Publications by authors named "Coleen A McNamara"

68 Publications

Identification of human immune cell subtypes most responsive to IL-1β-induced inflammatory signaling using mass cytometry.

Sci Signal 2021 03 9;14(673). Epub 2021 Mar 9.

Carter Immunology Center, University of Virginia, Charlottesville, VA 22908, USA.

IL-1β is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1β blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4CD8CD161 T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1β. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16CD56CD161 and CD16CD56CD161), and lineage (Lin) cells expressing CD161 and CD25. IL-1β also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1β stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1β-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1β in CCR6 T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1β-neutralizing therapies.
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http://dx.doi.org/10.1126/scisignal.abc5763DOI Listing
March 2021

Chemokine Receptor-6 Promotes B-1 Cell Trafficking to Perivascular Adipose Tissue, Local IgM Production and Atheroprotection.

Front Immunol 2021 19;12:636013. Epub 2021 Feb 19.

Carter Immunology Center, University of Virginia, Charlottesville, VA, United States.

Chemokine receptor-6 (CCR6) mediates immune cell recruitment to inflammatory sites and has cell type-specific effects on diet-induced atherosclerosis in mice. Previously we showed that loss of CCR6 in B cells resulted in loss of B cell-mediated atheroprotection, although the B cell subtype mediating this effect was unknown. Perivascular adipose tissue (PVAT) harbors high numbers of B cells including atheroprotective IgM secreting B-1 cells. Production of IgM antibodies is a major mechanism whereby B-1 cells limit atherosclerosis development. Yet whether CCR6 regulates B-1 cell number and production of IgM in the PVAT is unknown. In this present study, flow cytometry experiments demonstrated that both B-1 and B-2 cells express CCR6, albeit at a higher frequency in B-2 cells in both humans and mice. Nevertheless, B-2 cell numbers in peritoneal cavity (PerC), spleen, bone marrow and PVAT were no different in compared to mice. In contrast, the numbers of atheroprotective IgM secreting B-1 cells were significantly lower in the PVAT of compared to mice. Surprisingly, adoptive transfer (AT) of CD43 splenic B cells into B cell-deficient μ mice repopulated the PerC with B-1 and B-2 cells and reduced atherosclerosis when transferred into mice only when those cells expressed both CCR6 and sIgM. CCR6 expression on circulating human B cells in subjects with a high level of atherosclerosis in their coronary arteries was lower only in the putative human B-1 cells. These results provide evidence that B-1 cell CCR6 expression enhances B-1 cell number and IgM secretion in PVAT to provide atheroprotection in mice and suggest potential human relevance to our murine findings.
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http://dx.doi.org/10.3389/fimmu.2021.636013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933012PMC
February 2021

Quantitative Measurement of IgG to Severe Acute Respiratory Syndrome Coronavirus-2 Proteins Using ImmunoCAP.

Int Arch Allergy Immunol 2021 Feb 23:1-8. Epub 2021 Feb 23.

Division of Allergy & Clinical Immunology, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA,

Background: Detailed understanding of the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the cause of coronavirus disease 2019 (CO-VID-19) has been hampered by a lack of quantitative antibody assays.

Objective: The objective was to develop a quantitative assay for IgG to SARS-CoV-2 proteins that could be implemented in clinical and research laboratories.

Methods: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding domain (RBD) or nucleocapsid protein to the solid phase of the ImmunoCAP. Plasma and serum samples from patients hospitalized with COVID-19 (n = 60) and samples from donors banked before the emergence of COVID-19 (n = 109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform.

Results: At a cutoff of 2.5 μg/mL, the assay demonstrated sensitivity and specificity exceeding 95% for IgG to both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 μg/mL (IQR 18-52) and 24.5 μg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples, there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 μg/mL, or 1% of total IgG.

