Molecular Immunology 47 (2010) 1161–1170
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Short communication
The RNA binding protein tristetraprolin influences the activation state of murine dendritic cells Matthias Bros a,∗ , Nadine Wiechmann a , Verena Besche a , Julia Art b , Andrea Pautz b , Stephan Grabbe a , Hartmut Kleinert b , Angelika B. Reske-Kunz a a b
Clinical Research Unit Allergology, Department of Dermatology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany Department of Pharmacology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
a r t i c l e
i n f o
Article history: Received 1 May 2009 Received in revised form 15 September 2009 Accepted 3 November 2009 Available online 28 November 2009 Keywords: Dendritic cells Tristetraprolin Tolerance Autoimmunity
a b s t r a c t Dendritic cells (DCs) serve to maintain peripheral tolerance under steady state conditions. Upon triggering by activation signals they initiate strong immune responses. The activation of DCs is accompanied by a rapid upregulation of proinflammatory cytokines, which were shown in other cell types to be regulated by mechanisms at the transcriptional and posttranscriptional level. Tristetraprolin (TTP), an important RNA binding protein, is involved in the regulation of mRNA stability of such cytokines. In this study we analyzed the significance of TTP for mouse DCs, which were derived from TTP−/− and WT bone marrow progenitor cells (BM-DCs). Unstimulated BM-DCs of TTP−/− mice expressed lower levels of mRNAs encoding the costimulatory molecules CD40 and CD86 and surprisingly also the canonical TTP targets TNF-␣ and IL-10 as compared with WT DCs. On the protein level, both DC populations expressed comparable amounts of CD80 and CD86 and of either cytokine, but TTP−/− DCs expressed less MHCII than WT DCs. On the other hand, TTP−/− DCs displayed elevated expression of other TTP target mRNAs like IL-1, c-fos and Mkp-1. Stimulation of BM-DCs of either genotype with lipopolysaccharide resulted in a rapid upregulation to a comparable extent of all molecules monitored so far, except for c-fos mRNA. Subsequent mRNA decay analysis revealed gene-specific differences in mRNA stability, which was influenced by the presence of TTP and the activation state of the DCs. Unstimulated TTP−/− DCs exerted a markedly lower allogeneic T cell stimulatory potential than WT DCs. Moreover, TTP−/− DCs induced an altered cytokine pattern in cocultures of DCs and T cells. However, allogeneic T cells primed by unstimulated DCs of either genotype were equally refractory to restimulation and suppressed the proliferation of naive T cells to the same extent. Thus, the findings of this study lend support to the interpretation that without external stimulation antigen presenting activity in DCs in the presence of TTP is more pronounced than in its absence and that posttranscriptional regulation contributes to the control of gene expression in DCs. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction RNA binding proteins (RNA-BPs) were shown to control the expression of numerous proteins by binding to the respective mRNA species encoding proto-oncogenes, growth factors, cytokines, transcription factors and others in various cell types (Eberhardt et al., 2007). Several RNA-BPs recognize AU-rich elements (AREs) which are found to be located in the 3 -untranslated region (UTR) of their target mRNA, thus exerting posttranscriptional control of expression of the respective gene. This mode of control may be crucial for limiting immune responses as was described for tristetraprolin knockout (TTP−/− ) mice (Carballo et al., 1997). Such mice suffer from slow growth, underweight and
∗ Corresponding author. Tel.: +49 61313933473; fax: +49 61313933360. E-mail address:
[email protected] (M. Bros). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.11.002
the development of polyarticular arthritis due to excessive production of the proinflammatory cytokine TNF-␣ by macrophages. In wild type (WT) mice, activation-induced TNF-␣ production is under negative control of the likewise induced RNA-BP tristetraprolin (TTP). TTP binds to several TNF-␣ mRNA ARE sites, thus mediating mRNA destabilization (Carballo et al., 1998). The capacity of TTP to bind mRNA is negatively regulated by p38 MAPK-dependent phosphorylation (Mahtani et al., 2001). JNK activation has been shown recently to modify the translation of the TTP mRNA (Korhonen et al., 2007). Moreover, via binding to TTP mRNA ARE sequences, TTP down-regulates its own expression (Tchen et al., 2004). Dendritic cells (DCs) act in concert with regulatory T cells (Tregs) to maintain peripheral tolerance under steady state conditions (Tang and Bluestone, 2008). On the other hand, upon activation DCs constitute the most potent antigen presenting cells (APCs) known being capable of activating naive T cells. The APC activity of the DCs correlates with high level expression of surface bound costim-
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ulatory molecules and of secreted immunomodulatory mediators, with the latter shaping the character of the T cell response (Adams et al., 2005). In activated macrophages several of the immunomodulatory factors, including TNF-␣ (Carballo et al., 1997) and IL-1 (Chen et al., 2006), were shown to be regulated posttranscriptionally by TTP as well as by other RNA-BPs (Eberhardt et al., 2007), while in DCs posttranscriptional control of gene expression has yet been scarcely addressed. Emmons et al. (2008) show in a study published recently that TTP binds numerous mRNA species in human monocyte-derived DCs. To elucidate the role of TTP in mouse DCs, we used myeloid DCs differentiated in tissue culture from bone marrow-derived progenitor cells (BM-DCs) of TTP−/− (Carballo et al., 1997) and WT mice and compared their gene expression signature as well as APC capacity. Our findings indicate that unstimulated BM-DCs from TTP−/− as