Sara McArdle

Dynamic T cell–APC interactions sustain chronic inflammation in atherosclerosis

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries characterized by leukocyte accumulation in the vessel wall. Both innate and adaptive immune responses contribute to atherogenesis, but the identity of atherosclerosis-relevant antigens and the role of antigen presentation in this disease remain poorly characterized. We developed live-cell imaging of explanted aortas to compare the behavior and role of APCs in normal and atherosclerotic mice. We found that CD4+ T cells were capable of interacting with fluorescently labeled (CD11c-YFP+) APCs in the aortic wall in the presence, but not the absence, of cognate antigen. In atherosclerosis-prone Apoe-/-CD11c-YFP+ mice, APCs extensively interacted with CD4+ T cells in the aorta, leading to cell activation and proliferation as well as secretion of IFN-γ and TNF-α. These cytokines enhanced uptake of oxidized and minimally modified LDL by macrophages. We conclude that antigen presentation by APCs to CD4+ T cells in the arterial wall causes local T cell activation and production of proinflammatory cytokines, which promote atherosclerosis by maintaining chronic inflammation and inducing foam cell formation.

Patrolling monocytes control tumor metastasis to the lung

Monocytes block tumor access to the lung Metastatic cancer is especially hard to treat. In order to find potential new therapeutic targets, scientists are trying to understand the cellular events that promote or prevent metastasis. Hanna et al. now report a role for patrolling monocytes in blocking tumor metastasis to the lungs in mice. Tumors in mice engineered to lack patrolling monocytes showed increased metastasis to the lung but not to other tissues. Patrolling monocytes resided in the microvasculature of the lung, where they engulfed tumor material, which may explain how these cells prevent tumors from colonizing the lung. Science, this issue p. 985 An immune cell subset called patrolling monocytes prevents tumor lung metastasis in mice. The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical “patrolling” monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

Endothelial Protective Monocyte Patrolling in Large Arteries Intensified by Western Diet and AtherosclerosisNovelty and Significance

Rationale: Nonclassical mouse monocyte (CX3CR1high, Ly-6Clow) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. Objective: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. Methods and Results: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 &mgr;m/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1+/GFP/apoE−/− mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and &agr;4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1−/− apoE−/− mice leads to pronounced endothelial apoptosis. Conclusions: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.

CCR5+T-bet+FoxP3+ Effector CD4 T Cells Drive Atherosclerosis

RATIONALE CD4 T cells are involved in the pathogenesis of atherosclerosis, but atherosclerosis-specific CD4 T cells have not been described. Moreover, the chemokine(s) that regulates T-cell trafficking to the atherosclerotic lesions is also unknown. OBJECTIVE In Apoe(-/-) mice with mature atherosclerotic lesions (5 months of high fat diet), we find that most aortic T cells express CCR5 and interferon-γ with a unique combination of cell surface markers (CD4(+)CD25(-)CD44(hi)CD62L(lo)) and transcription factors (FoxP3(+)T-bet(+)). We call these cells CCR5Teff. We investigated the role of CCR5 in regulating T-cell homing to the atherosclerotic aorta and the functionality of the CCR5Teff cells. METHODS AND RESULTS CCR5Teff cells are exclusively found in the aorta and para-aortic lymph nodes of Apoe(-/-) mice. They do not suppress T-cell proliferation in vitro and are less potent than regulatory T cells at inhibiting cytokine secretion. Blocking or knocking out CCR5 or its ligand CCL5 significantly blocks T-cell homing to atherosclerotic aortas. Transcriptomic analysis shows that CCR5Teff cells are more similar to effector T cells than to regulatory T cells. They secrete interferon-γ, interleukin-2, interleukin-10, and tumor necrosis factor. Adoptive transfer of these CCR5Teff cells significantly increases atherosclerosis. CONCLUSIONS CCR5 is specifically needed for CD4 T-cell homing to the atherosclerotic plaques. CCR5(+)CD4 T cells express an unusual combination of transcription factors, FoxP3 and T-bet. Although CCR5Teff express FoxP3, we showed that they are not regulatory and adoptive transfer of these cells exacerbates atherosclerosis.

Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis

Ley et al. provide a review of the technology and accomplishments of dynamic imaging of myeloid cells in atherosclerosis.

Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces peritoneal IL-10-producing CD4 T cells

Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4+ T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-Ab by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model (Apoe-/- mice on Western diet). Immunization with each of the three peptides (1 time in complete Freunds adjuvant subcuntaneously and 4 time in incomplete Freunds adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4+ T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3+ regulatory CD4+ T cells and more than tripled the number of CCR5+FoxP3+ cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection.NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4+ T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.

Registering sequences of in vivo microscopy images for cell tracking using dynamic programming and minimum spanning trees

Registration of in vivo microscopy image sequences is important for tracking of cells. Registering a long sequence of in vivo microscopy images is particularly challenging for several reasons, which include motion artifacts created by the cardiac cycle and breathing movements of the living subject, occasional defocussing, illumination change, and noise in image acquisition. To accommodate these variations, we sample time points redundantly during microscopic image acquisition. Second, we use dynamic programming to select image frames with tolerable motion and eliminate those with large motion. Third, we employ a novel method based on the minimum spanning tree algorithm to register the selected image frames. Testing on actual in vivo image sequences reveals that our approach excels over three existing registration methods in terms of structural image similarity of the registered images.

Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues

BackgroundIntravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice.MethodsWe generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. ResultsDARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues.ConclusionsImmunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.

MISTICA: Minimum Spanning Tree-Based Coarse Image Alignment for Microscopy Image Sequences

Registration of an in vivo microscopy image sequence is necessary in many significant studies, including studies of atherosclerosis in large arteries and the heart. Significant cardiac and respiratory motion of the living subject, occasional spells of focal plane changes, drift in the field of view, and long image sequences are the principal roadblocks. The first step in such a registration process is the removal of translational and rotational motion. Next, a deformable registration can be performed. The focus of our study here is to remove the translation and/or rigid body motion that we refer to here as coarse alignment. The existing techniques for coarse alignment are unable to accommodate long sequences often consisting of periods of poor quality images (as quantified by a suitable perceptual measure). Many existing methods require the user to select an anchor image to which other images are registered. We propose a novel method for coarse image sequence alignment based on minimum weighted spanning trees (MISTICA) that overcomes these difficulties. The principal idea behind MISTICA is to reorder the images in shorter sequences, to demote nonconforming or poor quality images in the registration process, and to mitigate the error propagation. The anchor image is selected automatically making MISTICA completely automated. MISTICA is computationally efficient. It has a single tuning parameter that determines graph width, which can also be eliminated by the way of additional computation. MISTICA outperforms existing alignment methods when applied to microscopy image sequences of mouse arteries.

Selecting the Optimal Sequence for Deformable Registration of Microscopy Image Sequences Using Two-Stage MST-based Clustering Algorithm

We developed and implemented a novel two-stage Minimum Spanning Tree (MST)-based clustering method for deformable registration of microscopy image sequences. We first construct a MST for the input image sequence. MST mitigates the registration error propagation of time sequenced images by re-ordering the images in such a way where poor quality images appear at the end of the sequence. Then MST is clustered into several groups based on the similarity of the images. After that an optimal anchor image is selected automatically for each group through an iterative assessment of entropy and MSE based coarse registration error and the local deformable registration is performed within each group separately. Subsequently coarse registration is conducted to find the global anchor image selected among the whole time sequenced images and then a deformable registration is conducted on the whole sequence. Two-stage MST-based deformable registration algorithm can incorporate larger drifts and distortions more accurately than conventional one shot registration algorithm by fine-tuning the larger amount of deformation incrementally in a couple of stages. Our method outperforms other methods on both 2D and 3D in vivo microscopy image sequences of mouse arteries used in atherosclerosis study.