RESUMEN
BACKGROUND: B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. METHODS: Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS: Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-κB (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS: This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.
Asunto(s)
Aterosclerosis , Subgrupos de Linfocitos B , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Humanos , Inmunoglobulina M , Ratones , Ratones NoqueadosRESUMEN
OBJECTIVE: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. CONCLUSIONS: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.
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Tejido Adiposo/metabolismo , Linfocitos B/metabolismo , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/biosíntesis , Proteínas Inhibidoras de la Diferenciación/metabolismo , Paniculitis/metabolismo , Tejido Adiposo/inmunología , Animales , Linfocitos B/inmunología , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hialuronano Sintasas/genética , Proteínas Inhibidoras de la Diferenciación/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Paniculitis/genética , Paniculitis/inmunología , Fenotipo , Transducción de Señal , Regulación hacia ArribaRESUMEN
RATIONALE: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown. OBJECTIVE: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans. METHODS AND RESULTS: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE-/- mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased nontemplate-encoded nucleotide additions and a greater frequency of unique heavy chain complementarity determining region 3 sequences compared with peritoneal cavity B-1a cells. Some complementarity determining region 3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature peritoneal cavity B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM IgM production and plasma IgM levels were reduced in ApoE-/- mice with B-cell-specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased BM localization and plasma IgM against oxidation specific epitopes, including IgM specific for malondialdehyde-modified LDL (low-density lipoprotein). Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of IgM antibodies specific for malondialdehyde-modified LDL and inversely associates with human coronary artery plaque burden and necrosis. CONCLUSIONS: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and production of IgM against oxidation specific epitopes. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.
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Subgrupos de Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Enfermedad de la Arteria Coronaria/sangre , Inmunoglobulina M/sangre , Receptores CXCR4/biosíntesis , Receptores CXCR4/sangre , Animales , Enfermedad de la Arteria Coronaria/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
OBJECTIVE: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell-specific deletion of Id3 (Id3(Bcell KO)) to identify B-cell functions involved in the metabolic consequences of obesity. APPROACH AND RESULTS: Diet-induced obese Id3(Bcell KO) mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3(Bcell KO) mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1(-/-) VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1(-/-) hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. CONCLUSIONS: NAb-producing B-1b B cells are increased in Id3(Bcell KO) mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.
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Subgrupos de Linfocitos B/metabolismo , Intolerancia a la Glucosa/prevención & control , Cadenas mu de Inmunoglobulina/metabolismo , Inflamación/prevención & control , Resistencia a la Insulina , Grasa Intraabdominal/metabolismo , Obesidad/complicaciones , Traslado Adoptivo , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/trasplante , Biomarcadores/sangre , Glucemia/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Genotipo , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Cadenas mu de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/inmunología , Inflamación/sangre , Inflamación/genética , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Insulina/sangre , Grasa Intraabdominal/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/sangre , Obesidad/genética , Obesidad/inmunología , Fenotipo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
RATIONALE: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. OBJECTIVE: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. METHODS AND RESULTS: Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. CONCLUSIONS: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.
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Aorta/patología , Aterosclerosis/prevención & control , Aterosclerosis/fisiopatología , Linfocitos B/patología , Movimiento Celular/fisiología , Proteínas Inhibidoras de la Diferenciación/fisiología , Animales , Aorta/fisiopatología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/etiología , Linfocitos B/fisiología , Dieta/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Incidencia , Proteínas Inhibidoras de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/genética , Ratones , Ratones Noqueados , Placa Aterosclerótica/patología , Placa Aterosclerótica/fisiopatología , Receptores CCR6/fisiología , Transducción de Señal/fisiologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.
RESUMEN
OBJECTIVE: Inhibitor of differention-3 (Id3) promotes B cells homing to the aorta and atheroprotection in Apoe(-/-) mice. We sought to determine the impact of loss of Id3 in the Ldlr((-/-)) mouse model of diet-induced atherosclerosis and identify novel Id3 targets in the vessel wall. METHODS AND RESULTS: Ex vivo optical imaging confirmed that Id3((-/-)) Ldlr((-/-)) mice have significantly fewer aortic B cells than Id3((+/+)) Ldlr(-/-) mice. After 8 and 16 weeks of Western diet, Id3((-/-)) Ldlr((-/-)) mice developed significantly more atherosclerosis than Id3((+/+)) Ldlr((-/-)) mice, with Id3(+/-) Ldlr(-/-) mice demonstrating an intermediate phenotype. There were no differences in serum lipid levels between genotypes. Immunostaining demonstrated that aortas from Id3((-/-)) Ldlr((-/-)) mice had greater intimal macrophage density and C-C chemokine ligand 20 and vascular cell adhesion molecule 1 (VCAM-1) expression compared with Id3((+/+)) Ldlr(-/-) mice. Real-time polymerase chain reaction demonstrated increased VCAM-1 mRNA levels in the aortas of Id3(-/-) Ldlr(-/-) mice. Primary vascular smooth muscle cells from Id3((-/-)) mice expressed greater amounts of VCAM-1 protein compared with control. Gain and loss of function studies in primary vascular smooth muscle cells identified a role for Id3 in repressing VCAM-1 promoter activation. Chromatin immunoprecipitation demonstrated interaction of E12 with the VCAM-1 promoter, which is inhibited by Id3. CONCLUSIONS: Id3 is an atheroprotective transcription regulator with targets in both B cells and vessel wall cells leading to reduced macrophage accumulation and reduced atherosclerosis formation.