compared with WT mice showed an altered pattern of expression and stability of various mRNA species. In line with these findings the APC capacity of DCs derived from TTP−/− progenitors was less potent than that of WT DCs. However, stimulation of the DCs of either genotype with lipopolysaccharide (LPS) altered the stability of various mRNA species and largely abrogated the differences observed between TTP−/− and WT DCs in terms of mRNA expression and APC function. These results suggest that in mouse DCs regulation of gene expression at the posttranscriptional level plays an important role, with TTP being crucially involved in that regulation in unstimulated BM-DCs. 2. Materials and methods 2.1. Mice and cells Female C57BL/6 and BALB/c mice as well as TTP−/− mice on the C57BL/6 background (Carballo et al., 1997, a kind gift by Dr. Blackshear, NIEHS, National Institutes of Health, Research Triangle Park, NC, USA), were bred and maintained in the Central Animal Facilities of the University of Mainz under specific pathogen-free conditions on a standard diet. The “Principles of Laboratory Animal Care” (NIH publication no. 85–23, revised 1985) were followed. Bone marrow-derived DCs (BM-DCs) were generated as reported by Scheicher et al. (1992) with modifications as described (Gisch et al., 2007) except that DC culture supernatants were replenished on days 3 and 6, and part of the DC cultures was stimulated with LPS (1 g/ml) on day 7. BM-DC medium (IMDM with 10% FCS; [PAA, Cölbe, Germany], 2 mM l-glutamine [Biochrom AG, Berlin, Germany], 100 U/ml penicillin, 100 g/ml streptomycin [Gibco, Paisly, UK]) was supplemented with 5% of GM-CSF containing cell culture supernatant (Zal et al., 1994, a kind gift by Dr. Stockinger, MRC National Institute for Medical Research, Mill Hill, London). Immature BM-DCs harvested on day 8 of culture for experiments contained >85% CD11c+ cells as assessed by FACS analysis. To monitor alterations in mRNA expression levels in time course studies of stimulation as well as in decay experiments employing actinomycin D (Act D), BM-DCs (106 ) were reseeded in wells of 6-well tissue culture plates (Greiner, Frickenhausen, Germany) containing 5 ml of BM-DC medium supplemented with GM-CSF, and aliquots of these cultures were stimulated with LPS (1 g/ml) in the absence or presence of Act D (5 g/ml) as indicated. 2.2. T cell proliferation assays Nylon–wool-enriched BALB/c T cells (Bros et al., 2007) (3 × 105 ) were cocultured with graded numbers of irradiated (30 Gy) BM-DCs for 4 days on flat-bottom 96-well tissue culture plates in 200 l of BM-DC medium. To assess the restimulatory and suppressive func-
tion of primed T cells, BM-DCs (106 /well) were cocultured with nylon–wool-enriched BALB/c T cells (6 × 106 /well) on 6-well tissue culture plates in a volume of 4 ml for 7 days, and prestimulated T cells were harvested. Graded numbers of these prestimulated T cells were cocultured with C57BL/6 splenocytes, depleted of erythrocytes and ␥-irradiated (30 Gy), without (restimulation assay) or with (suppression assay) freshly isolated BALB/c T cells (3 × 105 each) for 6 days. In all coculture assays T cell proliferation was assessed by the uptake of [3 H] thymidine (0.25 Ci/well) during the last 16 h of culture. Cells were harvested onto glass fiber filters and retained radioactivity was measured in a liquid scintillation counter (1205 Betaplate, LKB Wallac, Turcu, Finland). 2.3. PCR and real time PCR analysis Total RNA was isolated from at least 5 × 105 BM-DCs by using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) as recommended. RNA was reverse-transcribed applying a 1:1 mix of oligo-dT and random hexamer primers using iScript (BioRad, Munich, Germany) as recommended. Primer sequences have been described (Bros et al., 2007). Additional primers were used to detect c-fos (5 -GGGGCAAAGTAGAGCAGCTA-3 , 5 GGCTGCCAAAATAAACTCCA-3 ), Cox-2 (5 -TCCTCCTGGAACATGGACTC-3 , 5 -TTCTGCAGCCATTTCCTTCT-3 ), Gapdh (5 -CCATCACCATCTTCCAGGAG-3 , 5 -TTTCTCGTGGTTCACACCC-3 ), and Mkp-1 (5 GAGCTGTGCAGCAAACAGTC-3 , 5 -CTTCCGAGAAGCGTGATAGG 3 ). PCR-mediated detection of TTP as well as TTP/neomycin mRNA which is apparent in TTP−/− cells due to the insertion of the neomycin cassette has been described (Carballo et al., 2000). All primers were purchased from Operon (Cologne, Germany). The house-keeping gene ubiquitin C served as internal control. For normalization of mRNA expression in mRNA decay analysis Gapdh was used instead. Real time PCR reactions included 200 ng of cDNA and SYBRGreen mastermix (ABgene, Hamburg, Germany) and were performed and analyzed as described (Bros et al., 2007). 2.4. Flow cytometry BM-DCs (5 × 105 ) were washed in staining buffer (PBS/2% FCS). To block Fc receptor-mediated staining, cells were incubated with rat anti-mouse CD16/CD32 (2.4.G2) (Dianova, Hamburg, Germany) for 15 min on ice. Afterwards, cells were incubated with FITCconjugated rat monoclonal antibodies (Abs) recognizing MHC class II I-A/I-E (2G9), CD80 (1G10), and CD86 (GL1) (BD Pharmingen, San Diego, CA) for 20 min on ice. Appropriate isotype controls were used. Flow cytometric analysis was performed using a FACScan flow cytometer (BD Biosciences) equipped with CellQuest Software. 2.5. Cytokine assays ELISA capture Abs binding to murine IFN-␥ (clone R4-6A2), IL-5 (TRFK5), and TNF-␣ (G281-2626) were purchased from BD Pharmingen and Abs to murine IL-10 (JES052A5) was obtained from R&D Systems. Biotinylated detection Abs to murine IFN-␥ (AN18.17.24), IL-5 (TRFK4), and TNF-␣ (MP6-XT3) were obtained from BD Pharmingen and Abs to murine IL-10 (BAF417) from R&D Systems. Abs were used as recommended by the manufacturer. Recombinant murine (rm) cytokines IFN-␥, IL-5, and TNF-␣ used for ELISA standards were purchased from BD Pharmingen, and rm IL-10 from R&D Systems. 2.6. Statistical analysis Data were analyzed for statistically significant differences by applying Student’s t-test.
