Publications

Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.

In Drosophila, the Toll family of proteins mediates the innate immune response. Toll is activated by Spaetzle, which is generated in response to pathogens via a serine protease cascade. We wished to investigate if lipopolysaccharides (LPS) might activate Toll-like receptor (TLR) 4 via a serine protease in humans. The serpin antithrombin III (ATIII) and the thrombin inhibitor hirudin both inhibited nuclear factor (NF)-kappaB activation by LPS and Lipid A. ATIII and hirudin were also able to inhibit LPS-induced NF-kappaB activation in cells stably transfected with TLR4. These results suggest that LPS may activate a mammalian serine protease, which generates a product required for TLR4 signalling.

No abstract is provided for this article.

: Endothelin‐1 (Et‐1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et‐1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et‐1 caused an early increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca 2+ , and the demonstration of an increase in phospholipase A 2 (PLA 2 ) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA 2 in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et‐1‐induced AA release but abolished that by phorbol 12‐myristate 13‐acetate, demonstrating that the Et‐1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca 2+ ] i triggered by Et‐1, whereas valinomycin, which causes K + efflux, not only caused a rapid rise in [Ca 2+ ] i but also caused AA mobilisation. The results therefore suggest that Et‐1 activation of PLA 2 in this cell type requires calcium influx dependent on K + efflux.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E 2 (PGE 2 ) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE 2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE 2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE 2 production with indomethacin enhances, whereas the prostaglandin E 1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE 2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21-/- macrophages display an increased bacterial burden with Listeria at 30 mins and 2h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21-/- macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21-/- BMDMs in response to LPS or Listeria. TNF- was increased in miR-21-/- BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21-/- cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 mins), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21-/- macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21-/- BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21-/- mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.

Introduction Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. Methods Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. Results In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b , Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1 . In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. Conclusions These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Macrophages activated by the TLR4 agonist LPS undergo dramatic changes in their metabolic activity. We here show that LPS induces expression of the key metabolic regulator Pyruvate Kinase M2 (PKM2). Activation of PKM2 using two well-characterized small molecules, DASA-58 and TEPP-46, inhibited LPS-induced Hif-1α and IL-1β, as well as the expression of a range of other Hif-1α-dependent genes. Activation of PKM2 attenuated an LPS-induced proinflammatory M1 macrophage phenotype while promoting traits typical of an M2 macrophage. We show that LPS-induced PKM2 enters into a complex with Hif-1α, which can directly bind to the IL-1β promoter, an event that is inhibited by activation of PKM2. Both compounds inhibited LPS-induced glycolytic reprogramming and succinate production. Finally, activation of PKM2 by TEPP-46 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1β production, while boosting production of IL-10. PKM2 is therefore a critical determinant of macrophage activation by LPS, promoting the inflammatory response.

Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1β responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma.

The induction of the immediate‐early genes coding for Interleukin 1β (IL‐1β) and Tumor Necrosis Factor α (TNFα) is dependent upon Toll‐Like receptor 4 (TLR4) detection of bacterial lipopolysaccharide (LPS). We have observed various distinctions for these genes that correlate with RNA Polymerase II (Pol II) pausing, specific transcription factor involvement, promoter architecture and histone epigenetics. In contrast to human TNF , which possesses paused Pol II, human IL1B requires lineage‐specific Spi‐1/PU.1 as an anchor for induction‐dependent interaction with C/EBPβ and NF‐κB, resulting in rapid LPS‐dependent de novo recruitment of TBP and Pol II in concert with a permissive state for elongation mediated by the rapid recruitment of elongation factor P‐TEFb. In contrast, we observed that LPS‐inducible binding of NF‐κB to TNF facilitates immediate recruitment of BRD4. Treatment of murine macrophages with the metabolic inhibitor 2‐deoxyglucose reduced Pol II levels at the Il1b gene, while Tnf remained unaffected. P‐TEFb recruitment, Pol II serine 2 phosphorylation and histone H3K36me3 modification were reduced more significantly on Il1b than Tnf . A similar effect is observed in the presence of a PI3‐kinase inhibitor. Consequently, IL‐1β gene transcription is both more sensitive to the metabolic state of the cell and a dependence upon P‐TEFb than the gene coding for TNFα.