Are toll-like receptors potential drug targets for atherosclerosis? Evidence from genetic studies to date.

Low-density lipoprotein cholesterol lowering, most notably via statin therapy, has successfully reduced the burden of coronary artery disease (CAD) in recent decades. However, the residual risk remaining even after aggressive lipid lowering has renewed interest in alternative targets. Anti-inflammatory drugs are thought to have much potential in this context, but side effects associated with long-term use of conventional anti-inflammatories, such as NSAIDs and glucocorticoids, preclude their use as preventive agents for CAD. Evidence from epidemiological studies and murine models of atherosclerosis suggests that toll-like receptors (TLRs) may have utility as targets for more focused anti-inflammatories, but it remains unclear if this pathway is causally related to CAD in man. Here, we review recent insight into this question gained from genetic studies of cardiovascular risk and innate immune function, focussing on the potential of Mendelian randomisation approaches based on intracellular-signalling pathways to identify and prioritise targets for drug development.

Genetic analysis of leukocyte type-I interferon production and risk of coronary artery disease.

OBJECTIVE: Patients with systemic lupus erythematosus are genetically predisposed to enhanced production of the type-I interferon IFN-α and are also at elevated risk of developing atherosclerosis compared with healthy subjects. We aimed to test whether genetic predisposition to increased type-I IFN production affects risk of coronary artery disease. APPROACH AND RESULTS: Using a list of 11 single nucleotide polymorphisms from the results of genome-wide association studies for systemic lupus erythematosus, which we hypothesised would be enriched in variants that regulate type-I IFN production, we identified a genetic risk score based on 3 single nucleotide polymorphisms (rs10516487, rs3131379 and rs7574865), which correlated significantly with production of IFN-α by human peripheral leukocytes stimulated with CpG-oligonucleotide (n=60, P=1.50 × 10(-5)). These single nucleotide polymorphisms explained 27.8% of variation in the CpG-oligonucleotide-induced IFN-α response and were also associated with Toll-like receptor-7/8- and Toll-like receptor-9-dependent IFN-α and IFN-ß responses, but were not associated with inflammatory cytokine production in response to Toll-like receptor-4 stimulation or risk of coronary artery disease in 22,233 cases and 64,762 controls (odds ratio 1.00, 95% CI 0.98-1.02) using Mendelian randomization-based analyses. Coronary artery disease risk was also not associated with the full panel of 11 systemic lupus erythematosus single nucleotide polymorphisms or loci responsible for the monogenic type-I interferonopathies Aicardi-Goutières syndrome and Spondyloenchondrodysplasia with immune dysregulation. CONCLUSIONS: The results argue against the potential utility of drugs targeting type-I IFN production for coronary artery disease. The use of genetic variants that modify leukocyte signaling pathways, rather than circulating biomarkers, as instruments in Mendelian randomization analyses may be useful for studies investigating causality of other candidate pathways of atherogenesis.

Toll-like receptor stimulants in processed meats promote lipid accumulation in macrophages and atherosclerosis in Apoe-/- mice.

Dietary intake of processed meat is a risk factor for cardiovascular disease. However, the effects of processed meats on lipid metabolism in macrophages, a key regulator of cardiovascular risk, have remained largely unexplored. Extracts of processed meats, but not their fresh non-processed equivalents, were found to promote a significant increase in macrophage lipid accumulation in vitro. Calibrated receptor-dependent reporter assays revealed that pro-inflammatory stimulants of Toll-like receptor (TLR)-2 and TLR4 were low or undetectable in fresh meats, but rose dramatically following chopping and storage at 4 °C. Lipid accumulation in response to processed meats correlated well with TLR-stimulant content, was significantly reduced in TLR4-deficient macrophages, and was absent in response to meats stored frozen to prevent bacterial growth. TLR-stimulation significantly increased the incorporation of 14C-acetate into cellular lipids, and induced lipid accumulation in the absence of exogenous lipoproteins, suggesting a key role for de novo lipid synthesis in this process. Aortic atherosclerosis was also significantly accelerated in Apoe-/- mice receiving a diet supplemented with TLR-stimulants at concentrations relevant to those measured in processed meats, compared to normal chow. The findings reveal novel mechanisms which may be of relevance to the observed connections between processed meat consumption, inflammatory markers and cardiovascular risk.