Asunto(s)
Aterosclerosis/fisiopatología , Movimiento Celular/fisiología , Proliferación Celular , Proteínas Inhibidoras de la Diferenciación/deficiencia , Macrófagos/patología , Receptores de LDL/deficiencia , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/epidemiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Linfocitos B/metabolismo , Linfocitos B/patología , Quimiocina CCL20/metabolismo , Modelos Animales de Enfermedad , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Prevalencia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Factores de RiesgoRESUMEN
OBJECTIVE: Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. METHODS AND RESULTS: Id3(-/-) mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3(-/-) mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. CONCLUSIONS: Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.
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Adiposidad/fisiología , Grasas de la Dieta/farmacología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Grasa Intraabdominal/metabolismo , Adipocitos/patología , Animales , Volumen Sanguíneo/fisiología , Proteínas Inhibidoras de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/genética , Grasa Intraabdominal/irrigación sanguínea , Grasa Intraabdominal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To determine whether increased 12/15-lipoxygenase (12/15LO) expression in vivo enhances neointimal formation in response to injury. METHODS AND RESULTS: 12/15LO expression in the vessel wall is increased in animal models of metabolic syndrome and diabetes mellitus. Increased expression of 12/15LO enhances cultured vascular smooth muscle cell (VSMC) proliferation, an effect mediated by the helix-loop-helix factor inhibitor of differentiation 3 (Id3). Carotid endothelial denudation was performed on apolipoprotein (Apo) E(-/-), ApoE(-/-)/12/15LO(-/-), C57BL/6, and 12/15LO-overexpressing transgenic mice. ApoE(-/-)/12/15LO(-/-) mice had attenuated and 12/15LO-overexpressing transgenic mice had enhanced neointimal formation compared with control mice. 12/15LO-overexpressing transgenic mice had greater postinjury carotid Id3 and Ki-67 expression, cell number, and fibronectin deposition compared with C57BL/6 mice. Loss of 12/15LO attenuated proliferation of cultured ApoE(-/-) VSMCs, whereas 12/15LO overexpression induced VSMC proliferation. Loss of Id3 enhanced immunoglobulin trascription factor (ITF)-2b binding to and activation of the p21(cip1) promoter and abrogated 12/15LO-induced VSMC proliferation. CONCLUSIONS: To our knowledge, these data are the first demonstration that increased expression of 12/15LO in the vessel wall enhances Id3-dependent cell proliferation, fibronectin deposition, and neointimal formation in response to injury. Results identify p21(cip1) as a potential target of the 12/15LO-Id3 pathway and suggest that modulation of this pathway may have therapeutic implications for targeting the increased risk of restenosis in patients with diabetes.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Traumatismos de las Arterias Carótidas/enzimología , Proliferación Celular , Fibronectinas/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Túnica Íntima/enzimología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Sitios de Unión , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Hiperplasia , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Regiones Promotoras Genéticas , Factores de Tiempo , Factor de Transcripción 4 , Túnica Íntima/patologíaRESUMEN
Immunoglobulin M (IgM) to oxidation specific epitopes (OSE) are inversely associated with atherosclerosis in mice and humans. The B-1b subtype of B-1 cells secrete IgM to OSE, and unlike B-1a cells, are capable of long-lasting IgM memory. What attributes make B-1b cells different than B-1a cells is unknown. Our objectives were to determine how B-1b cells produce more IgM compared to B-1a cells at homeostatic condition and to see the differences in the B-1a and B-1b cell distribution and IgM CDR-H3 sequences in mice with advanced atherosclerosis. Here, in-vivo studies demonstrated greater migration to spleen, splenic production of IgM and plasma IgM levels in ApoE-/-Rag1-/- mice intraperitoneally injected with equal numbers of B-1b compared to B-1a cells. Bulk RNA seq analysis and flow cytometry of B-1a and B-1b cells identified CCR6 as a chemokine receptor more highly expressed on B-1b cells compared to B-1a. Knockout of CCR6 resulted in reduced B-1b cell migration to the spleen. Moreover, B-1b cell numbers were significantly higher in spleen of aged atherosclerotic ApoE-/- mice compared to young ApoE-/- mice. Single cell sequencing results of IgHM in B-1a and B-1b cells from peritoneal cavity and spleen of atherosclerotic aged ApoE-/- mice revealed significantly more N additions at the V-D and D-J junctions, greater diversity in V region usage and CDR-H3 sequences in B-1b compared to B-1a cells. In summary, B-1b cells demonstrated enhanced CCR6-mediated splenic migration, IgM production, and IgM repertoire diversification compared to B-1a cells. These findings suggest that potential strategies to selectively augment B-1b cell numbers and splenic trafficking could lead to increased and more diverse IgM targeting OSE to limit atherosclerosis.