Conclusions: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to mutated COVID-19 proteins, has good performance characteristics, and has a readout in standardized units.
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http://dx.doi.org/10.1159/000514203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018212PMC
February 2021

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

Arterioscler Thromb Vasc Biol 2020 Dec 31:ATVBAHA120315588. Epub 2020 Dec 31.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville. (J.C.G., J.M.H., D.B.H., V.O., C.M., M.A.M., C.A.M.).

Objective: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed mice and circulating levels of HA were elevated. mRNA expression was increased in epididymal AT of mice. Luciferase promoter assays showed that Id3 suppressed promoter activity, while loss of stimulated promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of mice, an effect sensitive to hyaluronidase.

Conclusions: Our data demonstrate that loss of increases expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.
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http://dx.doi.org/10.1161/ATVBAHA.120.315588DOI Listing
December 2020

Quantitative measurement of IgG to SARS-CoV-2 proteins using ImmunoCAP.

medRxiv 2020 Nov 12. Epub 2020 Nov 12.

Background: Detailed understanding of the immune response to SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), has been hampered by a lack of quantitative antibody assays.

Objective: To develop a quantitative assay for IgG to SARS-CoV-2 proteins that could readily be implemented in clinical and research laboratories.

Methods: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding-domain (RBD) or nucleocapsid protein to the solid-phase of the ImmunoCAP resin. Plasma and serum samples from patients with COVID-19 (n=51) and samples from donors banked prior to the emergence of COVID-19 (n=109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform.

Results: Performance characteristics demonstrated 100% sensitivity and 99% specificity at a cut-off level of 2.5 µg/mL for both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG.

Conclusions: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to novel antigens, has good performance characteristics and a read-out in standardized units.
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http://dx.doi.org/10.1101/2020.11.09.20228411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668760PMC
November 2020

Naive CD8 T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

Arterioscler Thromb Vasc Biol 2020 12 15;40(12):2845-2859. Epub 2020 Oct 15.

Division of Inflammation Biology, La Jolla Institute for Immunology, CA (L.E.P., H.Q.D., R.W., D.E.G., D.J.A., H.W., C.C.H.).

Objective: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4 T cells have been extensively studied in CVD, the importance of CD8 T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8 T (T) cell population expressing CD95 (termed CD95CD8 stem cell memory T [CD8 T] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95 cells within the T compartment. We found that CD8 T cells positively correlated with CVD risk in humans, while CD8 T cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE) mice also displayed respective 7- and 2-fold increases in CD8 T frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8 T cells were 1.7-fold increased in aortas from western diet fed ApoE mice compared with normal laboratory diet-fed ApoE mice. Importantly, transfer of T cells into immune-deficient recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis.

Conclusions: CD8 T cells are increased in humans with high CVD. As these T cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.
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http://dx.doi.org/10.1161/ATVBAHA.120.315106DOI Listing
December 2020

Pre-operative aerobic exercise on metabolic health and surgical outcomes in patients receiving bariatric surgery: A pilot trial.

PLoS One 2020 2;15(10):e0239130. Epub 2020 Oct 2.

Department of Kinesiology, University of Virginia, Charlottesville, Virginia, United States of America.

Objective: Examine if adding aerobic exercise to standard medical care (EX+SC) prior to bariatric surgery improves metabolic health in relation to surgical outcomes.

Methods: Fourteen bariatric patients (age: 42.3±2.5y, BMI: 45.1±2.5 kg/m2) met inclusion criteria and were match-paired to pre-operative SC (n = 7) or EX+SC (n = 7; walking 30min/d, 5d/wk, 65-85% HRpeak) for 30d. A 120min mixed meal tolerance test was performed pre- and post-intervention (~2d prior to surgery) to assess insulin sensitivity (Matsuda Index) and metabolic flexibility (indirect calorimetry). Aerobic fitness (VO2peak), body composition (BodPod), and adipokines (adiponectin, leptin) were also measured. Omental adipose tissue was collected during surgery to quantify gene expression of adiponectin and leptin, and operating time and length of hospital stay were recorded. ANOVA and Cohen's d effect size (ES) was used to test group differences.

