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1.
Biology (Basel) ; 13(5)2024 May 01.
Article in English | MEDLINE | ID: mdl-38785798

ABSTRACT

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7-a commonly used experimental murine macrophage model-to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

2.
Biosci Rep ; 42(7)2022 07 29.
Article in English | MEDLINE | ID: mdl-35735109

ABSTRACT

Inflammation is central to several diseases. TLR4 mediates inflammation by recognising and binding to bacterial lipopolysaccharides and interacting with other proteins in the TLR4 signalling pathway. Although there is extensive research on TLR4-mediated inflammation, there are gaps in understanding its mechanisms. Recently, TLR4 co-localised with LPCAT2, a lysophospholipid acetyltransferase. LPCAT2 is already known to influence lipopolysaccharide-induced inflammation; however, the mechanism of LPCAT2 influencing lipopolysaccharide-mediated inflammation is not understood. The present study combined computational analysis with biochemical analysis to investigate the influence of LPCAT2 on lysine acetylation in LPS-treated RAW264.7 cells. The results suggest for the first time that LPCAT2 influences lysine acetylation in LPS-treated RAW264.7 cells. Moreover, we detected acetylated lysine residues on TLR4. The present study lays a foundation for further research on the role of lysine acetylation on TLR4 signalling. Moreover, further research is required to characterise LPCAT2 as a protein acetyltransferase.


Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Lipopolysaccharides , Toll-Like Receptor 4/metabolism , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Inflammation/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Lysine/metabolism , Mice , RAW 264.7 Cells , Toll-Like Receptor 4/genetics
3.
J Toxicol Environ Health A ; 82(20): 1076-1087, 2019.
Article in English | MEDLINE | ID: mdl-31797748

ABSTRACT

Contaminated marine bathing water has been reported to adversely affect human health. Our data demonstrated a correlation between total endotoxin (lipopolysaccharide; LPS) levels and degree of contamination of marine bathing waters. To assess the potential health implications of LPS present in marine bathing waters, the inflammation-inducing potency of water samples collected at different time points at multiple sampling sites were assessed using a cell culture-based assay. The numbers of fecal indicator bacteria (FIB) were also examined in the same samples. Water samples were used to stimulate two cell culture models: (1) a novel non-transformed continuously growing murine cell line Max Plank Institute (MPI) characteristic of alveolar macrophages and (2) human MonoMac 6 monocyte cell line. The inflammatory potential of the samples was assessed by measuring the release of inflammatory cytokines. The presence of high levels of LPS in contaminated bathing water led to induction of inflammatory response from our in vitro cell-based bioassays suggesting its potential health impact. This finding introduces an in vitro culture assay that reflects the level of LPS in water samples. These observations further promote previous finding that LPS is a reliable surrogate biomarker for fecal contamination of bathing water.


Subject(s)
Cytokines/immunology , Lipopolysaccharides/adverse effects , Macrophages/microbiology , Seawater/microbiology , Water Pollution/adverse effects , Animals , Bathing Beaches , Cell Line , England , Environmental Monitoring , Humans , Mice , Water Microbiology
4.
J Med Microbiol ; 66(7): 888-897, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28693666

ABSTRACT

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.


Subject(s)
Arthropod Proteins/metabolism , Endotoxins/analysis , Enzyme Precursors/metabolism , Lipopolysaccharides/analysis , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolism , Animals , Arthropod Proteins/genetics , Bacteria/chemistry , Chemistry Techniques, Analytical , Enzyme Precursors/genetics , Horseshoe Crabs , Humans , Recombinant Proteins/genetics , Serine Endopeptidases/genetics
5.
Arch Oral Biol ; 81: 41-47, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28472719

ABSTRACT

OBJECTIVE: The objective of the present study was to determine the effects of concurrent LPS and cytokine priming, reflective of the in vivo milieu, on macrophage production of key periodontitis associated cytokines TNF, IL-1ß and IL-6. DESIGN: THP-1 cells were pre-treated with combinations of Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS), concurrently with polarising cytokines IFNγ and IL-4, or PMA as a non-polarised control. Production of key periodontitis associated cytokines in response to subsequent LPS challenge were measured by enzyme - linked immunosorbent assay. RESULTS: Compared with cells incubated with IFNγ or IL-4 alone in the "polarisation" phase, macrophages that were incubated with LPS during the first 24h displayed a down-regulation of TNF and IL-1ß production upon secondary LPS treatment in the "activation" phase. In all three macrophage populations (M0, M1 and M2), pre-treatment with P. gingivalis LPS during the polarisation process led to a significant decrease in TNF production in response to subsequent activation by LPS (p=0.007, p=0.002 and p=0.004, respectively). Pre-treatment with E. coli LPS also led to a significant down-regulation in TNF production in all three macrophage populations (p<0.001). Furthermore, the presence of E. coli LPS during polarisation also led to the down-regulation of IL-1ß in the M1 population (p<0.001), whereas there was no measurable effect on IL-1ß production in M0 or M2 macrophages. There was no significant effect on IL-6 production. CONCLUSIONS: Macrophages become refractory to further LPS challenge, whereby production of key periodontitis associated cytokines TNF and IL-1ß is reduced after exposure to LPS during the polarisation phase, even in the presence of inflammatory polarising cytokines. This diminished cytokine response may lead to the reduced ability to clear infection and transition to chronic inflammation seen in periodontitis.


