Subcutaneous injection of dendritic cells aggravates atherosclerosis in ApoE?knockout mice by activation of TLR4.
ABSTRACT: Dendritic cells (DCs) are specialized antigen?presenting cells which are important in immune diseases, in particular atherosclerosis, a chronic inflammatory disease, however their role in atherosclerosis?associated immunity is unclear. To evaluate the role of DCs in atherosclerosis, exogenous bone marrow?derived DCs were transferred into ApoE?/? mice in the present study. The extent of disease was measured in the aorta and was compared with mice treated with phosphate?buffered saline (PBS) or left untreated and fed a western diet. Mice receiving exogenous DCs demonstrated significantly larger atherosclerotic lesions compared with the mice treated with PBS, with increasing numbers of mature DCs in circulation and enhanced DC infiltration into plaque lesions, in addition to activation of circulating inflammatory components and atherosclerotic lesions. Furthermore, it was demonstrated that exogenous DCs upregulated the expression of Toll?like receptor 4 (TLR4) on DCs, which may be an important mechanism to activate DCs and aggravate atherosclerosis. Therefore the present study concluded that exogenous DCs may induce maturation of endogenous DCs via upregulation of TLR4, further increasing the inflammatory response and accelerating atherosclerosis.
Project description:Venous grafts are often used to bypass occlusive atherosclerotic lesions; however, poor patency leads to vein graft disease. Deficiency of TLR4, an inflammatory regulator, reduces vein graft disease. Here, we investigate the effects of the accessory molecule and TLR4 analogue RadioProtective 105 (RP105) on vein graft disease. RP105 deficiency resulted in a 90% increase in vein graft lesion area compared to controls. In a hypercholesterolemic setting (LDLr(-/-)/RP105(-/-) versus LDLr(-/-) mice), which is of importance as vein graft disease is usually characterized by excessive atherosclerosis, total lesion area was not affected. However we did observe an increased number of unstable lesions and intraplaque hemorrhage upon RP105 deficiency. In both setups, lesional macrophage content, and lesional CCL2 was increased. In vitro, RP105(-/-) smooth muscle cells and mast cells secreted higher levels of CCL2. In conclusion, aggravated vein graft disease caused by RP105 deficiency results from an increased local inflammatory response.
Project description:Cellular fibronectin containing extra domain A (EDA(+)-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA(+)-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA(+)-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe(-/-)) mouse.The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA(-/-)Apoe(-/-) mice (which lack EDA(+)-FN), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA(-/-)Apoe(-/-) mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe(-/-), n=10-12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA(fl/fl)Apoe(-/-) mice (P<0.05) but had no effect on atherosclerotic lesions in EDA(-/-)Apoe(-/-) mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NF?B-mediated inflammation (increased phospho-NF?B p65/NF?B p65, tumor necrosis factor-?, and interleukin-1?) in bone marrow-derived macrophages from EDA(-/-)Apoe(-/-) mice but not from EDA(-/-)TLR4(-/-)Apoe(-/-) mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA(+)-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques.Our findings reveal that TLR4 signaling contributes to EDA(+)-FN-mediated exacerbation of atherosclerosis. We suggest that EDA(+)-FN could be a therapeutic target in atherosclerosis.
Project description:BACKGROUND:Atherosclerosis is a chronic inflammatory disease. Although Toll-like receptor 4 (TLR4) has been involved in inflammatory atherosclerosis, the exact mechanisms by which oxidized-low-density lipoproteins (ox-LDL) activates TLR4 and elicits inflammatory genesis are not fully known. Myeloid differentiation factor 2 (MD2) is an extracellular molecule indispensable for lipopolysaccharide recognition of TLR4. METHOD:Apoe-/-Md2-/- mice and pharmacological inhibitor of MD2 were used in this study. We also reconstituted Apoe-/- mice with either Apoe-/- or Apoe-/-Md2-/- marrow-derived cells. Mechanistic studies were performed in primary macrophages, HEK-293T cells, and cell-free system. FINDING:MD2 levels are elevated in atherosclerotic lesion macrophages, and MD2 deficiency or pharmacological inhibition in mice reduces the inflammation and stunts the development of atherosclerotic lesions in Apoe-/- mice fed with high-fat diet. Transfer of marrow-derived cells from Apoe-Md2 double knockout mice to Apoe knockout mice confirmed the critical role of bone marrow-derived MD2 in inflammatory factor induction and atherosclerosis development. Mechanistically, we show that MD2 does not alter ox-LDL uptake by macrophages but is required for TLR4 activation and inflammation via directly binding to ox-LDL, which triggers MD2/TLR4 complex formation and TLR4-MyD88-NF?B pro-inflammatory cascade. INTERPRETATION:We provide a mechanistic basis of ox-LDL-induced macrophage inflammation, illustrate the role of macrophage-derived MD2 in atherosclerosis, and support the therapeutic potential of MD2 targeting in atherosclerosis-driven cardiovascular diseases. FUNDING:This work was supported by the National Key Research Project of China (2017YFA0506000), National Natural Science Foundation of China (21961142009, 81930108, 81670244, and 81700402), and Natural Science Foundation of Zhejiang Province (LY19H020004).
