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.

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.

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.

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.

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.

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.

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.

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.

Maternal antibiotic-induced early changes in microbial colonization selectively modulate colonic permeability and inducible heat shock proteins, and digesta concentrations of alkaline phosphatase and TLR-stimulants in swine offspring.

Elevated intake of high energy diets is a risk factor for the development of metabolic diseases and obesity. High fat diets cause alterations in colonic microbiota composition and increase gut permeability to bacterial lipopolysaccharide, and subsequent low-grade chronic inflammation in mice. Chronic inflammatory bowel diseases are increasing worldwide and may involve alterations in microbiota-host dialog. Metabolic disorders appearing in later life are also suspected to reflect changes in early programming. However, how the latter affects the colon remains poorly studied. Here, we hypothesized that various components of colonic physiology, including permeability, ion exchange and protective inducible heat shock proteins (HSP) are influenced in the short- and long-terms by early disturbances in microbial colonization. The hypothesis was tested in a swine model. Offspring were born to control mothers (n = 12) or mothers treated with the antibiotic (ATB) amoxicillin around parturition (n = 11). Offspring were slaughtered between 14 and 42 days of age to study short-term effects. For long-term effects, young adult offspring from the same litters consumed a normal or a palm oil-enriched diet for 4 weeks between 140 and 169 days of age. ATB treatment transiently modified maternal fecal microbiota although the minor differences observed for offspring colonic microbiota were nonsignificant. In the short-term, consistently higher HSP27 and HSP70 levels and transiently increased horseradish peroxidase permeability in ATB offspring colon were observed. Importantly, long-term consequences included reduced colonic horseradish peroxidase permeability, and increased colonic digesta alkaline phosphatase (AP) and TLR2- and TLR4-stimulant concentrations in rectal digesta in adult ATB offspring. Inducible HSP27 and HSP70 did not change. Interactions between early ATB treatment and later diet were noted for paracellular permeability and concentrations of colonic digesta AP. In conclusion, our data suggest that early ATB-induced changes in bacterial colonization modulate important aspects of colonic physiology in the short- and long-terms.

Bacteria in the adventitia of cardiovascular disease patients with and without rheumatoid arthritis.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

High-fat meal induced postprandial inflammation.

Raised levels of circulating inflammatory markers are associated with coronary artery disease, obesity and type II diabetes. It has been proposed that the ingestion of high-fat meals may serve as a stimulus to raise systemic inflammatory tone, although interventional studies have yielded conflicting results. We here review 57 studies of high-fat meal induced acute postprandial inflammation to identify the most frequently reported markers of postprandial inflammation and to compare these results with the highly consistent low-grade endotoxaemia model in man. Most plasma borne markers of inflammation, such as cytokines and soluble adhesion molecules, were not consistently raised after a high-fat meal. However, pro-inflammatory leukocyte surface markers, mRNA and proteins were elevated in almost all studies in which they were measured. These markers followed kinetics similar to those observed following intravenous injection of low doses of endotoxin in man, were positively associated with likelihood of contamination of test meals with pro-inflammatory bacterial molecules and were reduced in several studies examining parallel meals supplemented with foodstuffs containing anti-inflammatory phytochemicals. Future studies of postprandial inflammation may yield more consistent evidence by focusing on leukocyte, rather than plasma-borne, markers of inflammation and by considering the test meal content of pro- and anti-inflammatory dietary constituents.

The 9p21 locus does not affect risk of coronary artery disease through induction of type 1 interferons.

OBJECTIVES: The study objective was to determine whether the coronary artery disease (CAD)-associated genotype at chromosome 9p21 modulates basal or induced expression of type I interferons (IFN-I). BACKGROUND: The mechanism responsible for the association between common variants in chromosome 9p21.3 and CAD remains unclear. It has been reported that the CAD risk locus is rich in enhancer-like elements and that chromosome looping can lead to its physical proximity with the IFN-I gene cluster, raising the possibility that the locus influences CAD risk by modulating expression of IFN-Is. METHODS: We examined whether genotype at the lead CAD-associated single nucleotide polymorphism (rs1333049) in 9p21 was associated with: 1) basal levels of IFN-I in plasma from 148 healthy male subjects; 2) induction of IFN-I by Toll-like receptor stimulants in peripheral blood mononuclear cells of 60 healthy volunteers assessed by enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, Western blot, and IFN-I bioassay; and 3) enhancer activity of predicted IFN regulatory factor 3/7 binding sites within the 9p21 CAD risk region in reporter assays. RESULTS: No significant effects of 9p21 genotype were observed for plasma levels of IFN-α, IFN-α21, or CXCL10, or leukocyte induction of IFN-α, IFN-α21, IFN-ß, CXCL10, or total IFN-I measured at the mRNA, protein, and biological activity levels. There was also no enhancement of reporter activity by predicted IFN regulatory factor 3/7 binding sites in the CAD risk locus of either genotype. CONCLUSIONS: The mechanism underlying the association between common 9p21 variants and CAD does not involve differential regulation of IFN-I responses.