Results: SC tended to increase percent body fat (P = 0.06) after the intervention compared to EX+SC. Although SC and EX+SC tended to raise insulin sensitivity (P = 0.11), EX+SC enhanced metabolic flexibility (P = 0.01, ES = 1.55), reduced total adiponectin (P = 0.01, ES = 1.54) with no change in HMW adiponectin and decreased the length of hospital stay (P = 0.05) compared to SC. Albeit not statistically significant, EX+SC increased VO2peak 2.9% compared to a 5.9% decrease with SC (P = 0.24, ES = 0.91). This increased fitness correlated to shorter operating time (r = -0.57, P = 0.03) and length of stay (r = -0.58, P = 0.03). Less omental total adiponectin (r = 0.52, P = 0.09) and leptin (r = 0.58, P = 0.05) expression correlated with shorter operating time, and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01), and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01).

Conclusion: Adding pre-operative aerobic exercise to standard care may improve surgical outcomes through a fitness and adipose tissue derived mechanism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239130PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531806PMC
November 2020

Preparation, Administration, and Assessment of In vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes.

J Vis Exp 2020 07 30(161). Epub 2020 Jul 30.

Robert M. Berne Cardiovascular Research Center, University of Virginia; Department of Medicine, Division of Cardiovascular Medicine, University of Virginia.

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.
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http://dx.doi.org/10.3791/61585DOI Listing
July 2020

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

Circulation 2020 Nov 17;142(21):2045-2059. Epub 2020 Jul 17.

Robert M. Berne Cardiovascular Research Center (G.F.A., K.M.O, S.K., A.N., C.M.W., S.S., C.A.H., R.H., R.A.B., C.A.M., E.R.Z., G.K.O.), University of Virginia, Charlottesville.

Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability.

Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis.

Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11, Lgals3 population with a chondrocyte-like gene signature that was markedly reduced with SMC- knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization.

Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682794PMC
November 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 Jul 16;127(3):402-426. Epub 2020 Jul 16.

La Jolla Institute for Immunology, CA (C.C.H., Y.G., H.Q.D., K.L.).

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371244PMC
July 2020

B Lymphocytes and Adipose Tissue Inflammation.

Arterioscler Thromb Vasc Biol 2020 05 5;40(5):1110-1122. Epub 2020 Mar 5.

From the Cardiovascular Research Center, Cardiovascular Division, Department of Medicine, University of Virginia, Charlottesville.

The immune system plays an important role in obesity-induced adipose tissue inflammation and the resultant metabolic dysfunction, which can lead to hypertension, dyslipidemia, and insulin resistance and their downstream sequelae of type 2 diabetes mellitus and cardiovascular disease. While macrophages are the most abundant immune cell type in adipose tissue, other immune cells are also present, such as B cells, which play important roles in regulating adipose tissue inflammation. This brief review will overview B-cell subsets, describe their localization in various adipose depots and summarize our knowledge about the function of these B-cell subsets in regulating adipose tissue inflammation, obesity-induced metabolic dysfunction and atherosclerosis.
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http://dx.doi.org/10.1161/ATVBAHA.119.312467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398177PMC
May 2020

liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects.

Theranostics 2020 1;10(2):585-601. Epub 2020 Jan 1.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia, 22908, USA.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an model of obesity-associated dysmetabolism. : Male leptin-deficient () mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. : Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. : These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations . These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.
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http://dx.doi.org/10.7150/thno.36572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929996PMC
January 2020

2019 Russell Ross Memorial Lecture in Vascular Biology: B Lymphocyte-Mediated Protective Immunity in Atherosclerosis.

Arterioscler Thromb Vasc Biol 2020 02 19;40(2):309-322. Epub 2019 Dec 19.