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Fig. 1. TTP mRNA and fused TTP/neo mRNA are expressed in unstimulated and LPS-stimulated WT and TTP−/− BM-DCs, respectively, each at comparable levels. BM-DCs were generated from bone marrow precursors of TTP−/− and WT mice and portions of the two cell populations were left unstimulated or were stimulated with LPS for 24 h. (A) Expression of intact WT TTP mRNA and of fused TTP/Neo mRNA in the case of TTP−/− mice was assessed qualitatively in the indicated DC populations by conventional PCR employing a TTP-specific sense primer in combination with a TTP- and TTP/neo-specific antisense primer, respectively. PCR products were separated electrophoretically on 1% agarose gels. M = molecular weight marker (band sizes are given in base pairs). The result is representative of 2 experiments performed. (B) The amount of TTP locus-derived mRNA of both TTP−/− and WT DCs after 24 h incubation with or without LPS was assessed quantitatively by real time PCR using a pair of primers, which allows to amplify a stretch of TTP sequence located downstream of the neo integration cassette. (C) Time dependent changes in TTP mRNA expression in LPS-stimulated WT DCs were monitored by real time PCR using the same primer pair as in (B). (B, C) Data indicate relative differences in mRNA expression compared with unstimulated WT DCs. Data represent (B) the mean ± SEM of 4 experiments performed in duplicate and (C) the mean ± SEM of 2 compiled experiments performed in duplicate. Statistically significant differences: * versus unstimulated WT DCs (**p < 0.01; ***p < 0.001).
3. Results 3.1. BM-DCs from TTP−/− and WT mice differ in their gene expression signatures For studies of the significance of TTP in DCs, myeloid DCs were grown from bone marrow progenitor cells (BM-DCs) prepared from TTP−/− and WT mice, respectively. WT DCs expressed intact TTP mRNA without and with prior LPS stimulation for 24 h (Fig. 1A). Because in TTP−/− mice the TTP gene locus is interrupted due to the integration of a neomycin resistance gene cassette (neo) into the second exon of TTP (Taylor et al., 1996), TTP−/− BM-DCs expressed a fused inactive TTP/neo mRNA, which was apparent at both states of activation. Quantitative assessment of the TTP and TTP/neo mRNAs in BM-DCs incubated for 24 h with or without LPS revealed largely stimulation-independent levels of mRNA expression (Fig. 1B). Kinetic measurements using LPS-stimulated WT DCs revealed a fast upregulation of TTP mRNA expression that reached its maximal level at 1 h after onset of LPS activation followed by a decline to the starting level within 24 h (Fig. 1C). The gene expression signatures of TTP−/− and WT BM-DCs were compared with a focus on mRNA species known to constitute TTP target genes in other cell types. Since a profound transcriptional reprogramming occurs in DCs in response to LPS activation, we analyzed mRNA levels both in the absence of activation and following stimulation with LPS for 24 h. Notably, unstimulated TTP−/− DCs
expressed lower levels of mRNA species encoding the costimulatory molecules CD40 and CD86 than did WT DCs, while mRNA of CD80 was expressed to a comparable extent in the two DC populations (Fig. 2A). However, stimulation of both DC populations for 1 day with LPS resulted in similar levels of the tested mRNAs. We observed lower surface expression of MHCII in TTP−/− than in WT DCs at unstimulated state, but expression adjusted to comparable levels in response to stimulation (Fig. 2D, left panel). CD80 and CD86 surface expression was comparable in both DC populations, when used unstimulated (Fig. 2D, middle and right panels). In both cases, expression was elevated to a comparable level upon stimulation. Using unstimulated DCs, the expression of TNF-␣ and IL-10 mRNA was markedly lower in TTP−/− as compared with WT DCs, while IL-1 mRNA was expressed at a higher level (Fig. 2B). The other mRNA species monitored encoding IL-6 and the IL-12 subunits IL12p35 and IL-12p40 were expressed at comparable levels in the two unstimulated DC populations. DCs of either origin responded to LPS stimulation with enhanced expression of these cytokine mRNAs to a comparable extent. Both TNF-␣ and IL-10 proteins were present at comparable low concentrations in culture supernatants of TTP−/− and WT DCs, and increased in response to stimulation (Fig. 2E). In either DC population, TNF-␣ concentrations reached their highest level at 3 h after the onset of stimulation, while IL-10 levels increased steadily within the stimulation period of 24 h. Moreover, we examined the expression of several other TTP target genes, whose products might modulate the APC activity of
