Post-transcriptional regulation of adapter molecules by IL-10 inhibits TLR-mediated activation of antigen-presenting cells.

Toll-like receptors (TLRs) act to sense the environment for microbial products and submit danger signals to antigen-presenting cells (APCs) resulting in activation of complex immune responses. In this study, we analyzed the function of human monocyte-derived APCs generated in vitro in the presence of interleukin (IL)-10 upon activation by TLR ligands. Exposure of these APCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by the TLR ligands, a reduced cytokine production, such as IL-12, IL-6 or tumor necrosis factor-alpha, and impaired capacity to stimulate T-cell activation. Furthermore, CCR7 upregulation in APCs exposed to TLR stimulation as well as migration towards CCL19/MIP-3beta were strongly reduced. IL-10 was found to downregulate MyD88, IRAK1 (IL-1 receptor-associated kinase) and tumor necrosis factor receptor-associated factor 6, essential adaptor molecules for TLR signaling, and to decrease TLR-induced nuclear expression of the nuclear factor-kappaB transcription factors c-Rel and Rel-B as well as interferon regulatory factor (IRF)-3 and IRF-8. This was not due to the inhibition of the mitogen-activated protein kinase pathway, but was rather mediated by the blockage of the PI3K signaling cascade. Interestingly, the inhibition of proteins involved in TLR signaling, such as MyD88, IRAK1 and mammalian target of rapamycin, was due to a selective post-transcriptional regulation.

CCAAT/enhancer-binding proteins alpha and beta negatively influence the capacity of tumor necrosis factor alpha to up-regulate the human cytomegalovirus IE1/2 enhancer/promoter by nuclear factor kappaB during monocyte differentiation.

Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.