Plasmacytoid dendritic cells: Biomarkers or potential therapeutic targets in atherosclerosis?

Pharmacology & Therapeutics 137 (2013) 172–182

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: R.M. Wadsworth

Plasmacytoid dendritic cells: Biomarkers or potential therapeutic targets in atherosclerosis? Gianluca Grassia a, b, Neil MacRitchie b, Andrew M. Platt b, James M. Brewer b, Paul Garside b, Pasquale Maffia b, a,⁎ a b

Department of Experimental Pharmacology, University of Naples Federico II, 80131 Naples, Italy Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8TA, UK

a r t i c l e

i n f o

Keywords: Atherosclerosis Plasmacytoid dendritic cells Interferons Autoimmunity

a b s t r a c t Plasmacytoid dendritic cells (pDCs) represent a unique subset of dendritic cells that play distinct and critical roles in the immune response. Importantly, pDCs play a pivotal role in several chronic autoimmune diseases strongly characterized by an increased risk of vascular pathology. Clinical studies have shown that pDCs are detectable in atherosclerotic plaques and others have suggested an association between reduced numbers of circulating pDCs and cardiovascular events. Although the causal relationship between pDCs and atherosclerosis is still uncertain, recent results from mouse models are starting to define the specific role(s) of pDCs in the disease process. In this review, we will discuss the role of pDCs in innate and adaptive immunity, the emerging evidence demonstrating the contribution of pDCs to vascular pathology and we will consider the possible impact of pDCs on the acceleration of atherosclerosis in chronic inflammatory autoimmune diseases. Finally, we will discuss how pDCs could be targeted for therapeutic utility. © 2012 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The phenotype and origin of plasmacytoid dendritic cells . . . . . . . . . . . . . . . . Function of plasmacytoid dendritic cells and their role in the inflammatory response . . . Plasmacytoid dendritic cells in human atherosclerosis . . . . . . . . . . . . . . . . . . Understanding the function of plasmacytoid dendritic cells in experimental atherosclerosis How plasmacytoid dendritic cells could influence co-morbidity between autoimmune and cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Concluding remarks and potential therapeutic immunomodulation . . . . . . . . . . . . Funding sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AMI, Acute myocardial infarction; ACPA, anti-citrullinated protein antibodies; APC, antigen presenting cells; apoE, apolipoprotein-E; BDCA, blood dendritic cell antigen; BST-2, bone marrow stromal antigen; CVD, cardiovascular diseases; CACs, circulating angiogenic cells; cDCs, conventional dendritic cells; CAD, coronary artery disease; DT, diphtheria toxin; ECs, endothelial cells; EPCs, endothelial precursor cells; G-CSF, granulocyte colony-stimulating factor; HEVs, high endothelial venules; HFD, high fat diet; IDO, indoleamine 2,3-dioxygenase; IFN, Interferon; IRF, Interferon Regulatory Factor; LDLr, low density lipoprotein receptor; LNs, lymph nodes; LAG-3, lymphocyte activation marker-3; MDPs, macrophage/dendritic cell precursors; MHC-II, major histocompatibility complex class II; NETs, neutrophil extracellular traps; NOD, non-obese diabetic; ODN, Oligonucleotides; ox-LDL, oxidized low density lipoprotein; pDCs, plasmacytoid dendritic cells; RA, rheumatoid arthritis; RF, rheumatoid factor; SLE, systemic lupus erythematosus; tDCs, total dendritic cells; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; T1D, type 1 diabetes; VEGF, vascular endothelial growth factor; VSMCs, vascular smooth muscle cells; WT, wild type. ⁎ Corresponding author at: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, 120 University Place, University of Glasgow, Glasgow G12 8TA, UK. Tel.: +44 141 330 7142; fax: +44 141 330 4297. E-mail address: Pasquale.Maffi[email protected] (P. Maffia). 0163-7258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pharmthera.2012.10.001

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1. Introduction

2. The phenotype and origin of plasmacytoid dendritic cells

The immune system is a key contributor to atherosclerosis-related cardiovascular diseases (CVD), therefore understanding the cellular and molecular mechanisms underlying immune responses is of central importance in current cardiovascular research (Hansson, 2005; Galkina & Ley, 2009; Libby et al., 2011). Multiple leukocyte subsets have been shown to play a significant role in cardiovascular diseases and among them, dendritic cells are receiving growing interest. Dendritic cells can be classified into two distinct lineages; conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). While the majority of studies have tended to focus on the role of cDCs (for review see Cybulsky & Jongstra-Bilen, 2010; Niessner & Weyand, 2010; Puddu et al., 2010; Koltsova & Ley, 2011; Manthey & Zernecke, 2011; Moore & Tabas, 2011; Butcher & Galkina, 2012; Cheong & Choi, 2012), pDCs are now emerging as important contributors to CVD (Sorrentino et al., 2010; Butcher & Galkina, 2012; Cheong & Choi, 2012; Döring & Zernecke, 2012). pDCs are innate immune cells whose name derives from their plasma cell-like morphology (Colonna et al., 2004). They are often referred to as ‘type 1 interferon (IFN) producing cells’ owing to their ability to rapidly secrete vast amounts of IFN-α/β following stimulation (Asselin-Paturel et al., 2005). Type 1 interferons possess intrinsic anti-viral activity (Gilliet et al., 2008) as well as driving antiviral responses such as the expansion of CD8+ cytotoxic T cells (Schlecht et al., 2004) and the induction of a Th1 polarized CD4+ T cell-mediated responses (Cella et al., 2000). Following stimulation, pDCs undergo phenotypic and morphological changes including a shift to a dendritic-like morphology coupled with upregulation of major histocompatibility complex class II (MHC-II) and T cell costimulatory molecules. Despite early controversies regarding the true professional antigen presenting cell (APC) functions of pDCs, there is now convincing evidence that pDCs can act as bona fide APCs in vivo (Villadangos & Young, 2008) with both immunogenic (Salio et al., 2004; Schlecht et al., 2004; Sapoznikov et al., 2007) and tolerogenic (De Heer et al., 2004; Ochando et al., 2006; Irla et al., 2010; Hadeiba et al., 2012) functions being described. Evidence is emerging that circulating pDCs are reduced in coronary artery disease (Van Vré et al., 2006; Yilmaz et al., 2006, 2009; Van Brussel et al., 2010) and patients with decompensated heart failure also have reduced blood pDC counts although these cells display an enhanced activation state (Sugi et al., 2011). The reduced number of circulating pDCs could reflect the mobilisation of pDCs to the site of inflammation as occurs in other autoimmune diseases such as systemic lupus erythematosus (Migita et al., 2005). Vascular pDCs have been identified in human and mouse Kawasaki disease, giant arteritis (Schulte et al., 2009; Yilmaz & Arditi, 2009) and in human atherosclerotic lesions (Niessner et al., 2006; Daissormont et al., 2011; Döring et al., 2012). Results from animal models are controversial but interesting. Recently, pDCs have been shown to protect against atherosclerosis by tuning T-cell proliferation/activity in an atherosclerotic lesioninduced model in low density lipoprotein receptor (LDLr)−/− mice (Daissormont et al., 2011). On the contrary, in apolipoprotein-E (apoE)−/− mice, pDC depletion inhibited plaque formation (Macritchie et al., 2012) and auto-antigenic protein–DNA complexes stimulated pDCs to promote atherosclerosis (Döring et al., 2012). Importantly, pDCs have been shown to play a pivotal role in several chronic autoimmune diseases, strongly characterized by an increased risk of CVD, such as systemic lupus erythematosus (SLE) (Chan et al., 2012), rheumatoid arthritis (RA) (Kavousanaki et al., 2010) and Type 1 diabetes (T1D) (Diana et al., 2011). In this review, we will describe the role of pDCs in innate and adaptive immunity, highlight their role in atherosclerosis and will discuss the impact of pDCs in chronic inflammatory autoimmune diseases on the acceleration of atherosclerosis. Finally, we will discuss how pDCs could be targeted for therapeutic utility in CVD.

