RESUMEN
Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/metabolismo , Secuencias de Aminoácidos , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Ratones , Especificidad de Órganos , Unión Proteica , Factores de Transcripción/metabolismo , Activación Transcripcional , Pez CebraRESUMEN
Macrophages are important for effective iron recycling and erythropoiesis, but they also play a crucial role in wound healing, orchestrating tissue repair. Recently, we demonstrated a significant accumulation of iron in healing wounds and a requirement of iron for effective repair. Herein, we sought to determine the influence of iron on macrophage function in the context of wound healing. Interestingly, wound macrophages extensively sequestered iron throughout healing, associated with a prohealing M2 phenotype. In delayed healing diabetic mouse wounds, both macrophage polarization and iron sequestration were impaired. In vitro studies revealed that iron promotes differentiation, while skewing macrophages toward a hypersecretory M2-like polarization state. These macrophages produced high levels of chemokine (C-C motif) ligands 17 and 22, promoting wound reepithelialization and extracellular matrix deposition in a human ex vivo wound healing model. Together, these findings reveal a novel, unappreciated role for iron in modulating macrophage behavior to promote subsequent wound repair. These findings support therapeutic evaluation of iron use to promote wound healing in the clinic.
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Quimiocina CCL17/fisiología , Quimiocina CCL22/fisiología , Hierro/farmacología , Macrófagos/efectos de los fármacos , Piel/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Hierro/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Leptina/genética , Piel/lesiones , Células THP-1RESUMEN
The regulated differentiation of macrophages (mφs) and their subsequent activation into proinflammatory or prohealing subtypes is critical for efficient wound healing. Chronic wounds such as diabetic (db) ulcers are associated with dysregulation of macrophage function. Whereas non-db mφs polarize to an M2-like, prohealing phenotype during the late stages of healing, db-derived mφs continue to display an M1-like, proinflammatory, or a mixed M1-like/M2-like phenotype. We have previously shown that sustained expression of Hoxa3 reduces the excessive number of leukocytes within the db wound; however, the effect of Hoxa3 on mφ polarization was unknown. In this study, we show that Hoxa3 protein transduction of mφs in vitro enhances macrophage maturation, inhibits M1 polarization, and promotes M2 polarization, in part via regulation of Pu.1/Spi1 and Stat6. Sustained expression of Hoxa3 in vivo in db wounds reduces the number of Nos2(+) (M1-like) mφs, increases the number of Arg1(+) and VEGF(+) (M2-like) mφs, and accelerates healing in a DNA-binding independent manner. Our findings suggest a role for Hox protein activity in promoting M1-to-M2-like phenotypic switching via interactions with myeloid transcription factors and provide insight into mechanisms regulating this process in db wound healing.
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Diferenciación Celular/inmunología , Proteínas de Homeodominio/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Cicatrización de Heridas/inmunología , Animales , Western Blotting , Complicaciones de la Diabetes/inmunología , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Inmunoprecipitación , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Reacción en Cadena de la Polimerasa , TranscriptomaRESUMEN
Diabetes can promote a state of chronic inflammation associated with serious complications that are difficult to treat, including ulceration of the lower extremities and chronic wounds. Chronic wounds are often incurable and contribute to both a reduced quality of life for patients and an enormous burden for healthcare services. In diabetes, the inflammatory response early in wound healing is inappropriately amplified and prolonged, leading to the persistent presence in the wound of vastly elevated numbers of dysfunctional, hyperpolarised macrophages that fail to transition to a pro-healing phenotype. Recent evidence suggests that systemic chronic inflammation induces intrinsic defects in monocytes via chromatin modifications that may pre-programme monocytes to a pro-inflammatory phenotype, while the local wound environment inhibits differentiation to a pro-healing phenotype. Current understanding remains incomplete, and careful dissection of how local and systemic inflammation combine to negatively influence myeloid cell development will be key to developing effective therapies aimed at healing the diabetic wound.
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Complicaciones de la Diabetes/inmunología , Células Mieloides/patología , Animales , Complicaciones de la Diabetes/patología , Humanos , Inflamación , Ratones , Células Mieloides/inmunología , Cicatrización de HeridasRESUMEN
Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair, but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization, but show altered recruitment/retention kinetics in the diabetic environment. Moreover, diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative, chemotaxis, adhesion, and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response, enhanced neovascularization, and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing.
