RESUMEN
AIMS: Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear. METHODS AND RESULTS: MiRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells (PBMCs) from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxLDL-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation (lL-1ß, TNF-a, IL-6). Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signaling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes. CONCLUSION: These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism.
RESUMEN
BACKGROUND AND AIMS: Accumulating evidence supports a critical role for CD4+ T cells as drivers and modifiers of the chronic inflammatory response in atherosclerosis. Effector T cells have pro-atherogenic properties, whereas CD4+ regulatory T cells (Tregs) exert suppressive activity in atherosclerosis through increased secretion of inhibitory cytokines such as transforming growth factor-ß or interleukin-10. In addition, Tregs have been shown to suppress inflammatory macrophages and promote the resolution of atherosclerosis plaques. Impaired Treg numbers and function have been associated with atherosclerosis plaque development. However, the underlying mechanisms remain unclear. METHODS AND RESULTS: Here, we investigated a cell-autonomous role of a transcription factor, Krüppel-like factor 10 (KLF10), in CD4+ T cells in regulating atherosclerosis progression. Using CD4+ T-cell-specific KLF10 knockout (TKO) mice, we identified exaggerated plaque progression due to defects in immunosuppressive functions of Tregs on macrophages. TKO mice exhibited increased lesion size as well as higher CD4+ T cells and macrophage content compared to WT mice. TKO plaques also showed increased necrotic cores along with defective macrophage efferocytosis. In contrast, adoptive cellular therapy using WT Tregs abrogated the accelerated lesion progression and deleterious effects in TKO mice. Intriguingly, RNA-seq analyses of TKO lesions revealed increased chemotaxis and cell proliferation, and reduced phagocytosis compared to WT lesions. Mechanistically, TKO-Tregs impaired the efferocytosis capacity of macrophages in vitro and promoted a pro-inflammatory macrophage phenotype via increased IFN-γ and decreased TGF-ß secretion. CONCLUSIONS: Taken together, these findings establish a critical role for KLF10 in regulating CD4+ Treg-macrophage interactions and atherosclerosis.
Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Linfocitos T CD4-Positivos , Citocinas , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Factor X , Interleucina-10 , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores , Factor de Crecimiento Transformador beta , Factores de Crecimiento TransformadoresRESUMEN
CD4+ T cells regulate inflammation and metabolism in obesity. An imbalance of CD4+ T regulatory cells (Tregs) is critical in the development of insulin resistance and diabetes. Although cytokine control of this process is well understood, transcriptional regulation is not. KLF10, a member of the Kruppel-like transcription factor family, is an emerging regulator of immune cell function. We generated CD4+-T-cell-specific KLF10 knockout (TKO) mice and identified a predisposition to obesity, insulin resistance, and fatty liver due to defects of CD4+ Treg mobilization to liver and adipose tissue depots and decreased transforming growth factor ß3 (TGF-ß3) release in vitro and in vivo. Adoptive transfer of wild-type CD4+ Tregs fully rescued obesity, insulin resistance, and fatty liver. Mechanistically, TKO Tregs exhibit reduced mitochondrial respiration and glycolysis, phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling, and consequently impaired chemotactic properties. Collectively, our study identifies CD4+ T cell KLF10 as an essential regulator of obesity and insulin resistance by altering Treg metabolism and mobilization.
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Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Hígado Graso/genética , Resistencia a la Insulina , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Obesidad/genética , Obesidad/metabolismo , Linfocitos T Reguladores/metabolismo , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Hígado Graso/metabolismo , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Inflamación/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
OBJECTIVE: The objective of this study was to investigate the role of Kruppel-like factor (KLF) 10, a zinc-finger transcription factor, in bone marrow (BM)-derived cell responses to arterial endothelial injury. Accumulating evidence indicates that BM-derived progenitors are recruited to sites of vascular injury and contribute to endothelial repair. APPROACH AND RESULTS: In response to carotid artery endothelial denudation, KLF10 mRNA expression was markedly increased in both BM and circulating lin(-) progenitor cells. To examine the specific role of KLF10 in arterial reendothelialization, we used 2 models of endothelial denudation (wire- and thermal-induced injury) of the carotid artery in wild-type (WT) and KLF10(-/-) mice. WT mice displayed higher areas of reendothelialization compared with KLF10(-/-) mice after endothelial injury using either method. BM transplant studies revealed that reconstitution of KLF10(-/-) mice with WT BM fully rescued the defect in reendothelialization and increased lin(-)CD34(+)kinase insert domain receptor(+) progenitors in the blood and injured carotid arteries. Conversely, reconstitution of WT mice with KLF10(-/-) BM recapitulated the defects in reendothelialization and peripheral cell progenitors. The media from cultured KLF10(-)/(-) BM progenitors was markedly inefficient in promoting endothelial cell growth and migration compared with the media from WT progenitors, indicative of defective paracrine trophic effects from KLF10(-)/(-) BM progenitors. Finally, BM-derived KLF10(-/-) lin(-) progenitors from reconstituted mice had reduced CXC-chemokine receptor 4 expression and impaired migratory responses. CONCLUSIONS: Collectively, these observations demonstrate a protective role for BM-derived KLF10 in paracrine and homing responses important for arterial endothelial injury and highlight KLF10 as a possible therapeutic target to promote endothelial repair in vascular disease states.
