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Introduction: Inhibition of STAT5 was recently reported to reduce murine atherosclerosis. However, the role of STAT5 isoforms, and more in particular STAT5A in macrophages in the context of human atherosclerosis remains unknown. Methods and results: Here, we demonstrate reciprocal expression regulation of STAT5A and STAT5B in human atherosclerotic lesions. The former was highly upregulated in ruptured over stable plaque and correlated with macrophage presence, a finding that was corroborated by the high chromosomal accessibility of STAT5A but not B gene in plaque macrophages. Phosphorylated STAT5 correlated with macrophages confirming its activation status. As macrophage STAT5 is activated by GM-CSF, we studied the effects of its silencing in GM-CSF differentiated human macrophages. STAT5A knockdown blunted the immune response, phagocytosis, cholesterol metabolism, and augmented apoptosis terms on transcriptional levels. These changes could partially be confirmed at functional level, with significant increases in apoptosis and decreases in lipid uptake and IL-6, IL-8, and TNFa cytokine secretion after STAT5A knockdown. Finally, inhibition of general and isoform A specific STAT5 significantly reduced the secretion of TNFa, IL-8 and IL-10 in ex vivo tissue slices of advanced human atherosclerotic plaques. Discussion: In summary, we identify STAT5A as an important determinant of macrophage functions and inflammation in the context of atherosclerosis and show its promise as therapeutic target in human atherosclerotic plaque inflammation.
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Aterosclerose , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Animais , Camundongos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Transativadores/genética , Fator de Transcrição STAT5/metabolismo , Interleucina-8/metabolismo , Transdução de Sinais , Macrófagos , Aterosclerose/metabolismo , Inflamação/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Monocytes circulate the vasculature at steady state and are recruited to sites of inflammation where they differentiate into macrophages (MФ) to replenish tissue-resident MФ populations and engage in the development of cardiovascular disease (CVD). Monocytes display considerable heterogeneity, currently reflected by a nomenclature based on their expression of cluster of differentiation (CD) 14 and CD16, distinguishing CD14++CD16- classical (cMo), CD14++CD16+ intermediate (intMo) and CD14+CD16++ non-classical (ncMo) monocytes. Several reports point to shifted subset distributions in the context of CVD, with significant association of intMo numbers with atherosclerosis, myocardial infarction, and heart failure. However, clear indications of their causal involvement as well as their predictive value for CVD are lacking. As recent high-parameter cytometry and single-cell RNA sequencing (scRNA-Seq) studies suggest an even higher degree of heterogeneity, better understanding of the functionalities of these subsets is pivotal. Considering their high heterogeneity, surprisingly little is known about functional differences between MФ originating from monocytes belonging to different subsets, and implications thereof for CVD pathogenesis. This paper provides an overview of recent findings on monocyte heterogeneity in the context of homeostasis and disease as well as functional differences between the subsets and their potential to differentiate into MФ, focusing on their role in vessels and the heart. The emerging paradigm of monocyte heterogeneity transcending the current tripartite subset division argues for an updated nomenclature and functional studies to substantiate marker-based subdivision and to clarify subset-specific implications for CVD.
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Aterosclerose , Doenças Cardiovasculares , Infarto do Miocárdio , Humanos , Monócitos/metabolismo , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/metabolismo , Macrófagos/metabolismo , Aterosclerose/metabolismo , Infarto do Miocárdio/metabolismo , Receptores de IgG/metabolismo , Receptores de LipopolissacarídeosRESUMO
Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1ß, IL-8, and TNF-α. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 103 cells/mm2), as visualized by principal component analysis. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments.
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Inflamassomos , Macrófagos , Humanos , Inflamassomos/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Fenótipo , LipídeosRESUMO
Acute myocardial infarction (AMI) is accompanied by a systemic trauma response that impacts the whole body, including blood. This study addresses whether macrophages, key players in trauma repair, sense and respond to these changes. For this, healthy human monocyte-derived macrophages are exposed to 20% human AMI (n = 50) or control (n = 20) serum and analyzed by transcriptional and multiparameter functional screening followed by network-guided data interpretation and drug repurposing. Results are validated in an independent cohort at functional level (n = 47 AMI, n = 25 control) and in a public dataset. AMI serum exposure results in an overt AMI signature, enriched in debris cleaning, mitosis, and immune pathways. Moreover, gene networks associated with AMI and with poor clinical prognosis in AMI are identified. Network-guided drug screening on the latter unveils prostaglandin E2 (PGE2) signaling as target for clinical intervention in detrimental macrophage imprinting during AMI trauma healing. The results demonstrate pronounced context-induced macrophage reprogramming by the AMI systemic environment, to a degree decisive for patient prognosis. This offers new opportunities for targeted intervention and optimized cardiovascular disease risk management.