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Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Células Mieloides/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígeno CD11b/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Células Mieloides/citología , Neovascularización Fisiológica , Receptores de Quimiocina/metabolismo , Receptores de Leptina/deficiencia , Receptores de Leptina/genética , TransfecciónRESUMEN
Diabetes is known to delay wound healing, and this delay is attributed to prolonged inflammation. We found that microRNAs (miRNAs) might be involved in the dysfunction of diabetic-derived neutrophils, and dynamics of neutrophil and chronic inflammation might be initiated by miRNA-regulated genes. Moreover, studies of miRNA function in nephropathy have suggested that circular RNAs (circRNAs), which function as sponges of miRNA to regulate their expression, are potential biomarkers and new therapeutic targets for the diagnosis of diabetic nephropathy. Accordingly, to investigate the molecular mechanism of the regulation of inflammation in diabetic-derived neutrophils, we identified circRNAs in diabetic-derived neutrophils obtained from BKS.Cg-Dock7m +/+ Leprdb/J (Leprdb/db and Leprdb/+) mice using microarrays. Neutrophils from pooled bone marrow of three diabetic and three non-diabetic mice were isolated and total RNA was extracted. Microarray analysis was performed using the Arraystar Mouse Circular RNA Array. The results showed that three circRNAs were significantly increased and six circRNAs were significantly decreased in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils. The expressions of some circRNAs in diabetic-derived neutrophils were more than double those in non-diabetic-derived neutrophils. The circRNAs contain binding sites of miRNAs, which were differentially expressed in diabetic-derived neutrophils. Our results suggest that circRNAs may be involved in the regulation of inflammation in diabetic-derived neutrophils.
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Ageing-related delays and dysregulated inflammation in wound healing are well-documented in both human and animal models. However, cellular and molecular changes underlying this impairment in healing progression are not fully understood. In this study, we characterised ageing-associated changes to macrophages in wounds of young and aged mice and investigated transcriptomic differences that may impact the progression of wound healing. Full-thickness wounds created on the dorsum of C57BL/6J young and aged mice were excised on Days 3 and 7 post-wounding for analysis by immunohistochemistry, flow cytometry, and RNA sequencing. Our data revealed that macrophages were significantly reduced in aged wounds in comparison to young. Functional transcriptomic analyses showed that macrophages from aged wounds exhibited significantly reduced expression of cell cycle, DNA replication, and repair pathway genes. Furthermore, we uncovered an elevated pro-inflammatory gene expression program in the aged macrophages correlated with poor inflammation resolution and excessive tissue damage observed in aged wounds. Altogether, our work provides insights into how poorly healing aged wounds are phenotypically defined by the presence of macrophages with reduced proliferative capacity and an exacerbated inflammatory response, both of which are pathways that can be targeted to improve healing in the elderly.
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Piel , Cicatrización de Heridas , Anciano , Animales , Humanos , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Piel/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Patients treated for cancer therapy using ionizing radiation (IR) have delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we here investigate the mechanisms by which IR exposure limits skin regeneration. Our data show that induction of the stromal cell-derived growth factor 1alpha (SDF-1alpha) is severely impaired in the wounded skin of irradiated, compared to non-irradiated, mice. Hence, we evaluated the potential of bone marrow-derived multipotent stromal cells (MSCs), which secrete high levels of SDF-1alpha, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. Injection of irradiated MSCs into the wound periphery of non-irradiated mice delayed wound closure, also suggesting an important role for the stromal microenvironment in skin repair. The beneficial actions of MSCs were mainly paracrine, as the cells did not differentiate into keratinocytes. Specific knockdown of SDF-1alpha expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF-1alpha secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF-1alpha enhances wound closure likely involves increased skin vascularization. Our findings collectively indicate that SDF-1alpha is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support.