Project description:Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr-/- mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses.
Project description:The liver is the key organ involved in systemic inflammation, but the relation between hepatic inflammation and atherogenesis is poorly understood. Since nuclear factor-?B (NF-?B) is a central regulator of inflammatory processes, we hypothesized that chronically enhanced hepatic NF-?B activation, through hepatocyte-specific expression of I?B kinase-? (IKK?) (LIKK), will aggravate atherosclerosis development in APOE*3-Leiden (E3L) mice.E3L.LIKK and E3L control littermates were fed a Western-type diet for 24 weeks. E3L.LIKK mice showed a 2.3-fold increased atherosclerotic lesion area and more advanced atherosclerosis in the aortic root with less segments without atherosclerotic lesions (11% vs. 42%), and more segments with mild (63% vs. 44%) and severe (26% vs. 14%) lesions. Expression of LIKK did not affect basal levels of inflammatory parameters, but plasma cytokine levels tended to be higher in E3L.LIKK mice after lipopolysaccharide (LPS) administration. E3L.LIKK mice showed transiently increased plasma cholesterol levels, confined to (V)LDL. This transient character resulted in a mild (+17%) increased cumulative plasma cholesterol exposure.We conclude that selective activation of NF-?B in hepatocytes considerably promotes atherosclerosis development which is (at least partly) explained by an increased sensitivity to proinflammatory triggers and transiently increased plasma cholesterol levels.
Project description:P-selectin and dendritic cells (DCs) are associated with atherosclerosis. However, their interactions in this setting are undefined. Herein, we investigated the role of P-selectin and its receptor P-selectin glycoprotein ligand (PSGL)-1 on atherosclerosis via activation of DCs. In the current study, a total of 34 patients with ST elevation myocardial infarction (STEMI) and 34 healthy control subjects were enrolled. Serum concentration of P-selectin was higher and the myeloid DC/plasmacytoid DC (mDC/pDC) ratio was lower in STEMI patients than in normal individuals. Interestingly, in STEMI patients, P-selectin was decreased and the mDC/pDC ratio was increased at 5-7 days after successful percutaneous coronary intervention, as compared with values on admission. Serum P-selectin was inversely correlated with the mDC/pDC ratio. Moreover, ApoE-/-P-/- and ApoE-/-PSGL-1-/- mice developed small atherosclerotic plaques after feeding of a western diet for 12 weeks and DC infiltration was significantly reduced. P-selectin stimulation markedly induced phenotypic maturation, enhanced secretion of inflammatory cytokines, communication with T cells, and the adhesion and migration of DCs. In vivo, DC maturation was significantly attenuated in P-selectin and PSGL1 knockout mice under hypercholesterolemic and inflammatory conditions. These effects were associated with the activation of myeloid differentiation primary response 88 (MYD88)-dependent and MyD88-independent Toll-like receptor 4 (TLR4) signaling pathways. Taken together, binding of P-selectin to PSGL-1 on DCs contributes to atherosclerosis progression via DC activation via the TLR4 signaling pathway.
Project description:TLR activation on CD11c+ DCs triggers DC maturation, which is critical for T cell activation. Given the expansion of CD11c+ DCs during the progression of atherosclerosis and the key role of T cell activation in atherogenesis, we sought to understand the role of TLR signaling in CD11c+ DCs in atherosclerosis. To this end, we used a mouse model in which a key TLR adaptor involved in DC maturation, MYD88, is deleted in CD11c+ DCs. We transplanted bone marrow containing Myd88-deficient CD11c+ DCs into Western diet-fed LDL receptor knockout mice and found that the transplanted mice had decreased activation of effector T cells in the periphery as well as decreased infiltration of both effector T cells and Tregs in atherosclerotic lesions. Surprisingly, the net effect was an increase in atherosclerotic lesion size due to an increase in the content of myeloid-derived inflammatory cells. The mechanism involves increased lesional monocyte recruitment associated with loss of Treg-mediated suppression of MCP-1. Thus, the dominant effect of MYD88 signaling in CD11c+ DCs in the setting of atherosclerosis is to promote the development of atheroprotective Tregs. In the absence of MYD88 signaling in CD11c+ DCs, the loss of this protective Treg response trumps the loss of proatherogenic T effector cell activation.