From the Robert M. Berne Cardiovascular Research Center (A.U., C.A.M.), University of Virginia School of Medicine, Charlottesville.

Atherosclerosis-the major underlying pathology of cardiovascular disease-is characterized by accumulation and subsequent oxidative modification of lipoproteins within the artery wall, leading to inflammatory cell infiltration and lesion formation that can over time result in arterial stenosis, ischemia, and downstream adverse events. The contribution of innate and adaptive immunity to atherosclerosis development is well established, and B cells have emerged as important modulators of both pro- and anti-inflammatory effects in atherosclerosis. Murine B cells can broadly be divided into 2 subsets: (1) B-2 cells, which are bone marrow derived and include conventional follicular and marginal zone B cells, and (2) B-1 cells, which are largely fetal liver derived and persist in adults through self-renewal. B-cell subsets are developmentally, functionally, and phenotypically distinct with unique subset-specific contributions to atherosclerosis development. Mechanisms whereby B cells regulate vascular inflammation and atherosclerosis will be discussed with a particular emphasis on B-1 cells. B-1 cells have a protective role in atherosclerosis that is mediated in large part by IgM antibody production. Accumulating evidence over the last several years has pointed to a previously underappreciated heterogeneity in B-1 cell populations, which may have important implications for understanding atherosclerosis development and potential targeted therapeutic approaches. This heterogeneity within atheroprotective innate B-cell subsets will be highlighted.
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http://dx.doi.org/10.1161/ATVBAHA.119.313064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398219PMC
February 2020

Importance of thorough tissue and cellular level characterization of targeted drugs in the evaluation of pharmacodynamic effects.

PLoS One 2019 14;14(11):e0224917. Epub 2019 Nov 14.

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, United States of America.

Targeted nanoparticle delivery is a promising strategy for increasing efficacy and limiting side effects of therapeutics. When designing a targeted liposomal formulation, the in vivo biodistribution of the particles must be characterized to determine the value of the targeting approach. Peroxisome proliferator-activated receptor (PPAR) agonists effectively treat metabolic syndrome by decreasing dyslipidemia and insulin resistance but side effects have limited their use, making them a class of compounds that could benefit from targeted liposomal delivery. The adipose targeting sequence peptide (ATS) could fit this role, as it has been shown to bind to adipose tissue endothelium and induce weight loss when delivered conjugated to a pro-apoptotic peptide. To date, however, a full assessment of ATS in vivo biodistribution has not been reported, leaving important unanswered questions regarding the exact mechanisms whereby ATS targeting enhances therapeutic efficacy. We designed this study to evaluate the biodistribution of ATS-conjugated liposomes loaded with the PPARα/γ dual agonist tesaglitazar in leptin-deficient ob/ob mice. The ATS-liposome biodistribution in adipose tissue and other organs was examined at the cellular and tissue level using microscopy, flow cytometry, and fluorescent molecular tomography. Changes in metabolic parameters and gene expression were measured by target and off-target tissue responses to the treatment. Unexpectedly, ATS targeting did not increase liposomal uptake in adipose relative to other tissues, but did increase uptake in the kidneys. Targeting also did not significantly alter metabolic parameters. Analysis of the liposome cellular distribution in the stromal vascular fraction with flow cytometry revealed high uptake by multiple cell types. Our findings highlight the need for thorough study of in vivo biodistribution when evaluating a targeted therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224917PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855449PMC
March 2020

Novel Autoimmune IgM Antibody Attenuates Atherosclerosis in IgM Deficient Low-Fat Diet-Fed, but Not Western Diet-Fed Mice.

Arterioscler Thromb Vasc Biol 2020 01 24;40(1):206-219. Epub 2019 Oct 24.

From the Robert M. Berne Cardiovascular Research Center (O.A.C., P.S., E.S.G., R.M.H., M.E.M., N.L., C.A.M., G.K.O.), University of Virginia, Charlottesville.