Asunto(s)
Células de la Médula Ósea/citología , Quimiocina CXCL12/metabolismo , Piel/patología , Piel/efectos de la radiación , Cicatrización de Heridas/efectos de la radiación , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/trasplante , Neovascularización Fisiológica/efectos de la radiación , Radiación Ionizante , Células del Estroma/metabolismo , Células del Estroma/efectos de la radiación , Células del Estroma/trasplante , Factores de TiempoRESUMEN
The regulated recruitment and differentiation of multipotent bone marrow-derived cells (BMDCs) to sites of injury are critical for efficient wound healing. Previously we demonstrated that sustained expression of HOXA3 both accelerated wound healing and promoted angiogenesis in diabetic mice. In this study, we have used green fluorescent protein-positive bone marrow chimeras to investigate the effect of HOXA3 expression on recruitment of BMDCs to wounds. We hypothesized that the enhanced neovascularization induced by HOXA3 is due to enhanced mobilization, recruitment, and/or differentiation of BMDCs. Here we show that diabetic mice treated with HOXA3 displayed a significant increase in both mobilization and recruitment of endothelial progenitor cells compared with control mice. Importantly, we also found that HOXA3-treated mice had significantly fewer inflammatory cells recruited to the wound compared with control mice. Microarray analyses of HOXA3-treated wounds revealed that indeed HOXA3 locally increased expression of genes that selectively promote stem/progenitor cell mobilization and recruitment while also suppressing expression of numerous members of the proinflammatory nuclear factor kappaB pathway, including myeloid differentiation primary response gene 88 and toll-interacting protein. Thus HOXA3 accelerates wound repair by mobilizing endothelial progenitor cells and attenuating the excessive inflammatory response of chronic wounds.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diabetes Mellitus/terapia , Proteínas de Homeodominio/fisiología , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/genética , Movimiento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Proteínas de Homeodominio/genética , Inmunohistoquímica , Leucocitos/citología , Leucocitos/fisiología , Masculino , Ratones , Ratones Mutantes , Análisis de Secuencia por Matrices de Oligonucleótidos , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: CXCL12 is a chemokine involved in postinjury leukocyte chemotaxis, migration, and homing of stem cells. We hypothesized that by increasing the level of the chemokine CXCL12 in wounds of diabetic mice, we would increase stem cell recruitment to the wound and, thus, accelerate time to wound closure. METHODS: Eighteen Lepr db-/db- (B6.Cg-m +/+ Leprdb/J; Jackson Labs, Bar Harbor, ME) and their nondiabetic littermates were wounded and treated either with an empty plasmid or a plasmid containing the CXCL12 gene. Wounds were measured approximately every 5 days until they closed completely and were analyzed using planimetry. Wounds were harvested, and relative expression of CXCL12 mRNA was measured using an ABI Prism SDS 7000. To study stem cells affected by this, the plasmid's affect on stem cell recruitment, we used flow cytometry. RESULTS: The diabetic wounds contain a significantly decreased level of CXCL12 mRNA at day 7 postwounding, and these wounds take 55 days to heal. Application of a CXCL12 plasmid to diabetic wounds significantly increases CXCL12 mRNA at day 7, and these wounds heal in 23 days. CONCLUSIONS: Lack of CXCL12 in diabetic wounds contributes to delayed wound healing and can be reversed via single application of a CXCL12-containing plasmid.
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Quimiocina CXCL12/farmacología , Complicaciones de la Diabetes/terapia , Terapia Genética/métodos , Plásmidos/farmacología , Cicatrización de Heridas/genética , Animales , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos , ARN Mensajero/análisis , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Bone marrow (BM)-derived classical monocytes are critical to wound repair, where they differentiate into macrophages and purge foreign materials and dead cells while also laying the framework for tissue repair and regeneration. A subset of this recruited population persists in the wound and acquires alternative activation states to promote cell proliferation and matrix remodeling. In diabetes, this phenotypic switch is impaired and inflammation persists in an elevated state, contributing to delayed wound healing. Long-term tissue-resident macrophages can also play a key role in the resolution of inflammation to varying degrees across different organs. In this study, we investigated different macrophage subpopulations in nondiabetic and diabetic wounds over time using Cx3CR1eGFP transgenic mice and BM transplants. We show Cx3CR1eGFP-hi macrophages in skin wounds are derived from long-term tissue-resident macrophages and predominantly exhibit an alternative activation state, whereas cells expressing low-intermediate Cx3CR1eGFP are derived from the BM, contribute to both early and later stages of wound healing, and show both classical and alternative activation states. Diabetic mice showed significant differences in the dynamics of these subpopulations, which likely contribute to elevated and persisting inflammatory states over time. In particular, failure of Cx3CR1int macrophages to mature into Cx3CR1hi links maturation to resolution of inflammation. Thus strategies to promote macrophage maturation may be effective therapeutic tools in chronic inflammatory environments.