pDCs, named because of their plasma cell-like morphology, have markedly distinct functions compared with cDCs. Although characterization of the pDC lineage has presented a considerable challenge as they express molecular markers expressed by other cell types, a definitive array of markers now clearly identifies this subset. In humans, pDCs circulate in the blood as lineage immature or pre-pDCs that express blood dendritic cell antigen-2 and 4 (BDCA-2, BDCA-4) (Dzionek et al., 2001; Colonna et al., 2004), ILT7 (Rissoan et al., 2002), MHC-II, CD4, CD45RA and CD123 (interleukin-3 receptor alpha) but lack expression of other T and B cell lineage markers and CD11c. Murine pDCs are characterized through expression of B220 (Bjorck, 2001), Ly6C, bone marrow stromal antigen (BST-2) (Blasius et al., 2006a), lymphocyte activation marker-3 (LAG-3) (Workman et al., 2009), CCR9 (Wendland et al., 2007), siglec-H, (Blasius et al., 2006b) in addition to low levels of CD11c. The immature phenotype is characterized by low expression of MHC-II and costimulatory molecules and a markedly limited T-cell stimulatory potential (Mittelbrunn et al., 2009). Pre-pDCs typically mature and produce large amounts of IFN-α/β in response to stimuli. Critically, pDCs express several unique markers and antibodies have been generated against these to deplete pDCs in mice as described below. The origin of pDCs remained contentious for several years as it was originally thought that cDCs and pDCs arose from distinct progenitors (Fogg et al., 2006; Waskow et al., 2008). However later studies using CX3CR1 reporter mice demonstrated that like cDCs and monocytes, pDCs arise from common CSF-1R+CX3CR1+Flt3 + macrophage/ dendritic cell precursors (MDPs) (Auffray et al., 2009). pDCs expand following treatment with Flt3L in humans and mice in vitro (Blom et al., 2000; Gilliet et al., 2002) and in vivo (Brawand et al., 2002; Pulendran et al., 2000). Furthermore, granulocyte colony-stimulating factor (G-CSF) can drive mobilization of pDCs from the bone marrow (Arpinati et al., 2000). Following their release from the bone marrow, pDCs recirculate through the blood and can be found in the spleen, lymph node, thymus (Bjorck, 2001) and also in peripheral tissues such as the liver, lung, skin (Blasius et al., 2004) and arteries (Daissormont et al., 2011; Döring et al., 2012; MacRitchie et al., 2012). pDCs have a turnover period of approximately 2 weeks, much longer than other subsets such as CD4−CD8+ cDCs which turnover every few days (O'Keeffe et al., 2002). From murine studies we now know that pDCs recirculating in blood enter lymph nodes via high endothelial venules (HEVs) (Liu et al., 2009), rather than through afferent lymphatics from peripheral tissues such as the intestine and liver (Yrlid et al., 2006). Consistent with this entry route, pDCs localize to areas of lymph nodes adjacent to HEVs (Cella et al., 1999). 3. Function of plasmacytoid dendritic cells and their role in the inflammatory response pDCs represent a unique subset of DCs which play distinct and critical roles in the immune response and these can differ depending on anatomical location and context. In original reports on the function of pDCs recovered from human blood, it was shown that after stimulation with influenza virus or CD40L, they are proficient producers of type I IFNs and thus are critical in the anti-viral response (Cella et al., 1999; Kadowaki et al., 2000). Consistent with this pDCs express TLR7 and TLR9 that recognizes single stranded viral RNA and CpG oligonucleotides (ODN) respectively, but lack expression of TLR2, 3, 4 or 5 (Kadowaki et al., 2001; Iwasaki & Medzhitov, 2004). TLR7 and TLR9 induce the production of type 1 IFNs and NFκB-dependent cytokines through constitutive expression of Interferon Regulatory Factor (IRF) 7 which binds to MyD88 forming a complex with IRAK1, IRAK4, TRAF3, TRAF6 and IKKα (Kawai & Akira, 2010). Phosphorylated IRF7 then translocates to the nucleus and facilitates the production of type 1 IFNs (Kawai & Akira, 2010). Expression of FcgammaRIIa (CD32), which modulates type I interferon production, by human pDCs ensures

