The regulation of IL-10 expression

Interleukin (IL)-10 is an important immunoregulatory cytokine and an understanding of how IL-10 expression is controlled is critical in the design of immune intervention strategies. IL-10 is produced by almost all cell types within the innate (including macrophages, monocytes, dendritic cells (DCs), mast cells, neutrophils, eosinophils and natural killer cells) and adaptive (including CD4(+) T cells, CD8(+) T cells and B cells) immune systems. The mechanisms of IL-10 regulation operate at several stages including chromatin remodelling at the Il10 locus, transcriptional regulation of Il10 expression and post-transcriptional regulation of Il10 mRNA. In addition, whereas some aspects of Il10 gene regulation are conserved between different immune cell types, several are cell type- or stimulus-specific. Here, we outline the complexity of IL-10 production by discussing what is known about its regulation in macrophages, monocytes, DCs and CD4(+) T helper cells

Production and characterisation of the TPL-2 / ABIN-2 / NF-κB1 p105 complex

TPL-2 functions as a MEK1/2 kinase that is an essential component of an ERK1/2 MAP kinase pathway activated by Toll-like receptors and members of the IL-1 and TNF receptor families. TPL-2 is required for production of the pro-inflammatory cytokine TNF by macrophages after lipopolysaccharide stimulation. Recent work from the Ley laboratory has demonstrated that TPL-2 induces TNF secretion by macrophages independently of MEK1/2 and ERK1/2 phosphorylation. However, the target protein that TPL-2 phosphorylates to control TNF production is not known. The aim of this project was to determine the primary amino acid sequence specificity of TPL-2 by peptide library screening to allow bioinformatic identification of novel TPL-2 substrates. To do this, a strategy was developed to purify TPL-2, complexed with its cellular binding partners ABIN-2 and NF-κB1 p105, from lysates of transiently transfected HEK293 cells. This method generated large amounts of highly purified TPL-2 complex, which was soluble, stable, and catalytically active. Interestingly, mass spectrometric analysis of highly purified TPL-2 complex identified several novel binding partners, including mTORC2, IRAK1, and TRIM25, which may be functionally linked to the TPL-2/ABIN-2/NF-κB1 p105 complex. Screening of a positional scanning peptide library demonstrated that TPL-2 complex had an unusual amino acid specificity, in which negatively charged amino acids were preferred at all five positions N-terminal to the phosphorylation site. A synthetic peptide based on this optimal motif was specifically phosphorylated by the TPL-2 complex much more efficiently than a positive control MEK peptide, strongly suggesting that the screen had worked. A Scansite search of protein sequence databases with the positional-specific scoring matrix for TPL-2 identified a number of potential novel substrates, including several proteins involved in intracellular membrane trafficking. A strategy for future validation of potential TPL-2 substrates and the possibility of using an optimal TPL-2 substrate peptide for high throughput inhibitor screening are discussed

Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs

I B Kinase 2 regulates TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct phosphorylation of TPL-2 serine 400

Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription

P38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses