Helga D. Manthey

Auto-Antigenic Protein-DNA Complexes Stimulate Plasmacytoid Dendritic Cells to Promote Atherosclerosis

Background— Inflammation has been closely linked to auto-immunogenic processes in atherosclerosis. Plasmacytoid dendritic cells (pDCs) are specialized to produce type-I interferons in response to pathogenic single-stranded nucleic acids, but can also sense self-DNA released from dying cells or in neutrophil extracellular traps complexed to the antimicrobial peptide Cramp/LL37 in autoimmune disease. However, the exact role of pDCs in atherosclerosis remains elusive. Methods and Results— Here we demonstrate that pDCs can be detected in murine and human atherosclerotic lesions. Exposure to oxidatively modified low-density lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses. Plasmacytoid DCs can be stimulated to produce interferon-&agr; by Cramp/DNA complexes, and we further identified increased expression of Cramp and formation of neutrophil extracellular traps in atherosclerotic arteries. Whereas Cramp/DNA complexes aggravated atherosclerotic lesion formation in apolipoprotein E–deficient mice, pDC depletion and Cramp-deficiency in bone marrow reduced atherosclerosis and anti–double-stranded DNA antibody titers. Moreover, the specific activation of pDCs and interferon-&agr; treatment promoted plaque growth, associated with enhanced anti–double-stranded–DNA antibody titers. Accordingly, anti–double-stranded DNA antibodies were elevated in patients with symptomatic versus asymptomatic carotid artery stenosis. Conclusions— Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti–double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.

Complement C5a inhibition reduces atherosclerosis in ApoE−/− mice

The complement C5a receptor, CD88, is present on many of the cells found within human atherosclerotic plaques, but little is known about the role of C5a in atherogenesis. Using real‐time PCR, we determined that ApoE–/– mice fed a normal diet express more aortic CD88 mRNA compared with controls, and this increase coincides with atherosclerotic lesion development (P< 0.001 for 3‐ vs. 25‐wk‐old animals). Conversely, mRNA expression of the alternative C5a receptor, C5L2, in aortas of ApoE–/– mice, was lower than controls at all time points. Using immunohistochemistry, we confirmed the presence of CD88 on macrophages, smooth muscle cells, and activated endothelial cells in plaques from brachiocephalic arteries. Treatment of ApoE–/– mice with a CD88 antagonist (PMX53; 3 mg/kg s.c. 3×/wk plus 1 mg/kg/d p.o.) for 25 wk reduced lesion size and lipid content in the plaque by ~40% (P<0.05). Our study provides evidence for a proatherogenic role for C5a and identifies the CD88 antagonist PMX53 as a potential antiatherosclerotic drug.—Manthey, H. D., Thomas, A. C., Shiels, I. A., Zernecke, A., Woodruff, T. M., Rolfe, B., Taylor, S. M. Complement C5a inhibition reduces atherosclerosis in ApoE–/– mice. FASEB J. 25, 2447–2455 (2011). www.fasebj.org

Dendritic cells in atherosclerosis: Functions in immune regulation and beyond

Chronic inflammation drives the development of atherosclerosis. Dendritic cells (DCs) are known as central mediators of adaptive immune responses and the development of immunological memory and tolerance. DCs are present in non-diseased arteries, and accumulate within atherosclerotic lesions where they can be localised in close vicinity to T cells. Recent work has revealed important functions of DCs in regulating immune mechanisms in atherogenesis, and vaccination strategies using DCs have been explored for treatment of disease. However, in line with a phenotypical and functional overlap with plaque macrophages vascular DCs were also identified to engulf lipids, thus contributing to lipid burden in the vessel wall and initiation of lesion growth. Furthermore, a function of DCs in regulating cholesterol homeostasis has been revealed. Finally, phenotypically distinct plasmacytoid dendritic cells (pDCs) have been identified within atherosclerotic lesions. This review will dissect the multifaceted contribution of DCs and pDCs to the initiation and progression of atherosclerosis and the experimental approaches utilising DCs in therapeutic vaccination strategies.

Visualization of Vascular Inflammation in the Atherosclerotic Mouse by Ultrasmall Superparamagnetic Iron Oxide Vascular Cell Adhesion Molecule-1–Specific Nanoparticles

Objective—Noninvasive imaging of atherosclerosis remains challenging in clinical applications. Here, we applied noninvasive molecular imaging to detect vascular cell adhesion molecule-1 in early and advanced atherosclerotic lesions of apolipoprotein E–deficient mice. Methods and Results—Ultrasmall superparamagnetic iron oxide particles functionalized with (P03011) or without (P3007) vascular cell adhesion molecule-1−binding peptide were visualized by ultra high-field (17.6 T) magnetic resonance. Injection of P03011 resulted in a marked signal loss in the aortic root of apolipoprotein E–deficient mice fed a Western diet for 8 and 26 weeks in vivo and ex vivo, compared with preinjection measurements, P3007-injected mice, and P03011- or P3007-injected age-matched C57BL/6 controls. Histological analyses revealed iron accumulations in the intima, in colocalization with vascular cell adhesion molecule-1−expressing macrophages and endothelial cells. Coherent anti-Stokes Raman scattering microscopy demonstrated iron signals in the intima and media of the aortic root in the P03011-injected but not untreated apolipoprotein E–deficient mice, localized to macrophages, luminal endothelial-like cells, and medial regions containing smooth muscle cells. Electron microscopy confirmed iron particles enclosed in endothelial cells and in the vicinity of smooth muscle cells. Conclusion—Using a combination of innovative imaging modalities, in this study, we demonstrate the feasibility of applying P03011 as a contrast agent for imaging of atherosclerosis.

The complement C3a receptor contributes to melanoma tumorigenesis by inhibiting neutrophil and CD4+ T cell responses

The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a–C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4+ T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4+ T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAFV600E mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers.

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice

Objective—Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)–mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1&agr; in APCs in atherosclerosis. Approach and Results—We found upregulated HIF1&agr; expression in CD11c+ APCs within atherosclerotic plaques of low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Conditional deletion of Hif1a in CD11c+ APCs in high-fat diet–fed Ldlr−/− mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1&agr; directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1&agr;-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr−/− mice. Notably, deletion of Hif1a in LysM+ bone marrow cells in Ldlr−/− mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1&agr;, STAT3, and interleukin-12 protein were found to colocalize with APCs. Conclusions—Our findings identify HIF1&agr; to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr−/− mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.

CCR6 selectively promotes monocyte mediated inflammation and atherogenesis in mice

The chemokine receptor CCR6 is expressed by various cell subsets implicated in atherogenesis, such as monocytes, Th17 and regulatory T cells. In order to further define the role of CCR6 in atherosclerosis, CCR6-deficient (Ccr6-/-) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr-/-) mice to generate atherosclerosis-prone mice deficient in CCR6. Compared to Ldlr-/- controls, atherosclerotic burden in the aortic sinus and aorta were reduced in Ccr6-/-Ldlr-/- mice fed a high fat diet, associated with a profound depression in lesional macrophage accumulation. Local and systemic distributions of T cells, including frequencies of Th1, Th17 and regulatory T cells were unaltered. In contrast, circulating counts of both Gr-1(high) and Gr1(low) monocytes were reduced in Ccr6-/-Ldlr-/- mice. Moreover, CCR6 was revealed to promote monocyte adhesion to inflamed endothelium in vitro and leukocyte adhesion to carotid arteries in vivo. Finally, CCR6 selectively recruited monocytes but not T cells in an acute inflammatory air pouch model. We here show that CCR6 functions on multiple levels and regulates the mobilisation, adhesion and recruitment of monocytes/macrophages to the inflamed vessel, thereby promoting atherosclerosis, but is dispensable for hypercholesterolaemia-associated adaptive immune priming. Targeting CCR6 or its ligand CCL20 may therefore be a promising therapeutic strategy to alleviate atherosclerosis.

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1β and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1β, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.

Effects of exercise training and RhoA/ROCK inhibition on plaque in ApoE−/− mice

BACKGROUND The molecular mechanisms of exercise-induced cardioprotection are poorly understood. We recently reported that exercise training down-regulated gene expression of the Ras homolog gene family member A (RhoA). RhoA and its first effectors, the Rho-kinases (ROCK), have already been implicated in the pathogenesis of cardiovascular disease. The aim of this study was to compare the effects of a RhoA/ROCK inhibitor (fasudil) and exercise in the Apolipoprotein E knockout (ApoE(-/-)) mouse model of atherosclerosis. METHODS Four groups of 14 week old ApoE(-/-) mice were randomised as follows (n=12/group): i) sedentary controls (Cont); ii) fasudil (Fas) treatment (100mg/kg bodyweight/day) for 8 weeks; iii) exercise intervention (Ex:free access to running wheel for 8 weeks) and iv) exercise intervention and fasudil treatment (ExFas) for 8 weeks. RESULTS Phosphorylation of myosin light chain was significantly reduced in the brachiocephalic artery of all treatment groups compared with sedentary controls, implying an inhibitory effect of exercise and fasudil on the RhoA/ROCK pathway. Furthermore, atherosclerotic lesions were significantly smaller in all treatment and intervention groups compared with the control group (Fas: 34.7%, Ex: 48.3%, ExFas: 40.9% less than Control). The intima:media ratio was reduced by both exercise intervention and fasudil treatment alone or in combination (Fas: 23.6%, Ex: 35.5%, ExFas: 43.9% less than Control). Exercise alone and fasudil treatment alone also showed similar effects on plaque composition, increasing both smooth muscle cell and macrophage density. CONCLUSION These results suggest that the protective effects of exercise on atherogenesis are similar to the inhibitory effects on the RhoA/ROCK signalling pathway.