Objective: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, , mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age.

Conclusions: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.
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http://dx.doi.org/10.1161/ATVBAHA.119.312771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006879PMC
January 2020

Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.

Circ Res 2019 10 24;125(10):e55-e70. Epub 2019 Sep 24.

From the Cardiovascular Research Center (A.U., P.S., H.M.P., A.N., C.M., M.A.M., J.C.G, A.M.T., C.A.M.), University of Virginia, Charlottesville.

Rationale: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown.

Objective: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans.

Methods And Results: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased nontemplate-encoded nucleotide additions and a greater frequency of unique heavy chain complementarity determining region 3 sequences compared with peritoneal cavity B-1a cells. Some complementarity determining region 3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature peritoneal cavity B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM IgM production and plasma IgM levels were reduced in ApoE mice with B-cell-specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased BM localization and plasma IgM against oxidation specific epitopes, including IgM specific for malondialdehyde-modified LDL (low-density lipoprotein). Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of IgM antibodies specific for malondialdehyde-modified LDL and inversely associates with human coronary artery plaque burden and necrosis.

Conclusions: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and production of IgM against oxidation specific epitopes. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830526PMC
October 2019

Cell- and Sex-Specific Role of FcγR (Fcγ Receptor) IIb in Experimental Atherosclerosis.

Arterioscler Thromb Vasc Biol 2019 07 26;39(7):1269-1271. Epub 2019 Jun 26.

From the Cardiovascular Research Center, University of Virginia Health Sciences Center, University of Virginia, Charlottesville.

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http://dx.doi.org/10.1161/ATVBAHA.119.312916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863628PMC
July 2019

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Cell Rep 2019 05;27(8):2304-2312.e6

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S1A1, Canada; Department of Immunology, University of Toronto, Toronto, ON M5S1A1, Canada; Toronto General Research Institute, University Health Network, Toronto, ON M5G1L7, Canada; Peter Munk Cardiac Centre, Toronto, ON M5G1L7, Canada. Electronic address:

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.
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http://dx.doi.org/10.1016/j.celrep.2019.04.090DOI Listing
May 2019

IgE, α-Gal and atherosclerosis.

Aging (Albany NY) 2019 04;11(7):1900-1902

Division of Allergy and Immunology, University of Virginia, Charlottesville, VA 22908, USA.

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http://dx.doi.org/10.18632/aging.101894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503887PMC
April 2019

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

Arterioscler Thromb Vasc Biol 2019 01;39(1):25-36

From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA (A.A.J.H., H.Q.D., G.D.T., P.M., A.B., C.S.N., C.C.H.).

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16 nonclassical monocyte population and 4 subsets belong to the CD14 classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a SlanCXCR6 nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.
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http://dx.doi.org/10.1161/ATVBAHA.118.311022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697379PMC
January 2019

A monoclonal antibody to assess oxidized cholesteryl esters associated with apoAI and apoB-100 lipoproteins in human plasma.

J Lipid Res 2019 02 18;60(2):436-445. Epub 2018 Dec 18.

Department of Medicine, University of California, San Diego, La Jolla, CA 92093

Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD.
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http://dx.doi.org/10.1194/jlr.D090852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358287PMC
February 2019

IgE to the Mammalian Oligosaccharide Galactose-α-1,3-Galactose Is Associated With Increased Atheroma Volume and Plaques With Unstable Characteristics-Brief Report.

Arterioscler Thromb Vasc Biol 2018 07 14;38(7):1665-1669. Epub 2018 Jun 14.

Division of Cardiology, Robert M. Berne Cardiovascular Center (A.T.N., A.M.T., C.A.M.), University of Virginia, Charlottesville

Objective: Emerging evidence suggests a link between coronary artery disease and type 2 immunity. We sought to test the hypothesis that IgE sensitization to the mammalian oligosaccharide galactose-α-1,3-galactose (α-Gal)-the target allergen of delayed anaphylaxis to red meat-is associated with coronary artery disease.

