Publications by authors named "Holger Winkels"

36 Publications

Normalization of cholesterol metabolism in spinal microglia alleviates neuropathic pain.

J Exp Med 2021 Jul 10;218(7). Epub 2021 May 10.

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

Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naive mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia or reverse CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling the TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.
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http://dx.doi.org/10.1084/jem.20202059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111462PMC
July 2021

The Enzymatic and Non-Enzymatic Function of Myeloperoxidase (MPO) in Inflammatory Communication.

Antioxidants (Basel) 2021 Apr 5;10(4). Epub 2021 Apr 5.

Department III of Internal Medicine, Heart Center, Faculty of Medicine and University Hospital of Cologne, 50937 North Rhine-Westphalia, Germany.

Myeloperoxidase is a signature enzyme of polymorphonuclear neutrophils in mice and humans. Being a component of circulating white blood cells, myeloperoxidase plays multiple roles in various organs and tissues and facilitates their crosstalk. Here, we describe the current knowledge on the tissue- and lineage-specific expression of myeloperoxidase, its well-studied enzymatic activity and incoherently understood non-enzymatic role in various cell types and tissues. Further, we elaborate on Myeloperoxidase (MPO) in the complex context of cardiovascular disease, innate and autoimmune response, development and progression of cancer and neurodegenerative diseases.
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http://dx.doi.org/10.3390/antiox10040562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066882PMC
April 2021

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

J Exp Med 2021 Mar;218(3)

La Jolla Institute for Immunology, La Jolla, CA.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.
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http://dx.doi.org/10.1084/jem.20200551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721910PMC
March 2021

Heterogeneity of T Cells in Atherosclerosis Defined by Single-Cell RNA-Sequencing and Cytometry by Time of Flight.

Arterioscler Thromb Vasc Biol 2021 Feb 3;41(2):549-563. Epub 2020 Dec 3.

Department of Cardiology, Clinic III for Internal Medicine, University of Cologne, Germany. Department of Cardiology and Angiology I, University Heart Center Freiburg, Faculty of Medicine, University of Freiburg, Germany.

The infiltration and accumulation of pro- and anti-inflammatory leukocytes within the intimal layer of the arterial wall is a hallmark of developing and progressing atherosclerosis. While traditionally perceived as macrophage- and foam cell-dominated disease, it is now established that atherosclerosis is a partial autoimmune disease that involves the recognition of peptides from ApoB (apolipoprotein B), the core protein of LDL (low-density lipoprotein) cholesterol particles, by CD4 T-helper cells and autoantibodies against LDL and ApoB. Autoimmunity in the atherosclerotic plaque has long been understood as a pathogenic T-helper type-1 driven response with proinflammatory cytokine secretion. Recent developments in high-parametric cell immunophenotyping by mass cytometry, single-cell RNA-sequencing, and in tools exploring antigen-specificity have established the existence of several unforeseen layers of T-cell diversity with mixed T1 and T regulatory cells transcriptional programs and unpredicted fates. These findings suggest that pathogenic ApoB-reactive T cells evolve from atheroprotective and immunosuppressive CD4 T regulatory cells that lose their protective properties over time. Here, we discuss T-cell heterogeneity in atherosclerosis with a focus on plasticity, antigen-specificity, exhaustion, maturation, tissue residency, and its potential use in clinical prediction.
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http://dx.doi.org/10.1161/ATVBAHA.120.312137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837690PMC
February 2021

Vaccination in Atherosclerosis.

Cells 2020 11 30;9(12). Epub 2020 Nov 30.

Department III of Internal Medicine III, Heart Center, Faculty of Medicine and University Hospital of Cologne, 50937 Cologne, Germany.

Atherosclerosis is the major underlying pathology of cardiovascular diseases that together are the leading cause of death worldwide. The formation of atherosclerotic plaques is driven by chronic vascular inflammation. Although several risk factors have been identified and significant progress in disease prevention and treatment has been made, no therapeutic agents targeting inflammation are clinically available. Recent clinical trials established the potential of anti-inflammatory therapies as a treatment of atherosclerosis. However, adverse impacts on host defense have raised safety concerns about these therapies. Scientific evidence during the past 40 years implicated an adaptive immune response against plaque-associated autoantigens in atherogenesis. Preclinical data have underscored the protective potential of immunization against such targets precisely and without the impairment of host defense. In this review, we discuss the current vaccination strategies against atherosclerosis, supposed mechanisms of action, therapeutic potential, and the challenges that must be overcome in translating this idea into clinical practice.
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http://dx.doi.org/10.3390/cells9122560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760548PMC
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

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

Eur Heart J 2020 08;41(31):2938-2948

Experimental Vascular Biology Division, Department of Medical Biochemistry, University of Amsterdam, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, Amsterdam, The Netherlands.