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Aterosclerosis , Anciano , Animales , Apolipoproteínas E , Aterosclerosis/genética , Homeostasis , Humanos , Inmunoglobulina M , Ratones , Ratones Endogámicos C57BLRESUMEN
Recent studies in rodent models suggest that liver X receptors (LXRs) may play an important role in the maintenance of glucose homeostasis and islet function. To date, however, no studies have comprehensively examined the role of LXRs in human islet biology. Human islets were isolated from non-diabetic donors and incubated in the presence or absence of two synthetic LXR agonists, TO-901317 and GW3965, under conditions of low and high glucose. LXR agonist treatment enhanced both basal and stimulated insulin secretion, which corresponded to an increase in the expression of genes involved in anaplerosis and reverse cholesterol transport. Furthermore, enzyme activity of pyruvate carboxylase, a key regulator of pyruvate cycling and anaplerotic flux, was also increased. Whereas LXR agonist treatment up-regulated known downstream targets involved in lipogenesis, we observed no increase in the accumulation of intra-islet triglyceride at the dose of agonist used in our study. Moreover, LXR activation increased expression of the genes encoding hormone-sensitive lipase and adipose triglyceride lipase, two enzymes involved in lipolysis and glycerolipid/free fatty acid cycling. Chronically, insulin gene expression was increased after treatment with TO-901317, and this was accompanied by increased Pdx-1 nuclear protein levels and enhanced Pdx-1 binding to the insulin promoter. In conclusion, our data suggest that LXR agonists have a direct effect on the islet to augment insulin secretion and expression, actions that should be considered either as therapeutic or unintended side effects, as these agents are developed for clinical use.
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Benzoatos/farmacología , Bencilaminas/farmacología , Ácidos Grasos no Esterificados/metabolismo , Glicéridos/metabolismo , Hidrocarburos Fluorados/farmacología , Islotes Pancreáticos/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Sulfonamidas/farmacología , Adolescente , Adulto , Núcleo Celular/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glucosa/farmacología , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Lipogénesis/efectos de los fármacos , Lipogénesis/fisiología , Receptores X del Hígado , Masculino , Persona de Mediana Edad , Receptores Nucleares Huérfanos/metabolismo , Regiones Promotoras Genéticas/fisiología , Piruvato Carboxilasa/metabolismo , Ácido Pirúvico/metabolismo , Edulcorantes/farmacología , Transactivadores/metabolismoRESUMEN
IL-1ß is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1ß blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4-CD8low/-CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1ß. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+), and lineage- (Lin-) cells expressing CD161 and CD25. IL-1ß also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1ß stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1ß-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1ß in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1ß-neutralizing therapies.