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Fig. 2. Gene expression signatures of TTP−/− and WT BM-DCs used without external stimulation differ from each other. BM-DCs were derived from BM cultures of TTP−/− and WT mice and aliquots were left untreated or were stimulated with LPS for 24 h. The relative mRNA expression level of genes encoding (A) costimulators, (B) cytokines and (C) other immunologically relevant molecules was assessed by quantitative real time PCR. Data indicate relative differences in mRNA expression of LPS-stimulated WT DCs and of unstimulated and LPS-stimulated TTP−/− DCs compared to unstimulated WT DCs. Data represent the mean ± SEM of 4 experiments performed in duplicates. (D) Surface protein expression of MHCII and the costimulatory molecules CD80 and CD86 was monitored by flow cytometry. Histograms represent specific Ab binding to WT (upper panel) and TTP−/− (lower panel) BM-DCs at unstimulated (thin lines) and LPS-stimulated (thick lines) state. Binding of isotype-matched control Ab is indicated (dotted line). Mean fluorescence intensities are indicated. Graphs are representative of 2 independent experiments. (E) Contents of TNF-␣ and IL-10 in supernatants of BM-DC cultures stimulated with LPS for the indicated lengths of time were assessed by ELISA. Data represent the mean ± SEM of 2 experiments. Statistically significant differences: * versus unstimulated WT DCs, + versus unstimulated TTP−/− DCs, # versus LPS-stimulated WT DCs (*, + , # p < 0.05; **, ++ p < 0.01; ***, +++ p < 0.001).
DCs as well. Of these representatives, mRNA encoding c-fos, an AP-1 transcription factor family member, as well as MAP kinase phosphatase-1 (Mkp-1) showed significantly higher expression in TTP−/− versus WT DCs in the absence of stimulation (Fig. 2C), while expression of Cox-2 (p = 0.25) and Ido mRNA was not significantly altered. Upon LPS stimulation, mRNA levels of the latter two genes were upregulated in both DC populations to a similar extent. In contrast, c-fos mRNA was down-regulated under these culture conditions. Downregulation was more pronounced in WT than in TTP−/− DCs. Finally, although Mkp-1 mRNA expression
increased in DCs of both genotypes in response to LPS stimulation, its expression level was higher in TTP−/− than in WT DCs. Taken together, divergent gene expression signatures were found in TTP−/− as compared with WT DCs in the absence of stimulation. However, differences noted with regard to CD86 and cytokine (TNF-␣, IL-10) mRNA expression were not apparent on the protein level. On the other hand, TTP−/− DCs expressed less MHCII. Upon LPS stimulation mRNA expression profiles and protein levels in the two DC populations largely converged. Thus, primarily
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Fig. 3. Stimulation-associated upregulation of mRNA expression of distinct costimulatory receptors and cytokines is faster in TTP−/− than in WT BM-DCs. BM-DCs of TTP−/− and WT genotype were stimulated with LPS for various lengths of time and the relative mRNA expression level of a number of immunorelevant genes was assessed by quantitative real time PCR. Data indicate relative differences in mRNA expression of stimulated TTP−/− and stimulated WT DCs compared to unstimulated WT DCs. Data represent the mean of 2 experiments performed in duplicate. Statistically significant differences: TTP−/− DCs ($) and WT DCs (*) from stimulation cultures versus unstimulated WT DCs, + TTP−/− DCs at stimulated versus unstimulated state, and # TTP−/− versus WT DCs at a given time point ($ ,* ,+,#p<0.05; $$ ,** ,++,##p<0.01; $$$ ,*** ,###p<0.001).
in unstimulated BM-DCs the presence of TTP has distinct consequences for the DC phenotype.
a stimulation-dependent regulatory activity of TTP and/or other interacting RNA-BPs.
