequences. Specifically, PPAR-c ligands include
*
Corresponding author. Fax: +44 1223 296063. E-mail address: [email protected] (P.W. Thompson).
1043-4666/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.07.191
endogenous fatty acids such as 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2), and the synthetic thiazolidinedione (TZD) class of insulin sensitizers, including rosiglitazone, which is currently being used for the treatment of type II diabetes [2]. Evidence suggests that PPAR ligands, especially those of PPAR-a and -c, are anti-inflammatory [3]. In vitro, TZDs and 15dPGJ2 inhibit the production of many pro-inflammatory cytokines and chemokines by macrophages and dendritic cells [4,5]. In contrast, the effects of PPAR-c ligands upon T cells have been less well characterised. However, both TZDs and endogenous ligands have been reported to decrease IFN-c and IL-2 release from murine and human T cells, with modest effects on proliferation [6,7], although the majority of these effects require high micromolar concentrations, which in the case of TZDs may not be therapeutically relevant. The propensity for PPAR-c ligands to downregulate Th1 responses has been demonstrated in many a range of in vivo experimental mod-
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els of disease [8–10]. Furthermore, PPAR-c-deficient heterozygous mice elicit augmented Th1 responses and develop experimental allergic encephalitis (EAE; [11]). In addition to down-regulating pro-inflammatory cytokines, it is possible that PPAR-c ligands may also mediate their anti-inflammatory effects by promoting the production of anti-inflammatory cytokines. A possible target of PPAR-c activity is the inhibitory cytokine IL-10, which inhibits the activation and effector function of T cells, monocytes and macrophages [12]. Indeed, TZD treatment of a mouse model of acute colitis was accompanied by a marked increase in IL-10 mRNA levels, concomitant with decreased IFN-c and TNF-a mRNA levels [13]. Most recently, IL-10 has been implicated in the anti-inflammatory effect of TZDs in airway inflammation [14]. However, the target cells of drug action and the mechanism by which IL-10 is elevated were not investigated. Here, we report that nanomolar concentrations of the thiazolidinedione rosiglitazone increase production of IL10 from mature dendritic cells and activated CD4+ T cells in a dose-dependent and PPAR-c-dependent manner. We also identify a new PPRE in the human IL-10 promoter, which is conserved in murine IL-10 and demonstrate that it is functionally active. Our findings confirm that IL-10 is upregulated by nM concentrations of rosiglitazone in cells of the immune system, and that this may, at least in part, explain the anti-inflammatory actions of PPAR-c agonists in humans. 2. Methods 2.1. Dendritic cell culture, activation and cytokine measurement Monocytes were isolated from human PBMCs by negative selection using MACS and seeded at 1 · 106 cells/ml in RPMI supplemented with 10% FCS. Differentiation was initiated by addition of rhGM-CSF (GSK; 30 ng/ml) and rhIL-4 (Becton–Dickinson; 10 ng/ml), and this was repeated every 2 days until day 6. At this point cells were checked using antibodies for the expression of immature monocyte-derived dendritic cell (MDDC) markers such as CD1a, CD83 and HLA-DR (Becton–Dickinson). Maturation of immature DCs1 was carried out by addition of either 10 ng/ml rhTNF-a (Becton–Dickinson) + 0.5 lg/ml rhCD40L (Alexis Biochemicals) or 10 ng/ml rhTNFa + 100 ng/ml LPS (Sigma), in the presence of different concentrations of rosiglitazone (5 lM, 500 nM or 10 nM). After 24 h, cells were pelleted and supernatants were removed for analysis using the BioPlex (Bio-Rad) fluid array system and the Bio-Rad custom 7-plex kit which 1
Abbreviations used: DC, dendritic cell; EMSA, electrophoretic mobility shift assay; IBD, inflammatory bowel disease; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; RXR, retinoic acid receptor; TZD, thiazolidinedione; VDR, vitamin D receptor; vitD3, 1,25 a-dihydroxyvitamin D3.