Aims: GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD).

Methods And Results: GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity.

Conclusion: Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.
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http://dx.doi.org/10.1093/eurheartj/ehaa484DOI Listing
August 2020

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B-Reactive CD4 T-Regulatory Cells.

Circulation 2020 Sep 24;142(13):1279-1293. Epub 2020 Jul 24.

Division of Vaccine Discovery (B.P., A.S.), La Jolla Institute for Immunology, CA.

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 T cells with an atheroprotective, regulatory T cell (T) phenotype in healthy individuals. Yet, the function of apoB-reactive T and their relationship with pathogenic T1 cells remain unknown.

Methods: To interrogate the function of autoreactive CD4 T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B (apoB) at the single-cell level.

Results: We found that apoB T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T-like transcriptome, although only 21% of all apoB T cells expressed the T transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB T cells formed several clusters with mixed T signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T1, T helper cell type 2 (T2), and T helper cell type 17 (T17), and of follicular-helper T cells. ApoB T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T1/T17-like cells with proinflammatory properties and only a residual T transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T1/T17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB T in lineage tracing of hyperlipidemic mice. In adoptive transfer experiments, converting apoB T failed to protect from atherosclerosis.

Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T as a novel cellular target in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.042863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515473PMC
September 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 07 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

Single Cell RNA Sequencing in Atherosclerosis Research.

Circ Res 2020 04 23;126(9):1112-1126. Epub 2020 Apr 23.

Laboratory of Inflammation Biology, La Jolla Institute for Immunology, CA (H.W., C.P.D., Y.G., K.L.).

Technological advances in characterizing molecular heterogeneity at the single cell level have ushered in a deeper understanding of the biological diversity of cells present in tissues including atherosclerotic plaques. New subsets of cells have been discovered among cell types previously considered homogenous. The commercial availability of systems to obtain transcriptomes and matching surface phenotypes from thousands of single cells is rapidly changing our understanding of cell types and lineage identity. Emerging methods to infer cellular functions are beginning to shed new light on the interplay of components involved in multifaceted disease responses, like atherosclerosis. Here, we provide a technical guide for design, implementation, assembly, and interpretations of current single cell transcriptomics approaches from the perspective of employing these tools for advancing cardiovascular disease research.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185048PMC
April 2020

T cell subsets and functions in atherosclerosis.

Nat Rev Cardiol 2020 07 16;17(7):387-401. Epub 2020 Mar 16.

Division of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, CA, USA.

Atherosclerosis is a chronic inflammatory disease of the arterial wall and the primary underlying cause of cardiovascular disease. Data from in vivo imaging, cell-lineage tracing and knockout studies in mice, as well as clinical interventional studies and advanced mRNA sequencing techniques, have drawn attention to the role of T cells as critical drivers and modifiers of the pathogenesis of atherosclerosis. CD4 T cells are commonly found in atherosclerotic plaques. A large body of evidence indicates that T helper 1 (T1) cells have pro-atherogenic roles and regulatory T (T) cells have anti-atherogenic roles. However, T cells can become pro-atherogenic. The roles in atherosclerosis of other T cell subsets such as T2, T9, T17, T22, follicular helper T cells and CD28 T cells, as well as other T cell subsets including CD8 T cells and γδ T cells, are less well understood. Moreover, some T cells seem to have both pro-atherogenic and anti-atherogenic functions. In this Review, we summarize the knowledge on T cell subsets, their functions in atherosclerosis and the process of T cell homing to atherosclerotic plaques. Much of our understanding of the roles of T cells in atherosclerosis is based on findings from experimental models. Translating these findings into human disease is challenging but much needed. T cells and their specific cytokines are attractive targets for developing new preventive and therapeutic approaches including potential T cell-related therapies for atherosclerosis.
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http://dx.doi.org/10.1038/s41569-020-0352-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872210PMC
July 2020

Migratory and Dancing Macrophage Subsets in Atherosclerotic Lesions.

Circ Res 2019 12 9;125(12):1038-1051. Epub 2019 Oct 9.