3.2. Stimulation triggers rapid adjustment of mRNA expression levels in TTP−/− and WT BM-DCs
3.3. The stability of mRNA species in BM-DCs correlates with the presence of TTP and the state of DC activation
To identify the time point(s) at which the mRNA expression levels adjusted in the course of LPS stimulation, we assessed the kinetics of stimulation-associated changes in gene expression in DCs of either genotype for selected mRNA species. In TTP−/− DCs the mRNA level of CD40 was rapidly upregulated in response to stimulation and reached similar levels as detected in WT DCs within the first 0.5 h and peaked by 3 h poststimulation (Fig. 3). On the other hand, the expression of IL-1 mRNA, being higher in unstimulated TTP−/− DCs than in their WT counterparts, remained essentially unchanged within the first 0.5 h of stimulation, but was then steadily upregulated until 3 h of stimulation. IL-12p40 subunit mRNA levels increased in both TTP−/− and WT DCs immediately after the onset of stimulation and reached maximal expression by 3 h of stimulation. In contrast, c-fos mRNA levels, which were higher in unstimulated TTP−/− than in WT DCs, increased in response to LPS stimulation in both DC populations within 1 h of stimulation onset, and declined thereafter. Taken together, the adjustment of mRNA expression levels in TTP−/− and WT DCs in response to LPS stimulation points to
Because TTP is known to affect the stability of target mRNAs, we assessed the decay rate of several mRNA species as described in Section 2 both in TTP−/− and WT DCs with and without prior LPS activation. CD40 mRNA displayed a rather high degree of stability, which was essentially stimulation-independent and was largely comparable in DCs of either genotype (Fig. 4). Similar results were obtained for CD86 mRNA (data not shown). On the other hand, the stability of IL-1 and IL-12p40 mRNA was markedly higher in unstimulated TTP−/− DCs than in their unstimulated WT counterparts. Moreover, transcripts of the two cytokines were more stable in stimulated WT DCs than in the absence of LPS stimulation. This pronounced stimulation-dependent difference in mRNA stability was not observed in TTP−/− DCs. Yet another mode of regulation was noted for c-fos mRNA, which displayed similar stability in unstimulated DCs of either origin. In WT DCs the stability of cfos mRNA was not strongly affected by LPS stimulation, while in stimulated TTP−/− DCs it was subjected to a more rapid decay. Finally, in the absence of stimulation Mkp-1 mRNA was more rapidly degraded in WT than in TTP−/− DCs. The opposite trend
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Fig. 4. TTP−/− BM-DCs as compared with WT DCs display an altered mRNA stability in a gene-specific manner. BM-DCs from BM cultures of TTP−/− and WT mice were incubated without or with LPS for 24 h. The DC cultures at either activation state were treated with actinomycin D (5 g/ml) for the indicated times. The relative mRNA expression level of various immunorelevant genes was assessed by quantitative real time PCR. Data indicate relative mRNA expression normalized to the content of Gapdh mRNA and are given as fold of expression level in the individual DC portions prior to treatment with actinomycin D (time point 0 h). Data represent the mean of 2 experiments performed in duplicate.
was seen in WT DCs following LPS stimulation. In this case Mkp-1 mRNA stability was increased in WT DCs, while in TTP−/− DCs it was essentially unaltered. Thus, the results of the mRNA decay experiments show that in BM-DCs the stability of the tested mRNA species is differentially regulated. It is dependent on the expression of TTP and on the state of activation of the cells.
3.4. Allogeneic T cell activation triggered by unstimulated TTP−/− BM-DCs as compared with WT BM-DCs is impaired Due to divergent phenotypes of TTP−/− and WT DCs, we asked whether these findings might have an impact on the functional state of the DCs and therefore assessed the primary allogeneic T cell stimulatory capacity of the DC populations. TTP−/− DCs from
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Fig. 5. Attenuated allogeneic T cell stimulatory activity shown by unstimulated TTP−/− BM-DCs. BM-DCs were generated from BM cultures of TTP−/− and WT mice and culture aliquots were stimulated with LPS for 24 h. (A) 5 × 104 irradiated DCs were cocultured with 3 × 105 nylon–wool-enriched allogeneic T cells from BALB/c mice in 0.2 ml culture medium in triplicate cultures for 4 days. T cell proliferation was assessed by measuring the uptake of [3 H] thymidine during the final 16 h of culture. T cell proliferation induced by unstimulated WT BM-DCs was set arbitrarily to one. Data represent the mean ± SEM of 4 independent experiments performed in triplicates. (B–D) Irradiated DCs (5 × 105 ) from unstimulated and stimulated TTP−/− and WT cultures were cocultured with 3 × 106 nylon–wool-enriched allogeneic BALB/c T cells in 2 ml culture medium. IFN-␥ (B), IL-5 (C), and IL-10 (D) in supernatants harvested on day 3 of coculture were quantified by ELISA. Data represent the mean ± SEM of 3–4 experiments performed in duplicate and were normalized to the content in WT DC/T cell cocultures set to one. Statistically significant differences: * versus unstimulated WT DCs, + versus unstimulated TTP−/− DCs, $ versus stimulated WT DCs (*p < 0.05; **p < 0.01; ***, +++ , $$$ p < 0.001).
unstimulated cultures induced significantly less T cell proliferation than did unstimulated WT DCs (Fig. 5A). On the other hand, LPS-stimulated DCs of either genotype acquired a significantly enhanced T cell stimulatory capacity with similar efficiency. Moreover, in cocultures of T cells and unstimulated DCs the Th1 marker cytokine IFN-␥ was found in comparable quantities irrespective of the genotype of the DCs (TTP−/− or WT), and IFN-␥ levels raised to a similar extent in the cocultures, when the two DC populations were employed following LPS stimulation (Fig. 5B). Unstimulated TTP−/− DCs induced a lower production of the Th2 marker cytokine IL-5 by allogeneic T cells than did WT DCs, while in cocultures containing stimulated DCs IL-5 levels were similar (Fig. 5C). On the other hand, the Th2/Treg marker cytokine IL-10 was present at similar levels in cocultures containing unstimulated DCs of either genotype, but upon stimulation TTP−/− DCs induced higher IL-10 production than WT DCs (Fig. 5D). Due to the observation of comparable T cell proliferation induced by DCs of either genotype at stimulated state, the finding of higher IL-10 content in cocultures harbouring stimulated TTP−/− DCs may reflect stronger Th2 skewing of T effector cells, rather than the differentiation of regulatory T cells. In conclusion, the lack of TTP in unstimulated DCs diminishes their allogeneic T cell stimulatory capacity. Moreover, TTP activity appears to affect the T cell polarizing properties of BM-DCs.
