Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Oxidized low-density lipoprotein-induced apoptotic dendritic cells as a novel therapy for atherosclerosis.

Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This could result, in part, from decreased emigration of DCs from atherosclerotic lesions because of the high-cholesterol environment. Nonetheless, local induction of anti-inflammatory responses by apoptotic cell clearance seems to dampen atherosclerosis, because inhibition of apoptotic cell clearance worsens atherosclerosis. In this study, we assessed whether i.v. administration of oxLDL-induced apoptotic DCs (apop(ox)-DCs) and, as a control, unpulsed apoptotic DCs could modulate atherosclerosis by inducing tolerance. Adoptive transfer of apop(ox)-DCs into low-density lipoprotein receptor knockout mice either before or during feeding of a Western-type diet resulted in increased numbers of CD103(+) tolerogenic splenic DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic DCs induced an immediate 40% decrease in Ly-6C(hi) monocyte numbers and a 50% decrease in circulating CCL2 levels, but only apop(ox)-DC treatment resulted in long-term effects on monocytes and CCL2 levels. Although initial lesion development was reduced by 40% in both treatment groups, only apop(ox)-DC treatment prevented lesion progression by 28%. Moreover, progressed lesions of apop(ox)-DC-treated mice showed a robust 45% increase in collagen content, indicating an enhanced stability of lesions. Our findings clearly show that apoptotic DC treatment significantly decreases lesion development, but only apop(ox)-DCs can positively modulate lesion progression and stability. These findings may translate into a safe treatment for patients with established cardiovascular diseases using patient-derived apop(ox)-DCs.

Interruption of the OX40-OX40 ligand pathway in LDL receptor-deficient mice causes regression of atherosclerosis.

Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.

CXCR4 blockade induces atherosclerosis by affecting neutrophil function.

AIMS: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. METHODS AND RESULTS: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. CONCLUSION: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.

T-cell immunoglobulin and mucin domain 3 acts as a negative regulator of atherosclerosis.

OBJECTIVE: Atherosclerosis is a chronic autoimmune-like disease in which lipids and fibrous elements accumulate in the arterial blood vessels. T cells are present within atherosclerotic plaques, and their activation is partially dependent on costimulatory signals, which can either provide positive or negative signals that promote T-cell activation or limit T-cell responses, respectively. T-cell immunoglobulin and mucin domain 3 (Tim-3) is a coinhibitory type 1 transmembrane protein that affects the function of several immune cells involved in atherosclerosis, such as monocytes, macrophages, effector T cells, and regulatory T cells. In the present study, we determined the role of Tim-3 in the development of atherosclerosis. APPROACH AND RESULTS: Western-type diet-fed low-density lipoprotein receptor-deficient (LDLr(-/-)) mice were treated with an anti-Tim-3 antibody for 3 and 8 weeks. Anti-Tim-3 administration increased fatty streak formation with 66% and increased atherosclerotic plaque formation after 8 weeks with 35% in the aortic root and with 50% in the aortic arch. Furthermore, blockade of Tim-3 signaling increased percentages of circulating monocytes with 33% and lesional macrophages with 20%. In addition, anti-Tim-3 administration increased CD4(+) T cells with 17%, enhanced their activation status, and reduced percentages of regulatory T cells with 18% and regulatory B cells with 37%. CONCLUSIONS: It is known that Tim-3 acts as a negative regulator of both innate and adaptive immune responses, and in the present study, we show that anti-Tim-3 treatment augments lesion development, accompanied by an increase in the number of monocytes/macrophages and CD4(+) T cells and by decreased regulatory T cells and regulatory B cells.

A multivalent ligand for the mannose-6-phosphate receptor for endolysosomal targeting of an activity-based probe.

The ubiquitously expressed mannose-6-phosphate receptors (MPRs) are a promising class of receptors for targeted compound delivery into the endolysosomal compartments of a variety of cell types. The development of a synthetic, multivalent, mannose-6-phosphate (M6P) glycopeptide-based MPR ligand is described. The conjugation of this ligand to fluorescent DCG-04, an activity-based probe for cysteine cathepsins, enabled fluorescent readout of its receptor-targeting properties. The resulting M6P-cluster-BODIPY-DCG-04 probe was shown to efficiently label cathepsins in cell lysates as well as in live cells. Furthermore, the introduction of the 6-O-phosphates leads to a completely altered uptake profile in COS and dendritic cells compared to a mannose-containing ligand. Competition with mannose-6-phosphate abolished all uptake of the probe in COS cells, and we conclude that the mannose-6-phosphate cluster targets the MPR and ensures the targeted delivery of cargo bound to the cluster into the endolysosomal pathway.