Subject(s)
Cytokines/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Cell Polarity , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Humans , Interferon-gamma/metabolism , Interleukin-1beta/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Peptide Fragments/metabolism , Porphyromonas gingivalis , Real-Time Polymerase Chain Reaction , THP-1 Cells , Tumor Necrosis Factor-alpha/metabolism
6.
ACS Appl Mater Interfaces ; 7(42): 23527-37, 2015 Oct 28.
Article in English | MEDLINE | ID: mdl-26438964

ABSTRACT

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.


Subject(s)
Biosensing Techniques , Nanotubes, Carbon/chemistry , Reactive Oxygen Species/isolation & purification , Animals , Lipopolysaccharides/chemistry , Macrophages/drug effects , Mice , NADPH Oxidases/chemistry , Reactive Oxygen Species/chemistry , Toll-Like Receptor 4/chemistry
7.
Innate Immun ; 17(3): 245-55, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20418262

ABSTRACT

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.


Subject(s)
Actins/metabolism , Epithelial Cells/metabolism , Hepatocytes/metabolism , Spheroids, Cellular , Cell Adhesion/immunology , Cell Culture Techniques , Cytokines/metabolism , Epithelial Cells/immunology , Epithelial Cells/pathology , Hep G2 Cells , Hepatocytes/immunology , Hepatocytes/pathology , Humans , Inflammation , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/metabolism , Signal Transduction/immunology
8.
Environ Toxicol Chem ; 30(3): 602-6, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21154847

ABSTRACT

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.


Subject(s)
Air Pollutants/toxicity , Endotoxins/toxicity , Inflammation/metabolism , Respiratory Mucosa/drug effects , Biodegradation, Environmental , Cell Line , Humans , Inflammation/chemically induced , Interleukin-6/metabolism , Interleukin-8/metabolism , Models, Biological , Respiratory Mucosa/metabolism
9.
J Lipid Res ; 51(2): 334-44, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19648651

ABSTRACT

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.


Subject(s)
Epithelial Cells/metabolism , Interleukin-8/biosynthesis , Lipopolysaccharides/pharmacology , Lung/cytology , Membrane Microdomains/drug effects , Pulmonary Surfactants/pharmacology , Toll-Like Receptor 4/metabolism , 1,2-Dipalmitoylphosphatidylcholine/pharmacology , Biological Products/pharmacology , Cell Line , Cell Survival/drug effects , Down-Regulation/drug effects , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Inflammation/metabolism , Interleukin-8/metabolism , Membrane Microdomains/metabolism , Phospholipids/pharmacology , Protein Transport/drug effects
10.
Pharmacol Ther ; 119(1): 104-14, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18538854

ABSTRACT

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.


Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase/physiology , Enzyme Inhibitors/therapeutic use , Inflammation/etiology , Sepsis/drug therapy , 1-Acylglycerophosphocholine O-Acyltransferase/antagonists & inhibitors , Amino Acid Sequence , Animals , Apoptosis , Brain Injuries/metabolism , Drug Discovery , Humans , Inflammation/enzymology , Interferon-gamma/pharmacology , Lipopolysaccharides/toxicity , Lysophosphatidylcholines/metabolism , Molecular Sequence Data , Monocytes/drug effects , Monocytes/metabolism , Phospholipids/metabolism , Sepsis/metabolism
11.
J Leukoc Biol ; 84(1): 86-92, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18403647

ABSTRACT

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.


Subject(s)
Cell Membrane/metabolism , Inflammation/metabolism , Lysophospholipids/metabolism , Toll-Like Receptor 4/metabolism , 1-Acylglycerophosphocholine O-Acyltransferase/antagonists & inhibitors , 1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Cell Line , Cell Membrane/drug effects , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Lipopolysaccharides/metabolism , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Nystatin/pharmacology , Protein Transport/drug effects , Pyridines/pharmacology , Toll-Like Receptor 2/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
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