Activation of the Regulatory T-Cell/Indoleamine 2,3-Dioxygenase Axis Reduces Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice.

T-cell activation is characteristic during the development of atherosclerosis. While overall T-cell responses have been implicated in disease acceleration, regulatory T cells (Tregs) exhibit atheroprotective effects. The expression of the enzyme indoleamine 2,3-dioxygenase-1 (IDO1), which catalyzes the degradation of tryptophan (Trp) along the kynurenine pathway, has been implicated in the induction and expansion of Treg populations. Hence, Tregs can reciprocally promote IDO1 expression in dendritic cells (DCs) via reverse signaling mechanisms during antigen presentation. In this study, we hypothesize that triggering the "Treg/IDO axis" in the artery wall is atheroprotective. We show that apolipoprotein B100-pulsed tumor growth factor beta 2-treated tolerogenic DCs promote de novo FoxP3+ Treg expansion in vivo. This local increase in Treg numbers is associated with increased vascular IDO1 expression and a robust reduction in the atherosclerotic burden. Using human primary cell cultures, we show for the first time that IDO1 expression and activity can be regulated by cytotoxic T-lymphocyte associated protein-4, which is a constitutive molecule expressed and secreted by Tregs, in smooth muscle cells, endothelial cells, and macrophages. Altogether, our data suggest that Tregs and IDO1-mediated Trp metabolism can mutually regulate one another in the vessel wall to promote vascular tolerance mechanisms that limit inflammation and atherosclerosis.

The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE-/- mice.

Leukotriene B4 (LTB4) induces proinflammatory signaling through BLT receptors expressed in atherosclerotic lesions. Either genetic or pharmacological targeting of the high affinity LTB4 receptor, BLT1, reduces atherosclerosis in different mouse models. The low affinity BLT2 receptor for LTB4 may transduce additional pro-atherogenic signaling, but combined BLT1 and BLT2 receptor antagonism has not previously been explored in atherosclerosis. The aim of the present study was to unravel the effects of the BLT receptor antagonist BIIL284 in apolipoprotein E deficient mice in terms of atherosclerotic lesion size and composition, as well as on arterial matrixmetalloproteinase (MMP) activity and plasma cytokines. Oral administration of BIIL284 (0.3-3mg/kg) dose-dependently decreased atherosclerotic lesion size after 12 weeks. In addition, significantly smaller aortic lesions were observed in mice treated with BIIL284 (3mg/kg) for 24 weeks. The reduced atherosclerosis was associated with less lesion smooth muscle cells, less arterial MMP activities and lower plasma levels of TNF-α and IL-6. Taken together, these results suggest a therapeutic value of BLT receptor antagonism in atherosclerosis.

Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe(-/-) mice.

Discovery of novel biomarkers for atherosclerosis is important to aid in early diagnosis of pre-symptomatic patients at high risk of cardiovascular events. The aim of the present study was therefore to identify potential biomarkers in circulating cells reflecting atherosclerotic lesion progression in the vessel wall. We performed gene arrays on circulating leucocytes from atherosclerosis prone Apoe(-/-) mice with increasing ages, using C57BL/6 mice as healthy controls. We identified fatty acid binding protein 4 (FABP4) mRNA to be augmented in mice with established disease compared with young Apoe(-/-) or controls. Interestingly, the transcript FABP4 correlated significantly with lesion size, further supporting a disease associated increase. In addition, validation of our finding on protein level showed augmented FABP4 in circulating leucocytes whereas, importantly, no change could be observed in plasma. Immunofluorescence analysis demonstrated FABP4 to be present mainly in circulating neutrophils and to some extent in monocytes. Moreover, FABP4-positive neutrophils and macrophages could be identified in the subintimal space in the plaque. Using human circulating leucocytes, we confirmed the presence of FABP4 protein in neutrophils and monocytes. In conclusion, we have showed that cellular levels of FABP4 in circulating leucocytes associate with lesion development in the experimental Apoe(-/-) model. The increased expression is primarily localized to neutrophils, but also in monocytes. We have identified FABP4 in leucocytes as a potential and easy accessible biomarker of atherosclerosis which could be of future clinical relevance.

Platelets regulate CD4⁺ T-cell differentiation via multiple chemokines in humans.

