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Unformatted text preview: EBV-Induced Gene 3 Transcription Is Induced by TLR Signaling in Primary Dendritic Cells via NF- k B Activation Stefan Wirtz,* Christoph Becker,* Massimo C. Fantini,* Edward E. Nieuwenhuis, ‡ Ingrid Tubbe,* Peter R. Galle,* Hans-Jo ¨rg Schild, † Mark Birkenbach, § Richard S. Blumberg, ‡ and Markus F. Neurath 1 * The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8 2 , CD8 1 , and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF- k B and Ets binding sites at 2 90 and 2 73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF- k B p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBI3-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF- k B p50- deficient mice. In summary, these data suggest that EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF- k B. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell development. The Journal of Immunology, 2005, 174: 2814–2824. T he recognition of pathogenic microorganisms by specific pattern recognition receptors on dendritic cells (DCs) 2 and macrophages is essential for the initiation of innate immune responses. Presumably, DC-derived cytokines secreted at the earliest phases of infections are major factors influencing the type and quality of T cell development (1). IL-12 and the closely related heterodimeric cytokine IL-23 are dominant factors for the initiation and maintenance of Th1-type immune responses, which are of critical importance for effective elimination of intracellular pathogens (2, 3). Whereas IL-12 p35/p40 acts primarily on naive CD4 1 T cells, the IL-23 p19/p40 heterodimer preferentially acti- vates memory CD4 1 T cells (4–6). Recently, IL-27 has been identified as a new bioactive member of the IL-12 cytokine family (7, 8). The IL-27 heterodimer consists of an IL-12 p40-related polypeptide, denoted EBV-induced gene 3 (EBI3) (9, 10), and a novel p28 subunit with some similarities to IL-12 p35. IL-27 induces the proliferation of naive CD4 1 T cells and production of the Th1 cytokine IFN- g (7). However, in con- trast to IL-23, IL-27 seems to have no major effect on memory T cells. Recent studies of IL-27 signaling (8, 11, 12) have shown that it activates STAT transcription factors (STAT-1, STAT-3, STAT-4, and STAT-5) and the Th1-specific transcription factor...
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