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Células de la Médula Ósea/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Proliferación Celular , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Células Endoteliales/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Comunicación Paracrina , Células Madre/metabolismo , Lesiones del Sistema Vascular/metabolismo , Animales , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Trasplante de Médula Ósea , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Quimiotaxis , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción de la Respuesta de Crecimiento Precoz/deficiencia , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Transducción de Señal , Trasplante de Células Madre , Factores de Tiempo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB-responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b "mimics" reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB-mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.
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Células Endoteliales/metabolismo , MicroARNs/metabolismo , Vasculitis/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/terapia , Animales , Enfermedad Crítica , Selectina E/genética , Selectina E/inmunología , Selectina E/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/patología , Endotoxemia/genética , Endotoxemia/inmunología , Endotoxemia/metabolismo , Endotoxemia/patología , Endotoxemia/terapia , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , Lipopolisacáridos/toxicidad , Ratones , MicroARNs/genética , MicroARNs/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vasculitis/genética , Vasculitis/inmunología , Vasculitis/terapia , alfa Carioferinas/genética , alfa Carioferinas/inmunología , alfa Carioferinas/metabolismoRESUMEN
Clinical studies using bone marrow-derived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heterogeneous nature of this cell population. Herein, we found that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs compared with megakaryocyte-erythrocyte progenitors, hematopoietic stem cells, and common lymphoid progenitors. In vivo hindlimb ischemia studies and Matrigel plug assays verified the enhanced neovascularization properties uniquely associated with PACs derived from CMPs and GMPs. Taken together, these observations identify CMPs and GMPs as key bone marrow progenitors for optimal PAC function in vitro and in vivo and provide a foundation for novel therapeutic approaches to modulate angiogenesis.
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Células Progenitoras de Granulocitos y Macrófagos/fisiología , Isquemia/fisiopatología , Células Progenitoras Mieloides/fisiología , Neovascularización Fisiológica , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/fisiología , Células Cultivadas , Técnicas de Cocultivo , Miembro Posterior , Células Endoteliales de la Vena Umbilical Humana/fisiología , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Isquemia Miocárdica/fisiopatología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factores de TiempoRESUMEN
Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-ß1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, â¼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-ß responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-ß1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-ß1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Factores de Transcripción de la Respuesta de Crecimiento Precoz/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Neovascularización Fisiológica , Transducción de Señal , Factor de Crecimiento Transformador beta1/fisiología , Animales , Proteínas de Unión al ADN/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/fisiología , Miembro Posterior , Isquemia/metabolismo , Isquemia/patología , Isquemia/fisiopatología , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/fisiología , Enfermedad Arterial Periférica/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Flujo Sanguíneo Regional , Factor de Crecimiento Transformador beta1/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The Krüppel-like transcription factor (KLF) family participates in diverse aspects of cellular growth, development, differentiation, and activation. Recently, several groups have identified new connections between the function of these factors and leukocyte responses in health and disease. Gene targeting of individual KLFs in mice has uncovered novel and unexpected physiologic roles among myeloid and lymphocyte cell lineage maturation, particularly in the bone marrow niche and blood. In addition, several KLF family members are downstream targets of stimuli and signaling pathways critical to T-cell trafficking, T regulatory cell differentiation or suppressor function, monocyte/macrophage activation or renewal, and B memory cell maturation or activation. Indeed, KLFs have been implicated in subtypes of leukemia, lymphoma, autoimmunity, and in acute and chronic inflammatory disease states, such as atherosclerosis, diabetes, and airway inflammation, raising the possibility that KLFs and their upstream signals are of therapeutic interest. This review focuses on the relevant literature of Krüppel-like factors in leukocyte biology and their implications in clinical settings.
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Factores de Transcripción de Tipo Kruppel/metabolismo , Leucocitos/citología , Leucocitos/patología , Animales , Expresión Génica , Hematopoyesis , Humanos , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Transducción de SeñalRESUMEN
CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.
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Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/metabolismo , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismo , Animales , Aterosclerosis/sangre , Aterosclerosis/metabolismo , Separación Celular , Citocinas/metabolismo , Citometría de Flujo , Ratones , Ratones Transgénicos , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel-like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1-/- fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.
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Diferenciación Celular , Factores de Transcripción de Tipo Kruppel/metabolismo , Monocitos/citología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Linaje de la Célula , ADN/metabolismo , Sistema Hematopoyético/citología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Receptores de Lipopolisacáridos/genética , Ratones , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transactivadores/deficiencia , Activación Transcripcional/genéticaRESUMEN
Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), or tumor necrosis factor-alpha can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-beta1 (TGF-beta1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-gamma, LPS, or tumor necrosis factor-alpha and decreased by TGF-beta1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-beta1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-gamma, LPS, or IFN-gamma plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-beta1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at -95 and -212 bp, and mutation of these sites diminished induction by IFN-gamma and LPS. We further provide evidence that KLF4 interacts with the NF-kappaB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-beta1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.