The capacity of foodstuffs to induce innate immune activation of human monocytes in vitro is dependent on food content of stimulants of Toll-like receptors 2 and 4.

The ingestion of fatty meals is associated with a transient, low-grade systemic inflammatory response in human subjects, involving the activation of circulating monocytes and the secretion of pro-inflammatory cytokines. However, it is not yet clear how different foodstuffs may promote inflammatory signalling. In a screen of forty filter-sterilised soluble extracts from common foodstuffs, seven were found to induce the secretion of TNF-α and IL-6 from human monocytes in vitro. To investigate what may differentiate inflammatory from non-inflammatory food extracts, stimulants of Toll-like receptor (TLR) 2 and TLR4 were quantified using human embryonic kidney-293 cells transfected with each TLR, and calibrated with defined bacterial lipopeptide (BLP) and lipopolysaccharide (LPS) standards. These assays revealed that while most foods contained undetectable levels of TLR2 or TLR4 stimulants, all TNF-α-inducing foods contained stimulants of either TLR2 (up to 1100 ng BLP-equivalent/g) or TLR4 (up to 2700 ng LPS-equivalent/g) in both the soluble and insoluble fractions. TLR stimulants were present mainly in meat products and processed foods, but were minimal or undetectable in fresh fruit and vegetables. The capacity of food extracts to induce TNF-α secretion in monocytes correlated with the content of both TLR2 (r 0·837) and TLR4 stimulants (r 0·748), and was completely abolished by specific inhibition of TLR2 and TLR4. LPS and BLP were found to be highly resistant to typical cooking times and temperatures, low pH and protease treatment. In conclusion, apparently unspoiled foodstuffs can contain large quantities of stimulants of TLR2 and TLR4, both of which may regulate their capacity to stimulate inflammatory signalling.

Toll-like receptor-dependent lipid body formation in macrophage foam cell formation.

PURPOSE OF REVIEW: The differentiation of macrophages into lipid-laden foam cells is central to the development of atherosclerosis. Traditionally, it has been assumed that the uptake of oxidized low-density lipoprotein by macrophage scavenger receptors is largely responsible for this process. However, in light of recent evidence that these mechanisms may not play as large a role as previously thought, alternative mechanisms of foam cell formation are now being explored. RECENT FINDINGS: The stimulation of Toll-like receptor (TLR) signalling by bacterial molecules has been shown to promote the accumulation of lipid in macrophages in the form of intracellular inclusions termed 'lipid bodies'. Interactions between TLR-signalling pathways and the liver-X receptor and peroxisome proliferator-activated receptor-γ signalling pathways modulate the formation of lipid bodies in macrophages and thereby cellular accumulation of cholesterol and triglyceride. These pathways appear to involve TLR-mediated regulation of lipid-binding proteins, cellular cholesterol sensors, lipid-body-associated proteins and secreted autocrine factors, but are independent of scavenger receptor or lipoprotein oxidation-dependent pathways. SUMMARY: TLR stimulation promotes the accumulation of lipid bodies in macrophages and consequently foam cell formation. The pathways responsible for these processes may constitute novel therapeutic targets for atherosclerosis.

Reversal of Tetracycline Resistance by Cepharanthine, Cinchonidine, Ellagic Acid and Propyl Gallate in a Multi-drug Resistant Escherichia coli.

Bacterial resistance to antibiotics is an increasing threat to global healthcare systems. We therefore sought compounds with potential to reverse antibiotic resistance in a clinically relevant multi-drug resistant isolate of Escherichia coli (NCTC 13400). 200 natural compounds with a history of either safe oral use in man, or as a component of a traditional herb or medicine, were screened. Four compounds; ellagic acid, propyl gallate, cinchonidine and cepharanthine, lowered the minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol and tobramycin by up to fourfold, and when combined up to eightfold. These compounds had no impact on the MICs of ampicillin, erythromycin or trimethoprim. Mechanistic studies revealed that while cepharanthine potently suppressed efflux of the marker Nile red from bacterial cells, the other hit compounds slowed cellular accumulation of this marker, and/or slowed bacterial growth in the absence of antibiotic. Although cepharanthine showed some toxicity in a cultured HEK-293 mammalian cell-line model, the other hit compounds exhibited no toxicity at concentrations where they are active against E. coli NCTC 13400. The results suggest that phytochemicals with capacity to reverse antibiotic resistance may be more common in traditional medicines than previously appreciated, and may offer useful scaffolds for the development of antibiotic-sensitising drugs.

Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling.

Toll-like receptor 4 (TLR4)-mediated recognition of lipopolysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. Two commonly occurring mutations in the human TLR4 gene (Asp299Gly and Thr399Ile) have recently been shown to be associated with blunted physiological responses to inhaled LPS, and with increased risk of Gram-negative bacteraemia in sepsis patients and reduced risk of atherosclerosis in an Italian population. Here we show that monocytes from individuals heterozygous for both mutations in the TLR4 gene exhibit no deficit in recognition of LPS of Escherichia coli, Neisseria meningitidis, Bacteroides fragilis, Yersinia pestis, Chlamydia trachomatis, Porphyromonas gingivalis, or Pseudomonas aeruginosa. We propose that the relatively high frequency of these mutations in the Caucasian population may reflect modified responses of carriers to alternative TLR4 agonists.

Saturated fatty acids do not directly stimulate Toll-like receptor signaling.

OBJECTIVE: Toll-like receptors (TLRs) initiate inflammatory signaling in response to conserved microbial molecules. It has been proposed that dietary saturated fatty acids (SFAs) may also serve as endogenous ligands of TLR2 or TLR4, thereby promoting diseases associated with inflammation and dyslipidemia, including atherosclerosis and insulin resistance. METHODS AND RESULTS: We investigated the effects of SFAs on TLR-dependent signaling using a broad range of cell types and readouts. In HEK-293 cells transfected with TLR2, TLR4, or TLR5, SFAs complexed with fatty-acid-free bovine serum albumin (BSA)-stimulated TLR-dependent signaling. However, SFAs alone did not elicit a similar response. Further analysis showed that the effect seen with the complexed SFAs was attributable to LPS and lipopeptide contamination of fatty-acid-free BSA. Additional studies in macrophages, endothelial cells, smooth muscle cells, adipocytes, skeletal muscle cells, and human peripheral blood mononuclear cells confirmed the lack of stimulation of TLR-dependent signaling pathways or expression of TLR-target genes by SFAs. CONCLUSIONS: SFAs do not directly stimulate TLR-dependent signaling, suggesting that alternative mechanisms link dietary fat intake with TLR-associated pathologies. LPS and lipopeptide contamination of the widely used reagent fatty-acid-free BSA explains the previously reported stimulation of TLR2 and TLR4 by SFAs.

Bacteroides fragilis signals through Toll-like receptor (TLR) 2 and not through TLR4.

Although it is desirable to identify the interactions between endotoxin/LPS and the innate immune mechanism, it is often not possible to isolate these interactions from other cell wall-related structures of protein or polysaccharide origin. There is no universally accepted method to extract different LPSs from different bacteria, and their natural state will be influenced by their interactions with the associated molecules in the bacterial outer membrane. It is now believed that Toll-like receptor (TLR) 4 is the main signal transducer of classical LPS (i.e. Escherichia coli LPS), while TLR2 is used by certain non-classical LPSs. There are contradictory reports as to whether Bacteroides fragilis LPS, a non-classical LPS, signals primarily through TLR2 or TLR4. This study was designed to address this problem. Different non-purified and purified B. fragilis LPSs extracted by different methods together with different heat-killed, whole-cell populations of B. fragilis were used to elucidate the TLR specificity. All of these B. fragilis preparations showed a significant signalling specificity for TLR2 but not for TLR4. This indicates that changing the extraction methods, with or without applying a repurification procedure, and varying the cell populations do not alter the TLR specificity of B. fragilis LPS.

The roles of pathogen-associated molecular patterns in atherosclerosis.