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COVID-19/inmunología , Interleucina-1beta/sangre , Subgrupos de Linfocitos T/inmunología , COVID-19/sangre , COVID-19/complicaciones , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Interleucina-1beta/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Monocitos/clasificación , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/sangre , Pandemias , Fosforilación , Receptores CCR6/sangre , SARS-CoV-2 , Factores de Transcripción STAT/sangre , Factores de Transcripción STAT/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/sangreRESUMEN
AIMS: In light of recent data regarding inflammatory signalling pathways in cardiovascular disease and the recently demonstrated impact of pharmacologic inhibition of interleukin-1ß (IL-1ß) in heart failure, the primary aim was to assess the physiologic effects of cardiac resynchronization therapy (CRT) on the expression of systemic inflammatory, immune-modulatory, metabolic, and apoptotic genes in peripheral blood mononuclear cells (PBMCs) of patients with heart failure. METHODS AND RESULTS: We used RNA sequencing (RNA-Seq) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to identify gene expression changes in PBMCs in response to CRT. In total, 27 patients were analysed: 12 with heart failure undergoing CRT, 6 with heart failure undergoing standard implanted cardioverter defibrillators, and 9 with coronary artery disease but not heart failure. In CRT patients (median age 65.5 years, interquartile range 63.0-66.8 years, 33% female), RNA-Seq analysis identified 40 genes, including multiple genes associated with the IL-1ß pathway, with significant correlations (false discovery rate < 0.05) with four key CRT response measures. CRT was associated with suppression of PBMC expression of IL-1ß (1.80-fold decrease, P = 0.047), FOS proto-oncogene (FOS) (3.25-fold decrease, P = 0.01), dual specificity phosphatase 1 (DUSP1) (2.05-fold decrease, P = 0.001), and early growth response 1 (EGR1) (7.38-fold decrease, P = 0.03), and suppression was greater in responders vs. non-responders (P = 0.03 for IL-1ß, P = 0.02 for FOS, P = 0.02 for DUSP1, and P = 0.11 for EGR1). Baseline FOS and DUSP-1 levels were greater in responders vs. non-responders (6.15-fold higher, FOS, P = 0.002; 2.60-fold higher, DUSP1, P = 0.0001). CRT responders but not non-responders showed higher baseline gene expression of FOS (P = 0.04) and DUSP1 (P = 0.06) compared with control patients without heart failure. Baseline serum high-sensitivity C-reactive protein levels were 3.47-fold higher in CRT responders vs. non-responders (P = 0.008). CONCLUSION: Treatment of heart failure with CRT resulted in decreased PBMC expression of genes linked to inflammation. Moreover, CRT responders had higher expression of these inflammatory genes prior to CRT and greater suppression of these genes after CRT compared with non-responders.
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Terapia de Resincronización Cardíaca , Cardioversión Eléctrica , Insuficiencia Cardíaca/terapia , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Leucocitos Mononucleares/metabolismo , Anciano , Dispositivos de Terapia de Resincronización Cardíaca , Estudios de Casos y Controles , Desfibriladores Implantables , Regulación hacia Abajo , Cardioversión Eléctrica/instrumentación , Femenino , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Interleucina-1beta/genética , Masculino , Persona de Mediana Edad , Proto-Oncogenes Mas , RNA-Seq , Transcriptoma , Resultado del TratamientoRESUMEN
As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.
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Traslado Adoptivo , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Movimiento Celular , Receptores CXCR4/metabolismo , Retroviridae/metabolismo , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Diferenciación Celular , Inmunoglobulina M/inmunología , Antígenos Comunes de Leucocito , Ratones , Receptores CXCR4/genética , Transducción de SeñalRESUMEN
OBJECTIVE: Examine if adding aerobic exercise to standard medical care (EX+SC) prior to bariatric surgery improves metabolic health in relation to surgical outcomes. METHODS: Fourteen bariatric patients (age: 42.3±2.5y, BMI: 45.1±2.5 kg/m2) met inclusion criteria and were match-paired to pre-operative SC (n = 7) or EX+SC (n = 7; walking 30min/d, 5d/wk, 65-85% HRpeak) for 30d. A 120min mixed meal tolerance test was performed pre- and post-intervention (~2d prior to surgery) to assess insulin sensitivity (Matsuda Index) and metabolic flexibility (indirect calorimetry). Aerobic fitness (VO2peak), body composition (BodPod), and adipokines (adiponectin, leptin) were also measured. Omental adipose tissue was collected during surgery to quantify gene expression of adiponectin and leptin, and operating time and length of hospital stay were recorded. ANOVA and Cohen's d effect size (ES) was used to test group differences. RESULTS: SC tended to increase percent body fat (P = 0.06) after the intervention compared to EX+SC. Although SC and EX+SC tended to raise insulin sensitivity (P = 0.11), EX+SC enhanced metabolic flexibility (P = 0.01, ES = 1.55), reduced total adiponectin (P = 0.01, ES = 1.54) with no change in HMW adiponectin and decreased the length of hospital stay (P = 0.05) compared to SC. Albeit not statistically significant, EX+SC increased VO2peak 2.9% compared to a 5.9% decrease with SC (P = 0.24, ES = 0.91). This increased fitness correlated to shorter operating time (r = -0.57, P = 0.03) and length of stay (r = -0.58, P = 0.03). Less omental total adiponectin (r = 0.52, P = 0.09) and leptin (r = 0.58, P = 0.05) expression correlated with shorter operating time, and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01), and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01). CONCLUSION: Adding pre-operative aerobic exercise to standard care may improve surgical outcomes through a fitness and adipose tissue derived mechanism.