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measures rhTNF-a, IL-1b, IL-6, IL-8, IL-10, IL-12(p70) and GM-CSF. 2.2. CD4+ T cell purification and stimulation Human CD4+ T cells were isolated from peripheral blood of healthy volunteers by gradient centrifugation (Histopaque 1077; Sigma) by negative selection using MACS (Miltenyi Biotech). CD4+ T cell purities were always >96% as assessed by flow cytometry. Cells were resuspended at 1 · 106 cells/ml in RPMI-1640 (Gibco) supplemented with 10% FCS (Gibco) and plated into 12-well plates, uncoated or pre-coated with anti-CD3 antibody (1 lg/ml; R&D Systems). To these cultures soluble antiCD28 antibody (1 lg/ml; R&D Systems) was added together with vehicle or different concentrations (0.1 nM to 10 lM) of the PPAR-c agonist rosiglitazone and pioglitazone. In some experiments the PPAR-c antagonist GW9662 (500 nM) or 1,25 a-dihydroxyvitamin D3 (10 nM; Sigma) were also added to the cells at this point. Supernatants were collected at 48 h and the levels of IL10, TNF-a, IFN-c and IL-2 measured by ELISA (R&D Systems). Student’s t-tests were performed on all data to determine significance values for treatments compared to the vehicle controls, unless specified in figure legends. 2.3. EMSA, plasmids and luciferase assays The following oligonucleotides (ProOligo) were used for EMSAs; 10F 5 0 -TTGTCCACGTCACTGTGACCTAGGA ACAC-3 0 , 10R 5 0 -GTGTTCCTAGGTCACAGTGACGTG GACAA-3 0 ; 10Fshift 5 0 -ACGTCACTGTGACCTAGGA ACACGCGAATG-3 0 ; 10Rshift 5 0 -CATTCGCGTGTTC CTAGGTCACAGTGACGT-3 0 ; 102F 5 0 -GAAGGGAAGG TGAAGGCTCAATCAAAGG-3 0 , 102R 5 0 -CCT TTGAT TGAGCCTTCACCTTCCCCTTC-3 0 ; 103F 5 0 -TTTCAGG GAGCTCAAAGCTGATTCGGCAG-3 0 , 103R 3 0 -CTGC CGAATCAGCTTTGAGCTCCCTGAAA-3 0 . These oligonucleotides correspond to sequences within the human IL10 promoter (GenBank AF295024) with 5 0 end of forward primer positioned at 409 (10Fshift), 416 (10F), 1329 (102F), and 2267 (103F) nucleotides from the start of transcription. EMSAs were carried out as previously described [15]. For supershift assays, 1 ll of PPAR-c TransCruz GelShift reagent was used (Santa Cruz). Fragments of the human IL-10 promoter were cloned from human genomic DNA (Clontech) by PCR using the following primers; 384F 5 0 -GGGGTACCGAATGAGA ACCCACAGCTG-3 0 , 432F 5 0 -GGGGTACCGGCAATT TGTCCACGTCAC-3 0 , and 120R 5 0 -CCGCTCGAGGG CAGGTTGCCTGGGAAG-3 0 , yielding 2 fragments spanning from 384 to +120 and 432 to +120 of the promoter sequence. After fragments were subcloned into pCR2.1, sequences were checked and fragments were then ligated into the pGL3-Prom (Promega) vector to produce pGL3-P384 and pGL3-P432.
