Wondwossen Abate

Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids.

Lysophospholipid metabolism facilitates Toll‐like receptor 4 membrane translocation to regulate the inflammatory response

Sepsis, an overwhelming inflammatory response to infection, is a major cause of morbidity and mortality worldwide and has no specific therapy. Phospholipid metabolites, such as lysophospholipids, have been shown to regulate inflammatory responses in sepsis, although their mechanism of action is not well understood. The phospholipid‐metabolizing enzymes, lysophospholipid acyltransferases, control membrane phospholipid composition, function, and the inflammatory responses of innate immune cells. Here, we show that lysophosphatidylcholine acyltransferase (LPCAT) regulates inflammatory responses to LPS and other microbial stimuli. Specific inhibition of LPCAT down‐regulated inflammatory cytokine production in monocytes and epithelial cells by preventing translocation of TLR4 into membrane lipid raft domains. Our observations demonstrate a new regulatory mechanism that facilitates the innate immune responses to microbial molecular patterns and provide a basis for the anti‐inflammatory activity observed in many phospholipid metabolites. This provides the possibility of the development of new classes of anti‐inflammatory and antisepsis agents.

Lysophospholipid acyltransferases: novel potential regulators of the inflammatory response and target for new drug discovery.

Molecular and biochemical analyses of membrane phospholipids have revealed that, in addition to their physico-chemical properties, the metabolites of phospholipids play a crucial role in the recognition, signalling and responses of cells to a variety of stimuli. Such responses are mediated in large part by the removal and/or addition of different acyl chains to provide different phospholipid molecular species. The reacylation reactions, catalysed by specific acyltransferases control phospholipid composition and the availability of the important mediators free arachidonic acid and lysophospholipids. Lysophospholipid acyltransferases are therefore key control points for cellular responses to a variety of stimuli including inflammation. Regulation or manipulation of lysophospholipid acyltransferases may thus provide important mechanisms for novel anti-inflammatory therapies. This review will highlight mammalian lysophospholipid acyltransferases with particular reference to the potential role of lysophosphatidylcholine acyltransferase and its substrates in sepsis and other inflammatory conditions and as a potential target for novel anti-inflammatory therapies.

Three-dimensional spheroid cultures of A549 and HepG2 cells exhibit different lipopolysaccharide (LPS) receptor expression and LPS-induced cytokine response compared with monolayer cultures

Lipopolysaccharide (LPS) is a potent modulator of pathogen-induced host inflammatory responses. Lipopolysaccharide signaling to host cells is correlated with the expression of well-characterized LPS receptors. We have developed three-dimensional (3-D) cell cultures (spheroids) that are more representative of in vivo conditions than traditional monolayer cultures and may provide novel in vitro models to study the inflammatory response. In this work, we have compared F-actin organization, LPS-induced pro-inflammatory cytokine response and LPS receptor expression between spheroid and monolayer cultures from A549 lung epithelial cells and HepG2 hepatocytes. Significant junctional F-actin was seen at the cell—cell contact points throughout the spheroids, while monolayer cells showed stress fibers of actin and more prominent F-actin localized at the cell base. A time course of cytokine release in response to LPS showed that A549 spheroids secreted persistently higher levels of interleukin (IL)-6 and IL-8 compared with monolayer cultures. Unlike monolayer cultures, HepG2 spheroids responded to LPS by releasing a significant level of IL-8. We identified a significant increase in the expression of CD14 and MD2 in these spheroids compared with monolayers, which may explain the enhanced cytokine response to LPS. Thus, we suggest that 3-D spheroid cell cultures are more typical of in vivo cell responses to LPS during the development of inflammation and would be a better in vitro model in inflammation studies.

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system.

Evaluation of inflammatory effects of airborne endotoxin emitted from composting sources

Because of the lack of effective methodology, the biological effects of environmental endotoxin have not been assessed. Here we have collected and measured airborne endotoxin at different locations around composting sites. Increased endotoxin concentrations were observed close to composting activities and also at nearby boundary areas. Analysis of proinflammatory effects of the environmental endotoxin on interleukin (IL)-8 and IL-6 release from human D562 pharyngeal epithelial and MM6 monocytic cell cultures showed an association between endotoxin level and cytokine induction. The cytokine-inducing effect of bioaerosol extracts was inhibited by polymyxin B, indicating that endotoxin was the cause of cytokine responses we found. The environmental endotoxin was also more active for stimulating cytokines in airway epithelial cells than commercially purified Escherichia coli endotoxin. Our results suggest that these in vitro inflammatory cell models may contribute to the assessment of health impacts of environmental endotoxin.

Fast, Ultrasensitive Detection of Reactive Oxygen Species Using a Carbon Nanotube Based-Electrocatalytic Intracellular Sensor.

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular “pulse” of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection.

Evaluation of recombinant factor C assay for the detection of divergent lipopolysaccharide structural species and comparison with Limulus amebocyte lysate-based assays and a human monocyte activity assay

Purpose. The Limulus amebocytelysate (LAL) assay is widely used for the screening of lipopolysaccharide (LPS) in parenteral pharmaceuticals. However, correlation of LPS in Gram‐negative bacterial infections by LAL assay has been problematic, partly due to the variable reactivity of different LPS structures. Recombinant factor C (rFC) has allowed the development of a new simple, specific and sensitive LPS detection system (PyroGene). In this work, the potential of the new assay for detecting various LPS structures has been investigated and compared with two LAL‐based assays and a human monocyte activity assay. Methodology. The activity of the various LPS structures has been investigated by PyroGene and two LAL‐based assays and a human monocyte activity assay. Results. The rFC assay detected most LPS structures in picogram quantities and the potency of E. coli, B. cepacia, Salmonella smooth and Salmonella R345 LPS was no different when measured with PyroGene or LAL assays. However, the reactivity of K. pneumoniae, S. marcescens, B. pertussis and P. aeruginosa LPS differed significantly between these assays. Importantly, pairwise correlation analysis revealed that only the PyroGene assay produced a significant positive correlation with the release of IL‐6 from a monocytic cell line. Conclusion. We conclude that the rFC‐based assay is a good replacement for conventional LAL assays and as it correlates significantly with IL‐6 produced by a human monocyte cell line it could potentially be more useful for detecting LPS in a clinical setting.