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Macrófagos , Infarto do Miocárdio , Humanos , Macrófagos/metabolismo , Infarto do Miocárdio/metabolismo , Prognóstico , Redes Reguladoras de GenesRESUMO
In response to inflammatory activation by pathogens, macrophages accumulate triglycerides in intracellular lipid droplets. The mechanisms underlying triglyceride accumulation and its exact role in the inflammatory response of macrophages are not fully understood. Here, we aim to further elucidate the mechanism and function of triglyceride accumulation in the inflammatory response of activated macrophages. Lipopolysaccharide (LPS)-mediated activation markedly increased triglyceride accumulation in macrophages. This increase could be attributed to up-regulation of the hypoxia-inducible lipid dropletassociated (HILPDA) protein, which down-regulated adipose triglyceride lipase (ATGL) protein levels, in turn leading to decreased ATGL-mediated triglyceride hydrolysis. The reduction in ATGL-mediated lipolysis attenuated the inflammatory response in macrophages after ex vivo and in vitro activation, and was accompanied by decreased production of prostaglandin-E2 (PGE2) and interleukin-6 (IL-6). Overall, we provide evidence that LPS-mediated activation of macrophages suppresses lipolysis via induction of HILPDA, thereby reducing the availability of proinflammatory lipid precursors and suppressing the production of PGE2 and IL-6.
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Gotículas Lipídicas , Metabolismo dos Lipídeos , Humanos , Inflamação/metabolismo , Gotículas Lipídicas/metabolismo , Lipídeos , Macrófagos/metabolismo , Proteínas de Neoplasias/metabolismo , Triglicerídeos/metabolismoRESUMO
Platelet-derived growth factor B (PDGF-B) is a mitogenic, migratory and survival factor. Cell-associated PDGF-B recruits stabilizing pericytes towards blood vessels through retention in extracellular matrix. We hypothesized that the genetic ablation of cell-associated PDGF-B by retention motif deletion would reduce the local availability of PDGF-B, resulting in microvascular pericyte loss, microvascular permeability and exacerbated atherosclerosis. Therefore, Ldlr-/-Pdgfbret/ret mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of Pdgfbret/ret mice, microvessel density and intraplaque hemorrhage were unexpectedly unaffected. Plaque macrophage content was reduced, which is likely attributable to increased apoptosis, as judged by increased TUNEL+ cells in Pdgfbret/ret plaques (2.1-fold) and increased Pdgfbret/ret macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, Pdgfbret/ret plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain Pdgfbret/ret collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in Pdgfbret/ret liver and adipose tissue. While dampening plaque inflammation, Pdgfbret/ret paradoxically induced systemic leukocytosis. The increased incorporation of 5-ethynyl-2'-deoxyuridine indicated increased extramedullary hematopoiesis and the increased proliferation of circulating leukocytes. We concluded that Pdgfbret/ret confers vascular and metabolic effects, which appeared to be protective against diet-induced cardiovascular burden. These effects were unrelated to arterial mesenchymal cell content or adventitial microvessel density and leakage. In contrast, the deletion drives splenic hematopoiesis and subsequent leukocytosis in hypercholesterolemia.
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Aterosclerose/metabolismo , Hematopoese Extramedular , Proteínas Proto-Oncogênicas c-sis/metabolismo , Animais , Apoptose , Peso Corporal , Movimento Celular , Proliferação de Células , Leucócitos/patologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , SolubilidadeRESUMO
BACKGROUND: Merkel cell carcinoma (MCC) is a highly malignant skin cancer. Despite major treatment improvements during the last decade, up to 50% of patients do not respond to therapy or develop recurrent disease. For these patients, alternative treatment options are urgently needed. Here, we assessed the efficacy of the combination of the BCL-2 inhibitor Navitoclax and the PI3K p110α inhibitor Alpelisib in MCC cell lines. METHODS: The expression of BCL-2 was assessed by immunohistochemistry in MCC and MCC cell lines. Treatment with Navitoclax and Alpelisib alone and in combination was performed on four MCC cell lines. The decrease of cell viability during treatment was assessed by XTT assay and visualized for the combinations by 3D combinatorial index plotting. The increase of apoptotic cells was determined by cleaved PARP Western blotting and Annexin V staining. RESULTS: Some 94% of MCCs and all three MCPyV-positive cell lines showed BCL-2 expression. Navitoclax monotreatment was shown to be highly effective when treating BCL-2-positive cell lines (IC50-values ranging from 96.0 to 323.0 nM). The combination of Alpelisib and Navitoclax resulted in even stronger synergistic and prolonged inhibitions of MCC cell viability through apoptosis up to 4 days. DISCUSSION: Our results show that the anti-apoptotic BCL-2 is frequently expressed in MCC and MCC cell lines. Inhibition of BCL-2 by Navitoclax in combination with Alpelisib revealed a strong synergy and prolonged inhibition of MCC cell viability and induction of apoptosis. The combination of Navitoclax and Alpelisib is a novel potential treatment option for MCC patients.