Interference of the CD30-CD30L pathway reduces atherosclerosis development.

OBJECTIVE: Costimulatory molecules tightly control immune responses by providing positive signals that promote T-cell activation or by transducing inhibitory signals that limit T-cell responses. CD30 and CD30L are members of the tumor necrosis factor receptor superfamily and are involved in the activation and proliferation of T and B cells, which have been implicated in the initiation and progression of atherosclerosis. In the present study, we thus aimed to determine the role of the CD30-CD30L pathway in the development of atherosclerosis. METHODS AND RESULTS: Western-type diet-fed low-density lipoprotein receptor-deficient mice were treated with an anti-CD30L antibody for 8 weeks, which resulted in a reduction of atherosclerotic lesion formation in the aortic root by 35%. Reduced numbers of adventitial CD3(+) T cells were found in anti-CD30L-treated mice, whereas no differences were observed in collagen and macrophage content of the atherosclerotic lesions. B-cell and mast cell responses were also not affected on anti-CD30L treatment. Interestingly, splenocyte proliferation was reduced by 53%, whereas T-cell numbers were concomitantly reduced in anti-CD30L-treated mice compared with control mice. These data thus indicate that the CD30-CD30L pathway solely exerts its function via inhibition of T-cell responses. CONCLUSIONS: In the present study, we are the first to show that interruption of the CD30-CD30L pathway reduced initial atherosclerosis development by modulating T-cell function.

Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice.

OBJECTIVE: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis. METHODS AND RESULTS: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population. CONCLUSIONS: We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.

Tim-1 mucin domain-mutant mice display exacerbated atherosclerosis.

BACKGROUND AND AIMS: Increasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous studies of the role of Tim-1 in atherosclerosis using anti-Tim-1 antibodies have yielded contradictory results. We thus aimed to investigate atherosclerosis development in Tim-1 deficient mice. METHODS: Mice with a specific loss of the Tim-1 mucin-domain (Tim-1Δmucin) and C57BL/6 (WT) mice received a single injection of a recombinant adeno-associated virus encoding murine Pcsk9 (rAAV2/8-D377Y-mPcsk9) and were fed a Western type diet for 13 weeks to introduce atherosclerosis. RESULTS: Tim-1Δmucin mice developed significantly larger lesions in the aortic root compared to WT mice, with significantly more macrophages and a trend towards a larger necrotic core. Furthermore, Tim-1Δmucin mice showed a significant loss of IL-10+ B cells and regulatory B cell subsets and increased pro-atherogenic splenic follicular B cells compared to WT mice. Moreover, Tim-1Δmucin mice displayed a dramatic reduction in Th2-associated immune response compared to controls but we did not observe any changes in humoral immunity. CONCLUSIONS: In summary, Tim-1Δmucin mice displayed a profound impairment in IL-10+ B cells and an imbalance in the Th1/Th2 ratio, which associated with exacerbated atherosclerosis.

IFNγ-Stimulated B Cells Inhibit T Follicular Helper Cells and Protect Against Atherosclerosis.

B and T cells are interconnected in the T follicular helper-germinal center B cell (TFH-GC B cell) axis, which is hyperactive during atherosclerosis development and loss of control along this axis results in exacerbated atherosclerosis. Inhibition of the TFH-GC B cell axis can be achieved by providing negative co-stimulation to TFH cells through the PD-1/PD-L1 pathway. Therefore, we investigated a novel therapeutic strategy using PD-L1-expressing B cells to inhibit atherosclerosis. We found that IFNγ-stimulated B cells significantly enhanced PD-L1 expression and limited TFH cell development. To determine whether IFNγ-B cells can reduce collar-induced atherosclerosis, apoE -/- mice fed a Western-type diet were treated with PBS, B cells or IFNγ-B cells for a total of 5 weeks following collar placement. IFNγ-B cells significantly increased PD-L1hi GC B cells and reduced plasmablasts. Interestingly, IFNγ-B cells-treated mice show increased atheroprotective Tregs and T cell-derived IL-10. In line with these findings, we observed a significant reduction in total lesion volume in carotid arteries of IFNγ-B cells-treated mice compared to PBS-treated mice and a similar trend was observed compared to B cell-treated mice. In conclusion, our data show that IFNγ-stimulated B cells strongly upregulate PD-L1, inhibit TFH cell responses and protect against atherosclerosis.

Diet-induced dyslipidemia induces metabolic and migratory adaptations in regulatory T cells.