Project description:Background:As reported previously by our group, medium-chain triglycerides can ameliorate atherosclerosis. Given that TLR4 is closely related to atherosclerosis, we hypothesized herein that caprylic acid (C8:0) would suppress inflammation via TLR4/NF-κB signaling and further promote the amelioration of atherosclerosis in apoE- deficient (apoE-/-) mice. Methods:Fifty 6-week male apoE-/- mice were randomly allocated into five diet groups: a high-fat diet (HFD) without or with 2% caprylic acid (C8:0), capric acid (C10:0), stearic acid (C18:0), or linolenic acid (C18:3). RAW246.7 cells were treated with caprylic acid (C8:0), docosahexenoic acid (DHA), palmitic acid (C16:0), and lipopolysaccharide (LPS) with or without TLR4 knock-down (TLR4-KD). The serum lipid profiles, inflammatory biomolecules, and mRNA and protein expression levels were measured. Atherosclerotic lesions that occurred in the aorta and aortic sinuses were evaluated and quantified. Results:Our results indicated that C8:0 reduced body fat, improved the lipid profiles, suppressed inflammatory cytokine production, downregulated aortic TLR4, MyD88, NF-κB, TNF-α, IKKα, and IKKβ mRNA expression, and alleviated atherosclerosis in the apoE-/- mice (P < 0.05). In RAW 264.7 cells, C8:0 diminished the inflammatory response and both mRNA and protein expression of TLR4, MyD88, NF-κB, and TNF-α compared to those in the LPS and C16:0 groups (P < 0.05). However, in the TLR4-KD RAW 264.7 cells, C8:0 significantly upregulated NF-κB mRNA and protein expression compared to those in the C16:0 and DHA groups. Conclusions:These results suggest that C8:0 functions via TLR4/NF-κB signaling to improve the outcomes of apoE-/- mice through suppressing inflammation and ameliorating atherosclerosis. Thus, C8:0 may represent as a promising nutrient against chronic inflammatory diseases.
Project description:OBJECTIVE:Fibronectin containing extra domain A (Fn-EDA) is an endogenous ligand of TLR4 (toll-like receptor 4) and is abundant in the extracellular matrix of advanced atherosclerotic lesions in human and mice. Irrespective of sex, deletion of Fn-EDA reduces early atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. However, the contribution of Fn-EDA in advanced atherosclerosis remains poorly characterized. We determined the contribution of Fn-EDA in advanced atherosclerotic lesions of aged (1-year-old) Apoe-/- mice. APPROACH AND RESULTS:Plaque composition was determined in the innominate artery, a plaque instability site that is known to mimic several histological features of vulnerable human plaques. Female Apoe-/-, Fn-EDA-/-Apoe-/-, TLR4-/-Apoe-/-, and Fn-EDA-/-TLR4-/-Apoe-/- mice were fed a high-fat Western diet for 44 weeks. Fn-EDA-/-Apoe-/- mice exhibited reduced plaque size characterized by smaller necrotic cores, thick fibrous caps containing abundant vascular smooth muscle cells and collagen, reduced CD68/MMP9 (matrix metalloproteinase 9)-positive content, less accumulation of MMP-cleaved extracellular matrix aggrecan, and decreased vascular smooth muscle cell and macrophage apoptosis (P<0.05 versus Apoe-/- mice). Together these findings suggest that Fn-EDA induces plaque destabilization. Deletion of TLR4 reduced histological features of plaque instability in Apoe-/- mice but did not further reduce features of plaque destabilization in Fn-EDA-/-Apoe-/- mice, suggesting that TLR4 may contribute to Fn-EDA-induced plaque destabilization. Fn-EDA potentiated TLR4-dependent MMP9 expression in bone marrow-derived macrophages, suggesting that macrophage TLR4 may contribute to Fn-EDA-mediated plaque instability. CONCLUSIONS:Fn-EDA induces histological features of plaque instability in established lesions of aged Apoe-/- mice. The abundance of Fn-EDA in advanced atherosclerotic lesions may increase the risk of plaque destabilization.
Project description:The pharmacological manipulation of liver X receptors (LXRs) has been an attractive therapeutic strategy for atherosclerosis treatment as they control reverse cholesterol transport and inflammatory response. This study presents the development and efficacy of nanoparticles (NPs) incorporating the synthetic LXR agonist GW3965 (GW) in targeting atherosclerotic lesions. Collagen IV (Col IV) targeting ligands are employed to functionalize the NPs to improve targeting to the atherosclerotic plaque, and formulation parameters such as the length of the polyethylene glycol (PEG) coating molecules are systematically optimized. In vitro studies indicate that the GW-encapsulated NPs upregulate the LXR target genes and downregulate proinflammatory mediator in macrophages. The Col IV-targeted NPs encapsulating GW (Col IV-GW-NPs) successfully reaches atherosclerotic lesions when administered for 5 weeks to mice with preexisting lesions, substantially reducing macrophage content (?30%) compared to the PBS group, which is with greater efficacy versus nontargeting NPs encapsulating GW (GW-NPs) (?18%). In addition, mice administered the Col IV-GW-NPs do not demonstrate increased hepatic lipid biosynthesis or hyperlipidemia during the treatment period, unlike mice injected with the free GW. These findings suggest a new form of LXR-based therapeutics capable of enhanced delivery of the LXR agonist to atherosclerotic lesions without altering hepatic lipid metabolism.