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For luciferase assays, COS7 cells were seeded into 24well plates at 50% confluence. Cells were then transfected with either pGL3-P384 or pGL3-P432 (1 lg/well each) and phRL-TK (200 ng/well) alone or in conjunction with either pSG5-PPAR-c (100 ng) alone or pSG5-PPAR-c and pSG5-RXR-c (100 ng) together. Cells were left to express exogenous proteins overnight before treatment with vehicle or rosiglitazone (10 lM or 100 nM) for 24 h. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) using a luminometer and results were normalized against the Renilla luciferase control. A student’s t-test was performed to determine significance values for treatments compared to the vehicle controls. 3. Results 3.1. Rosiglitazone induces IL-10 production by activated mature MDDCs With the possible exception of regulatory T cells, cells of the myeloid lineage are the most potent producers of IL-10 in vivo, in particular the monocyte/macrophage and dendritic cells [12]. Therefore we tested whether rosiglitazone was capable of inducing IL-10 production from activated
mature monocyte-derived dendritic cells (MDDCs). MDDCs were generated by monocyte differentiation in the presence of GM-CSF and IL-4. Cells were characterised by morphology and the expression of cell-surface antigens typical of MDDCs (i.e., CD83, CD1a and HLA-DR; data not shown). MDDCs were activated with either LPS (100 ng/ml) or soluble rhCD40L (0.5 lg/ml) and supernatants tested for a panel of cytokines using the BioPlex system. Low nM concentrations of rosiglitazone specifically induced IL-10 production by approximately 75% compared to control treatments (Fig. 1a and b). These results were repeated using either CD40L or LPS as the primary stimulus (Fig. 1a: CD40L DMSO vs. 10 nM rosiglitazone, 360.6 ± 41.1 pg/ml vs. 633.0 + 68.4 pg/ml (n = 5, p < 0.05): Fig. 1b: LPS DMSO vs. 10 nM rosiglitazone, 599.2 ± 89.1 pg/ml vs. 1053.4 ± 146.6 pg/ml (n = 5, p < 0.01)). IL-12, a known target for PPAR-c-induced suppression, was dose dependently inhibited by rosiglitazone (Fig. 1c and d), once again proving the selectivity of the IL-10 response. No significant changes were observed in other cytokines measured by the BioPlex system (data not shown). Consistent with previously published data [5], rosiglitazone did alter the phenotype of mature MDDC with respect to expression of CD80 and CD86 (data not shown). Thus IL-10 is a target of PPAR-c activation in activated mature MDD.
Fig. 1. Rosiglitazone increases IL-10 production from both LPS- and CD40L-activated MMDDCs. MMDDCs were activated by either rhCD40L (a and c; 0.5 lg/ml) or LPS (b and d; 100 ng/ml) in the presence of different concentrations of rosiglitazone for 24 h. Supernatants were tested for the presence of IL-10 (a and b) and IL-12 (c and d) using the BioPlex system (n = 5). *p < 0.05, **p < 0.01, ***p < 0.005 relative to control IL-10 or IL-12 secretion.
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3.2. Rosiglitazone increases IL-10 production in human CD4+ T cells in a PPAR-c-dependent manner To investigate the potential of the PPAR-c ligand rosiglitazone to modulate the function of TCR-activated CD4+ T cells from human donors, the compound was added over a broad range of concentrations (0.1 nM–10 lM—to ensure that the lowest concentration did not affect baseline cytokine levels)and cytokine secretion was determined. In agreement with the data from mature dendritic cells, the addition of rosiglitazone moderately, but significantly, enhanced IL-10 production (Fig. 2a). Individual donor data is shown in Fig. 2b. A bell-shaped dose–response curve was observed, with a concentration of 1 nM rosiglitazone inducing a 39% (501.3 ± 77.9 pg/ml; n = 5, p < 0.005) increase in IL-10 production compared to control levels (366.8 ± 63.6 pg/ml). A significant increase in IL-10 production also occurred in response to 10 nM rosiglitazone (465.5 ± 69.92 pg/ml; 29%, p < 0.01). The data fits to the quadratic model with an adjusted R2 value of 0.84 (data analysed using repeated mixed model ANOVA test). In contrast lM concentrations of rosiglitazone had no effect on IL-10 production. Bell-shaped dose–response curves for rosiglitazone on target genes have previously been shown [16]. We also noted that at high lM concentrations, rosiglitazone showed an anti-proliferative activity (data not shown), which may contribute to the shape of the response curve. Next, we used the specific PPAR-c antagonist GW9662 [15] to confirm that the enhancement of IL-10 production by rosiglitazone was specific to activation of PPAR-c. Treatment with 500 nM GW9662 abolished the effects of 10 nM rosiglitazone upon IL-10 production (Fig. 2c), thereby confirming that rosiglitazone-induced IL-10 production from CD4+ T cells is PPAR-c-specific. Treatment with GW9662 alone had no effect upon IL-10 production (Fig. 2c). In parallel, the effect of rosiglitazone on the production of Th1-type pro-inflammatory cytokines was also measured. Production of IFN-c by activated CD4+ T cells remained unchanged in the presence of 5 nM rosiglitazone concentrations which increased IL-10 secretion (Fig. 2d), thereby emphasising the selectivity of the IL-10 increase. IL-10 produced by CD4+ T cells in