The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE2, in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A–dependent pathway. Both TLR agonists and PGE2 promote the phosphorylation of the transcription factor CREB on Ser133. However, although CREB regulates IL-10 transcription, the mutation of Ser133 to Ala in the endogenous CREB gene did not prevent the ability of PGE2 to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser343, inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE2 on IL-10 production.

Macrophages are an important source of cytokines following infection. Stimulation of macrophages with TLR agonists results in the secretion of TNFα, IL-6 and IL-12, and the production of these cytokines is controlled by multiple feedback pathways. Macrophages also produce IL-10, which acts to inhibit pro-inflammatory cytokine production by macrophages via a JAK/STAT3 dependent pathway. We show here that, Ruxolitinib, a recently described selective inhibitor of JAKs, increases TNF, IL-6 and IL-12 secretion in mouse bone marrow derived macrophages stimulated with LPS This effect is largely due to its ability to block IL-10 mediated feedback inhibition on cytokine transcription in macrophages. Similar results were also obtained with a second structurally unrelated Jak inhibitor, Tofacitinib. In addition, LPS induced the production of IFNβ, which was then able to activate JAKs in macrophages resulting in the stimulation of STAT1 phosphorylation. The initial induction of IL-10 was independent of JAK signaling, however inhibition of JAKs did reduce IL-10 secretion at later time points. This reflected a requirement for the IFNβ feedback loop to sustain IL-10 transcription following LPS stimulation. In addition to IL-10, IFNβ also helped sustain IL-6 and IL-12 transcription. Overall, these results suggest that inhibition of JAKs may increase the inflammatory potential of macrophages stimulated with TLR4 agonists.

The Janus Kinases (JAKs) and their downstream effectors Signal Transducer and Activator of Transcription proteins (STATs) form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation and hematopoiesis and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity-relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å co-crystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8D270A/D270A mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8D270A mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes. Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.

The protein kinase ERK5 (MAPK7) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role mediating cell proliferation, survival, epithelial–mesenchymal transition, and angiogenesis. To date, no three-dimensional structure has been published that would allow rational design of inhibitors. To address this, we determined the X-ray crystal structure of the human ERK5 kinase domain in complex with a highly specific benzo[e]pyrimido[5,4-b]diazepine-6(11H)-one inhibitor. The structure reveals that specific residue differences in the ATP-binding site, compared to the related ERKs p38s and JNKs, allow for the development of ERK5-specific inhibitors. The selectivity of previously observed ERK5 inhibitors can also be rationalized using this structure, which provides a template for future development of inhibitors with potential for treatment of disease.

There is an imbalance in extracellular matrix turnover with minimal contribution of adaptive immune responses at this stage of trichiasis. There was little evidence of broad differential expression in genes characteristic of polar responses of adaptive T cells or macrophages. The control of the MMP7 response and its activity appears significant in the fibrotic changes observed in TT.

IL-10 is an important anti-inflammatory cytokine that plays important roles in controlling inflammatory responses and keeping the immune system in check following activation. Loss of IL-10 function in mice or humans results in the development of inflammatory bowel disease in response to an elevated immune response to the gut flora. IL-10 also acts to prevent excessive inflammation during the course of infection and has been implicated in a variety of autoimmune conditions. In response to inflammatory signals, IL-10 can be produced by a number of immune cells including T cells, B cells, macrophages, and dendritic cells. Distinct mechanisms control the production of IL-10 in these different cells types. In this review, we describe recent studies that have looked at the signaling pathways that regulate IL-10 production in these cells. Given the number of cell types that produce IL-10, it is perhaps not surprising that the in vivo source of IL-10 can vary in different immune models. We also describe how work using conditional IL-10 knockout mice or adoptive transfer of IL-10-deficient cells has begun to further our understanding regarding which specific immune cells are required for IL-10 production in vivo under different conditions.

HighlightsMCP-1 mRNA levels and protein secretion in macrophages are induced by TLR activation.In response to LPS, the initial induction of MCP-1 mRNA is IFNβ independent.The sustained production of MCP-1 by LPS requires an IFNβ mediated feedback loop.The sustained production of MCP-1 by poly IC also requires IFNβ.