Stimulation of Toll-like receptors, which serve to initiate inflammatory signaling in response to the detection of conserved microbial pathogen-associated molecular patterns (PAMPs), has been shown to play a central role in the development of atherosclerosis. In this review, the recent evidence supporting a role for both infection- and commensal-derived PAMPs in the pathogenesis of atherosclerosis will be discussed. Potential sources of PAMPs, their routes of delivery to the artery wall and the mechanisms by which PAMPs may affect vascular function independently of bacteremia or infection of the artery wall with viable organisms will be examined. Finally, the recent evidence that obesity and high-fat diets may each promote translocation of commensal-derived endotoxin from the gut into the circulation to induce inflammation, insulin resistance and atherosclerosis will be discussed.

Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production.

Background: The mechanisms connecting dietary intake of processed foods with systemic inflammatory markers and cardiovascular risk remain poorly defined. We sought to compare the abundance of pro-inflammatory stimulants of innate immune receptors in processed foods with those produced by the murine ileal and caecal microbiota, and to explore the impact of their ingestion on systemic inflammation and lipid metabolism in vivo. Methods and results: Calibrated receptor-dependent reporter assays revealed that many processed foods, particularly those based on minced meats, contain pro-inflammatory stimulants of Toll-like receptor (TLR)-2 and TLR4 at concentrations which greatly exceed those produced by the endogenous murine ileal microbiota. Chronic dietary supplementation with these stimulants, at concentrations relevant to those measured in the Western diet, promoted hepatic inflammation and reduced several markers of reverse cholesterol transport (RCT) in mice. Hepatocytes were found to be insensitive to TLR2- and TLR4-stimulants directly, but their secretion of functional cholesterol acceptors was impaired by interleukin (IL)-1ß released by TLR-responsive hepatic macrophages. Hepatic macrophage priming by high-fat diet enhanced the impairment of RCT by ingested endotoxin, and this was reversed by macrophage depletion via clodronate liposome treatment, or genetic deficiency in the IL-1 receptor. Conclusion: These findings reveal an unexpected mechanism connecting processed food consumption with cardiovascular risk factors, and introduce the food microbiota as a potential target for therapeutic regulation of lipid metabolism.

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

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSION: Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Regulation of low-density lipoprotein cholesterol by intestinal inflammation and the acute phase response.

Systemic inflammation, induced by disease or experimental intervention, is well established to result in elevated levels of circulating triglycerides, and reduced levels of high-density lipoprotein-cholesterol (HDL-C), in most mammalian species. However, the relationship between inflammation and low-density lipoprotein-cholesterol (LDL-C) concentrations is less clear. Most reports indicate that systemic inflammation, as observed during sepsis or following high dose experimental endotoxaemia, lowers total, and LDL-C in man. However, isolated reports have suggested that certain inflammatory conditions are associated with increased LDL-C. In this review, we summarize the emerging evidence that low-grade inflammation specifically of intestinal origin may be associated with increased serum LDL-C levels. Preliminary insights into potential mechanisms that may mediate these effects, including those connecting inflammation to trans-intestinal cholesterol efflux (TICE), are considered. We conclude that this evidence supports the potential downregulation of major mediators of TICE by inflammatory mediators in vitro and during intestinal inflammation in vivo. The TICE-inflammation axis therefore merits further study in terms of its potential to regulate serum LDL-C, and as a readily druggable target for hypercholesterolaemia.

The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker.

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that toll-like receptor (TLR)-2 signaling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n = 21), in both relapse and remission, compared to healthy controls (HC) (n = 24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and HC were preserved when the data from the two cohorts were pooled together (n = 41 RRMS and 44 HC, P = 0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK)-293 cells transfectant reporter assay, were significantly higher in urine of MS patients than HC. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signaling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity-urinary TLR2-stimulants and serum sTLR2 levels-are significantly elevated in MS patients compared to HC.

A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation.