response to rosiglitazone in the absence of TCR-activation was also investigated. Although a similar dose–response curve for IL-10 secretion was noted with nM concentrations of compound, the absolute IL-10 concentrations produced were only between 3 and 15 pg/ml (data not shown). Rosiglitazone had no significant effect on the secretion of IL-2 or T cell proliferation at all doses tested (data not shown). IL-10 production by TCR-activated CD4+ T cells was increased by 35% after treatment with 100 nM pioglitazone (data not shown). High concentrations (10 lM) of pioglitazone markedly decreased IL-10 production, although this was accompanied by some cell toxicity (data not shown). The difference in optimal concentrations of rosiglitazone and pioglitazone required for the maximal IL-10 effect is reflected in the
Fig. 2. PPAR-c regulates IL-10 production by human CD4+ T cells. CD4+ T cells were stimulated with CD3/CD28 antibodies in the presence of different rosiglitazone concentrations, or vehicle control, for 48 h. (a) Supernatants were tested for IL-10 levels using ELISAs (n = 5; error bars = SEM). *p < 0.05, **p < 0.01, ***p < 0.005 relative to control IL-10 secretion. (b) Separate donor results for part (a). (c) CD4+ T cells were stimulated with CD3/CD28 with 10 nM rosiglitazone with or without GW9662 (500 nM), and supernatants were analysed for IL-10 levels (n = 5). (d) Supernatants of stimulated cells treated with rosiglitazone in the same experiments were analysed for both IL-10 and IFN-c levels (n = 3).
approximate 10-fold difference in affinity for PPAR-c between these two TZDs [17] Taken together this data strongly suggests that rosiglitazone and pioglitazone can
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selectively increase IL-10 production by CD4+ T cells in a dose-dependent PPAR-c-dependent manner. 3.3. A functional PPRE is present in the IL-10 promoter The classical mechanism for PPAR-driven gene transcription requires the presence of a consensus DNA sequence, specific for PPARs, the PPRE. A PPRE was predicted to be present in the human IL-10 promoter (GenBank U63015), situated 407 bp from the start site of transcription (Fig. 3a). The IL-10 PPRE contains a high degree of sequence identity to the consensus PPRE (Fig. 3a), and is also conserved at the same position in the mouse IL-10 promoter. EMSAs revealed that the human IL-10 PPRE could bind PPAR-c/RXR-c heterodimers in vitro (Fig. 3b; arrow). Binding of the IL-10 PPRE to PPAR-c/RXR-c heterodimers was blocked by co-incubation with unlabelled IL-10 PPRE oligonucleotide. Furthermore, the IL-10 PPRE/PPAR complex was further retarded by co-incubation with an anti-PPAR-c antibody, proving the specificity of the interaction (Fig. 3b; arrowhead). Three other putative PPRE sequences were tested
for PPAR interaction by EMSAs (10shift, 102, and 103). However binding of PPAR-c/RXR-c heterodimers was not detectable for these sequences (data not shown). In order to test whether the IL-10 PPRE was functional in cells, 2 human IL-10 promoter luciferase-reporter gene constructs were synthesized, the first containing the IL-10 PPRE (pGL3-P432) and the second lacking the PPRE (pGL3-P384). After transfection of the IL-10 promoter constructs into COS7 cells, together with constructs encoding PPAR-c and RXR-c, the ability of rosiglitazone to increase luciferase transcription was assessed. After 24 h of treatment with rosiglitazone (10 lM), only transcription from pGL3-P432 was enhanced, increasing by 41% (p < 0.01), whereas transcription from pGL3-P384 remained unaffected (Fig. 3c). Specificity for the increased transcription is shown by the removal of RXR-c from the assay, which resulted in abrogation of the rosiglitazone-induced transcription increase. It is important to note that a concentration of rosiglitazone in the nanomolar range (100 nM) also induced a 25% increase in transcriptional activity that showed a trend towards significance (p = 0.09). In summary, we have identified a functional PPRE in the IL-10 gene promoter, to our knowledge this is the first demonstration of the presence of a functional PPRE in a cytokine gene promoter which is capable of transcriptional activation. 3.4. Rosiglitazone co-operates with vitamin D3 for enhanced IL-10 production
Fig. 3. A functional PPRE is present in the human IL-10 gene promoter. (a) The putative PPRE sequence in the human IL-10 gene is shown, aligned to the consensus PPRE motif, incorporating essential nucleotides at the 5 0 end of the sequence. (b) 33P-labelled IL-10 PPRE oligonucleotides were incubated with PPAR-c/RXR-c alone (-), PPAR-c/RXR-c plus unlabelled IL-10 PPRE oligonucleotides (Cold), or PPAR-c/RXR-c plus anti-PPAR-c antibody (PPAR-c Ab). Mixtures were analysed for interaction using gel-electrophoresis. Arrow indicates IL-10 PPRE/transcription factor complex, arrowhead indicates super-shifted complex. (c) Two human IL-10 promoter constructs, pGL3-P384 and pGL3-P432, were synthesized and transfected into COS7 cells alone, or together with PPAR-c/RXR-c or PPAR-c alone, and left to express overnight. After stimulation with vehicle control or rosiglitazone (10 lM or 100 nM) for 24 h, luciferase activity was measured. *p < 0.05, **p < 0.01, relative to control treatments.