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multiple pathways can trigger type I IFN production by this subset (Bave et al., 2003). Importantly, pDCs are able to participate in antigen presentation to T cells (Grouard et al., 1997; Bjorck, 2001; Villadangos & Young, 2008). However immature or pre-pDCs do not prime effector T cells but instead drive IL-10-producing T cells in humans and mice (Kuwana et al., 2001; Martín et al., 2002) which can exhibit a regulatory T cell phenotype (Moseman et al., 2004). Additionally, pDCs produce the cytotoxic serine protease granzyme B and indoleamine 2,3-dioxygenase (IDO), which have been described to be involved in the suppression of effector T cell responses (Jahrsdörfer et al., 2010; Fabricius & Jahrsdörfer, 2011). Consistent with this, pDCs are critical for the induction of tolerance to cardiac allografts (Ochando et al., 2006) and prevention of inflammatory responses to inhaled antigen (de Heer et al., 2004). In contrast to these seemingly tolerogenic properties, pDCs play critical roles during inflammation and this is evident by their accumulation in inflammatory sites such as allergic mucosa (Jahnsen et al., 2000) and skin during contact dermatitis, psoriasis vulgaris and SLE (Farkas et al., 2001; Wollenberg et al., 2002) and within lymph nodes draining sites of inflammation (Cella et al., 1999). Although the roles of pDCs in these inflammatory sites remain ill defined, recent studies have shed some light on their functions. Firstly, through the production of copious amounts of type 1 IFN, pDCs drive NK cell cytolytic activity and Th1 differentiation (Rogge et al., 1998). Following activation, pDCs upregulate MHC-II and co-stimulatory molecules enabling their ability to directly stimulate T cells and drive Th1 polarization (Cella et al., 2000; Asselin-Paturel et al., 2001). Type 1 IFNs also promote maturation of monocyte-derived DCs and endow them with a greater capacity to prime T cells (Santini et al., 2000), suggesting pDCs may augment the function of other DCs. In addition, pDC-derived type 1 IFNs and IL-6 induce B cells to differentiate into immunoglobulin-producing plasma cells (Jego et al., 2003). Moreover, pDCs produce large amounts of inflammatory chemokines such as CXCL10, and CCL3, 4 and 5 which can augment inflammation by recruiting activated lymphocytes (Krug et al., 2002). A major advance in understanding the roles of pDCs in vivo has been the use of specific antibody-mediated depletion and genetic targeting systems. While the mPDCA-1 antibody has been used to specifically deplete pDCs in mice (Krug et al., 2004), antibodies directed against markers such as Gr-1, and BST-2 (120 G8 and mPDCA-1) have been problematic because Gr-1 expression is not specific to pDCs and BST-2 is induced on other cell types after activation (Swiecki & Colonna, 2010). Recently, Siglec-H-DTR mice were generated and following treatment of these mice with Diphtheria toxin (DT), pDCs were efficiently and selectively depleted (Takagi et al., 2011). Levels of inflammatory cytokines in the serum following administration of TLR ligands were reduced in pDC-ablated mice suggesting that pDCs are critical for the initiation of inflammation in this scenario (Takagi et al., 2011). These experiments also reinforced in vitro human data that pDCs augment the antigen-presenting capacity of other DCs (Santini et al., 2000) as cDCs had reduced levels of MHC-II and co-stimulatory molecules in pDC-ablated mice. That accumulation and activation of cDCs in mesenteric lymph nodes after feeding a TLR7/8 ligand is reduced after pDC ablation lends further credence to this hypothesis (Swiecki et al., 2010). Indeed, together these reports suggest that pDCs are required for optimal accumulation and activation of cDCs in secondary lymphoid organs. Nevertheless, CD4+ T cell responses were enhanced after pDC depletion in Siglec-H-DTR mice and generation of inducible T regulatory cells was suppressed indicating a negative effect of pDCs on CD4 + T cell responses in this experimental model (Takagi et al., 2011). In contrast, pDCs were required for the generation of cytolytic CD8+ T cell responses (Takagi et al., 2011). The suppression of CD4+ T cell responses by pDCs demonstrated using cell depletion is consistent with targeting antigen to pDCs with an anti-Siglec H antibody, which lead to T cell hyporesponsiveness

and reduced antibody responses to associated antigen (Loschko et al., 2011). In addition to Siglec-H-DTR mice, BDCA-2-DTR mice were generated and here it was shown that pDCs are critical for the survival and accumulation of vesicular stromatitis virus (VSV)-specific CD8+ T cells (Swiecki et al., 2010). Together, these findings indicate that upon activation pDCs are critical in determining the intensity of T and B cell responses. This is likely mediated through their production of inflammatory cytokines and chemokines, antigen presenting capacity and importantly, their capacity to provide unique signals to other DC populations that dictate priming of adaptive immunity. Interestingly, pDCs are now emerging as key contributors to cardiovascular diseases (Sorrentino et al., 2010; Butcher & Galkina, 2012; Cheong & Choi, 2012; Döring & Zernecke, 2012). 4. Plasmacytoid dendritic cells in human atherosclerosis 4.1. Analysis of plasmacytoid dendritic cells in human arteries The presence of pDCs in human arteries has been confirmed by electron microscopy (Van Vré et al., 2011a) and immunohistochemistry (Niessner et al., 2006; Yilmaz et al., 2006; Van Vré et al., 2011b; Daissormont et al., 2011; Döring et al., 2012). The population of pDCs present in plaques was analyzed by immunohistochemistry on frozen sections from carotid artery specimens collected from patients undergoing endarterectomy (Niessner et al., 2006). To assess the relationship between plaque vulnerability and pDC content, authors graded endarterectomy samples according to the presence of thrombus, lipid content, and density of the inflammatory infiltrate (TLI score). The investigators identified CD123+ cells in 16 of 30 atherosclerotic plaques and these were located in the shoulder region or at the base of the plaque, with IFN-α-producing cells also restricted to these atheroma regions. Importantly, the number of pDCs was 10-fold higher in unstable versus stable plaques, and IFN-α mRNA levels were markedly higher in plaques with high TLI scores than in non-inflamed, non-thrombosed plaques (Niessner et al., 2006). Whereas CpG ODN stimulation of lipid-rich plaques in organ culture increased IFN-α mRNA levels and secretion of IFN-α, this failed to induce IFN-α secretion from stable plaques. Additionally, IFN-α produced by pDCs induced the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on CD4+ T cells (Niessner et al., 2006). Of note, adoptive transfer of human plaque-isolated CD4+ T cells into immunodeficient mice engrafted with human atherosclerotic plaque resulted in apoptosis of vascular smooth muscle cells (VSMCs) in a TRAIL-dependent manner (Sato et al., 2006). Taken together, these studies could suggest a link between aortic pDCs and possible TRAIL-dependent T cell-induced fibrous cap thinning and plaque destabilization. Interestingly, TRAIL is also directly secreted by pDCs (Riboldi et al., 2009; Kalb et al., 2012), however the evaluation of the net contribution of TRAIL-producing pDCs in atherosclerosis still needs further investigation. Indeed, TRAIL, has been shown to exert opposite effects on the vasculature, inhibiting experimental atherosclerosis in mouse models (Secchiero et al., 2006; Di Bartolo et al., 2011; Watt et al., 2011) on one hand, but also promoting VSMC proliferation (Kavurma et al., 2008; Chan et al., 2010) and activation (Song et al., 2011) on the other. Recently, CD123 expression by endothelial cells, macrophages and VSMCs has been identified in human samples (Van Vré et al., 2011b; Daissormont et al., 2011). The presence of these cell types in advanced plaques makes identification of pDCs by CD123 expression debatable. Van Vré et al. (2011b), analyzing 11 complicated lesions from carotid endarterectomy frozen samples, demonstrated that all samples contained CD123 expressing cells, whereas BDCA-2+ cells were detectable only in 82% of lesions. As a result, the actual plaque pDC content may be lower than originally envisioned by Niessner et al. (2006). The same pDC marker, BDCA-2, was used in the analysis of the carotid atheromata (Yilmaz et al., 2006). Plaques of 34 patients with stable morphology and of 31 patients with vulnerable