3.5. The potency of unstimulated BM-DCs to induce Treg is TTP-independent It has been shown previously that naive T cells acquire an anergic state in cocultures with unstimulated DCs. Restimulation of those anergic T cells results in a hypoproliferative response as compared with the response shown by stimulated naive T cells (Lu et al., 1995). We therefore compared the functional state of T cells following coculture with unstimulated DCs of either genotype. As depicted in Fig. 6, T cells recovered from a prior coculture with unstimulated DCs of either genotype congruently showed a significantly lower proliferative response than naive T cells upon restimulation with allogeneic spleen cells. Moreover, when these DC-pretreated T cells were added to cocultures of syngeneic naive T cells and allogeneic spenocytes, they inhibited the proliferation of the naive T cells to a comparable extent, lending support to the conclusion that unstimulated TTP−/− DCs and WT DCs induced differentiation of the interacting T cells into Tregs with congruent efficiency. Moreover, in these cultures the patterns of cytokine production regarding IFN-␥, IL-5 and IL-10 were comparable (data not shown). These results show that unstimulated TTP−/− DCs and unstimulated WT DCs do not differ in their Treg inducing potential upon coculture with naive T cells.
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Fig. 6. Unstimulated TTP−/− BM-DCs resemble their WT counterpart DCs in their tolerogenic potential towards naive T cells. BM-DCs (106 ) derived from BM cultures of TTP−/− and WT mice were irradiated and cocultured with nylon–wool-enriched T cells (6 × 106 ) of BALB/c mice in a volume of 4 ml in wells of 6-well cluster plates. After 7 days of coculture, precultured T cells (pTC(TTP−/− ), pTC(WT)) were harvested. The two pTC populations (3 × 105 ) were cocultured with 3 × 105 irradiated C57BL/6 spleen cells alone or with 3 × 105 irradiated C57BL/6 spleen cells in the presence of 3 × 105 enriched naive BALB/c T cells (nTC) in a volume of 0.2 ml for 6 days in triplicate cultures. T cell proliferation was assessed as described in the legend to Fig. 5. The proliferation of 3 × 105 nTCs induced by allogeneic stimulation with 3 × 105 irradiated C57BL/6 spleen cells was arbitrarily set to one. Data represent mean ± SEM of 4 independent experiments performed in triplicates. Statistically significant differences: * versus nTCs, + pTC/nTC cocultures versus corresponding pTC cultures (+ p < 0.05; ***p < 0.001).
4. Discussion Immature DCs constantly probe their microenvironment for antigen. By inducing Tregs they essentially contribute to the induction and maintenance of peripheral tolerance (Smits et al., 2005). Such DCs become activated by contact with pathogen-derived molecules. Activation can also be accomplished by proinflammatory cytokines, which serve as danger signals (Adams et al., 2005). Activation of the DCs causes fundamental changes of their gene expression pattern and is accompanied by rapid upregulation of several mRNA species known to constitute targets of ARE binding proteins (Eberhardt et al., 2007). Recently, the prototypic ARE-BP TTP was demonstrated to bind differentially to numerous immunorelevant mRNA species in human monocyte-derived DCs depending on the DC activation state (Emmons et al., 2008). The relevance of TTP activity for immune responses was substantiated by the fact that TTP−/− mice develop severe autoimmune diseases caused by excessive TNF-␥ production (Carballo et al., 1997). TTP−/− mice as compared with WT mice, exhibit major changes in the composition of their immune cell compartment, with decreased B and T cell numbers, but enhanced numbers of NK cells in addition to myeloid progenitors as well as granulocytes and macrophages. The composition of the myeloid cell populations and their differentiation state in TTP−/− mice might be due to the enhanced production of TNF-␣ (Carballo et al., 1997) and GMCSF by macrophages (Carballo et al., 2000). Meanwhile TTP target mRNA species have been identified for several other immune cell types. For instance, in B cells TTP binds to transcripts of the transcription factor E47 (Frasca et al., 2007), in T cells it targets mRNA of the survival and activation cytokine IL-2 (Ogilvie et al., 2005) and also other mRNA species like c-fos, IL-3, TNF-␣, and GM-CSF
(Raghavan et al., 2001), and finally in mast cells mRNA of TNF-␣ (Suzuki et al., 2003). These findings suggest that the altered composition of the immune cell compartment shown by TTP−/− mice is associated with abnormal gene expression in various immune cell types. To analyze the role of TTP in mouse myeloid DCs, we differentiated BM-DCs by in vitro culture from bone marrow progenitor cells of TTP−/− and WT mice. Expression of TTP transcripts was detected in WT DCs in the absence of external stimuli (see Fig. 1A) which is in line with its rather ubiquitous occurrence (Eberhardt et al., 2007). The lower expression level of mRNA species encoding costimulatory molecules (CD40 and CD86) in unstimulated DCs of TTP−/− mice as compared with their WT counterparts suggests that TTP activity positively regulates their expression. Our finding of comparable stability of either mRNA species in DCs irrespective of the presence or absence of TTP suggests that TTP might enhance the expression of these costimulatory molecules indirectly on the transcriptional level via (a) factor(s) whose mRNA constitute(s) a TTP target. However, the finding of