Approach And Results: Total IgE and specific IgE to α-Gal were assayed on sera from 118 subjects who presented for cardiac catheterization and underwent intravascular ultrasound. IgE to α-Gal was detected in 26%, and atheroma burden was higher in sensitized subjects (=0.02). Because α-Gal sensitization relates to an environmental exposure that could be a risk factor for early-onset coronary artery disease (ie, tick bites), we age stratified the cohort. In subjects ≤65 years of age, the strength of the association with atheroma burden was stronger (<0.001), and plaques in the sensitized group had less stable features based on intravascular ultrasound. To address the specificity of the association with IgE to α-Gal, IgE to inhalants and peanut were assayed and were not associated with coronary artery disease. Total IgE and α-Gal-specific IgE were strongly associated with each other, but the strength of the relationship with atheroma burden was stronger for α-Gal-specific IgE. This association was significant when adjusted for sex, diabetes mellitus, hypertension, statin use, and total IgE (regression coefficient, 12.2; SE, 5.2; =0.02).

Conclusions: Increased atheroma burden and plaques with more unstable features were associated with IgE to α-Gal-an effect most pronounced in subjects ≤65 years of age. IgE sensitization to α-Gal may represent a novel, and potentially modifiable, risk factor for coronary atherosclerosis.
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http://dx.doi.org/10.1161/ATVBAHA.118.311222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039405PMC
July 2018

Apolipoprotein AI prevents regulatory to follicular helper T cell switching during atherosclerosis.

Nat Commun 2018 03 15;9(1):1095. Epub 2018 Mar 15.

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA, 92037, USA.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.
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http://dx.doi.org/10.1038/s41467-018-03493-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854619PMC
March 2018

Association of D-dimer with Plaque Characteristics and Plasma Biomarkers of Oxidation-Specific Epitopes in Stable Subjects with Coronary Artery Disease.

J Cardiovasc Transl Res 2018 06 17;11(3):221-229. Epub 2018 Jan 17.

Cardiovascular Research Center, University of Virginia, 415 Lane Road, Bldg MR-5, Rm-G231, Charlottesville, VA, 22908, USA.

D-dimer has emerged as a biomarker of cardiovascular event risk, yet pathophysiological factors associated with plasma D-dimer levels in stable coronary artery disease (CAD) subjects are poorly understood. In 106 stable CAD subjects undergoing intravascular ultrasound with virtual histology (IVUS-VH), we measured D-dimer, lipoprotein(a) (Lp(a)), plasminogen, biomarkers reflecting oxidation-specific epitopes (OSE) such as oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), OxPL on plasminogen (OxPL-PLG), and autoantibodies to phosphorylcholine-BSA [PC-BSA] and a malondialdehyde [MDA] mimotope. In univariate analysis, D-dimer was positively associated with Lp(a), OxPL-apoB, OxPL-PLG, and coronary artery calcium, and inversely associated with autoantibodies to OSE and plaque fibrosis. D-dimer levels > 500 ng/ml also showed positive association with plaque necrosis. After multivariate analysis, D-dimer remained significantly associated with Lp(a) and plaque calcium. While further studies are needed, results provide evidence that plasma D-dimer levels are associated with levels of OxPLs and IVUS-VH indices linked to plaque erosion and rupture.
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http://dx.doi.org/10.1007/s12265-018-9790-4DOI Listing
June 2018

Perivascular Adipose Tissue Harbors Atheroprotective IgM-Producing B Cells.

Front Physiol 2017 22;8:719. Epub 2017 Sep 22.

Cardiovascular Research Center, University of VirginiaCharlottesville, VA, United States.