AIMS: A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T (Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cell migration by affecting the expression of specific membrane proteins, thereby decreasing Treg cell migration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has been shown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemia contributes to altered migration of Treg cells, in part, by affecting cellular metabolism. METHODS AND RESULTS: Dyslipidemia was induced by feeding Ldlr-/- mice a western-type diet for 16-20 weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined. Dyslipidemia was associated with altered mTORC2 signalling in Treg cells, decreased expression of membrane proteins involved in migration, including CD62L, CCR7, and S1Pr1, and decreased Treg cell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemia inhibited mTORC1 signalling, induced PPARδ activation and increased fatty acid (FA) oxidation in Treg cells. Moreover, mass-spectrometry analysis of serum from Ldlr-/- mice with normolipidemia or dyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with a synthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FA oxidation-dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cell migration into the inflamed peritoneum and into atherosclerotic lesions in vitro. CONCLUSION: Altogether, our findings implicate that dyslipidemia does not contribute to atherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-like migratory phenotype in Treg cells.

Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells.

B- and T-lymphocyte attenuator stimulation protects against atherosclerosis by regulating follicular B cells.

AIMS: The immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B- and T-lymphocyte attenuator (BTLA) is a novel co-receptor that negatively regulates the activation of B and T cells; however, there have been no reports of BTLA and its function in atherosclerosis or cardiovascular disease (CVD). We aimed to assess the dominant BTLA expressing leucocyte in CVD patients and to investigate whether BTLA has a functional role in experimental atherosclerosis. METHODS AND RESULTS: We show that BTLA is primarily expressed on B cells in CVD patients and follicular B2 cells in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We treated Ldlr-/- mice that were fed a western-type diet (WTD) with phosphate-buffered saline, an isotype antibody, or an agonistic BTLA antibody (3C10) for 6 weeks. We report here that the agonistic BTLA antibody significantly attenuated atherosclerosis. This was associated with a strong reduction in follicular B2 cells, while regulatory B and T cells were increased. The BTLA antibody showed similar immunomodulating effects in a progression study in which Ldlr-/- mice were fed a WTD for 10 weeks before receiving antibody treatment. Most importantly, BTLA stimulation enhanced collagen content, a feature of stable lesions, in pre-existing lesions. CONCLUSION: Stimulation of the BTLA pathway in Ldlr-/- mice reduces initial lesion development and increases collagen content of established lesions, presumably by shifting the balance between atherogenic follicular B cells and atheroprotective B cells and directing CD4+ T cells towards regulatory T cells. We provide the first evidence that BTLA is a very promising target for the treatment of atherosclerosis.

Effect of natural killer T cell activation on the initiation of atherosclerosis.

It has been shown that natural killer T (NKT) cell activation accelerates atherosclerosis in apoE(-/-) mice. ApoE is, however, an important mediator in the presentation of lipids which may complicate conclusions on the role of NKT cells in atherosclerosis. Treatment of LDLr(-/-) mice with alpha-GalCer during Western-type diet feeding is therefore of interest. Atherosclerosis was induced by Western-type diet feeding and collar placement around the carotid arteries in both LDLr(-/-) and apoE(-/-) mice. Subsequently, the mice were treated twice a week with alpha-GalCer. This resulted in an 84% reduction in plaque size in LDLr(-/-) mice (P < 0.05), while no effect was observed in apoE(-/-) mice. In-vitro incubation of splenocytes with alpha-GalCer showed that LDLr(-/-) splenocytes proliferated stronger than apoE(-/-) splenocytes. This is reflected in a larger increase in production of cytokines and especially IL-10 after in-vitro stimulation with alpha-GalCer in LDLr(-/-) mice compared with apoE(-/-) splenocytes. Additionally, feeding a Western-type diet for 1.5 weeks induced a strong increase in the number of NKT cells in LDLr(-/-) mice and this increase was slower and less prominent in apoE(-/-) mice. Administration of alpha-GalCer to LDLr(-/-) mice in combination with Western-type diet feeding reduced plaque formation, but this effect was not seen in apoE(-/-) mice. This may be explained by the decreased presentation of lipids on CD1d molecules due to the lack of apoE. In this study we proved for the first time that NKT cells may also act in an atheroprotective manner.

Bidirectional effects of IL-10+ regulatory B cells in Ldlr-/- mice.