comparable CD86 protein surface expression in TTP−/− and WT DCs suggests that at least one additional factor, which affects translational efficiency or protein half life may counteract this TTP-dependent mechanism. On the other hand, we noted largely comparable CD80 mRNA and protein expression in both DC populations. With regard to differences in translational efficiency in DCs of both genotypes, for example the RNA-BP HuR shares common mRNA targets with TTP including TNF-␣ (Eberhardt et al., 2007). Besides its well known mRNA stabilizing function, HuR was also shown to modulate the translational efficiency of mRNA targets without affecting their stability (Hinman and Lou, 2008). In this regard, HuR was reported to increase the nuclear export of mRNA species like CD83 and thereby their availability for translation (Prechtel et al., 2006). In addition, HuR was demonstrated to specifically recruit distinct mRNA species into translationally active polysomes (Hinman and Lou, 2008). It is also noteworthy that unstimulated TTP−/− BM-DCs expressed higher mRNA levels of c-fos, a member of the AP-1 transcription factor family, than did WT DCs. c-fos was reported to inhibit NF-B activity in macrophages (Ray et al., 2006). The elevated level of c-fos expression in unstimulated TTP−/− DCs may contribute to the repressed expression of NF-B responsive genes encoding MHCII as assessed on the protein as well as CD86 as noted on the transcriptional level (Li et al., 2008). Furthermore, c-fos mRNA was shown to be destabilized by TTP in T cells (Raghavan et al., 2001). This might be the case in DCs as well, since c-fos mRNA abundance was higher in TTP−/− than in WT DCs in the absence of external stimulation. Notably, of all mRNA species monitored, only c-fos expression was down-regulated upon stimulation concomitant with a stimulation-associated decrease in mRNA stability, which was more pronounced in TTP−/− than in WT DCs. This finding indicates that in stimulated DCs TTP may serve to support stabilization of c-fos mRNA. Indeed, although TTP is largely recognized as a ARE-BP destabilizing mRNA (Eberhardt et al., 2007), there are examples that it could also be involved in enhancing mRNA stability. Analysing the mRNA expression of several genes in LPS-treated macrophages from TTP−/− mice, Stoecklin et al. (2008) reported reduced expression of the LIF- and IL-15 mRNA in these cells compared with macrophages from WT mice. This TTP-related mRNA stabilizing effect may be due to the formation of complexes with other RNA-BPs, as we have demonstrated for human inducible nitric oxide synthetase mRNA (Fechir et al., 2005). In this case the mRNA stabilizing property of TTP is not accomplished by direct mRNA binding, but relies on protein–protein interactions with other ARE-BPs like KSRP and HuR (Fechir et al., 2005; Linker et al., 2005). On the other hand, the finding of higher levels of c-fos mRNA expression in TTP−/− than in WT DCs upon stimulation contrasts with the respective mRNA stability in the two DC populations.
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Taken together, these findings suggest that in stimulated DCs TTP inhibits c-fos mRNA expression by indirect mechanisms probably on the transcriptional level, but at the same time positively affects c-fos mRNA stability. We also noted a higher mRNA content of Mkp-1, a recently identified TTP target (Lin et al., 2008; Emmons et al., 2008) in TTP−/− as compared with WT DCs irrespective of the cell’s state of activation. MKP-1 acts as a stimulation-induced endogenous inhibitor of MAPK activation (Wang and Liu, 2007), and BM-derived macrophages of MKP-1−/− mice were shown to express higher levels of CD40 and CD86 proteins in response to stimulation with LPS than did WT macrophages (Salojin et al., 2006). Therefore, enhanced Mkp-1 mRNA expression as apparent in TTP−/− DCs as compared with WT DCs may be responsible for diminished transcriptional expression of either of the two costimulator encoding mRNA species in unstimulated DCs. The observation of a higher Mkp-1 mRNA stability in unstimulated TTP−/− than in WT DCs suggests a destabilizing effect of TTP on this mRNA species and might explain the higher level of Mkp-1 mRNA expression in TTP−/− DCs as compared with WT DCs. Upon stimulation, solely WT DCs, but not TTP−/− DCs, displayed an increased Mkp-1 mRNA stability. Therefore, the observation of a higher Mkp-1 mRNA expression level in stimulated TTP−/− DCs than in their WT counterparts might be due to indirect regulatory effects of TTP as suggested above for the expression of c-fos. In accordance with the reported role of TTP to destabilize AREcontaining mRNA species (Eberhardt et al., 2007), unstimulated TTP−/− DCs expressed higher amounts of IL-1 mRNA than did unstimulated WT DCs, which is in agreement with the higher half life of IL-1 mRNA in TTP−/− DCs. Unexpectedly, the mRNA levels of TNF-␣ and IL-10 were lower in unstimulated TTP−/− than in unstimulated WT DCs, which is in contrast to the findings reported for TTP−/− macrophages (Carballo et al., 1997; Stoecklin et al., 2008). However, TTP−/− and WT DCs produced comparable amounts of TNF-␣ and IL-10 protein at unstimulated state, whereby the levels of both cytokines were rather low, but increased in the course of stimulation. As suggested above as an explanation for the repressed expression of CD40 and CD86 mRNA in TTP−/− DCs, both c-fos and MKP-1 represent candidate molecules which might mediate transcriptional repression