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Receptor 1 de Quimiocinas CX3C/metabolismo , Diabetes Mellitus Experimental/metabolismo , Macrófagos/metabolismo , Cicatrización de Heridas/fisiología , Animales , Células de la Médula Ósea , Trasplante de Médula Ósea , Receptor 1 de Quimiocinas CX3C/genética , Diferenciación Celular , Proliferación Celular , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/metabolismo , Inflamación/metabolismo , Macrófagos/clasificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Monocitos/metabolismo , Receptores CCR2/metabolismo , Receptores de Leptina/deficienciaRESUMEN
Chronic inflammation is a hallmark of impaired healing in a plethora of tissues, including skin, and is associated with aging and diseases such as diabetes. Diabetic chronic skin wounds are characterized by excessive myeloid cells that display an aberrant phenotype, partially mediated by stable intrinsic changes induced during hematopoietic development. However, the relative contribution of myeloid cell-intrinsic factors to chronic inflammation versus aberrant signals from the local environmental was unknown. Moreover, identification of myeloid cell intrinsic factors that contribute to chronic inflammation in diabetic wounds remained elusive. Here we show that Gr-1+CD11b+ myeloid cells are retained specifically within the presumptive granulation tissue region of the wound at a higher density in diabetic mice and associate with endothelial cells at the site of injury with a higher frequency than in nondiabetic mice. Adoptive transfer of myeloid cells demonstrated that aberrant wound retention is due to myeloid cell intrinsic factors and not the local environment. RNA sequencing of bone marrow and wound-derived myeloid cells identified Selplg as a myeloid cell intrinsic factor that is deregulated in chronic wounds. In vivo blockade of this protein significantly accelerated wound healing in diabetic mice and may be a potential therapeutic target in chronic wounds and other chronic inflammatory diseases.
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Inflamación/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Cicatrización de Heridas , Traslado Adoptivo , Animales , Células de la Médula Ósea/metabolismo , Antígeno CD11b/genética , Enfermedad Crónica , Diabetes Mellitus Experimental , Células Endoteliales/metabolismo , Femenino , Masculino , Ratones , Fenotipo , Análisis de Secuencia de ARNRESUMEN
Neutrophils are involved in the first stage of acute inflammation. After injury, they are mobilized and recruited to the injured tissue. In diabetes, wound healing is delayed and aberrant, leading to excessive recruitment and retention of neutrophils that fail to promote angiogenesis and prolong inflammation. However, the exact pathological mechanisms of diabetic-derived neutrophils in chronic inflammation remain unclear. Here, miRNA profiling of neutrophils from bone marrow in type 2 diabetic mice was performed using a microarray. miRNAs regulate the posttranscriptional expression of target mRNAs and are important in countering inflammation-related diseases. Our study revealed that miRNAs exhibit differential expression in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils, especially miR-129 family members. miR-129-2-3p directly regulated the translation of Casp6 and Ccr2, which are involved in inflammatory responses and apoptosis. Furthermore, miR-129-2-3p overexpression at the wound site of type 2 diabetic mice accelerated wound healing. These results suggest possible involvement of miR-129-2-3p in diabetic-derived neutrophil dysfunction and that retention kinetics of neutrophils and chronic inflammation may be initiated through miR-129-2-3p-regulated genes. This study characterizes changes in global miRNA expression in diabetic-derived neutrophils and systematically identifies critical target genes involved in certain biological processes related to the pathology of diabetic wound healing.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , MicroARNs/metabolismo , Neutrófilos/metabolismo , Cicatrización de Heridas/fisiología , Células 3T3 , Animales , Linfocitos B/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Células HL-60 , Humanos , Hibridación in Situ , Inflamación/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Mutantes , Mutación/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Cellular senescence can be broadly defined as a stable, but essentially irreversible, loss of proliferative capacity. Historically, senescence has been described as a negative outcome of advanced cellular age. It is now clear, however, that senescence represents a dynamic autonomous stress response, integral to long-term tumor suppression. Transient induction of a senescent phenotype has actually been suggested to promote regeneration in both liver and skin. Here, we explored the role of senescence in pathological aged and diabetic murine wound healing. Aged and diabetic wounds had greater numbers of senescent cells, and diabetic macrophages maintained altered retention of polarization and produced a CXCR2-enriched senescence-associated secretory phenotype (i.e., SASP). Of translational relevance, targeted expression of CXCR2 in primary human dermal fibroblasts led to paracrine induction of nuclear p21. Furthermore, a selective agonist to CXCR2 was able to reverse delayed healing in diabetic mice and accelerate ex vivo human skin wound healing. Collectively, these data suggest a hitherto unappreciated role for CXCR2 in mediating cellular senescence in pathological wound repair.