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morphology were compared. Only a few pDC precursors were found in 53 of all 65 atherosclerotic plaques. In contrast to Niessner et al. (2006) pDC numbers did not significantly differ between the 2 groups. Similar results were recently obtained using BDCA-4 as a pDC marker (Daissormont et al., 2011). BDCA-4+ cell expression did not differ between stable (n = 22) and unstable (n = 22) advanced atherosclerotic lesions from carotid endoarterectomy, and showed a sparse but consistent presence of pDCs in human plaques. In agreement, microarray and real-time PCR analysis failed to demonstrate differential expression of established pDC markers between the 2 groups (Daissormont et al., 2011). Döring et al. (2012) suggested that the presence of pDCs correlated with plaque progression in human atherosclerosis. Through a comprehensive analysis of carotid artery specimens at all stages of the pathology (from intimal xanthoma to fibrous cap atheroma with plaque rupture; n=3–6 per plaque type), the authors demonstrated that BDCA2+ pDCs were detected in advanced human carotid plaques but less frequently in early lesions. Indeed, gene expression microarrays revealed an increase in expression of pDC markers in advanced versus early lesions (Döring et al., 2012). In conclusion, immunohistochemical analysis of vascular pDCs is problematic due to the diversity of different markers expressed by these cells at different stages of maturation. Although the presence of pDCs into the human atherosclerosis plaque is well documented, the numbers and correlation with plaque stability remains contentious. 4.2. Evaluation of circulating plasmacytoid dendritic cells The evaluation of pDC numbers in the blood of patients with stable and unstable coronary disease in comparison with controls has been used as an indirect measure to determine a possible role of pDCs in coronary artery disease (CAD). Absolute and relative numbers of circulating pDC precursors (BDCA-2+) were significantly lower in patients with CAD (>50% stenosis in one or more coronary arteries; n = 18) compared with age- and sex-matched controls (n= 18) (Van Vré et al., 2006). pDCs were decreased in patients with troponin-positive unstable coronary syndromes compared with patients with low troponin values, and tended to be lower in more extensive CAD (Van Vré et al., 2006). Furthermore, circulating cDC and pDC precursors were analyzed by flow cytometry in healthy controls (n= 19), CAD patients with stable (n= 20), unstable angina pectoris (n= 19), and acute myocardial infarction (AMI; n = 17) (Yilmaz et al., 2006). The authors observed a marked reduction in cDC precursors in CAD patients, however; the decline in circulating pDCs precursors (BDCA2+ cells) was less pronounced. Patients with AMI had the most dramatic decline (63%) in circulating cDCs, but showed no differences in pDCs (Yilmaz et al., 2006). Shi et al. (2007) analyzed the blood from 32 men who underwent coronary artery angiography for chest pain, divided into the CAD group (>50% stenosis in one or more coronary arteries; n = 21) and the control group (normal coronary angiography results, n = 11). cDCs were defined as Lin1 −HLA-DR+CD11c +; and pDCs as Lin1 − HLA-DR+CD123 +. Here, the absolute number of peripheral-blood DCs was significantly higher in the CAD group compared with the control group. Both percentage and absolute number of cDCs was also significantly increased in the CAD group compared with the control group, whereas the pDC fraction was similar between the 2 groups. These differences may, at least in part, originate from the use of different markers to identify DCs in these studies. The absence of a gold standard for subtype enumeration complicates cross-study comparisons, and may contribute to conflicting data on changes in blood DCs in CAD patients (van Brussel et al., 2010). In a subsequent, more extended study, the numbers of pDC (BDCA2+ cells), cDC (BDCA1+ cells), and total DC (tDCs) precursors were found to decline as the extent of coronary atherosclerosis