Adipose tissue surrounding major arteries (Perivascular adipose tissue or PVAT) has long been thought to exist to provide vessel support and insulation. Emerging evidence suggests that PVAT regulates artery physiology and pathology, such as, promoting atherosclerosis development through local production of inflammatory cytokines. Yet the immune subtypes in PVAT that regulate inflammation are poorly characterized. B cells have emerged as important immune cells in the regulation of visceral adipose tissue inflammation and atherosclerosis. B cell-mediated effects on atherosclerosis are subset-dependent with B-1 cells attenuating and B-2 cells aggravating atherosclerosis. While mechanisms whereby B-2 cells aggravate atherosclerosis are less clear, production of immunoglobulin type M (IgM) antibodies is thought to be a major mechanism whereby B-1 cells limit atherosclerosis development. B-1 cell-derived IgM to oxidation specific epitopes (OSE) on low density lipoproteins (LDL) blocks oxidized LDL-induced inflammatory cytokine production and foam cell formation. However, whether PVAT contains B-1 cells and whether atheroprotective IgM is produced in PVAT is unknown. Results of the present study provide clear evidence that the majority of B cells in and around the aorta are derived from PVAT. Interestingly, a large proportion of these B cells belong to the B-1 subset with the B-1/B-2 ratio being 10-fold higher in PVAT relative to spleen and bone marrow. Moreover, PVAT contains significantly greater numbers of IgM secreting cells than the aorta. ApoE mice with B cell-specific knockout of the gene encoding the helix-loop-helix factor Id3, known to have attenuated diet-induced atherosclerosis, have increased numbers of B-1b cells and increased IgM secreting cells in PVAT relative to littermate controls. Immunostaining of PVAT on human coronary arteries identified fat associated lymphoid clusters (FALCs) harboring high numbers of B cells, and flow cytometry demonstrated the presence of T cells and B cells including B-1 cells. Taken together, these results provide evidence that murine and human PVAT harbor B-1 cells and suggest that local IgM production may serve to provide atheroprotection.
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http://dx.doi.org/10.3389/fphys.2017.00719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609437PMC
September 2017

Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis.

Arterioscler Thromb Vasc Biol 2017 11 21;37(11):2043-2052. Epub 2017 Sep 21.

From the Department of Medicine, University of California San Diego School of Medicine, La Jolla (P.M.M., Y.I.M.); Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, CA (P.M.M., G.D.T., Z.M., E.E., K.A.L.M., A.B., R.W., K.L., C.C.H.); Department of Cardiology and Circulatory Diseases, Internal Medicine Clinic III, Eberhard Karls University Tübingen, Germany (K.A.L.M.); and Robert M. Berne Cardiovascular Research Center, Division of Cardiology, University of Virginia, Charlottesville (A.T.N., A.M.T., C.A.M.).

Objective: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature.

Approach And Results: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36 mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein.

Conclusions: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.
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http://dx.doi.org/10.1161/ATVBAHA.117.309123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930003PMC
November 2017

Human Blood Monocyte Subsets: A New Gating Strategy Defined Using Cell Surface Markers Identified by Mass Cytometry.

Arterioscler Thromb Vasc Biol 2017 08 8;37(8):1548-1558. Epub 2017 Jun 8.

From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, CA (G.D.T., A.A.J.H., C.N., P.M., C.C.H.); and Division of Cardiology and Robert M. Berne Cardiovascular Center, University of Virginia, Charlottesville (A.M.T., C.M., A.T.N., C.A.M.).

Objective: Human monocyte subsets are defined as classical (CD14CD16), intermediate (CD14CD16), and nonclassical (CD14CD16). Alterations in monocyte subset frequencies are associated with clinical outcomes, including cardiovascular disease, in which circulating intermediate monocytes independently predict cardiovascular events. However, delineating mechanisms of monocyte function is hampered by inconsistent results among studies.