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Envejecimiento/genética , Senescencia Celular/genética , Receptores de Interleucina-8B/genética , Cicatrización de Heridas/genética , Análisis de Varianza , Animales , Biopsia con Aguja , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Modelos Animales de Enfermedad , Fibroblastos/citología , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-8B/metabolismo , Valores de Referencia , Úlcera Cutánea/genética , Úlcera Cutánea/patología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/genética , Heridas y Lesiones/patologíaRESUMEN
Iron is crucial for maintaining normal bodily function with well-documented roles in erythropoiesis, hemostasis, and inflammation. Despite this, little is known about the temporal regulation of iron during wound healing, or how iron contributes to wound biology and pathology. In this study, we profiled tissue iron levels across a healing time-course, identifying iron accumulation during late-stage repair. Diabetic murine wounds displayed significantly reduced iron levels, delayed extracellular matrix deposition, and dysregulation of iron gene expression. In vitro studies revealed important cellular roles for iron, promoting both the deposition and remodeling of extracellular proteins. Functional studies identified oxidative stress-dependent upregulation of the iron-converting metalloreductase, STEAP3, as a key mediator of extracellular matrix production. Taken together, these data reveal a mechanistic role for iron in facilitating the remodeling stage of wound healing. Indeed, targeting tissue iron could be a promising future strategy to tackle the development and progression of chronic wounds.
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Proteínas de Ciclo Celular/metabolismo , Complicaciones de la Diabetes/metabolismo , Matriz Extracelular/metabolismo , Hierro/metabolismo , Oxidorreductasas/metabolismo , Piel/metabolismo , Heridas y Lesiones/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Oxidorreductasas/genética , ARN Interferente Pequeño/genética , Receptores de Leptina/genética , Piel/patología , Cicatrización de HeridasRESUMEN
Controlled inflammatory responses of myeloid cells recruited to wounds are essential for effective repair. In diabetes, the inflammatory response is prolonged and augmented over time, with increased myeloid cells present in the wound that fail to switch from a pro-inflammatory phenotype to a pro-healing phenotype. These defects lead to delayed angiogenesis and tissue repair and regeneration, and contribute to chronic wound formation. In mouse models of diabetes, this aberrant phenotype is partially mediated by stable intrinsic changes to the developing myeloid cells in the bone marrow, affecting their maturation and polarization potential. Previous studies have shown that freshly isolated peripheral blood mononuclear cells from diabetic patients are more inflammatory than non-diabetic counterparts. However, the phenotype of macrophages from human diabetic patients has not been well characterized. Here we show that diabetic-derived human macrophages cultured for 6 days in vitro maintain a pro-inflammatory priming and hyperpolarize to a pro-inflammatory phenotype when stimulated with LPS and INF-É£ or TNF. In addition, diabetic-derived macrophages show maturation defects associated with reduced expression of the RUNX1 transcription factor that promotes myeloid cell development. Targeting intrinsic defects in myeloid cells by protein transduction of the Hoxa3 transcription factor can rescue some inflammation and maturation defects in human macrophages from diabetic patients via upregulation of Runx1. In addition, Hoxa3 can modulate the levels of p65/NF-κB and histone acetyltransferase and deacetylase activity, as well as inhibit acetylation of the TNF promoter. Altogether, these results show a link between myeloid cell maturation and inflammatory responses, and that diabetes induces intrinsic changes to human myeloid cells that are maintained over time, as well as potentially therapeutic Hoxa3-mediated mechanisms of controlling the inflammatory response in diabetes.