Division of Inflammation Biology (S.M., K.B., Y.G., A.B.P., J.M., H.W., D.W., K.L.), La Jolla Institute for Immunology, San Diego, CA.

Rationale: Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging.

Objective: Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes.

Methods And Results: mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques.

Conclusions: The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201888PMC
December 2019

PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function.

Sci Immunol 2019 06;4(36)

Institute for Cardiovascular Prevention, Ludwig-Maximilians-Universität (LMU), 80336 Munich, Germany.

The role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1 NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1 NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.
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http://dx.doi.org/10.1126/sciimmunol.aar3054DOI Listing
June 2019

CX3CL1-Fc treatment prevents atherosclerosis in Ldlr KO mice.

Mol Metab 2019 02 2;20:89-101. Epub 2018 Dec 2.

Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA, USA. Electronic address:

Objective: Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist.

Methods: In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet.

Results: CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion.

Conclusion: These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis.
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http://dx.doi.org/10.1016/j.molmet.2018.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358552PMC
February 2019

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Eur Heart J 2019 01;40(4):372-382

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences (ACS), Amsterdam University Medical Centers, University of Amsterdam, Room K1-110, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.

Methods And Results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells.

Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.
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http://dx.doi.org/10.1093/eurheartj/ehy714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340101PMC
January 2019

Atherosclerosis in the single-cell era.

Curr Opin Lipidol 2018 10;29(5):389-396

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Purpose Of Review: The immune system plays a critical role in the development and modulation of atherosclerosis. New high-parameter technologies, including mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), allow for an encompassing analysis of immune cells. Unexplored marker combinations and transcriptomes can define new immune cell subsets and suggest their functions. Here, we review recent advances describing the immune cells in the artery wall of mice with and without atherosclerosis. We compare technologies and discuss limitations and advantages.

Recent Findings: Both CyTOF and scRNAseq on leukocytes from digested aortae show 10-30 immune cell subsets. Myeloid, T, B and natural killer cells were confirmed. Although cellular functions can be inferred from RNA-Seq data, some subsets cannot be identified based on current knowledge, suggesting they may be new cell types. CyTOF and scRNAseq each identified four B-cell subsets and three macrophage subsets in the atherosclerotic aorta. Limitations include cell death caused by enzymatic digestion and the limited depth of the scRNAseq transcriptomes.

Summary: High-parameter methods are powerful tools for uncovering leukocyte diversity. CyTOF is currently more powerful at discerning leukocyte subsets in the atherosclerotic aorta, whereas scRNAseq provides more insight into their likely functions.
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http://dx.doi.org/10.1097/MOL.0000000000000537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314310PMC
October 2018

A clinically applicable adjuvant for an atherosclerosis vaccine in mice.

Eur J Immunol 2018 09 12;48(9):1580-1587. Epub 2018 Aug 12.

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA.

Vaccination with MHC-II-restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen-specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe-deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene-based oil-in-water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL-10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene-based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.
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http://dx.doi.org/10.1002/eji.201847584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281392PMC
September 2018

Regulatory CD4 T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

Circulation 2018 09;138(11):1130-1143

Department of Bioengineering, University of California, San Diego, La Jolla (K.L.).

Background: CD4 T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules.

Methods: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined.

Results: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4 T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3 regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4 T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-A-p18 tetramer identified the expansion of p18-specific CD4 T cells on vaccination, which were enriched for interleukin-10-producing Tregs.

Conclusions: These findings show that APOB p18-specific CD4 T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.031420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160361PMC
September 2018

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

Circ Res 2018 06 15;122(12):1675-1688. Epub 2018 Mar 15.

Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.)

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.

Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.

Methods And Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed and mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic and mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.

Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993603PMC
June 2018

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

Circ Res 2018 06 15;122(12):1661-1674. Epub 2018 Mar 15.

From the Institute of Experimental Biomedicine (C.C., A.Z.)

Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they have been defined by the expression of a restricted number of markers.

Objective: We have applied single-cell RNA sequencing as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis.

Method And Results: We performed single-cell RNA sequencing of total aortic CD45 cells extracted from the nondiseased (chow fed) and atherosclerotic (11 weeks of high-fat diet) aorta of low-density lipoprotein receptor-deficient () mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortas, whereas monocytes, monocyte-derived dendritic cells, and 2 populations of macrophages were almost exclusively detectable in atherosclerotic aortas, comprising inflammatory macrophages showing enrichment in and previously undescribed TREM2 (triggered receptor expressed on myeloid cells 2) macrophages showing enrichment in . Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these 3 macrophage subsets and monocyte-derived dendritic cells and uncovered putative functions of each cell type. Notably, TREM2 macrophages seemed to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in aortas, indicating relevance of our findings in different stages of atherosclerosis and mouse models.

Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and monocyte-derived dendritic cells in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312509DOI Listing
June 2018

CD40L Deficiency Protects Against Aneurysm Formation.

Arterioscler Thromb Vasc Biol 2018 05 8;38(5):1076-1085. Epub 2018 Mar 8.

From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands (P.J.H.K., T.T.P.S., L.B., D.L., H.W., V.d.W., E.L.)

Objective: The mechanisms underlying formation of arterial aneurysms remain incompletely understood. Because inflammation is a common feature during the progressive degeneration of the aortic wall, we studied the role of the costimulatory molecule CD40L, a major driver of inflammation, in aneurysm formation.

Approach And Results: Transcriptomics data obtained from human abdominal aortic aneurysms and normal aortas revealed increased abundance of both CD40L and CD40 in media of thrombus-free and thrombus-covered human abdominal aortic aneurysms samples. To further unravel the role of CD40L in aneurysm formation, apolipoprotein E-deficient () and mice were infused with angiotensin II for 7 and 28 days. Only a minority of mice (33% and 17%) developed (dissecting) aneurysms compared with 75% and 67% of littermates after 7 and 28 days of infusion, respectively. Total vessel area of the aorta at the suprarenal level was 52% smaller in angiotensin II-infused mice compared with that in angiotensin II-infused mice. Chimeric mice repopulated with bone marrow afforded a similar protection against dissecting aneurysm formation. Moreover, lack of CD40L protected mice from fatal aneurysm rupture. T helper cell and macrophage accumulation in aneurysmal tissue was reduced in mice with a concomitant decrease in expression of proinflammatory chemo- and cytokines. In addition, aneurysms of mice displayed reduced abundance of matrix metalloproteinase-13 and an increase in tissue inhibitor of metalloproteinase-3 while activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was diminished.

Conclusions: Deficiency of (hematopoietic) CD40L protects against dissecting aneurysm formation and reduces the incidence of fatal rupture. This is associated with a decreased accumulation and activation of inflammatory cells and a dampened protease activity in the arterial wall.
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http://dx.doi.org/10.1161/ATVBAHA.117.310640DOI Listing
May 2018

A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense.

Nat Commun 2018 02 6;9(1):525. Epub 2018 Feb 6.

Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, 92037, USA.

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.
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http://dx.doi.org/10.1038/s41467-018-02896-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802769PMC
February 2018

Natural Killer Cells at Ease: Atherosclerosis Is Not Affected by Genetic Depletion or Hyperactivation of Natural Killer Cells.

Circ Res 2018 01;122(1):6-7

From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, CA (H.W., K.L.); and Department of Bioengineering, University of California San Diego, La Jolla (K.L.).

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http://dx.doi.org/10.1161/CIRCRESAHA.117.312289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761686PMC
January 2018

IL-1 family cytokines in cardiovascular disease.

Cytokine 2019 10 1;122:154215. Epub 2017 Dec 1.

Division of Cardiology, Pulmonology, and Vascular Medicine, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany; Institute for Cardiovascular Prevention (IPEK), LMU Munich, Munich, Germany. Electronic address:

The interleukin (IL)-1 family is a group of cytokines crucially involved in regulating immune responses to infectious challenges and sterile insults. The family consists of the eponymous pair IL-1α and IL-1β, IL-18, IL-33, IL-37, IL-38, and several isoforms of IL-36. In addition, two endogenous inhibitors of functional receptor binding, IL-1R antagonist (IL-1Ra) and IL-36Ra complete the family. To gain biological activity IL-1β and IL-18 require processing by the protease caspase-1 which is associated with the multi-protein complex inflammasome. Numerous clinical association studies and experimental approaches have implicated members of the IL-1 family, their receptors, or component of the processing machinery in underlying processes of cardiovascular diseases (CVDs). Here we summarize the current state of knowledge regarding the pro-inflammatory and disease-modulating role of the IL-1 family in atherosclerosis, myocardial infarction, aneurysm, stroke, and other CVDs. We discuss clinical evidence, experimental approaches and lastly lend a perspective on currently developing therapeutic strategies involving the IL-1 family in CVD.
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http://dx.doi.org/10.1016/j.cyto.2017.11.009DOI Listing
October 2019

CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice.