BACKGROUND: Bacterial endotoxin is a potently inflammatory antigen that is abundant in the human gut. Endotoxin circulates at low concentrations in the blood of all healthy individuals, although elevated concentrations are associated with an increased risk of atherosclerosis. OBJECTIVE: We sought to determine whether a high-fat meal or smoking increases plasma endotoxin concentrations and whether such concentrations are of physiologic relevance. DESIGN: Plasma endotoxin and endotoxin neutralization capacity were measured for 4 h in 12 healthy men after no meal, 3 cigarettes, a high-fat meal, or a high-fat meal with 3 cigarettes by using the limulus assay. RESULTS: Baseline endotoxin concentrations were 8.2 pg/mL (interquartile range: 3.4-13.5 pg/mL) but increased significantly (P < 0.05) by approximately 50% after a high-fat meal or after a high-fat meal with cigarettes but not after no meal or cigarettes alone. These results were validated by the observations that a high-fat meal with or without cigarettes, but not no meal or smoking, also significantly (P < 0.05) reduced plasma endotoxin neutralization capacity, which is an indirect measure of endotoxin exposure. Human monocytes, but not aortic endothelial cells, were responsive to transient (30 s) or low-dose (10 pg/mL) exposure to endotoxin. However, plasma from whole blood treated with as little as 10 pg endotoxin/mL increased the endothelial cell expression of E-selectin, at least partly via tumor necrosis factor-alpha-induced cellular activation. CONCLUSIONS: Low-grade endotoxemia may contribute to the postprandial inflammatory state and could represent a novel potential contributor to endothelial activation and the development of atherosclerosis.

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

OBJECTIVE: 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. METHODS AND RESULTS: Unlike established TLR agonists, OxPAPC did not induce NF-kappaB-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. CONCLUSIONS: 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.

Acinetobacter baumannii lipopolysaccharides are potent stimulators of human monocyte activation via Toll-like receptor 4 signalling.

Acinetobacter baumannii is a major nosocomial pathogen and frequent cause of hospital-acquired pneumonia, surgical wound infections and sepsis. As very little is known of the endotoxic potential of A. baumannii lipopolysaccharide (LPS) with respect to human cells or of its ability to stimulate inflammatory signalling via human Toll-like receptors (TLRs), the biological activity of these endotoxins was investigated in human monocytic THP-1 cells and in TLR-deficient HEK-293 cells transfected with human TLR2 and TLR4 constructs. Endotoxins derived from five clinical isolates of A. baumannii and one of Acinetobacter 'genomospecies 9' showed high potency, which was comparable to that of Escherichia coli strain R1 NCTC 13114 LPS, in the induction of the Limulus amoebocyte reaction and interleukin 8 and tumour necrosis factor alpha release from THP-1 cells. Whole UV-killed cells of A. baumannii and Acinetobacter 'genomospecies 9' stimulated both TLR2- and TLR4-dependent signalling, whereas pure endotoxins of all investigated strains induced signalling via TLR4, but not TLR2.

25-Hydroxycholesterol, 7beta-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), 7beta-hydroxycholesterol (7beta-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 IkappaBalpha degradation or tumour necrosis factor-alpha 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.

Host defenses against metabolic endotoxaemia and their impact on lipopolysaccharide detection.

Bacterial endotoxin (lipopolysaccharide, LPS), is one of the most potent inducers of inflammatory signaling, yet it is abundant in the human gut and the modern diet. Small quantities of LPS routinely translocate from the gut lumen to the circulation (so-called metabolic endotoxaemia), and elevated plasma LPS concentrations are reported in a variety of chronic non-communicable diseases, including obesity, non-alcoholic fatty liver disease, atherosclerosis and type II diabetes. Murine models of experimentally-induced endotoxaemia and Toll-like receptor-4 deficiency suggest that endotoxin may promote the metabolic disturbances that underpin these diseases. However, as bioactive LPS is cleared rapidly from the circulation, and reported levels of endotoxin in human plasma vary widely, the potential relevance of metabolic endotoxaemia to human disease remains unclear. We here review insight into these questions gained from human and murine models of experimental endotoxaemia, focusing on the kinetics of LPS neutralization and its clearance from blood, the limitations of the widely used limulus assay and alternative methods for LPS quantitation. We conclude that although new methods for LPS measurement will be required to definitively quantify the extent of metabolic endotoxaemia in man, evidence from numerous approaches suggests that this molecule may play a key role in the development of diverse metabolic diseases.