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Cirugía Bariátrica , Terapia por Ejercicio/métodos , Ejercicio Físico , Obesidad Mórbida/cirugía , Obesidad Mórbida/terapia , Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Adulto , Composición Corporal , Femenino , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Obesidad Mórbida/metabolismo , Aptitud Física , Proyectos Piloto , Cuidados Preoperatorios/métodos , Periodo Preoperatorio , Resultado del TratamientoRESUMEN
Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.
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Alcanosulfonatos/farmacología , Riñón/efectos de los fármacos , Liposomas/administración & dosificación , Hígado/efectos de los fármacos , Macrófagos/efectos de los fármacos , Obesidad/tratamiento farmacológico , PPAR alfa/agonistas , PPAR gamma/agonistas , Fenilpropionatos/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Inflamación/metabolismo , Riñón/metabolismo , Liposomas/química , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
The endocrine pancreas comprises the islets of Langerhans, tiny clusters of cells that contribute only about 2% to the total pancreas mass. However, this little endocrine organ plays a critical role in maintaining glucose homeostasis by the regulated secretion of insulin (by beta-cells) and glucagon (by alpha-cells). The rapid increase in the incidence of diabetes worldwide has spurred renewed interest in islet cell biology. Some of the most widely prescribed oral drugs for treating type 2 diabetes include agents that bind and activate the nuclear hormone receptor, peroxisome proliferator-activated receptor-gamma. As a first step in addressing potential roles of peroxisome proliferator-activated receptor-gamma and other nuclear hormone receptors (NHRs) in the biology of the endocrine pancreas, we have used quantitative real-time PCR to profile the expression of all 49 members of the mouse NHR superfamily in primary islets, and cell lines that represent alpha-cells (alphaTC1) and beta-cells (betaTC6 and MIN6). In summary, 19 NHR members were highly expressed in both alpha- and beta-cell lines, 13 receptors showed predominant expression (at least an 8-fold difference) in alpha- vs. beta-cell lines, and 10 NHRs were not expressed in the endocrine pancreas. In addition we evaluated the relative expression of these transcription factors during hyperglycemia and found that 16 NHRs showed significantly altered mRNA levels in mouse islets. A similar survey was conducted in primary human islets to reveal several significant differences in NHR expression between mouse and man. These data identify potential therapeutic targets in the endocrine pancreas for the treatment of diabetes mellitus.
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Islotes Pancreáticos/metabolismo , Receptores Citoplasmáticos y Nucleares/clasificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Línea Celular , Femenino , Expresión Génica , Glucosa/metabolismo , Homeostasis , Humanos , Islotes Pancreáticos/citología , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Glucose is the primary regulator of insulin granule release from pancreatic islets. In rodent islets, the role of glucose in the acute regulation of insulin gene transcription has remained unclear, primarily because the abundance and long half-life of insulin mRNA confounds analysis of transcription by traditional methods that measure steady-state mRNA levels. To investigate the nature of glucose-regulated insulin gene transcription in human islets, we first quantitated the abundance and half-lives of insulin mRNA and pre-mRNAs after addition of actinomycin D (to stop transcription). Our results indicated that intron 1-and intron 2-containing pre-mRNAs were approximately 150- and 2,000-fold less abundant, respectively, than mature mRNA. 5' intron 2-containing pre-mRNAs displayed half-lives of only approximately 60 min, whereas all other transcripts displayed more extended lifetimes. In response to elevated glucose, pre-mRNA species increased within 60 min, whereas increases in mature mRNA did not occur until 48 h, suggesting that measurement of mature mRNA species does not accurately reflect the acute transcriptional response of the insulin gene to glucose. The acute increase in pre-mRNA species was preceded by a sixfold increase in histone H4 acetylation and a twofold increase in RNA polymerase II recruitment at the insulin promoter. Taken together, our data suggest that pre-mRNA species may be a more reliable reflection of acute changes to human insulin gene transcriptional rates and that glucose acutely enhances insulin transcription by a mechanism that enhances chromatin accessibility and leads to recruitment of basal transcriptional machinery.
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Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/fisiología , Humanos , Insulina/genéticaRESUMEN
The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in beta-cells (betaTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in betaTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the beta-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1.
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Cromatina/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/genética , Transactivadores/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Cromatina/química , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eucromatina/química , Eucromatina/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/análisis , Ratones , Nucleosomas/química , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Transactivadores/análisis , Transcripción Genética , Activación TranscripcionalRESUMEN
In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, we identify a novel beta-cell-specific enhancer element in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element. We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. We show by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in betaTC3 cells. We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events.