Eur Heart J 2017 12;38(48):3590-3599

Institute for Cardiovascular Prevention (IPEK), LMU Munich, Pettenkoferstrasse 9, 80336 Munich, Germany.

Aims: The co-stimulatory receptor CD27 modulates responses of T cells, B cells, and NK cells. Various T cell subsets participate in atherogenesis. However, the role of CD27 in atherosclerosis remains unexplored.

Methods And Results: Here we investigated the effect of bone marrow-derived and systemic CD27 deficiency in Apolipoprotein E-deficient (Apoe-/-) mice in early and advanced stages of atherosclerosis. Lethally-irradiated Apoe-/- mice reconstituted with Cd27-/-Apoe-/- bone marrow and consuming an atherogenic diet displayed a markedly increased plaque size and lesional inflammation compared to mice receiving Cd27+/+Apoe-/- bone marrow. Accordingly, chow diet-fed Cd27-/-Apoe-/- mice showed exacerbated lesion development and increased inflammation at the age of 18 weeks. At a more advanced stage of atherosclerosis (28 weeks), lesion size and phenotype did not differ between the two groups. Systemic and bone marrow-derived CD27 deficiency reduced the abundance of regulatory T cells (Treg) in blood, lymphoid organs, and the aorta. Numbers of other immune cells were not affected while expression of inflammatory cytokine genes (e.g. IL-1β and IL-6) was increased in the aorta when haematopoietic CD27 was lacking. In vitro, Tregs of CD27-deficient mice showed similar suppressive capacity compared with their wild-type controls and migrated equally towards CCL19 and CCL21. However, thymic Cd27-/- Tregs underwent increased apoptosis and expressed fewer markers of proliferation in vivo. Reconstitution of Cd27-/-Apoe-/- mice with Cd27+/+Apoe-/- Tregs reversed the increase in atherosclerosis.

Conclusion: We demonstrate that CD27 co-stimulation increases the number of Tregs and limits lesion development and inflammation in experimental atherosclerosis, particularly during early stages of disease. Thus, our study suggests that promotion of CD27 function may mitigate atherosclerosis.
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http://dx.doi.org/10.1093/eurheartj/ehx517DOI Listing
December 2017

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption.

Am J Pathol 2017 Dec 19;187(12):2912-2919. Epub 2017 Sep 19.

Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilian University of Munich, Munich, Germany. Electronic address:

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.
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http://dx.doi.org/10.1016/j.ajpath.2017.08.016DOI Listing
December 2017

ATVB Distinguished Scientist Award: How Costimulatory and Coinhibitory Pathways Shape Atherosclerosis.

Arterioscler Thromb Vasc Biol 2017 05 30;37(5):764-777. Epub 2017 Mar 30.

From the Division of Inflammation Biology, La Jolla Institute for Allergy & Immunology, CA (K.L., H.W.); Division of Cardiology, Pulmonology, and Vascular Medicine, Medical Faculty, University Hospital Düsseldorf, Germany (N.G.); and Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU), Munich, Germany (N.G.).

Objective: Immune cells play a critical role in atherosclerosis. Costimulatory and coinhibitory molecules of the tumor necrosis factor receptor and CD28 immunoglobulin superfamilies not only shape T-cell and B-cell responses but also have a major effect on antigen-presenting cells and nonimmune cells.

Approach And Results: Pharmacological inhibition or activation of costimulatory and coinhibitory molecules and genetic deletion demonstrated their involvement in atherosclerosis. This review highlights recent advances in understanding how costimulatory and coinhibitory pathways shape the immune response in atherosclerosis.

Conclusions: Insights gained from costimulatory and coinhibitory molecule function in atherosclerosis may inform future therapeutic approaches.
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http://dx.doi.org/10.1161/ATVBAHA.117.308611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424816PMC
May 2017

CD70 limits atherosclerosis and promotes macrophage function.

Thromb Haemost 2017 01 27;117(1):164-175. Epub 2016 Oct 27.