Atherosclerosis is an inflammatory and thrombotic disease. Both platelets and lymphocytes play important roles in atherogenesis. However, information on their interaction is limited. We therefore studied how platelets regulate CD4+ T cell activation and differentiation. Human CD4+ T cells and autologous platelets were co-cultured. Platelets concentration-dependently enhanced anti-CD3/CD28-induced IFNγ production by CD4+ T cells, but attenuated their proliferation. Abrogation of heterotypic cell-cell contact partially reversed the enhancement, and supernatant from activated platelets partially mimicked the enhancement, suggesting that platelets exert their effects via both soluble mediators and direct cell-cell contact. Platelets enhanced the production of IL-10 and cytokines characteristic for type 1 T helper (TH1) (IFNγ/TNFα) and TH17 (IL-17) cells, but influenced TH2 cytokines (IL-4/IL-5) little. The cytokine responses were accompanied by enhanced TH1/TH17/TReg differentiation. Using neutralising antibodies and recombinant PF4, RANTES, and TGFß, we found that platelet-derived PF4 and RANTES enhanced both pro- and anti-inflammatory cytokine production, whilst recombinant TGFß enhanced IL-10 but not TNFα production. In conclusion, platelets enhance the differentiation and cytokine production of anti-CD3/CD28-stimulated CD4+ T cells via both multiple chemokines and direct cell-cell contact. Our study provides new insights into the cross-talk between thrombosis and adaptive immunity, and indicates that platelets can enhance T-effector cell development.

Immunotherapy with tolerogenic apolipoprotein B-100-loaded dendritic cells attenuates atherosclerosis in hypercholesterolemic mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease characterized by a massive intimal accumulation of low-density lipoprotein that triggers chronic vascular inflammation with an autoimmune response to low-density lipoprotein components. METHODS AND RESULTS: To dampen the inflammatory component of atherosclerosis, we injected hypercholesterolemic huB100(tg) × Ldlr(-/-) mice (mice transgenic for human apolipoprotein B100 [ApoB100] and deficient for the low-density lipoprotein receptor) intravenously with dendritic cells (DCs) that had been pulsed with the low-density lipoprotein protein ApoB100 in combination with the immunosuppressive cytokine interleukin-10. DCs treated with ApoB100 and interleukin-10 reduced proliferation of effector T cells, inhibited production of interferon-γ, and increased de novo generation of regulatory T cells in vitro. Spleen cells from mice treated with DCs plus ApoB100 plus interleukin-10 showed diminished proliferative responses to ApoB100 and significantly dampened T-helper 1 and 2 immunity to ApoB100. Spleen CD4(+) T cells from these mice suppressed activation of ApoB100-reactive T cells in a manner characteristic of regulatory T cells, and mRNA analysis of lymphoid organs showed induction of transcripts characteristic of these cells. Treatment of huB100(tg) × Ldlr(-/-) mice with ApoB100-pulsed tolerogenic DCs led to a significant (70%) reduction of atherosclerotic lesions in the aorta, with decreased CD4(+) T-cell infiltration and signs of reduced systemic inflammation. CONCLUSIONS: Tolerogenic DCs pulsed with ApoB100 reduced the autoimmune response against low-density lipoprotein and may represent a novel possibility for treatment or prevention of atherosclerosis.

The immune system in atherosclerosis.

Cardiovascular disease, a leading cause of mortality worldwide, is caused mainly by atherosclerosis, a chronic inflammatory disease of blood vessels. Lesions of atherosclerosis contain macrophages, T cells and other cells of the immune response, together with cholesterol that infiltrates from the blood. Targeted deletion of genes encoding costimulatory factors and proinflammatory cytokines results in less disease in mouse models, whereas interference with regulatory immunity accelerates it. Innate as well as adaptive immune responses have been identified in atherosclerosis, with components of cholesterol-carrying low-density lipoprotein triggering inflammation, T cell activation and antibody production during the course of disease. Studies are now under way to develop new therapies based on these concepts of the involvement of the immune system in atherosclerosis.

Inhibition of T cell response to native low-density lipoprotein reduces atherosclerosis.

