The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

Vascular cell responsiveness to toll-like receptor ligands in carotid atheroma

Background Atherosclerosis is potentiated by stimulation of Toll-like receptors (TLRs), which serve to detect pathogen associated molecular patterns (PAMPs). However little is known of which PAMPs may be present in atheroma, or capable of stimulating inflammatory signalling in vascular cells. Materials and Methods DNA extracted from human carotid atheroma samples was amplified and sequenced using broad-range 16S gene specific primers to establish historical exposure to bacterial PAMPs. Responsiveness of primary human arterial and venous endothelial and smooth muscle cells to PAMPs specific for each of the TLRs was assessed by measurement of interleukin-8 secretion and E-selectin expression. Results Extracts of atheromatous tissue stimulated little or no signalling in TLR-transfected HEK-293 cells. However, sequencing of bacterial DNA amplified from carotid atheroma revealed the presence of DNA from 17 different bacterial genera, suggesting historical exposure to bacterial lipopeptide, lipopolysaccharide and flagellin. All cells examined were responsive to the ligands of TLR3 and TLR4, poly inosine:cytosine and lipopolysaccharide. Arterial cells were responsive to a wider range of PAMPs than venous cells, being additionally responsive to bacterial flagellin and unmethylated cytosine-phosphate-guanosine DNA motifs, the ligands of TLR5 and TLR9, respectively. Cells were generally unresponsive towards the ligands of human TLR7 and TLR8, loxoribine and single stranded RNA. Only coronary artery endothelial cells expressed TLR2 mRNA and responded to the TLR2 ligand Pam3CSK4. Conclusions Vascular cells are responsive to a relatively diverse range of TLR ligands and may be exposed, at least transiently, to ligands of TLR2, TLR4, TLR5 and TLR9 during the development of carotid atheroma.

Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2

Lipopolysaccharide (LPS), which generally activates Toll-like receptor 4 (TLR4), is expressed on commensal colonic bacteria. In a number of tissues, LPS can act directly on epithelial cells to increase paracellular permeability. Such an effect in the colon would have an important impact on the understanding of normal homeostasis and of pathology. Our aim was to use a novel primary culture of colonic epithelial cells grown on Transwells to investigate whether LPS, or Pam(3)CSK( 4), an activator of TLR2, affected paracellular permeability. Consequently, [(14)C]-mannitol transfer and transepithelial electrical resistance (TEER) were measured. The preparation consisted primarily of cytokeratin-18 positive epithelial cells that produced superoxide, stained for mucus with periodic acid-Schiff reagent, exhibited alkaline phosphatase activity and expressed TLR2 and TLR4. Tight junctions and desmosomes were visible by transmission electron microscopy. Basally, but not apically, applied LPS from Escherichia coli increased the permeability to mannitol and to a 10-kDa dextran, and reduced TEER. The LPS from Helicobacter pylori increased paracellular permeability of gastric cells when applied either apically or basally, in contrast to colon cells, where this LPS was active only from the basal aspect. A pan-caspase inhibitor prevented the increase in caspase activity caused by basal E. coli LPS, and reduced the effects of LPS on paracellular permeability. Synthetic Pam(3)CSK(4) in the basal compartment prevented all effects of basal E. coli LPS. In conclusion, LPS applied to the base of the colonic epithelial cells increased paracellular permeability by a mechanism involving caspase activation, suggesting a process by which perturbation of the gut barrier could be exacerbated. Moreover, activation of TLR2 ameliorated such effects.

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4 roles for cd14, lps-binding protein, and md2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor- production, IB degradation, p38 MAPK phosphorylation, and NF-B-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from 30 µM. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Oxidised phospholipid regulation of toll-like receptor signalling

Toll-like receptors (TLRs) serve to initiate inflammatory signalling in response to the detection of conserved microbial molecules or products of host tissue damage. Recent evidence suggests that TLR-signalling plays a considerable role in a number of inflammatory diseases, including atherosclerosis and arthritis. Agents which modulate TLR-signalling are, therefore, receiving interest in terms of their potential to modify inflammatory disease processes. One such family of molecules, the oxidised phospholipids (OxPLs), which are formed as a result of inflammatory events and accumulate at sites of chronic inflammation, have been shown to modulate TLR-signalling in both in vitro and in vivo systems. As the interaction between OxPLs and TLRs may play a significant role in chronic inflammatory disease processes, consideration is given in this review to the potential role of OxPLs in the regulation of TLR-signalling.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Toll-like receptor 4 signalling is neither sufficient nor required for oxidised phospholipids mediated induction of interleukin-8 expression

