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CCL17 is produced by conventional dendritic cells (cDCs), signals through CCR4 on regulatory T cells (Tregs), and drives atherosclerosis by suppressing Treg functions through yet undefined mechanisms. Here we show that cDCs from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed Treg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4+ T cells reduced CCL3 secretion, boosted FoxP3+ Treg numbers, and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained Treg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, while FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective Tregs.
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The benefits of current state-of-the-art treatments to combat atherosclerotic cardiovascular disease (ASCVD) have stagnated. Treatments are mostly based on controlling cardiovascular risk factors, especially hyperlipidemia. Although the most recent advances with PCSK-9 inhibitors support the hyperlipidemia aspect of ASCVD, several lines of experimental evidence have outlined that atherosclerosis is also driven by inflammation. In the past years, phase 1, 2, and 3 clinical trials targeting inflammation to combat ASCVD have revealed that patients do tolerate such immune therapies, show decreases in inflammatory markers, and/or have reductions in cardiovascular endpoints. However, the search for the optimal anti-inflammatory or immune-modulating strategy and the stratification of patients who would benefit from such treatments and appropriate treatment regimens to combat ASCVD is only just beginning. In this review, we focus on immune checkpoint-based therapeutics (costimulation and coinhibition), many of which are already approved by the U.S. Food and Drug Administration for the treatment of cancer or autoimmune diseases, and discuss their use as a novel immunotherapeutic strategy to treat ASCVD.
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Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.
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Aterosclerose , Linfoma , Placa Aterosclerótica , Animais , Camundongos , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Linfócitos T CD8-Positivos , Camundongos Knockout , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.
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Aterosclerose , Água Potável , Placa Aterosclerótica , Aminoácidos , Animais , Apolipoproteínas E , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Feminino , Homoarginina/farmacologia , Camundongos , Cadeias Pesadas de Miosina , Linfócitos T/metabolismoRESUMO
Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.
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Aterosclerose/patologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Interferon gama/metabolismo , Placa Aterosclerótica/patologia , Animais , Plaquetas/metabolismo , Linfócitos T CD4-Positivos/citologia , Doenças Cardiovasculares/patologia , Células Dendríticas/imunologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Transdução de Sinais/fisiologia , Trombose/patologiaRESUMO
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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Aterosclerose , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Placa Aterosclerótica , Animais , Apolipoproteínas E/genética , Modelos Animais de Doenças , Glucocorticoides , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores do Fator de Necrose TumoralRESUMO
As a leading cause of death worldwide, cardiovascular disease is a global health concern. The development and progression of atherosclerosis, which ultimately gives rise to cardiovascular disease, has been causally linked to hypercholesterolemia. Mechanistically, the interplay between lipids and the immune system during plaque progression significantly contributes to the chronic inflammation seen in the arterial wall during atherosclerosis. Localized inflammation and increased cell-to-cell interactions may influence polarization and proliferation of immune cells via changes in amino acid metabolism. Specifically, the amino acids l-arginine (Arg), l-homoarginine (hArg) and l-tryptophan (Trp) have been widely studied in the context of cardiovascular disease, and their metabolism has been established as key regulators of vascular homeostasis, as well as immune cell function. Cyclic effects between endothelial cells, innate, and adaptive immune cells exist during Arg and hArg, as well as Trp metabolism, that may have distinct effects on the development of atherosclerosis. In this review, we describe the current knowledge surrounding the metabolism, biological function, and clinical perspective of Arg, hArg, and Trp in the context of atherosclerosis.
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Aminoácidos/metabolismo , Aterosclerose/metabolismo , Animais , Arginase/antagonistas & inibidores , Arginase/metabolismo , Arginina/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/etiologia , Aterosclerose/imunologia , Progressão da Doença , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Homoarginina/metabolismo , Humanos , Hipercolesterolemia/complicações , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Subpopulações de Linfócitos/imunologia , Terapia de Alvo Molecular , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Triptofano/metabolismo , Vasculite/metabolismoRESUMO
OBJECTIVE: Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. APPROACH AND RESULTS: Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6Chigh monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6Chigh monocytes were CCR2high, whereas only a minority was CCR1high and CCR5high. We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6Chigh monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6Chigh monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6Chigh monocyte infiltration after ZT13 MI but not ZT5 MI. CONCLUSIONS: CCR2 surface expression on Ly6Chigh monocytes changes in a time-of-day-dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.
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Antígenos Ly/imunologia , Quimiotaxia de Leucócito , Ritmo Circadiano , Monócitos/imunologia , Infarto do Miocárdio/imunologia , Miocárdio/imunologia , Adulto , Animais , Antígenos Ly/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imunofenotipagem/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Monócitos/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Fenótipo , Receptores CCR1/imunologia , Receptores CCR1/metabolismo , Receptores CCR2/genética , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Transdução de Sinais , Fatores de Tempo , Adulto JovemRESUMO
Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.
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Sistema do Grupo Sanguíneo Duffy , Eritroblastos , Hematopoese , Células-Tronco Hematopoéticas , Neutropenia , Neutrófilos , Receptores de Superfície Celular , Animais , Humanos , Camundongos , População Negra/genética , Medula Óssea/patologia , Células da Medula Óssea/metabolismo , Proliferação de Células , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/metabolismo , Eritroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Microscopia Confocal , Neutropenia/genética , Neutrófilos/citologia , Neutrófilos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
Myocardial infarction (MI) is the leading cause of death in Western countries. Epidemiological studies show acute MI to be more prevalent in the morning and to be associated with a poorer outcome in terms of mortality and recovery. The mechanisms behind this association are not fully understood. Here, we report that circadian oscillations of neutrophil recruitment to the heart determine infarct size, healing, and cardiac function after MI Preferential cardiac neutrophil recruitment during the active phase (Zeitgeber time, ZT13) was paralleled by enhanced myeloid progenitor production, increased circulating numbers of CXCR2(hi) neutrophils as well as upregulated cardiac adhesion molecule and chemokine expression. MI at ZT13 resulted in significantly higher cardiac neutrophil infiltration compared to ZT5, which was inhibited by CXCR2 antagonism or neutrophil-specific CXCR2 knockout. Limiting exaggerated neutrophilic inflammation at this time point significantly reduced the infarct size and improved cardiac function.