Diverse bacteria promote macrophage foam cell formation via Toll-like receptor-dependent lipid body biosynthesis.

AIM: Atherosclerotic lesions contain DNA signatures from a wide variety of bacteria, although little is known of how exposure to these organisms may modulate the accumulation of lipids in macrophages. METHODS: To address this, a panel of nine bacteria representing those most frequently reported to be present in human atheroma were examined for their potential to promote lipid accumulation in human primary monocytes and murine J774 macrophages. RESULTS: All bacteria examined, and defined stimulants of Toll-like receptors (TLRs) 2, 3, 4, 5 and 9, induced lipid body formation and cholesterol ester accumulation in a dose-dependent manner. The mechanisms of bacteria-mediated foam cell formation were found to be dependent on TLR2 and/or TLR4 signalling, but independent of lipoprotein oxidation pathways, since lipid accumulation was significantly inhibited by the TLR4 inhibitors polymyxin-B and TAK-242, or the TLR2 and TLR4 inhibitor oxidised palmitoyl-arachidonyl-phosphatidyl-choline, but not by the scavenger receptor blocker polyinosinic acid or the antioxidant butylated hydroxytoluene. A number of genes involved in lipid body biosynthesis, including perilipin-A, stearoyl-coenzyme-A desaturase 1, fatty acid synthase and HMG-CoA reductase were upregulated in response to TLR4 stimulation. CONCLUSIONS: The bacterial debris observed in human atheroma, which is currently considered to be harmless, may have potential to contribute to disease progression via TLR-dependent lipid body formation in macrophages.

Accumulation of stimulants of Toll-like receptor (TLR)-2 and TLR4 in meat products stored at 5 °C.

UNLABELLED: Recent evidence suggests that exposure to stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 may contribute to the development of atherosclerosis and insulin resistance. We showed recently that common foodsuffs can contain TLR-stimulants, and that the greatest concentrations were present in meat-based products. Using a recently developed quantitative bioassay, we here examined the kinetics of accumulation of TLR2- and TLR4-stimulants in a variety of meat products held at 5 °C in air or under a modified atmosphere for up to 8 d. Meat content of TLR-stimulants increased with time in each meat examined and was paralleled by growth of pseudomonads and Enterobacteriaceae, suggesting that bacterial lipopeptides and lipopolysaccharides are the likely sources of TLR2- and TLR4-stimulants, respectively. TLR-stimulants reached the highest levels (approximately 80 µg lipopeptide-equivalents per gramme and approximately 7 µg lipopolysaccharide-equivalents per gram) in meat that was minced rather than intact, and when stored in air rather than under a modified atmosphere. TLR2- and TLR4-stimulants in meat products cooked for 1 h retained approximately 20% and approximately 40% of their bioactivity, respectively. In summary, storage conditions and microbial flora critically regulate the kinetics of TLR2- and TLR4-stimulant accumulation in meat products and these may retain biological activity after cooking. PRACTICAL APPLICATION: The novel assays presented in this work could be used to predict the potential of foodstuffs to promote inflammatory signaling in human subjects, which may be deleterious to health. These assays may also be used to monitor the historical microbial flora in food products after cooking or other forms of food processing may have rendered the original microflora nonviable.

Stimulants of Toll-like receptor (TLR)-2 and TLR-4 are abundant in certain minimally-processed vegetables.

Stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 have been shown to promote insulin resistance and atherosclerosis in animal models of these diseases. As minimally processed vegetables (MPV) can contain a relatively large bacterial load compared to other foodstuffs, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in MPV using a transfection-based bioassay calibrated with Escherichia coli LPS and the synthetic lipopeptide Pam(3)CSK(4). Of 5 classes of MPV and 3 classes of related vegetable products considered to be likely to contain a high microbial load, diced onion and bean sprouts contained the highest levels of stimulants of TLR2 (up to 18.5 µg Pam(3)CSK(4)-equivalents per g) and TLR4 (up to 11.4 µg LPS-equivalents per g). By contrast, the majority of fresh whole vegetables examined reproducibly contained minimal or undetectable levels of TLR2- or TLR4-stimulants. The accumulation of TLR-stimulants in MPVs correlated well with growth of enterobacterial spoilage organisms. In conclusion, the modern trend towards eating minimally processed vegetables rather than whole foods is likely to be associated with increased oral exposure to stimulants of TLR2 and TLR4.

Diet, commensals and the intestine as sources of pathogen-associated molecular patterns in atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease.

Stimulation of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 has been shown to promote the development of a variety of diseases involving dysregulated metabolism, including atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease. However, the origin and nature of the agents responsible for stimulating TLR2 or TLR4 signalling in these conditions remain to be clearly identified. This review summarises the evidence supporting the proposal that 'pathogen-associated molecular patterns' (PAMPs) derived from dietary and commensal sources may contribute to the chronic inflammatory processes that underpin the development of these diseases via stimulation of TLR2 and TLR4. In particular, insights gained from recent studies employing TLR-transfectant based bioassays to quantify the abundance of PAMPs in foodstuffs and specific commensal compartments are discussed. Finally, the major mechanisms by which TLR-stimulants may gain access to the circulation to promote systemic low-grade inflammation are considered.

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.

Endogenous ligands of TLR2 and TLR4: agonists or assistants?

The mammalian TLRs serve as key sensors of PAMPs, such as bacterial LPS, lipopeptides, and flagellins, which are present in microbial cells but not host cells. TLRs have therefore been considered to play a central role in the discrimination between "self" and "non-self". However, since the discovery of their microbial ligands, many studies have provided evidence that host-derived molecules may also stimulate TLR2- or TLR4-dependent signaling. To date, more than 20 of these endogenous TLR ligands have been proposed, which have tended to fall into the categories of released intracellular proteins, ECM components, oxidatively modified lipids, and other soluble mediators. This review aims to summarize the evidence supporting the intrinsic TLR-stimulating capacity of each of these proposed endogenous ligands with a particular emphasis on the measures taken to exclude contaminating LPS and lipopeptides from experimental systems. The emerging evidence that many of these molecules may be more accurately described as PAMP-binding molecules (PBMs) or PAMP-sensitizing molecules (PSMs), rather than genuine ligands of TLR2 or TLR4, is also summarized. The relevance of this possibility to the pathogenesis of chronic inflammatory diseases, tumor surveillance, and autoimmunity is discussed.

The induction of colitis and ileitis in mice is associated with marked increases in intestinal concentrations of stimulants of TLRs 2, 4, and 5.

BACKGROUND: Inflammatory bowel diseases (IBDs) appear to be modulated by the interaction of pathogen-associated molecular patterns (PAMPs) derived from intestinal bacteria with their respective innate immune receptors, including Toll-like receptors (TLRs). We aimed to establish if intestinal concentrations of proinflammatory bacterial ligands of TLR2, TLR4, or TLR5 may be altered in murine IBD models, and to characterize which of the major bacterial groups may contribute to each signal. METHODOLOGY/PRINCIPAL FINDINGS: PAMPs specific for TLR2 (lipopeptide equivalents), TLR4 (lipopolysaccharide equivalents), and TLR5 (flagellin equivalents) in human and murine fecal and intestinal samples were quantified using HEK-293 cells transfected with respective TLRs and calibrated with defined standard PAMPs. The induction of colitis in mice by dextran-sodium-sulphate treatment significantly increased colonic lipopeptide (fourfold) and LPS equivalent (550-fold) concentrations, while flagellin equivalent concentrations remained similar. The induction of ileitis by oral infection with Toxoplasma gondii dramatically increased ileal concentrations of lipopeptide (370-fold), LPS (3,300-fold), and flagellin equivalents (38-fold), all P<0.01. Analysis of representative strains of the major bacterial groups of the human intestine revealed that enterobacterial species are likely to be more significant contributors of soluble TLR2 and TLR4 stimulants to the intestinal milieu than Bacteroides species or Gram-positive Firmicutes. CONCLUSIONS/SIGNIFICANCE: We conclude that the induction of colitis or ileitis in mice is associated with significant disease-specific alterations to the PAMP profile of the gut microbiota.