BACKGROUND AND AIMS: Limiting the pro-inflammatory immune response is critical for the treatment of atherosclerosis. Regulatory B cells (Bregs) can modulate the immune response through interleukin-10 (IL-10). Current data regarding Bregs and atherosclerosis is scarce and conflicting. METHODS: In this study, we investigated the frequency of IL-10+ B cells during the development of atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr-/-) mice and studied the effect of adoptive transfer of IL-10+ B cells on atherosclerosis. RESULTS: We found a very strong inverse correlation between atherosclerosis severity and the frequency of IL-10+ B cells. This effect was cholesterol-independent and observed in spleen, draining lymph nodes and peritoneal cavity. To directly assess the effects of IL-10+ B cells on atherosclerosis, we expanded IL-10+ B cells ex vivo with anti-CD40 and selected pure and viable IL-10-secreting B cells and IL-10- B cells and adoptively transferred them to Ldlr-/- mice, respectively. While IL-10- B cells were strongly atherogenic compared to control-treated mice, IL-10+ B cells did not affect lesion size. Adoptive transfer of IL-10+ B cells strongly reduced circulating leukocyte numbers and inflammatory monocytes. In addition, they decreased CD4+ T cell activation and increased IL-10+ CD4+ T cell numbers. Interestingly, both IL-10+ and IL-10- B cells exacerbated serum cholesterol levels and resulted in fatty livers, which potentially masked the beneficial effects of IL-10+ B cells on atherosclerosis. CONCLUSIONS: These findings underscore the strong immune-regulating function of IL-10+ B cells and provide additional incentives to explore effective strategies that expand IL-10+ B cells in atherosclerosis.

Disruption of a CD1d-mediated interaction between mast cells and NKT cells aggravates atherosclerosis.

BACKGROUND AND AIMS: The development of atherosclerosis is tightly regulated by the innate and adaptive immune system. Communication between these two compartments occurs, among others, upon presentation of lipid antigens to the NKT cell population by CD1d-expressing antigen-presenting cells. Recent evidence states that also mast cells express CD1d and can directly communicate with NKT cells. However, no such relationship has been reported in atherosclerosis. Here, we aimed to elucidate in vivo the CD1d-mediated interaction between mast cells and NKT cells upon atherosclerosis progression. METHODS: We adoptively transferred CD1d-/- or control mast cells to mast cell-deficient apoE-/-KitW-sh/W-sh mice and subsequently placed the animals on a Western-type diet for 10 weeks. RESULTS: At the end of the Western-type diet period, the aortic root of CD1d-/- mast cell-reconstituted mice displayed increased plaque size, with less collagen deposition and higher intraplaque CD4+ T cells, as compared to control mice. In addition, T cells inside the aortic arch showed higher pro-inflammatory cytokine production in the form of IFNγ, TNFα and IL-17. Finally, T-bet expression was found elevated in both CD4+ and CD8+ circulating T cells. CONCLUSIONS: This study is the first to illustrate that disruption of the CD1d communication pathway between mast cells and NKT cells aggravates atherosclerosis, through a shift towards pro-inflammatory T cell responses. This ability of mast cell action during plaque progression sheds new light on their role in atherosclerosis.

Induction of HLA-A2 restricted CD8 T cell responses against ApoB100 peptides does not affect atherosclerosis in a humanized mouse model.

Cardiovascular diseases form the most common cause of death worldwide, with atherosclerosis as main etiology. Atherosclerosis is marked by cholesterol rich lipoprotein deposition in the artery wall, evoking a pathogenic immune response. Characteristic for the disease is the pathogenic accumulation of macrophages in the atherosclerotic lesion, which become foam cells after ingestion of large quantities of lipoproteins. We hypothesized that, by inducing a CD8 T cell response towards lipoprotein derived apolipoprotein-B100 (ApoB100), lesional macrophages, that are likely to cross-present lipoprotein constituents, can specifically be eliminated. Based on in silico models for protein processing and MHC-I binding, 6 putative CD8 T cell epitopes derived from ApoB100 were synthesized. HLA-A2 binding was confirmed for all peptides by T2 cell binding assays and recall responses after vaccination with the peptides proved that 5 of 6 peptides could induce CD8 T cell responses. Induction of ApoB100 specific CD8 T cells did not impact plaque size and cellular composition in HLA-A2 and human ApoB100 transgenic LDLr-/- mice. No recall response could be detected in cultures of cells isolated from the aortic arch, which were observed in cell cultures of splenocytes and mesenteric lymph nodes, suggesting that the atherosclerotic environment impairs CD8 T cell activation.

Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) pathway attenuates atherogenesis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Atherosclerosis is a chronic (auto-)inflammatory disease and T cell activation is an important factor in this process. Tnfrsf4 (OX40) and Tnfsf4 (OX40 ligand) are members of the tumor necrosis factor (TNF) and TNF receptor family and OX40/OX40L mediated signaling is important in co-activation of T cells and facilitates B-T cell interaction. In this study we assessed the role of the OX40/OX40L pathway in atherosclerosis and the effect of interruption of the OX40/OX40L pathway on lesion development. METHODS AND RESULTS: We treated low-density lipoprotein receptor-deficient (LDLr-/-) mice with an anti-OX40L antibody which lead to a 53% decrease in atherosclerotic lesion formation. Treatment resulted in inhibition of Th2 mediated isotype switching by decreasing interleukin (IL)-4 secretion and subsequent low IgG1 serum levels against oxLDL, whereas protective anti-oxLDL specific IgM titers were increased in treated mice compared with control. CONCLUSIONS: We conclude that blocking the OX-40/OX40L interaction reduced atherogenesis by inhibition of IL-4 mediated Th2 induced isotype switching and subsequent increased levels of anti-oxLDL IgM.

Vaccination against VEGFR2 attenuates initiation and progression of atherosclerosis.

OBJECTIVE: Vascular endothelial growth factor receptor 2 (VEGFR2)-overexpressing cells may form an interesting target for the treatment of atherosclerosis because of their involvement in processes that contribute to this disease, such as angiogenesis. METHODS AND RESULTS: We vaccinated mice against VEGFR2 by an orally administered DNA vaccine, comprising a plasmid, encoding murine VEGFR2, carried by live attenuated Salmonella typhimurium. This vaccine induces cellular immunity against cells that overexpress VEGFR2. Vaccination of hypercholesterolemic mice against VEGFR2 resulted in a marked induction of CD8+ cytotoxic T cells specific for VEGFR2 and led to an inhibition of angiogenesis in a hindlimb ischemia model. Interestingly, VEGFR2 vaccination attenuated the progression of preexisting advanced atherosclerotic lesions in the brachiocephalic artery of apoE-/- mice. Furthermore, VEGFR2 vaccination strongly reduced the initiation of collar-induced atherosclerosis in the carotid arteries of LDLr-/- mice. In addition, denudation of the carotid artery, as a model for postinterventional lesion formation, resulted in delayed endothelial replacement and significantly increased neointima formation on VEGFR2 vaccination. CONCLUSIONS: These data indicate the prominent role of VEGFR2+ cells in cardiovascular diseases and show that induction of cellular immunity against atherosclerosis-associated cells by means of DNA vaccination may contribute to the development of novel therapies against atherosclerosis.

Defective Autophagy in T Cells Impairs the Development of Diet-Induced Hepatic Steatosis and Atherosclerosis.

Macroautophagy (or autophagy) is a conserved cellular process in which cytoplasmic cargo is targeted for lysosomal degradation. Autophagy is crucial for the functional integrity of different subsets of T cells in various developmental stages. Since atherosclerosis is an inflammatory disease of the vessel wall which is partly characterized by T cell mediated autoimmunity, we investigated how advanced atherosclerotic lesions develop in mice with T cells that lack autophagy-related protein 7 (Atg7), a protein required for functional autophagy. Mice with a T cell-specific knock-out of Atg7 (Lck-Cre Atg7f/f) had a diminished naïve CD4+ and CD8+ T cell compartment in the spleen and mediastinal lymph node as compared to littermate controls (Atg7f/f). Lck-Cre Atg7f/f and Atg7f/f mice were injected intravenously with rAAV2/8-D377Y-mPCSK9 and fed a Western-type diet to induce atherosclerosis. While Lck-Cre Atg7f/f mice had equal serum Proprotein Convertase Subtilisin/Kexin type 9 levels as compared to Atg7f/f mice, serum cholesterol levels were significantly diminished in Lck-Cre Atg7f/f mice. Histological analysis of the liver revealed less steatosis, and liver gene expression profiling showed decreased expression of genes associated with hepatic steatosis in Lck-Cre Atg7f/f mice as compared to Atg7f/f mice. The level of hepatic CD4+ and CD8+ T cells was greatly diminished but both CD4+ and CD8+ T cells showed a relative increase in their IFNγ and IL-17 production upon Atg7 deficiency. Atg7 deficiency furthermore reduced the hepatic NKT cell population which was decreased to < 0.1% of the lymphocyte population. Interestingly, T cell-specific knock-out of Atg7 decreased the mean atherosclerotic lesion size in the tri-valve area by over 50%. Taken together, T cell-specific deficiency of Atg7 resulted in a decrease in hepatic steatosis and limited inflammatory potency in the (naïve) T cell compartment in peripheral lymphoid tissues, which was associated with a strong reduction in experimental atherosclerosis.