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increased (Yilmaz et al., 2009). 290 patients with suspected CAD were enrolled for this study. Exclusion criteria were acute coronary syndromes and non-cardiac diseases that could interfere with the analysis. A coronary angiogram was used to evaluate a CAD score for each patient as follows: (i) CAD excluded (n = 57); (ii) early CAD (n = 63); (iii) moderate CAD (n = 85); and (iv) advanced CAD (n = 85). In advanced CAD, a significant decrease in circulating pDCs (BDCA-2+ cells) was observed, with a significant inverse correlation observed between the CAD score and pDC, cDC and tDC numbers. Patients who required percutaneous coronary intervention or coronary artery bypass grafting had less circulating pDCs, cDCs and tDCs than controls. The decrease in DC numbers was an independent predictor of the presence of CAD when several risk factors (age, gender, diabetes, and hypertension) were included (Yilmaz et al., 2009). Similarly, the numbers of circulating pDCs (BDCA-2+) were reduced in CAD patients (n = 46) compared with control patients (n = 15); however no differences were observed between stable and unstable CAD groups (Van Vré et al., 2010). Interestingly, the use of β-blockers or lipid-lowering drugs was associated with increased cDCs, whereas pDCs were unaffected (Van Vré et al., 2010). Importantly, van Brussel et al. (2010) investigated whether the discrepancies in the results between different research groups were due to flow cytometric enumeration of pDC using BDCA-2 versus CD123 as a marker. A total of 10 adult healthy subjects that had no known cardiovascular risk factors were examined. In addition, 10 patients with stable angina pectoris and angiographically proven significant CAD (>50% lumen reduction in at least one coronary artery) participated in this study. 94.7 ± 1.0% of the pDCs were double positive for BDCA-2 and CD123, indicating that both pDC populations overlap significantly. Moreover, absolute and relative numbers of circulating pDCs were significantly decreased in CAD patients, irrespective of the subset marker (BDCA-2 or CD123). The expression of BDCA-2 and CD123 was also analyzed in vitro on fresh blood samples. The expression of BCDA-2 was significantly downregulated after stimulation with the TLR-7 ligand imiquimod, as already reported (Dzionek et al., 2000; Wu et al., 2008). Conversely, the expression of CD123 was significantly upregulated during TLR-triggered maturation of pDCs. To assess the activation state of circulating pDCs in healthy subjects and CAD patients, the authors compared their CD123/BDCA-2 ratio. No significant differences were found between both study groups, which suggests that the activation status of circulating pDCs from CAD patients was not raised as compared to healthy individuals (van Brussel et al., 2010). Overall these data suggest that the number of circulating pDCs decreases in coronary artery disease; however, the possibility of using blood pDC counts as independent predictors of future cardiovascular events requires further investigation. The net contribution of pDCs to atherosclerosis formation and development has been recently investigated in mouse models. 5. Understanding the function of plasmacytoid dendritic cells in experimental atherosclerosis The anatomical location of pDCs in mouse atherosclerotic arteries was investigated by immunohistochemistry. Few scattered pDCs (Siglec-H+) were detectable in the adventitia of carotid arteries subjected to collar-induced atherosclerosis in LDLr −/− mice (Daissormont et al., 2011). pDCs were scarce in the aortic sinus of standard chow diet fed apoE −/− mice (Daissormont et al., 2011; Döring et al., 2012) and few siglec-H + pDCs accumulated at the plaque shoulder and in the proximity of the necrotic core in the aortic sinus of apoE −/− mice fed a high fat diet (HFD) (Döring et al., 2012; Macritchie et al., 2012). In addition, mRNA transcripts of siglec-H and IFN-α were increased in apoE −/− mice fed HFD compared with apoE −/− on chow diet (Döring et al., 2012). Using flow cytometry, our lab examined total numbers of pDCs (CD11c lowB220 + PDCA-1+) in wild type controls (C57BL/6; WT) and apoE −/− mice on chow diet.

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pDCs comprised approximately 0.2% of the total aortic CD45+ leukocyte population in both non-inflamed wild type and atherosclerotic arteries. Similar numbers of aortic and splenic pDCs were observed between the two groups (Macritchie et al., 2012). In summary pDCs are present in both normal and inflamed aortae and accumulate primarily at the plaque shoulder in atherosclerotic/hyperlipidemic mice. Antibody-mediated pDC depletion has produced controversial results, with pDCs either reducing or promoting plaque formation. These conflicting data are mainly attributable to the use of different mouse models and blocking antibodies. Daissormont et al. (2011) reported that pDC depletion with a specific antibody (120 G8; Biceros) against BST-2, aggravated atherosclerotic lesion development in a particular model of carotid collar-induced atherosclerosis in LDLr−/− mice. Likewise, pDC depletion induced a less stable plaque phenotype (increased lesional T cell accumulation, necrotic core expansion and reduced cap VSMC content). Similarly, pDC depletion aggravated lesion formation in the aortic root of the same mice subjected to vascular injury (Daissormont et al., 2011). On the contrary, pDC depletion induced by using a different antibody against BST-2 (PDCA-1; Miltenyi Biotec) significantly decreased plaque formation and macrophage content in the aortic root of apoE−/− mice fed HFD (Döring et al., 2012). Notably, the two experimental approaches differ in a number of ways. The placement of a perivascular collar (Daissormont et al., 2011) is mostly employed for studying vascular response to arterial injury and the mechanical manipulation could explain, for example, the unusual location of pDCs observed in the adventitia of the injured carotid arteries. Whereas, the mouse model used by Döring et al. (2012) is the model of choice for investigating the role of pDCs in early atherosclerosis formation (Daugherty & Rateri, 2005), however the depletion antibody regime chosen by the investigators is debatable. Daissormont et al. (2011) reported a repopulation of pDCs at 72 h after antibody depletion and have therefore applied repetitive 120 G8 antibody injections four times a week throughout the study period (3.5 weeks). In contrast, Döring et al. (2012) injected the monoclonal antibody PDCA-1 (500 μg/mouse) only at days 1 and 7 during 32 days of HFD therefore, only partially depleting pDCs throughout the feeding experiment. By using the same antibody (PDCA-1; 250 μg/mouse), we demonstrated that pDCs were significantly depleted in the spleen, lymph nodes (LNs), blood and aorta of apoE−/− mice at 24 h after treatment. Depletion remained evident at 72 h in spleen, LNs and blood. Interestingly, while lymphoid, aortic and blood pDCs had returned to near control levels by day 5, the splenic pDC population was still less than 50% recovered. In order to continuously deplete pDCs and to limit the amount of antibody administered, we settled the frequency of dosing to every 5 days (Macritchie et al., 2012). We found that continuous pDC depletion also reduced atherosclerosis formation in the aortic sinus of apoE−/− mice fed HFD for 4 weeks, leading to a more stable plaque phenotype (Macritchie et al., 2012). Our data would therefore favor a detrimental role for pDCs in early lesion formation and are supported by the observation that the specific activation of pDCs and IFN-α treatment promoted plaque growth in apoE−/− mice, an effect abrogated by additional depletion of pDCs (Döring et al., 2012). Although informative, one limitation of antibody depletion studies is that the BST-2 Ag is expressed on pDCs and plasma cells in naive mice but is induced on most cell types after stimulation with IFN-I or IFN-γ (Blasius et al., 2006a; Swiecki et al., 2010). Therefore, pDC-depleting Abs could deplete or affect additional cell types (other than leukocytes) during chronic inflammation, thus confounding the interpretation of these studies. Alternative approaches are therefore needed to validate the role of pDCs in atherosclerosis. For example, the use of recently developed transgenic mouse models, such as the above mentioned Siglec-H-DTR mice (Takagi et al., 2011) or the BDCA-2-DTR mice (Swiecki et al., 2010), in which pDCs can be selectively depleted by injection of DT, could help to unequivocally evaluate the role of pDCs in lesion formation and development. However the multiple injections of diphtheria toxin required for long-term depletion