Immune responses to oxidized low-density lipoprotein (oxLDL) are proposed to be important in atherosclerosis. To identify the mechanisms of recognition that govern T cell responses to LDL particles, we generated T cell hybridomas from human ApoB100 transgenic (huB100(tg)) mice that were immunized with human oxLDL. Surprisingly, none of the hybridomas responded to oxidized LDL, only to native LDL and the purified LDL apolipoprotein ApoB100. However, sera from immunized mice contained IgG antibodies to oxLDL, suggesting that T cell responses to native ApoB100 help B cells making antibodies to oxLDL. ApoB100 responding CD4(+) T cell hybridomas were MHC class II-restricted and expressed a single T cell receptor (TCR) variable (V) beta chain, TRBV31, with different Valpha chains. Immunization of huB100(tg)xLdlr(-/-) mice with a TRBV31-derived peptide induced anti-TRBV31 antibodies that blocked T cell recognition of ApoB100. This treatment significantly reduced atherosclerosis by 65%, with a concomitant reduction of macrophage infiltration and MHC class II expression in lesions. In conclusion, CD4(+) T cells recognize epitopes on native ApoB100 protein, this response is associated with a limited set of clonotypic TCRs, and blocking TCR-dependent antigen recognition by these T cells protects against atherosclerosis.

Intranasal immunization with an apolipoprotein B-100 fusion protein induces antigen-specific regulatory T cells and reduces atherosclerosis.

OBJECTIVE: Atherosclerosis is an inflammatory disease. Autoimmune responses to low-density lipoproteins (LDL) contribute to its progression, whereas immunization with LDL may induce atheroprotective or proatherogenic responses. The objective of this study was to develop an atheroprotective vaccine by targeting a peptide of the LDL protein constituent apolipoprotein B-100 (apoB-100) to the nasal mucosa to induce a protective mucosal immune response. METHODS AND RESULTS: A peptide comprising amino acids 3136 to 3155 of apoB-100 (p210) was fused to the B subunit of cholera toxin (CTB), which binds to a ganglioside on mucosal epithelia. The effect of nasal administration of the p210-CTB fusion protein on atherogenesis was compared with that of an ovalbumin peptide fused to CTB and with untreated controls. Immunization with p210-CTB for 12 weeks caused a 35% reduction in aortic lesion size in Apoe(-/-) mice. This effect was accompanied by induction of regulatory T cells that markedly suppressed effector T cells rechallenged with apoB-100 and increased numbers of interleukin (IL)-10(+) CD4(+) T cells. Furthermore, a peptide-specific antibody response was observed. Atheroprotection was also documented in apoe(-/-) mice lacking functional transforming growth factor-beta receptors on T cells. CONCLUSION: Nasal administration of an apoB-100 peptide fused to CTB attenuates atherosclerosis and induces regulatory Tr1 cells that inhibit T effector responses to apoB-100.

Dendritic cells pulsed with malondialdehyde modified low density lipoprotein aggravate atherosclerosis in Apoe(-/-) mice.

OBJECTIVE: Atherosclerosis is an inflammatory disease involving activation of adaptive and innate immune responses to modified lipoproteins. Dendritic cells (DCs), which are professional antigen-presenting cells that activate T cells, are present in atherosclerotic lesions but their role for atherosclerosis-related immunity is unclear. METHODS AND RESULTS: To evaluate the role of DC in atherosclerosis, DCs pulsed with malondialdehyde modified low density lipoprotein (MDA-LDL) were transferred into Apoe(-/-) mice. The extent of disease was measured in the aortic root and was compared to that in animals treated with Keyhole Limpet Hemocyanin (KLH) pulsed DCs and to untreated animals. Mice receiving MDA-LDL pulsed DCs showed significantly larger atherosclerotic lesions compared to controls, with increased inflammation in the lesions and antigen-specific immune responses. Furthermore, MDA-LDL administration in complete Freund's adjuvant, which is atheroprotective, led to the induction of regulatory T cells whereas MDA-LDL-DCs treatment did not, suggesting that modulation of immune properties can result in different effects on atherosclerosis. CONCLUSIONS: DCs presenting components of LDL promote specific immunity to their antigen, increase lesion inflammation and accelerate atherosclerosis.

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain.

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. Providing a link between miRNA-mediated regulation of gene expression and inflammation, our results suggest that the formation of miRNAs may be regulated by 5LO in leukocytes and cancer cells expressing this lipoxygenase.