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OBJECTIVE: Long noncoding RNAs (lncRNAs) are an emergent class of molecules with diverse functional roles, widely expressed in human physiology and disease. Although some lncRNAs have been identified in cardiovascular disease, their potential as novel targets in the prevention of atherosclerosis is unknown. We set out to discover important lncRNAs in unstable plaque and gain insight into their functional relevance. Approach and Results: Analysis of RNA sequencing previously performed on stable and unstable atherosclerotic plaque identified a panel of 47 differentially regulated lncRNAs. We focused on LINC01272, a lncRNA upregulated in unstable plaque previously detected in inflammatory bowel disease, which we termed PELATON (plaque enriched lncRNA in atherosclerotic and inflammatory bowel macrophage regulation). Here, we demonstrate that PELATON is highly monocyte- and macrophage-specific across vascular cell types, and almost entirely nuclear by cellular fractionation (90%-98%). In situ hybridization confirmed enrichment of PELATON in areas of plaque inflammation, colocalizing with macrophages around the shoulders and necrotic core of human plaque sections. Consistent with its nuclear localization, and despite containing a predicted open reading frame, PELATON did not demonstrate any protein-coding potential in vitro. Functionally, knockdown of PELATON significantly reduced phagocytosis, lipid uptake and reactive oxygen species production in high-content analysis, with a significant reduction in phagocytosis independently validated. Furthermore, CD36, a key mediator of phagocytic oxLDL (oxidized low-density lipoprotein) uptake was significantly reduced with PELATON knockdown. CONCLUSIONS: PELATON is a nuclear expressed, monocyte- and macrophage-specific lncRNA, upregulated in unstable atherosclerotic plaque. Knockdown of PELATON affects cellular functions associated with plaque progression.
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Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica , RNA Longo não Codificante/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Macrófagos/patologia , Masculino , Necrose , Fagocitose , RNA Longo não Codificante/genética , Espécies Reativas de Oxigênio/metabolismo , Ruptura EspontâneaRESUMO
The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1-/-) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1-/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1-/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1-/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.
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Apoptose , Células Gigantes/citologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células 3T3 , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Diferenciação Celular , Deleção de Genes , Humanos , Imuno-Histoquímica , Lipídeos/química , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neutrófilos/metabolismo , Fenótipo , Placa Aterosclerótica/metabolismoRESUMO
Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim -/- or wild type bone marrow transplanted ldlr -/- mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim -/- transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim -/- mice. Bim -/- mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim -/- mice.
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Aterosclerose/genética , Doenças Autoimunes/etiologia , Proteína 11 Semelhante a Bcl-2/deficiência , Inflamação/etiologia , Leucócitos/imunologia , Leucócitos/metabolismo , Receptores de LDL/deficiência , Animais , Apoptose/genética , Doenças Autoimunes/patologia , Proteína 11 Semelhante a Bcl-2/genética , Transplante de Medula Óssea , Modelos Animais de Doenças , Hiperlipidemias , Imunidade Humoral , Imunoglobulinas/imunologia , Inflamação/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Esplenomegalia , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr(-/-) mice were transplanted with batf3(-/-) or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8α(+) DC numbers in spleen and lymph nodes (>80%; P < 0,001). Concordantly, batf3(-/-) chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3(-/-) chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3(-/-) chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3(-/-) chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8α(+) DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8α(+) DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability.
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Aterosclerose/imunologia , Antígenos CD8/imunologia , Hiperlipidemias/imunologia , Placa Aterosclerótica/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Epitopos/imunologia , Humanos , Hiperlipidemias/genética , Hiperlipidemias/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Receptores de LDL/genética , Proteínas Repressoras/genética , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Insulin-like growth factor 1 (IGF-1) is a potent enhancer of tissue regeneration, and its overexpression in muscle injury leads to hastened resolution of the inflammatory phase. Here, we show that monocytes/macrophages constitute an important initial source of IGF-1 in muscle injury, as conditional deletion of the IGF-1 gene specifically in mouse myeloid cells (ÏIGF-1 CKO) blocked the normal surge of local IGF-1 in damaged muscle and significantly compromised regeneration. In injured muscle, Ly6C+ monocytes/macrophages and CD206+ macrophages expressed equivalent IGF-1 levels, which were transiently upregulated during transition from the inflammation to repair. In injured ÏIGF-1 CKO mouse muscle, accumulation of CD206+ macrophages was impaired, while an increase in Ly6C+ monocytes/macrophages was favored. Transcriptional profiling uncovered inflammatory skewing in ÏIGF-1 CKO macrophages, which failed to fully induce a reparative gene program in vitro or in vivo, revealing a novel autocrine role for IGF-1 in modulating murine macrophage phenotypes. These data establish local macrophage-derived IGF-1 as a key factor in inflammation resolution and macrophage polarization during muscle regeneration.