Prevention of radiotherapy-induced arterial inflammation by interleukin-1 blockade.

AIMS: Radiotherapy-induced cardiovascular disease is an emerging problem in a growing population of cancer survivors where traditional treatments, such as anti-platelet and lipid-lowering drugs, have limited benefits. The aim of the study was to investigate vascular inflammatory patterns in human cancer survivors, replicate the findings in an animal model, and evaluate whether interleukin-1 (IL-1) inhibition could be a potential treatment. METHODS AND RESULTS: Irradiated human arterial biopsies were collected during microvascular autologous free tissue transfer for cancer reconstruction and compared with non-irradiated arteries from the same patient. A mouse model was used to study the effects of the IL-1 receptor antagonist, anakinra, on localized radiation-induced vascular inflammation. We observed significant induction of genes associated with inflammasome biology in whole transcriptome analysis of irradiated arteries, a finding supported by elevated protein levels in irradiated arteries of both, pro-caspase and caspase-1. mRNA levels of inflammasome associated chemokines CCL2, CCL5 together with the adhesion molecule VCAM1, were elevated in human irradiated arteries as was the number of infiltrating macrophages. A similar pattern was reproduced in Apoe-/- mouse 10 weeks after localized chest irradiation with 14 Gy. Treatment with anakinra in irradiated mice significantly reduced Ccl2 and Ccl5 mRNA levels and expression of I-Ab. CONCLUSION: Anakinra, administered directly after radiation exposure for 2 weeks, ameliorated radiation induced sustained expression of inflammatory mediators in mice. Further studies are needed to evaluate IL-1 blockade as a treatment of radiotherapy-induced vascular disease in a clinical setting.

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid-rich necrotic cores in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10-week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid-rich necrotic cores in Ldlr(-/-) mice. Myeloid-specific ablation of NOD2, but not its downstream kinase, receptor-interacting serine/threonine-protein kinase 2, restrained the expansion of the lipid-rich necrotic core in Ldlr(-/-) chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low-density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP-binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF-κB-mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid-rich necrotic core and promotes vascular inflammation in atherosclerosis.

α7 Nicotinic acetylcholine receptor is expressed in human atherosclerosis and inhibits disease in mice--brief report.

OBJECTIVE: Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr(-/-) mouse model. APPROACH AND RESULTS: α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr(-/-) mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling. CONCLUSIONS: The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Lack of invariant natural killer T cells affects lipid metabolism in adipose tissue of diet-induced obese mice.

OBJECTIVE: Obesity promotes a chronic inflammatory condition in adipose tissue (AT). Impairment of insulin sensitivity coincides with infiltration of T cells into AT in early stages of obesity, when macrophages are not yet present. Here, we examine the role of invariant natural killer T (iNKT) cells, a subtype of T cells activated by lipid antigens, on glucose and lipid metabolism in obesity. APPROACH AND RESULTS: Jα18(-/-) mice, specifically lacking iNKT cells, and wild-type mice consumed a chow or high-fat diet for 10 weeks. One third of all T lymphocytes in the liver of wild-type mice were iNKT cells, whereas few were detected in AT. Diet-induced obesity increased blood glucose in both genotypes of mice, whereas glucose tolerance test revealed similar kinetics of glucose clearance in Jα18(-/-) and wild-type mice. Under obese conditions, expression of inflammatory cytokines in AT did not differ between the groups, although the number of T cells and macrophages was lower in Jα18(-/-) mice. Nonetheless, AT homeostasis in Jα18(-/-) mice was altered evidenced by lower AT weight, smaller adipocytes, accelerated lipogenesis, increased expression of hormone-sensitive lipase, and accelerated basal lipolysis. CONCLUSIONS: iNKT cells do not affect glucose clearance but rather modulate lipid metabolism in both liver and AT. Only few iNKT cells are found in AT under lean and obese conditions, suggesting that their effects on lipid metabolism are mainly mediated in the liver, their primary host organ.