could represent a limitation to the application of this approach in experimental atherosclerosis. Mechanistic data emphasize the complexity of pDC functions in atherosclerosis. On one hand, pDCs have been shown to suppress T cell proliferation in an indoleamine 2,3-dioxygenase (IDO)-dependent manner in collar-induced atherosclerosis in LDLr−/− mice (Daissormont et al., 2011), suggesting a potential tolerogenic effect of pDCs in response to vascular injury, as already demonstrated in chronic inflammatory and autoimmune diseases (Arpinati et al., 2003; de Heer et al., 2004; Fallarino et al., 2005; Manches et al., 2008; Jongbloed et al., 2009; Nikolic et al., 2009; Mueller et al., 2010). Opposite results were obtained in the study of early lesion formation in apoE−/− mice where pDC depletion induced a robust systemic anti-inflammatory effect (Döring et al., 2012; Macritchie et al., 2012). T cell activation was decreased in the spleen of pDC-depleted mice compared to controls with splenic T cells from depleted mice producing significantly less TNF-α (Macritchie et al., 2012). Additionally, pDC depletion induced a systemic reduction of pro-atherosclerotic circulating mediators (Macritchie et al., 2012) such as IL-12, which is produced by DCs and monocytes/macrophages and plays a critical role in Th1 polarization (Ait-Oufella et al., 2011). Similarly, pDC ablation caused a reduction in CXCL1 (Zhou et al., 2011), MIG/CXCL9, IP-10/CXCL10, IFN-γ-induced chemokines which play important roles in the vascular recruitment of immune cells in atheromas (Mach et al., 1999), and vascular endothelial growth factor (VEGF), a potent angiogenic factor involved in atherosclerosis (Couffinhal et al., 1997; Trapé et al., 2006). Despite the observed contribution of pDCs to atherosclerosis, we demonstrated that lymphoid, splenic and aortic pDCs showed a similar level of activation between WT and apoE −/− mice. CD40 and CD86 were detectable on a minority of aortic pDCs indicating that the majority were in an immature state (Macritchie et al., 2012). Although these data are somewhat surprising, similar results were recently obtained in vitro (Döring et al., 2012). Intriguingly, oxidized low density lipoprotein (ox-LDL) enhanced phagocytosis by sorted pDCs and their capacity to prime antigen-specific T cell responses, while not altering surface expression of MHC-II and CD86, nor secretion of IFN-αβ (Döring et al., 2012). The enhanced phagocytic capacity upon exposure to oxLDL may boost uptake of antigenic complexes by pDCs and their activation in atherosclerosis. Interestingly, we demonstrated that aortic pDCs from apoE −/− mice were able to uptake and present antigen in the context of MHC-II in vivo. In contrast, aortic pDCs from WT mice were not, or at least less, capable of presenting systemically administered antigen (Macritchie et al., 2012). Our results highlight an important functional difference in pDCs between mice with and without atherosclerosis. pDCs could play a key role as APCs in situ in the atherosclerotic vessel where they may be exposed to several disease-related stimuli including viral antigens (Epstein et al., 2000) and modified-self nucleic acid complexes that may be released from dying cells (Gilliet et al., 2008; Döring et al., 2012), contributing to the breakdown of tolerance to self-DNA (Lovgren et al., 2004; Means et al., 2005; Lande et al., 2007). Of relevance is the fact that pDCs have recently been shown to specifically promote anti-dsDNA antibody formation in early atherosclerosis (Döring et al., 2012), and Cramp (an antimicrobial peptide released from neutrophils)/ self-DNA complex-mediated pDC activation drives atherosclerotic plaque development (Döring et al., 2012). Collectively, these data support the hypothesis that pDCs are sensors of self-DNA complexes derived from damaged cells that accumulate after vessel injury and that they could orchestrate the response of the immune system to vessel damage. The cytokine-mediated effect of pDCs through the release of IFN-α does not seem to play a pivotal role in experimental atherosclerosis. Atherogenic stimuli do not induce pDC activation with baseline plasma IFN-α levels remaining unchanged under conditions of hyperlipidemia or after pDC depletion (Daissormont et al., 2011). Furthermore, IFN-α

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was below the level of detection in both aortic tissue and serum samples in HFD fed apoE−/− mice irrespective of pDC depletion (Macritchie et al., 2012). Only Döring et al. (2012) observed a reduction of IFN-α serum levels in apoE−/− mice depleted of pDCs compared to isotype-injected controls, with levels of circulating IFN-α just above the assay detection limit. These data suggest that large increases in IFN-α production are unlikely to be responsible for the pDC-mediated effect in experimental atherosclerosis and are in line with the fact that activated murine pDCs secrete large amounts of IL-12 but relatively low levels of type I IFNs (Reizis et al., 2011). Although the current data regarding the role of pDCs in mouse models of atherosclerosis are controversial, they clearly show that pDCs demonstrate significant plasticity, with the capacity to produce high levels of cytokines and activate the adaptive immune system on one hand, and to regulate inflammation by inhibiting effector T cell responses on the other. New studies are required to understand how increased levels of signals from the plaque core may affect DC functions during atherogenesis and whether antigen-presenting functions of pDCs will be a key component in the regulation of the atherosclerotic immune response. Clarification of these points would also be fundamental for a better understanding of how pDCs could influence co-morbidity between autoimmune and cardiovascular disease.