Analysis of the human IL-10 promoter revealed the presence of a potential vitamin D receptor (VDR) element (data not shown). The vitamin D receptor [18] belongs to the same nuclear receptor superfamily as the PPARs, and also heterodimerizes with the retinoic acid receptor RXR, suggesting that there could be co-operation between the PPARs and VDRs during IL-10 transcription. Therefore we investigated the effects of vitamin D3 on CD4+ T cell IL-10 production. Levels of TNF-a were also monitored to ensure that responses were selective for IL-10. Treatment with 1,25 a-dihydroxyvitamin D3 (vitD3; 10 nM) significantly increased IL-10 by 52% (Fig. 4a, n = 7; p < 0.05), but did not significantly affect the production of TNF-a (Fig. 4b). The combination of vitD3 and rosiglitazone strongly increased IL-10 levels by 103% compared to control levels (Fig. 4a, n = 7; p < 0.01). The proliferation of cells in response to vitD3 and rosiglitazone remained unchanged from vitD3 alone (Fig. 4c). 4. Discussion Ligands of PPAR-c have been demonstrated to be antiinflammatory in many in vitro cell systems and in vivo disease models. Although the mechanism for PPAR-mediated down-regulation of pro-inflammatory cytokine production from immune cells is unclear, much evidence supports a transrepressive mechanism in which transcription factors
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Fig. 4. Combination of rosiglitazone and Vitamin D3 further enhance IL-10 production. CD4+ T cells were stimulated as previously in the presence of either vehicle, 10 nM 1,25 a-dihydroxyvitamin D3, or 1 nM rosiglitazone and 1,25 a-dihydroxyvitamin D3. IL-10 (a) and TNF-a (b) levels were measured by ELISA (n = 7). **p < 0.01, of combination relative to vitamin D3 alone. (c) Re-stimulation assays for combination treatments were performed as for rosiglitazone treatments in Fig. 3 (n = 4). *p < 0.05, relative to control treatments.
such as NF-KB, NF-AT, Sp1 and AP-1 are inhibited by binding of activated PPAR [19–21]. Indeed, an additional consequence of these transrepressive mechanisms is that cell proliferation is blunted and autocrine growth factor production is inhibited (i.e., IL-2 for T cells in ref 20). We did not observe anti-proliferative effects at our highest concentration of rosiglitazone reported here, although we have observed anti-proliferative effects using concentrations greater than 25 lM. It is likely that either different concentrations used, different TZDs or PPAR-c agonist used, or the nature of cell stimulus employed may account for contrasting data produced by different research groups on PPAR-c ligands. Transrepression may well occur in vivo and explain observed reductions in TNF-a, IFN-c, and IL1b upon PPAR-c ligand treatment of mouse inflammatory disease models. However, in this study we have described a PPRE-dependent mechanism for PPAR-c-mediated upregulation of the anti-inflammatory cytokine IL-10 in both mature MDDCs and human CD4+ T cells. Increased production of IL-10 was only observed at low nM concentrations of rosiglitazone and slightly higher concentrations of pioglitazone and showed different magnitudes in the 2 different cell types. Very recently, rosiglitazone has been shown to increase IL-10 production in a mouse model of asthma [14]. Moreover, treatment with a soluble IL-10 receptor partially blocks the inhibitory effect of PPAR-c agonists on airway inflammation. A further report also showed a similar rosiglitazone-induced bell-shaped dose– response curve for IL-10 production from LPS-stimulated murine macrophages, although with higher concentrations