Toll-like receptor 7 protects from atherosclerosis by constraining "inflammatory" macrophage activation.

BACKGROUND: Toll-like receptors (TLRs) have long been considered to be major culprits in the development of atherosclerosis, contributing both to its progression and clinical complications. However, evidence for most TLRs beyond TLR2 and TLR4 is lacking. METHODS AND RESULTS: We used experimental mouse models, human atheroma cultures, and well-established human biobanks to investigate the role of TLR7 in atherosclerosis. We report the unexpected finding that TLR7, a receptor recognizing self-nucleic acid complexes, is protective in atherosclerosis. In Apoe(-/-) mice, functional inactivation of TLR7 resulted in accelerated lesion development, increased stenosis, and enhanced plaque vulnerability as revealed by Doppler ultrasound and/or histopathology. Mechanistically, TLR7 interfered with macrophage proinflammatory responses to TLR2 and TLR4 ligands, reduced monocyte chemoattractant protein-1 production, and prevented expansion of Ly6C(hi) inflammatory monocytes and accumulation of inflammatory M1 macrophages into developing atherosclerotic lesions. In human carotid endarterectomy specimens TLR7 levels were consistently associated with an M2 anti-inflammatory macrophage signature (interleukin [IL]-10, IL-1RA, CD163, scavenger and C-type lectin receptors) and collagen genes, whereas they were inversely related or unrelated to proinflammatory mediators (IL-12/IL-23, interferon beta, interferon gamma, CD40L) and platelet markers. Moreover, in human atheroma cultures, TLR7 activation selectively suppressed the production of key proatherogenic factors such as monocyte chemoattractant protein-1 and tumor necrosis factor without affecting IL-10. CONCLUSIONS: These findings provide evidence for a beneficial role of TLR7 in atherosclerosis by constraining inflammatory macrophage activation and cytokine production. This challenges the prevailing concept that all TLRs are pathogenic and supports the exploitation of the TLR7 pathway for therapy.

Rip2 deficiency leads to increased atherosclerosis despite decreased inflammation.

RATIONALE: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. OBJECTIVE: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. METHODS AND RESULTS: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2(-/-) bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2(-/-) macrophages. Further analysis in Rip2(-/-) macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. CONCLUSIONS: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2(-/-) macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.

The tryptophan metabolite 3-hydroxyanthranilic acid lowers plasma lipids and decreases atherosclerosis in hypercholesterolaemic mice.

AIMS: Cardiovascular disease is the most common cause of death in the world and atherosclerosis, an inflammatory process in the vessel wall, accounts for the majority of these deaths. The tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) has been shown to inhibit inflammation in different experimental autoimmune disease models. However, the effect of 3-HAA in atherosclerosis has never been explored. METHODS AND RESULTS: In this study, we used the atherosclerosis prone Ldlr-/- mice, and cell culture experiments to evaluate the role of 3-HAA in atherosclerosis. Eight weeks treatment with 3-HAA significantly reduced the lesion size in the aorta, and modulated local and systemic inflammatory responses. 3-hydroxyanthranilic acid inhibited the uptake of oxLDL by macrophages, an initiating event in the formation of foam cells, a major cellular component of atherosclerotic lesions. Surprisingly, 3-HAA significantly affected plasma cholesterol and triglyceride levels in Ldlr-/- mice, likely due to modulation of signalling through peroxisome proliferator-activated receptors. CONCLUSION: 3-Hydroxyanthranilic acid inhibits atherosclerosis by regulating lipid metabolism and inflammation, two major components of this disease.

High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor.

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain-containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain-containing adaptor-inducing interferon-ß [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

Innate immune recognition receptors and damage-associated molecular patterns in plaque inflammation.