6. How plasmacytoid dendritic cells could influence co2morbidity between autoimmune and cardiovascular disease It is over 35 years since the first association between autoimmune diseases and increased cardiovascular mortality was made with the pivotal discovery that mortality in patients with late-stage SLE was mostly due to AMI secondary to atherosclerotic heart disease (Urowitz et al., 1976). A number of studies support the concept that an increase in inflammatory markers in SLE patients correlates with increased atherosclerotic burden. These include C-reactive protein and fibrinogen (Manzi et al., 1999), IL-6 and MCP-1 (Asanuma et al., 2006), TNF-α (Svenungsson et al., 2003; Rho et al., 2008), VCAM-1 and CD40L (Lee et al., 2006). As the major producers of IFN-α, pDCs have come under scrutiny as potential inducers of tissue destructive autoimmunity. A role for type I IFNs (IFN-α and IFN-β) in the pathophysiology of SLE was first suggested in the 1970s when elevated levels of IFN-α were detected in the serum of SLE patients and positively correlated with disease severity (Hooks et al., 1979). Further studies revealed that IFN-α induced a global induction of type I IFN-responsive genes in a subgroup of SLE patients, which was associated with a more progressive disease phenotype (Kirou et al., 2004, 2005). Elevated levels of IFN-α have been implicated in the vascular endothelial cell injury that precedes atherosclerosis. A loss of vascular integrity in SLE arises from an acceleration of endothelial cell apoptosis (Rajagopalan et al., 2004) and a reduction in vascular repair. Vasculogenesis depends, in part, on the contribution of bone marrow-derived endothelial precursor cells (EPCs) and circulating angiogenic cells (CACs), both of which are impaired in SLE patients (Westerweel et al., 2007; Moonen et al., 2007; Grisar et al., 2008). High serum type I IFN levels are associated with a reduction in EPCs and enhanced endothelial dysfunction in SLE patients (Lee et al., 2007) whereas Ab-mediated blockade of IFN-α restores the normal angiogenic phenotype of EPCs/CACs (Denny et al., 2007). Additionally, IFN-α was found to promote foam cell formation through enhancement of ox-LDL uptake via macrophage scavenger receptors (Li et al., 2011) and a type I interferon gene signature in platelets has been associated with their aberrant activation in SLE patients (Lood et al., 2010). Importantly, Sifalimumab, a monoclonal Ab inhibitor of IFN-α, has been tested in clinical trials and shown to be effective at reducing the severity of SLE while being generally well tolerated (Merrill et al., 2011).

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While the precise mechanism responsible for the increased levels of IFN-α in SLE is not known, evidence strongly points towards DNA containing immune complexes, which can activate pDCs via TLR9 to release large quantities of IFN-α (Bave et al., 2001; Means et al., 2005). Of particular interest is the recent data showing that neutrophil-derived antimicrobial peptides and self-DNA are released from neutrophils as web-like structures called neutrophil extracellular traps (NETs), detectable in the sera of SLE patients and potent activators of pDCs (Lande et al., 2011). NETs have been recently identified in murine atherosclerotic arteries where they were shown to promote atherosclerosis through pDC activation and subsequent IFN-α release (Döring et al., 2012). This may be indicative of a converging mechanism of action whereby pDCs chronically activated by extracellular DNA-Ab immune complexes secret IFN-α and promote systemic autoimmunity and atherosclerotic lesion formation. Rheumatoid Arthritis, the most prevalent autoimmune condition, also increases the likelihood of CVD (van Zonneveld et al., 2010; Full & Monaco, 2011; Kitas & Gabriel, 2011). In contrast to the well established role of type I IFNs in the pathology of SLE, their role in RA is more controversial. A subgroup of RA patients express systemic upregulation of type I IFN-regulated genes (van der Pouw Kraan et al., 2007; Higgs et al., 2012) and autoantibody positive patients with athralgia, who displayed a type I IFN gene signature, were at a greater risk of advancing to RA (van Baarsen et al., 2010). Additional evidence for a causative role for IFN-α in RA comes from case reports of patients receiving IFN-α therapy for other conditions who subsequently developed RA with symptoms reversible on cessation of treatment (Passos de Souza et al., 2001; Ionescu et al., 2008; Izumi et al., 2011). RA patients have reduced numbers of pDCs in peripheral blood while their numbers at the site of inflammation are increased, most likely through chemotactic migration from the circulation (Van Krinks et al., 2004; Lande et al., 2004). pDCs that have migrated into the synovial tissue show surface marker changes consistent with maturation and these cells co-localize with IFN-α-induced proteins, implicating pDCs as local producers of type I IFNs (Lande et al., 2004). In support of this view, pDCs migrating into the synovial tissue of RA patients actively produce IFN-α and IFN-β. Strikingly, the number of pDCs accumulating within synovial tissue outnumbers cDCs and they are frequently found in aggregates with lymphocytes including T cells, CD19+ B cells and CD38+ plasma cells, suggesting possible regulation of autoantibody formation (Lande et al., 2004). Indeed, pDC numbers were higher in rheumatoid factor (RF)-positive and anti-citrullinated protein antibodies (ACPA)-positive patients and positively correlated with serum ACPA levels consistent with a pro-inflammatory phenotype (Lebre et al., 2008). IFN-α in synovial tissue may provide a key link between innate and adaptive immunity. Roelofs et al. (2009) demonstrated co-expression of IFN-α with TLR3/7 in the synovial tissue of RA patients and IFN-α released following TLR3/7 stimulation augments TLR4 signaling on peripheral blood mononuclear cells and synovial fibroblasts, thereby increasing release of pro-inflammatory cytokines including TNF-α. TNF-α, which plays a pivotal role in the tissue destructive process in RA (Feldmann & Maini, 2003), is also strongly pro-atherogenic and blockade of TNF-α reduces atherosclerosis in animal models (Branen et al., 2004). Upregulation of TNF-α in RA may contribute to the formation of atherosclerosis and evidence from clinical studies offers evidence to support this hypothesis. Anti-TNF-α therapies in RA patients improves endothelial function (Hurlimann et al., 2004; Gonzalez-Juanatey et al., 2004), reduces carotid arterial intima-media thickness (Del Porto et al., 2007) and reduces the likelihood of CVD events (Jacobsson et al., 2005; McKellar et al., 2009). Intriguingly, TNF-α levels in atherosclerotic plaques closely correlate with plaque IFN-α levels where IFN-α performs a similar function to that described above in synovial tissue, namely an amplification of TLR4 signaling on monocytes/cDCs and release of pro-inflammatory cytokines including TNF-α which contribute to plaque destabilization (Niessner et al., 2007). Intriguingly, pDCs and type I IFNs have also