Approach And Results: We use cytometry by time-of-flight mass cytometry to profile human monocytes using a panel of 36 cell surface markers. Using the dimensionality reduction approach visual interactive stochastic neighbor embedding (viSNE), we define monocytes by incorporating all cell surface markers simultaneously. Using viSNE, we find that although classical monocytes are defined with high purity using CD14 and CD16, intermediate and nonclassical monocytes defined using CD14 and CD16 alone are frequently contaminated, with average intermediate and nonclassical monocyte purity of ≈86.0% and 87.2%, respectively. To improve the monocyte purity, we devised a new gating scheme that takes advantage of the shared coexpression of cell surface markers on each subset. In addition to CD14 and CD16, CCR2, CD36, HLA-DR, and CD11c are the most informative markers that discriminate among the 3 monocyte populations. Using these additional markers as filters, our revised gating scheme increases the purity of both intermediate and nonclassical monocyte subsets to 98.8% and 99.1%, respectively. We demonstrate the use of this new gating scheme using conventional flow cytometry of peripheral blood mononuclear cells from subjects with cardiovascular disease.

Conclusions: Using cytometry by time-of-flight mass cytometry, we have identified a small panel of surface markers that can significantly improve monocyte subset identification and purity in flow cytometry. Such a revised gating scheme will be useful for clinical studies of monocyte function in human cardiovascular disease.
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http://dx.doi.org/10.1161/ATVBAHA.117.309145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828170PMC
August 2017

B cells and atherosclerosis.

Am J Physiol Heart Circ Physiol 2017 May 17;312(5):H1060-H1067. Epub 2017 Mar 17.

Cardiovascular Research Center, Charlottesville, Virginia; and.

B cells have emerged as important immune cells in cardiovascular disease. Initial studies have suggested that B cells protect against atherosclerosis development. However, subsequent studies demonstrating aggravation of atherosclerosis by B-2 cells have shed light on the subset-dependent effects of B cells. Here, we review the literature that has led to our current understanding of B cell regulation of atherosclerosis, touching on the importance of subsets, local regulation, human translation, and therapeutic potential.
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http://dx.doi.org/10.1152/ajpheart.00859.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451581PMC
May 2017

Regulation of Transcription Factors by Reactive Oxygen Species and Nitric Oxide in Vascular Physiology and Pathology.

Antioxid Redox Signal 2017 05 4;26(13):679-699. Epub 2017 Jan 4.

1 IUF-Leibniz Research Institute for Environmental Medicine , Düsseldorf, Germany .

Significance: Cardiovascular diseases are the main cause of death worldwide and pose an immense economical burden. In most cases, the underlying problem is vascular occlusion by atherosclerotic plaques. Importantly, different cell types of the vascular wall and the immune system play crucial roles in atherosclerosis at different stages of the disease. Furthermore, atherosclerosis and conditions recognized as risk factors are characterized by a reduced availability of the vasoprotective molecule nitric oxide and an increase in reactive oxygen species, so-called oxidative stress. Transcription factors function as intracellular signal integrators and relays and thus, play a central role in cellular responses to changing conditions. Recent Advances: Work on specific transcriptional regulators has uncovered many of their functions and the upstream pathways modulating their activity in response to reactive oxygen and nitrogen species. Here, we have reviewed for a few selected examples how this can contribute not only to protection against atherosclerosis development but also to disease progression and the occurrence of clinical manifestations, such as plaque rupture.

Critical Issues: Transcription factors have pleiotropic outputs and often also divergent functions in different cell types and tissues. Thus, in light of potential severe adverse side effects, a global activation or inhibition of particular transcriptions factors does not seem a feasible therapeutic option.

Future Directions: A further in-depth characterization of the cell- and stage-specific actions and regulation of transcription factors in atherosclerosis with respect to protein-protein interactions and target genes could open up new avenues for prevention or therapeutic interventions in this vascular disease. Antioxid. Redox Signal. 26, 679-699.
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http://dx.doi.org/10.1089/ars.2016.6946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421514PMC
May 2017