PURPOSE OF REVIEW: To highlight critical advances achieved over the last year in the study of endogenous proatherogenic danger signals and corresponding molecular mechanism of innate immune signalling in atherosclerosis. RECENT FINDINGS: The identity and signalling mechanisms of LDL-derived inflammatory components are central in understanding the pathogenic role of modified LDL in the development of atherosclerosis. Studies in the preceding years have revealed LDL-derived phospholipids and cholesterol crystals as endogenous danger signals. These danger signals trigger Toll-like receptors and nucleotide-binding oligomerization domain-like receptors inflammasome respectively, thereby instigating inflammatory responses and promoting disease progression. SUMMARY: Recent understandings of the causal role of LDL in atherosclerosis provide a new perspective on modified LDL-derived danger signals. These insights suggest dysregulated Toll-like receptor and nucleotide-binding oligomerization domain inflammasome signalling as an important mechanism underlying atherogenesis.

Innate immune signals in atherosclerosis.

Atherosclerosis is a chronic disease characterised by lipid retention and inflammation in the arterial intima. Innate immune mechanisms are central to atherogenesis, involving activation of pattern-recognition receptors (PRRs) and induction of inflammatory processes. In a complex tissue, such as the atherosclerotic lesion, innate signals can originate from several sources and promote atherogenesis through ligation of PRRs. The receptors recognise conserved molecular patterns on pathogens and endogenous products of tissue injury and inflammation. Activation of PRRs might affect several aspects of atherosclerosis by acting on lesion resident cells. Scavenger receptors mediate antigen uptake and clearance of lipoproteins, thereby promoting foam cell formation. Signalling receptors, such as Toll-like receptors (TLRs), lead to induction of pro-inflammatory cytokines and antigen-specific immune responses. In this review we describe the innate mechanisms present in the plaque. We focus on TLRs, their cross-talk with other PRRs, and how their signalling cascades influence inflammation within the atherosclerotic lesion.

Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice.

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC(1)-R, VPAC(2)-R and PAC(1)-R. Based on beneficial effects of VIP and VPAC(1)-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice. METHODS AND RESULTS: Contrary to our hypothesis, administration of a VPAC(1)-R agonist, (Ala(11,22,28))-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A(b), and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala(11,22,28))-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease. CONCLUSION: Despite beneficial effects in murine models of several inflammatory disorders, VPAC(1)-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.

Role of IRAK4 and IRF3 in the control of intracellular infection with Chlamydia pneumoniae.

TLR signal transduction involves a MyD88-mediated pathway, which leads to recruitment of the IL-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-beta-mediated pathway, resulting in the activation of IFN regulatory factor (IRF)3. Both pathways can lead to expression of IFN-beta. TLR-dependent and -independent signals converge in the TNF receptor-associated factor 6 (TRAF6) adaptor, which mediates the activation of NF-kappaBeta. Infection of murine bone marrow-derived macrophages (BMM) with Chlamydia pneumoniae induces IFN-alpha/beta- and NF-kappaBeta-dependent expression of IFN-gamma, which in turn, will control bacterial growth. The role of IRAK4 and IRF3 in the regulation of IFN-alpha/beta expression and NF-kappaBeta activation was studied in C. pneumoniae-infected BMM. We found that levels of IFN-alpha, IFN-beta, and IFN-gamma mRNA were reduced in infected IRAK4(-/-) BMM compared with wild-type (WT) controls. BMM also showed an IRAK4-dependent growth control of C. pneumoniae. No increased IRF3 activation was detected in C. pneumoniae-infected BMM. Similar numbers of intracellular bacteria, IFN-alpha, and IFN-gamma mRNA titers were observed in C. pneumoniae-infected IRF3(-/-) BMM. On the contrary, IFN-beta(-/-) BMM showed lower IFN-alpha and IFN-gamma mRNA levels and higher bacterial titers compared with WT controls. C. pneumoniae infection-induced activation of NF-kappaBeta and expression of proinflammatory cytokines were shown to be TRAF6-dependent but did not require IRAK4 or IRF3. Thus, our data indicate that IRAK4, but not IRF3, controls C. pneumoniae-induced IFN-alpha and IFN-gamma secretion and bacterial growth. IRAK4 and IRF3 are redundant for infection-induced NF-kappaB activation, which is regulated by TRAF6.