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been postulated to have anti-inflammatory effects in RA. In contrast to IFN-α, IFN-β appears to have protective anti-inflammatory properties in RA patients and has undergone clinical trials with mixed results to date (Conigliaro et al., 2010; Crow, 2010). An additional immunosuppressive role for pDCs in RA has been suggested by a study utilizing a breach of tolerance model of arthritis whereby pDC depletion was associated with a worsening of joint inflammation, expansion of antigen-specific T cells and an increase in autoantibody titers (Jongbloed et al., 2009). This possible tolerogenic role of pDCs in arthritis may suggest a dichotomy in pDC function, whereby pDCs can stimulate adaptive immunity and autoantibody formation through IFN-α, or act to promote tolerance through inhibition of T-effector function or induction of Tregs. These apparent contradictory effects ascribed to pDCs and type I IFNs in the pathology of RA will require further clarification and should provide valuable data not just on the role of these systems in RA but also in the accompanying vascular pathology. Circulating pDC numbers are also increased at the onset of type 1 diabetes; however serum IFN-α levels and IFN-α release from isolated pDCs following TLR-9 stimulation was unchanged in diabetic

patients (Allen et al., 2009). Using an in vitro culture system, the authors were able to demonstrate that pDCs can present islet autoantigens to T cells in the presence of autoantibody containing sera from diabetic patients. The enhanced antigen presentation was not associated with IFN-α release (Allen et al., 2009). This offers a novel mechanism of action in the context of T1D whereby pDCs can acquire and present β-cell autoantigen complexes to autoreactive T-cells. The role of type I IFNs in T1D in humans is unclear (Fabris et al., 2003; Rother et al., 2009; Yamazaki et al., 2010). The lack of consensual data on whether pDCs and type I IFN are pro-inflammatory in T1D leaves the question of whether they contribute to the atherosclerotic comorbidity unanswered. 7. Concluding remarks and potential therapeutic immunomodulation This review summarizes recent evidence showing the contribution of pDCs to atherosclerosis (Fig. 1). The presence of pDCs in the human atherosclerosis plaque is well documented and the number

Fig. 1. pDCs in atherosclerosis. pDCs accumulate at the plaque shoulder and in the proximity of the necrotic core. They are also detectable in the adventitia of arteries subjected to injury. (A) pDCs activated by nucleic acids of viral, bacterial or self-origin via TLR7 and TLR9 produce IFN-α. IFN-α could induce the expression of TRAIL on CD4+ T cells causing apoptosis of VSMCs and ECs, fibrous cap thinning and plaque destabilization. In addition, through the production of type 1 IFNs, pDCs drive NK cell cytolytic activity, maturation of monocyte-derived DCs, expansion of CD8+ cytotoxic T cells and Th1 differentiation. (B) pDCs, directly or via activation of other immune cells, produce large amounts of pro-inflammatory/atherosclerotic mediators such as CXCL10, and CCL3,4 and 5, IL-12, CXCL1, MIG and VEGF which can augment vascular inflammation by recruiting activated leukocytes. (C) pDCs produce the cytotoxic serine protease granzyme B and IDO, which have been described to be involved in the suppression of effector T cell responses. On the other hand, ox-LDL enhance phagocytosis of pDCs, and their capacity to prime antigen-specific T cell responses. (D) pDC-derived type 1 IFNs and IL-6 induce B cells to undergo isotype-switching and to mature into antibody-secreting cells.

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Table 1 pDCs in atherosclerosis: key findings. pDCs are present in human plaques pDC number increases in unstable plaques Same number of pDCs in stable vs unstable plaques Number of circulating pDCs decreases in CAD patients compared to controls Same number of circulating pDCs in CAD patients compared to controls pDCs inhibit injury-induced lesion formation in LDLr−/− mice pDCs aggravate early lesion formation in apoE−/− mice

of circulating pDCs decreases in coronary artery disease. pDCs contribute to early lesion formation in mouse models, but have also been shown to protect against injury-induced atherosclerosis (Table 1). Current data clearly show that pDCs display a high degree of plasticity in vascular pathology with the capacity to produce high levels of cytokines and activate the adaptive immune system on one hand and to regulate inflammation by inhibiting effector T cell responses on the other. A detailed understanding of the mechanisms triggering inflammatory versus protective immune responses by pDCs in the context of atherosclerosis, and a better characterization of pDCs and their potential subsets in steady state and during lesion evolution should be an important objective in future research. Crucial to our understanding of pDC function in the context of co-morbidity between autoimmunity and CVD will be unraveling the nature of pDC activation by disease-specific stimuli, how the location and activation state of pDCs affects their functional properties and the respective contribution of pDC-dependent antigen presentation and type I IFN release to disease pathology. Importantly, pDCs play very specific roles in immune processes and for this reason, they may turn out to be effective therapeutic targets. Blocking antibodies specific for IFN-α (sifalimumab and rontalizumab) are currently being tested as potential treatments in SLE (Moldovan & Katsaros, 2012). Given the experimental evidence implicating IFN-α in the vascular damage associated with SLE and the recent demonstration that type I IFN aggravates experimental atherosclerosis (Goossens et al., 2010), it would be important that clinical studies would include appropriate evaluation of these compounds on vascular dysfunction. Intriguingly, inhibition of IFN-α signaling may explain the dual role that statins have in ameliorating SLE and improving endothelial function, an effect that is independent of circulating lipid levels (Ferreira et al., 2007). Simvastatin and pitavastatin reduce IFN-α release from pDCs derived from SLE patients and from pDCs from healthy subjects that have been treated with SLE serum (Amuro et al., 2010). Functional blockade or antibody-mediated ablation of pDCs may provide an attractive alternative to blocking type I IFNs, without impairing antiviral defenses in general. For example, antagonists of TLR7 and TLR9 inhibits SLE in animal models (Hennessy et al., 2010) and have recently entered clinical trials (NCT00547014) with the aim to reduce chronic IFN production by pDCs (Hennessy et al., 2010; Reizis et al., 2011). Finally, the ability of pDCs to induce immune tolerance might have an important role in preventing vascular inflammation. So far several attempts to treat atherosclerosis have involved modulating cDC function (Cheong & Choi, 2012). The administration of DCs pulsed with oxidized low density lipoprotein to LDLr −/− mice reduced atherosclerosis and increased plaque stability (Habets et al., 2010). More recently, the injection of IL-10 was found to induce tolerogenic DCs loaded with apolipoprotein B100, which attenuated atherosclerosis by inducing the generation of antigen-specific Treg cells (Hermansson et al., 2011). It would be interesting to test the efficacy of pDCs in similar experimental approaches, especially in view of recent data showing that Ag delivery to pDCs can be harnessed to inhibit T cell-mediated immunity (Loschko et al., 2011). As these studies progress we can

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