Norbert Gerdes, PhD, Institute for Cardiovascular Prevention, Ludwig-Maximilians University Munich, Pettenkoferstrasse 9, 80336 Munich, Germany, Tel.: +49 89 4400 54672, Email:

The co-stimulatory molecule CD70 is expressed on activated immune cells and is known to modulate responses of T, B, and NK cells via its receptor CD27. Until now, there is only limited data describing the role of CD70 in atherosclerosis. We observed that ruptured human carotid atherosclerotic plaques displayed higher CD70 expression than stable carotid atherosclerotic plaques, and that CD70 expression in murine atheroma localized to macrophages. Lack of CD70 impaired the inflammatory capacity (e. g. reactive oxygen species and nitric oxide production) of bone marrow-derived macrophages, increased both M1-like and M2-like macrophage markers, and rendered macrophages metabolically inactive and prone to apoptosis. Moreover, CD70-deficient macrophages expressed diminished levels of scavenger receptors and ABC-transporters, impairing uptake of oxidised low-density lipoprotein (oxLDL) and cholesterol efflux, respectively. Hyperlipidaemic Apoe mice reconstituted with CD70-deficient bone marrow displayed a profound increase in necrotic core size, plaque area, and number of lesional macrophages as compared to mice receiving control bone marrow. Accordingly, 18 week-old, chow diet-fed CD70-deficient Apoe mice displayed larger atheroma characterised by lower cellularity and more advanced plaque phenotype than Apoe mice. In conclusion, CD70 promotes macrophage function and viability and is crucial for effective phagocytosis and efflux of oxLDL. Deficiency in CD70 results in more advanced atheroma. Our data suggest that CD70 mitigates atherosclerosis at least in part by modulating macrophage function.
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http://dx.doi.org/10.1160/TH16-04-0318DOI Listing
January 2017

Alterations in systemic levels of Th1, Th2, and Th17 cytokines in overweight adolescents and obese mice.

Pediatr Diabetes 2017 Dec 6;18(8):714-721. Epub 2016 Sep 6.

Department of Medicine, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.

Background: Obesity represents a major problem for patients and health care systems in most industrialized countries. A chronic inflammatory state in obese individuals leads to disease conditions associated with activation of cellular immune mechanisms. Here, we sought to investigate the role of Th1-, Th2-, and Th17-related cytokines in overweight adolescents and mice on a high-fat diet.

Methods: Plasma samples were obtained from 79 male adolescents aged 13-17 years. Thirty-seven of them had a body mass index (BMI) above the 90th age-specific percentile. Th1, Th2, and Th17 cytokines were measured using Bio-Plex multiplex technology (Bio-Rad, Hercules, USA). In an experimental approach, mice were fed with high-fat (HFD) or normal chow for 15 weeks.

Results: Interleukin (IL)-17 concentrations were significantly decreased in overweight adolescents compared to lean controls [99.8 ± 7.3 pg/mL standard error of the mean (SEM) vs 146.6 ± 11.5 pg/mL SEM P = .001]. Levels of IL-17 correlated significantly with anthropometrical parameters of obesity. A concordant response was found in mice consuming a HFD for 15 weeks compared to controls (861 ± 165 pg/mL SEM vs 1575 ± 187 pg/ml SEM, P = .0183). However, a biphasic response was evident for most Th1, Th2, and Th17 cytokines as levels initially increased within the first 5 weeks on HFD and showed a decline afterwards.

Conclusions: In contrast to previous studies showing elevated levels of IL-17 in obese adults, we found a decreasing trend in overweight adolescents. This difference could possibly be related to the fact that disease conditions associated with obesity such as hypertension, vascular pathologies, diabetes, and a triggering of the Th1/Th17 axis were not yet present in overweight teenagers.
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http://dx.doi.org/10.1111/pedi.12435DOI Listing
December 2017

Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4+ T-Cell Responses-Brief Report.

Arterioscler Thromb Vasc Biol 2016 09 21;36(9):1748-52. Epub 2016 Jul 21.

From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands (S.M., E.S., H.W., A.S., L.B., E.L.); Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University, Munich, Germany (S.M., H.W., C.W., N.G., E.L.); Sanquin Research, Department of Hematopoiesis, Amsterdam, The Netherlands (M.F.P., M.A.N.); and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany (C.W.).

Objective: Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4(+) effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood.

Approach And Results: Low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) were lethally irradiated and reconstituted with either bone marrow from B-cell-restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr(-/-) Gitrl(tg) mice showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr(-/-) Gitrl(tg) chimeras contained more total CD3(+) T cells as well as Foxp3(+) regulatory T cells overall, leading to significantly less severe atherosclerosis.

Conclusions: These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells.
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http://dx.doi.org/10.1161/ATVBAHA.116.307354DOI Listing
September 2016