Toll-like receptor (TLR)-4 signalling has been shown to accelerate atherosclerosis. As oxidised phospholipids are present in atherosclerotic plaque and have been shown to modulate TLR4 signalling, we investigated the role of oxidised 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) in the regulation of TLR 1, 2, 4 and 6 signalling. Unlike established TLR agonists, OxPAPC did not induce NF-?B-dependent gene expression in monocytic THP-1 cells, human aortic endothelial cells or TLR-deficient HEK-293 cells transfected with TLRs 1, 2, 4 or 6. OxPAPC induction of IL-8 was not blocked by the TLR4 specific antagonist Rhodobacter sphaeroides LPS in human aortic endothelial cells, though OxPAPC potently inhibited TLR4 mediated IL-8 induction in these cells. OxPAPC upregulated IL-8 production in TLR4 deficient HEK-293 cells and this was not increased following TLR4 overexpression. Lipids extracted from carotid atherectomy samples did not stimulate TLR 1, 2, 4 or 6 signalling in a HEK-293 transfection assay. TLR4 signalling does not contribute to OxPAPC induced IL-8 expression in human epithelial HEK-293, monocytic THP-1 or aortic endothelial cells. As lipids extracted from diseased human artery also induced no TLR signalling, it is likely that the TLR-activating materials contributing to atherosclerosis are not of endogenous lipid origin.

CuII chain compound showing a ferromagnetic coupling through triple N1,N2-1,2,4-triazole bridges

[Cu(hyetrz)3](CF3SO3)2·H2O [hyetrz = 4-(2′-hydroxyethyl)-1,2,4-triazole] represents the first structurally characterised ferromagnetically coupled CuII chain compound containing triple N1,N2-1,2,4-triazole bridges. catena-[μ-Tris{4-(2′-hydroxyethyl)-1,2,4-triazole-N1,N2}copper(II)] bis(trifluoromethanesulfonate) hydrate (C14H23F6S2O10CuN9) crystallises in the triclinic space group Pl, a = 13.54(3), b = 14.37(3), c = 15.61(4) Å, α = 95.9(1), β = 104.9(1), γ = 106.5(1)°, V = 2763(11) Å3, Z = 4 (CuII units). The CuII ions are linked by triple N1,N2-1,2,4-triazole bridges yielding an alternating chain with Cu1−Cu2 = 3.8842(4) Å and Cu2−Cu3 = 3.9354(4) Å. Analysis of the magnetic data according to a high-temperature series expansion gives a J value of +1.45(3) cm−1. The nature and the magnitude of the ferromagnetic exchange have been discussed on the basis of the structural features. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

Building bridges: the third sector responding locally to diversity

This paper focuses on a hitherto unstudied segment of the broad 'third sector': organisations and groupings that aim to build bridges (that is, increase interpersonal contacts) between people of different faiths and/or ethnic groups. We draw on the findings of an empirical study, conducted in three diverse urban areas of England, of community-level projects with bridge building as an explicit aim. We describe the characteristics of bridge-building activities and the challenges they face; both the organisational challenges and those that arise from the nature of bridge building itself. We conclude by exploring the implications of our findings for an understanding of the third sector generally and for the potential role of the sector in responding to our diverse society.

Building bridges for future sustainability? Breaching the research-teaching nexus in engineering education

The relationship between research and learning and teaching represents what has been described as amongst the most intellectually tangled, managerially complex and politically contentious issues in mass higher education (Scott, 2005, p 53). Despite this, arguments that in order to achieve high quality scholarly outcomes, university teachers need to adopt an approach to teaching similar to that of research (founded upon academic rigour and evidence), has long been discussed in the literature. However, the practicalities of promoting an empirical and evidence-based approach to teaching in engineering education make dealing with the research / teaching nexus a somewhat challenging proposition. Using a phenomenographic approach, bringing together and applying the findings of a mixed methodological study, the workshop will adopt an activity based, interactive approach to encourage staff to consider the challenges and benefits of adopting an evidence-based approach to learning and teaching through the utilisation of research to inform their own practice. © 2009 Authors.

Building bridges for innovation in ageing:synergies between action groups of the EIP on AHA

The Strategic Implementation Plan of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA) proposed six Action Groups. After almost three years of activity, many achievements have been obtained through commitments or collaborative work of the Action Groups. However, they have often worked in silos and, consequently, synergies between Action Groups have been proposed to strengthen the triple win of the EIP on AHA. The paper presents the methodology and current status of the Task Force on EIP on AHA synergies. Synergies are in line with the Action Groups’ new Renovated Action Plan (2016-2018) to ensure that their future objectives are coherent and fully connected. The outcomes and impact of synergies are using the Monitoring and Assessment Framework for the EIP on AHA (MAFEIP). Eight proposals for synergies have been approved by the Task Force: Five cross-cutting synergies which can be used for all current and future synergies as they consider overarching domains (appropriate polypharmacy, citizen empowerment, teaching and coaching on AHA, deployment of synergies to EU regions, Responsible Research and Innovation), and three cross-cutting synergies focussing on current Action Group activities (falls, frailty, integrated care and chronic respiratory diseases).