of rosiglitazone [22]. On the other hand, a previous report has shown that IL-10 production was dramatically reduced from dendritic cells cultured with high lM concentrations of rosiglitazone and other PPAR-c ligands [23], although these ligands were supplied at the beginning of monocyteto-DC differentiation (i.e., day 1). An earlier study reported no effect of 10 lM rosiglitazone on immature DC IL-10 production [5], which our data is in agreement with. Both of these PPAR-c/dendritic cell reports do not fully explore the relationship of IL-10 expression and concentration of the PPAR-c agonists. Therefore, a key issue with the majority of in vitro data generated with PPAR-c agonists is that of the concentration used. Concentrations of TZDs greater than 1 lM are not achieved systemically after daily 8 mg treatment of diabetic patients with rosiglitazone [24]. Moreover, with a half-life of 3–4 h, the concentration of systemic rosiglitazone falls below 20 nM approx 12 h after dosing with 2 mg rosiglitazone, with no accumulation of rosiglitazone occurring after repeat dosing. Thus, whilst in vitro data using lM concentrations may be more dynamic, translation of this data to the in vivo situation is less likely to be relevant. As previously mentioned, the ability of activated PPARs to transrepress various transcription factors as well as activate transcription through PPRE binding may result in complexed dose–response relationships, such as those observed in our study (Fig. 1). Furthermore, the magnitude of the PPAR response is likely to be determined by the strength and nature of the accompanying stimulus used, especially when considering PPRE-dependent responses
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in which PPARs would play a direct role in the formation of the multimeric transcriptional activation complex. Our data would suggest that the identified IL-10 PPRE is comparatively weak when compared to PPREs that are present in lipid metabolism gene promoters [25], and that this would impose a limit on the possible magnitude of IL-10 response to PPAR-c. The maximal upregulation of IL-10 observed in CD40L-stimulated MMDDCs and TCR-activated CD4+ T cells correlates well with the IL-10 PPRE driven transcription observed in the luciferase assays. Critically, the nature of the IL-10 upregulation in terms of concentration of rosiglitazone required and selectivity was repeated in different cell-types, stimulation protocols and measurement methods, thus emphasizing the importance and reproducibility of this finding. Our data clearly demonstrate that rosiglitazone (and probably pioglitazone) can selectively increase IL-10 production by mature MDDCs and CD4+ T cells in a bellshaped dose-dependent and PPAR-c-dependent manner. The ability of dendritic cells and activated T cells to synthesize increased amounts of IL-10 protein in response to PPAR-c ligands may be a potential explanation for their mechanism of action in inflammatory disease. Of further interest is the co-operation shown between rosiglitazone and 1,25 a-dihydroxyvitamin D3 towards IL-10 production from TCR-activated CD4+ T cells. Co-operation between vitamin D3 and other nuclear receptor activators (dexamethasone) towards IL-10 production has been previously described [26] In conclusion we have shown that IL-10 is significantly upregulated by nanomolar rosiglitazone in cells of the human immune system. This regulation may be a critical part of the mechanism of action of PPAR-c ligands in the potential resolution of inflammatory disease in humans. Acknowledgments We thank Filippo Volpe (Bioinformatics group, GlaxoSmithKline) for bioinformatics analysis of the IL-10 promoter, Liang Ye for technical assistance, Steve Smith for suggestions and advice, and Bill Davis for helpful discussions. References [1] Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 2002;53:409–35. [2] van Bilsen M, van der Vusse GJ, Gilde AJ, Lindhout M, van der Lee KA. Peroxisome proliferator-activated receptors: lipid binding proteins controling gene expression. Mol Cell Biochem 2002;239:131–8. [3] Daynes RA, Jones DC. Emerging roles of PPARs in inflammation and immunity. Nat. Rev Immunol 2002;2:748–59. [4] Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 1998;391:82–6. [5] Gosset P, Charbonnier AS, Delerive P, Fontaine J, Staels B, et al. Peroxisome proliferator-activated receptor gamma agonists affect the maturation of human monocyte-derived dendritic cells. Eur J Immunol 2001;31:2857–65.
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