of TNF-␣ and IL-10 mRNA. This notion is substantiated by the observation that upon stimulation macrophages derived from c-fos−/− (Ray et al., 2006) or MKP-1−/− (Chi et al., 2006; Salojin et al., 2006) mice produced excessive amounts of TNF-␣ and other proinflammatory cytokines, including IL-10 in the case of MKP-1−/− macrophages. In response to stimulation with LPS, expressional differences of mRNA content between unstimulated DCs of both genotypes decreased below significancy within 30 min, which suggests a concomitant decrease in regulatory activity of TTP for the respective mRNA species and/or an increase in activity of other competitively acting RNA-BPs. Since other genuine TTP targets like c-fos and Mkp1 mRNA were differentially regulated in stimulated TTP−/− versus WT DCs, the regulatory activity of TTP for those targets might be diminished after stimulation in a target-specific manner. In mouse macrophages a stimulation-dependent rapid increase in TTP expression and activity was demonstrated, which limits the increase and persistance of stimulation-induced expression of mRNA species encoding inflammation-associated factors like TNF␣ (Carballo et al., 1997). Similarly, we noted a rapid increase in TTP mRNA expression in WT DCs in response to LPS stimulation although at a much lower rate than noted for macrophages. In either cell type, the stimulation-associated increase in TTP mRNA expression was followed by a rapid decline. In macrophages TTP negatively regulates expression of its own RNA by mRNA destabilization (Tchen et al., 2004) and TTP mRNA translation may be enhanced by activated JNK (Korhonen et al., 2007). In addition, activated p38 MAPK impairs TTP function by phosphorylation, which
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may then affect its expression (Mahtani et al., 2001), its ARE binding affinity (Hitti et al., 2006), and its sequestration by complexing 14-3-3 proteins (Stoecklin et al., 2004). Since increased p38 MAPK activity is a hallmark of DC stimulation (Yu et al., 2004), a reduction in TTP activity in stimulated WT DCs may be attributable to p38 MAPK-dependent mechanisms. In accordance with their diminished expression of MHCII, unstimulated TTP−/− DCs induced less primary allogeneic T cell proliferation than WT DCs and exhibited altered T cell polarizing properties as deduced from the cytokine patterns monitored in primary DC/T cell cocultures. Irrespective of the lower T cell activation potential shown by unstimulated TTP−/− as compared with WT BM-DCs, T cells primed by either BM-DC population were equally hyporesponsive to restimulation by allogeneic splenocytes. They suppressed the proliferation of naive T cells in suppression assays to a similar extent and evoked production of comparable patterns of cytokines (data not shown). In summary, our results suggest that TTP activity regulates gene expression in mouse myeloid BM-DCs in a gene-specific manner both by directly binding to target mRNAs and also by indirect mechanisms. On the functional level TTP activity contributes to an overall higher degree of activation in the absence of external stimuli, but in stimulated DCs TTP seems to play a minor role. In contrast, in macrophages TTP serves to control the extent of activation. Therefore TTP exerts qualitatively distinct tasks in the two cell types, which both share the same progenitor (Randolph et al., 2008). Our findings raise the question as to which extent activation-dependent alterations of posttranscriptional regulation in DCs constitute an essential step for the acquisition of potent APC activity. Finally, besides TTP other RNA-BPs (Eberhardt et al., 2007) have to be considered to contribute to the phenotype and function of DCs at either state of activation. Further studies aimed at clarifying their role in DCs may not only improve our insight into DC biology, but also open up new possibilities to genetically modify DCs for therapeutic applications. Acknowledgements We thank Dr. P.J. Blackshear (NIEHS, National Institutes of Health, Research Triangle Park, NC, USA) for providing the TTP−/− mice. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 548, project B5, and the Stiftung Rheinland-Pfalz für Innovation (15212-386261/761). References Adams, S., O’Neill, D.W., Bhardwaj, N., 2005. Recent advances in dendritic cell biology. J. Clin. Immunol. 25, 87–98. Bros, M., Jährling, F., Renzing, A., Wiechmann, N., Dang, N.A., Sutter, A., Ross, R., Knop, J., Sudowe, S., Reske-Kunz, A.B., 2007. A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid. Blood 109, 3820–3829. Carballo, E., Gilkeson, G.S., Blackshear, P.J., 1997. Bone marrow transplantation reproduces the tristetraprolin-deficiency syndrome in recombination activating gene-2 (−/−) mice. Evidence that monocyte/macrophage progenitors may be responsible for TNFalpha overproduction. J. Clin. Invest. 100, 986–995. Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 281, 1001–1005. Carballo, E., Lai, W.S., Blackshear, P.J., 2000. Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood 95, 1891–1899. Chen, Y.L., Huang, Y.L., Lin, N.Y., Chen, H.C., Chiu, W.C., Chang, C.J., 2006. Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells. Biochem. Biophys. Res. Commun. 346, 160–168. Chi, H., Barry, S.P., Roth, R.J., Wu, J.J., Jones, E.A., Bennett, A.M., Flavell, R.A., 2006. Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc. Natl. Acad. Sci. U.S.A. 103, 2274–2279.
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