Augmented Th17 differentiation in Trim21 deficiency promotes a stable phenotype of atherosclerotic plaques with high collagen content.

Aims: Patients with hyperlipidemia are at risk of atherosclerosis, but not all develop cardiovascular disease, highlighting the importance of other risk factors such as inflammation. Both the innate and adaptive arms of the immune system have been suggested in the initiation and propagation of plaque formation. Tri-partite motif (TRIM) 21 is a regulator of tissue inflammation and pro-inflammatory cytokine production, and has been implicated in chronic inflammatory disease. Here, we investigate a potential role for TRIM21 in coronary artery disease. Methods and results: Trim21-deficient or wild-type bone marrow was transplanted into Ldlr-/- mice fed a hypercholesterolemic diet. The Trim21-/-->Ldlr-/- mice developed larger atherosclerotic plaques, with significantly higher collagen content compared to mice transplanted with wild-type cells. High collagen content of the atheroma is stabilizing, and has recently been linked to IL-17. Interestingly, Trim21-/-->Ldlr-/- mice had elevated CD4 and IL-17 mRNA expression in plaques, and increased numbers of activated CD4+ T cells in the periphery. An increased differentiation of naïve T cells lacking Trim21 into Th17 cells was confirmed in vitro, with transcriptomic analysis revealing upregulation of genes of a non-pathogenic Th17 phenotype. Also, decreased expression of matrix metalloproteinases (MMPs) was noted in aortic plaques. Analysis of human carotid plaques confirmed that TRIM21 expression negatively correlates with the expression of key Th17 genes and collagen, but positively to MMPs also in patients, linking our findings to a clinical setting. Conclusion: In this study, we demonstrate that TRIM21 influences atherosclerosis via regulation of Th17 responses, with TRIM21 deficiency promoting IL-17 expression and a more fibrous, stable, phenotype of the plaques.

Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn's disease.

Both NOD2 (CARD15) alleles are mutated in roughly 15% of patients with Crohn's disease, but functional effects are unclear. We analysed the cytokine response of peripheral blood mononuclear cells to muramyl dipeptide (MDP), the ligand for NOD2. MDP induced little TNFalpha or interleukin 1beta, but strong interleukin-8 secretion. MDP also substantially upregulated secretion of TNFalpha and interleukin 1beta induced by toll-like receptor ligands. These effects were abolished by the most common Crohn's NOD2 double mutant genotypes at low nanomolar MDP concentrations, and provide the basis to develop a test of NOD2 functional deficiency. In Crohn's disease, there are defects in neutrophil recruitment driven by NOD2 and interleukin 8 and in cross talk between the NOD2 and toll-like receptor pathways, which suggests that the immune system fails to receive an early priming signal.

Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice.

Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe(-/-)Neil3(-/-) mice on high-fat diet showed accelerated plaque formation as compared to Apoe(-/-) mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe(-/-)Neil3(-/-) mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage.

TRIF adaptor signaling is important in abdominal aortic aneurysm formation.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is characterized by inflammation, loss of smooth muscle cells (SMCs), and degradation of the extracellular matrix in the vessel wall. Innate immune receptors such as Toll-like receptors (TLRs) were recently shown to regulate immunological processes leading to the formation and progression of atherosclerotic plaques as well as to other cardiovascular pathologies. Our aim was to investigate whether blockage of TLR signaling, under the control of TIR domain-containing adaptor protein including IFN-ß (TRIF), could inhibit the inflammatory response and AAA development in mice. RESULTS: In human AAA, an increased TLR3 and TLR4 expression in association with macrophages and T lymphocytes was demonstrated with immunohistochemical analysis. Angiotensin (Ang) II-induced aneurysm formation was significantly reduced by 30% in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice. Morphologically, AngII-infused ApoE(-/-)Trif(-/-) mice had a more intact cellular and extracellular matrix while ApoE(-/-) mice infused with AngII displayed an increased medial thickness associated with aortic dissection, thrombus formation, and a more disorganized vessel wall. Gene expression analysis of the abdominal aorta revealed a profound decrease of the inflammatory genes CD68 (P < 0.05), CD11b (P < 0.05), and TNF-α (P < 0.05) and the protease gene MMP-12 (P < 0.01) in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice infused with AngII. CONCLUSION: Our results suggest that signaling through TRIF is important for the inflammatory response of AngII-induced AAA and that blockage of the TRIF pathway reduces vascular inflammation and protects against AAA formation.

