Elsevier

Molecular Immunology

Volume 47, Issue 5, February 2010, Pages 1161-1170
Molecular Immunology

Short communication
The RNA binding protein tristetraprolin influences the activation state of murine dendritic cells

https://doi.org/10.1016/j.molimm.2009.11.002Get rights and content

Abstract

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-α 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-1β, 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.

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 the development of polyarticular arthritis due to excessive production of the proinflammatory cytokine TNF-α by macrophages. In wild type (WT) mice, activation-induced TNF-α production is under negative control of the likewise induced RNA-BP tristetraprolin (TTP). TTP binds to several TNF-α 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 costimulatory 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-α (Carballo et al., 1997) and IL-1β (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.

Section snippets

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

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

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

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).

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