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Diabetes Mellitus Tipo 2/patología , Proteínas de Homeodominio/metabolismo , Macrófagos/metabolismo , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Estudios de Casos y Controles , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Medios de Cultivo Condicionados/química , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Proteínas de Homeodominio/genética , Humanos , Interleucina-6/análisis , Leucocitos Mononucleares/citología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Fenotipo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factores de Necrosis Tumoral/análisis , Factores de Necrosis Tumoral/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Recruitment of innate immune cells from the bone marrow (BM) to an injury site is required for effective repair. In diabetes, this process is altered, leading to excessive recruitment and retention of dysfunctional myeloid cells that fail to promote angiogenesis, prolong inflammation, and block healing. The aberrant myeloid phenotype is partially mediated by stable intrinsic changes to developing cells in the BM that are induced by the diabetic (db) environment, but the exact mechanisms remain largely unknown. Here, we show that the db-derived Gr-1(+)CD11b(+) immature myeloid population has widespread misexpression of chromatin-remodeling enzymes and myeloid differentiation factors. Crucially, diabetes represses transcription of the key myeloid transcription factor CEBPA via diminished H3 Lys 27 promoter acetylation, leading to a failure in monocyte and granulocyte maturation. Restoring Cebpa expression by granulocyte colony-stimulating factor reverses the db phenotype and rescues myeloid maturation. Importantly, our data demonstrate a possible link between myeloid cell maturation and chronic inflammation.
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Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Hematopoyesis , Inmunidad Innata , Células Mieloides/metabolismo , Adulto , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/agonistas , Proteínas Potenciadoras de Unión a CCAAT/sangre , Proteínas Potenciadoras de Unión a CCAAT/genética , Células Cultivadas , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Femenino , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Células Mieloides/inmunología , Células Mieloides/patología , Receptores de Quimiocina/sangre , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERß, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.
Asunto(s)
Receptor alfa de Estrógeno/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Cicatrización de Heridas/inmunología , Animales , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/inmunología , Receptor beta de Estrógeno/metabolismo , Estrógenos/metabolismo , Estrógenos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ovariectomía , Transducción de Señal/efectos de los fármacosRESUMEN
Acute inflammation in response to injury is a tightly regulated process by which subsets of leukocytes are recruited to the injured tissue and undergo behavioural changes that are essential for effective tissue repair and regeneration. The diabetic wound environment is characterised by excessive and prolonged inflammation that is linked to poor progression of healing and, in humans, the development of diabetic foot ulcers. However, the underlying mechanisms contributing to excessive inflammation remain poorly understood. Here we show in a murine model that the diabetic environment induces stable intrinsic changes in haematopoietic cells. These changes lead to a hyper-responsive phenotype to both pro-inflammatory and anti-inflammatory stimuli, producing extreme M1 and M2 polarised cells. During early wound healing, myeloid cells in diabetic mice show hyperpolarisation towards both M1 and M2 phenotypes, whereas, at late stages of healing, when non-diabetic macrophages have transitioned to an M2 phenotype, diabetic wound macrophages continue to display an M1 phenotype. Intriguingly, we show that this population predominantly consists of Gr-1(+) CD11b(+) CD14(+) cells that have been previously reported as 'inflammatory macrophages' recruited to injured tissue in the early stages of wound healing. Finally, we show that this phenomenon is directly relevant to human diabetic ulcers, for which M2 polarisation predicts healing outcome. Thus, treatments focused at targeting this inflammatory cell subset could prove beneficial for pathological tissue repair.
Asunto(s)
Células de la Médula Ósea/patología , Diabetes Mellitus Experimental/patología , Inflamación/patología , Cicatrización de Heridas , Animales , Adhesión Celular , Quimiotaxis , Enfermedad Crónica , Macrófagos/patología , RatonesRESUMEN
Analysis of tissue repair and regeneration in a variety of organisms has demonstrated that stem and progenitor cells play a critical role in the healing and regenerative response. In particular, during cutaneous wound healing bone marrow-derived cells are recruited to the site of injury in large numbers, often comprising over 50% of the cells within the wound milieu. These bone marrow-derived cells are comprised mostly of a heterogeneous mix of myeloid cells. In the early stages of wound healing, the most prominent subtypes are Gr-1(+)CD11b(+) cells that consist of progenitor cells and more differentiated granulocytes. Under certain conditions, these cells have the potential to strongly promote angiogenesis, and thus tissue repair and regeneration. This chapter provides methods by which one can isolate these cells from wound tissue and assess their pro-angiogenic capacity via gene expression analyses and functional in vivo angiogenesis assays.