Toll-Like Receptor 3 Influences Glucose Homeostasis and ß-Cell Insulin Secretion.

Toll-like receptors (TLRs) have been implicated in the pathogenesis of type 2 diabetes. We examined the function of TLR3 in glucose metabolism and type 2 diabetes-related phenotypes in animals and humans. TLR3 is highly expressed in the pancreas, suggesting that it can influence metabolism. Using a diet-induced obesity model, we show that TLR3-deficient mice had enhanced glycemic control, facilitated by elevated insulin secretion. Despite having high insulin levels, Tlr3(-/-) mice did not experience disturbances in whole-body insulin sensitivity, suggesting that they have a robust metabolic system that manages increased insulin secretion. Increase in insulin secretion was associated with upregulation of islet glucose phosphorylation as well as exocytotic protein VAMP-2 in Tlr3(-/-) islets. TLR3 deficiency also modified the plasma lipid profile, decreasing VLDL levels due to decreased triglyceride biosynthesis. Moreover, a meta-analysis of two healthy human populations showed that a missense single nucleotide polymorphism in TLR3 (encoding L412F) was linked to elevated insulin levels, consistent with our experimental findings. In conclusion, our results increase the understanding of the function of innate receptors in metabolic disorders and implicate TLR3 as a key control system in metabolic regulation.

Toll-like receptor 3 and 4 signalling through the TRIF and TRAM adaptors in haematopoietic cells promotes atherosclerosis.

AIMS: Members of the Toll-like receptor (TLR) family initiate innate immune responses and were recently shown to play a role in atherosclerosis. However, the mechanisms that link TLR ligation to vascular inflammation and atherogenesis remain unclear. To identify which signalling pathways downstream of TLRs in immune cells are pro-atherogenic, we analysed the role of the TLR-specific adaptors MyD88 adaptor-like (MAL), TRIF-related adaptor molecule (TRAM), and TIR-domain-containing adaptor-inducing interferon-ß (TRIF) in atherosclerosis. METHODS AND RESULTS: Using a bone-marrow transplantation strategy into low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, we could specifically study the absence of the TLR adaptors in immune cells. We showed that haematopoietic deficiency of TRAM and TRIF, but not MAL, reduces atherosclerosis without affecting cholesterol metabolism. This was mediated by decreased aortic inflammation, indicated by lower aortic levels of pro-inflammatory mediators, and reduced influx of macrophages and T cells. Furthermore, by studying Tlr3(-/-) chimeric Ldlr(-/-) mice, we found that deleting TLR3 in immune cells significantly reduced both aortic inflammation and atherosclerotic burden. CONCLUSIONS: By studying hypercholesterolaemic mice with defects in TLR-signalling adaptors, we demonstrated that deleting either TRAM or TRIF in immune cells is sufficient to attenuate vessel inflammation and protect against atherosclerosis. In addition, these adaptors elicit partly different sets of inflammatory mediators and can independently inhibit the disease process. Furthermore, we identify TLR3 as a pro-atherogenic receptor in haematopoietic immune cells. The identification of these pro-atherogenic pathways downstream of TLR3 and TLR4 contributes to a better understanding of TLRs and their signalling pathways in the pathogenesis of atherosclerosis.