RESUMEN
Fat is stored in distinct depots with unique features in both mice and humans and B cells reside in all adipose depots. We have shown that B cells modulate cardiometabolic disease through activities in two of these key adipose depots: visceral adipose tissue (VAT) and perivascular adipose tissue (PVAT). VAT refers to the adipose tissue surrounding organs, within the abdomen and thorax, and is comprised predominantly of white adipocytes. This depot has been implicated in mediating obesity-related dysmetabolism. PVAT refers to adipose tissue surrounding major arteries. It had long been thought to exist to provide protection and insulation for the vessel, yet recent work demonstrates an important role for PVAT in harboring immune cells, promoting their function and regulating the biology of the underlying vessel. The role of B-2 cells and adaptive immunity in adipose tissue biology has been nicely reviewed elsewhere. Given that, the predominance of B-1 cells in adipose tissue at homeostasis, and the emerging role of B-1 cells in a variety of disease states, we will focus this review on how B-1 cells function in VAT and PVAT depots to promote homeostasis and limit inflammation linked to cardiometabolic disease and factors that regulate this function.
Asunto(s)
Tejido Adiposo , Inmunidad Innata , Inflamación , Humanos , Animales , Inflamación/inmunología , Inflamación/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/inmunología , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Obesidad/inmunología , Obesidad/metabolismo , Grasa Intraabdominal/metabolismo , Grasa Intraabdominal/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Enfermedades Metabólicas/inmunología , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/etiología , InmunomodulaciónRESUMEN
Human recombinant B cell activating factor (BAFF) is secreted as 3-mers, which can associate to form 60-mers in culture supernatants. However, the presence of BAFF multimers in humans is still debated and it is incompletely understood how BAFF multimers activate the B cells. Here, we demonstrate that BAFF can exist as 60-mers or higher order multimers in human plasma. In vitro, BAFF 60-mer strongly induced the transcriptome of B cells which was partly attenuated by antagonism using a soluble fragment of BAFF receptor 3. Furthermore, compared to BAFF 3-mer, BAFF 60-mer strongly induced a transient classical and prolonged alternate NF-κB signaling, glucose oxidation by both aerobic glycolysis and oxidative phosphorylation, and succinate utilization by mitochondria. BAFF antagonism selectively attenuated classical NF-κB signaling and glucose oxidation. Altogether, our results suggest critical roles of BAFF 60-mer and its BAFF receptor 3 binding site in hyperactivation of B cells.
RESUMEN
B cell-activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Aneurisma de la Aorta Abdominal/terapia , Factor Activador de Células B/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/patología , Factor Activador de Células B/genética , Factor Activador de Células B/inmunología , Factor Activador de Células B/fisiología , Subgrupos de Linfocitos B/patología , Recuento de Células , Células Cultivadas , Quimiotaxis de Leucocito/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Adipose tissue macrophages (ATMs) adapt their metabolic phenotype either to maintain lean tissue homeostasis or drive inflammation and insulin resistance in obesity. However, the factors in the adipose tissue microenvironment that control ATM phenotypic polarization and bioenergetics remain unknown. We have recently shown that oxidized phospholipids (OxPL) uniquely regulate gene expression and cellular metabolism in Mox macrophages, but the presence of the Mox phenotype in adipose tissue has not been reported. Here we show, using extracellular flux analysis, that ATMs isolated from lean mice are metabolically inhibited. We identify a unique population of CX3CR1neg/F4/80low ATMs that resemble the Mox (Txnrd1+HO1+) phenotype to be the predominant ATM phenotype in lean adipose tissue. In contrast, ATMs isolated from obese mice had characteristics typical of the M1/M2 (CD11c+CD206+) phenotype with highly activated bioenergetics. Quantifying individual OxPL species in the stromal vascular fraction of murine adipose tissue, using targeted liquid chromatography-mass spectrometry, revealed that high fat diet-induced adipose tissue expansion led to a disproportional increase in full-length over truncated OxPL species. In vitro studies showed that macrophages respond to truncated OxPL species by suppressing bioenergetics and up-regulating antioxidant programs, mimicking the Mox phenotype of ATMs isolated from lean mice. Conversely, full-length OxPL species induce proinflammatory gene expression and an activated bioenergetic profile that mimics ATMs isolated from obese mice. Together, these data identify a redox-regulatory Mox macrophage phenotype to be predominant in lean adipose tissue and demonstrate that individual OxPL species that accumulate in adipose tissue instruct ATMs to adapt their phenotype and bioenergetic profile to either maintain redox homeostasis or to promote inflammation.
Asunto(s)
Tejido Adiposo , Antígenos de Diferenciación , Metabolismo Energético , Macrófagos , Obesidad , Fosfolípidos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Fosfolípidos/genética , Fosfolípidos/metabolismoRESUMEN
OBJECTIVE: Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1ß in the formation of murine experimental AAAs. Here, the hypothesis that IL-1ß-induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested. APPROACH AND RESULTS: NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1ß. In vitro, IL-1RA attenuated IL-1ß-induced NETosis in human neutrophils. Mechanistically, IL-1ß treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-ß-induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage-day 3 of aneurysm induction. IL-1ß-knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1ß-knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation. CONCLUSIONS: Altogether, the results suggest a dominant role of IL-1ß-induced NETosis in AAA formation.
Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Trampas Extracelulares/metabolismo , Interleucina-1beta/metabolismo , Neutrófilos/metabolismo , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/prevención & control , Ceramidas/metabolismo , Modelos Animales de Enfermedad , Trampas Extracelulares/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Interleucina-1beta/deficiencia , Interleucina-1beta/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente/métodos , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Neutrófilos/trasplante , Ornitina/análogos & derivados , Ornitina/farmacología , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Esfingosina N-Aciltransferasa/metabolismoRESUMEN
OBJECTIVE: Resolvins have been shown to attenuate inflammation, whereas NETosis, the process of neutrophils releasing neutrophil extracellular traps (NETs), produces increased inflammation. It is hypothesized that treatment of animals with resolvin D1 (RvD1) would reduce abdominal aortic aneurysm (AAA) formation by inhibiting NETosis. METHODS: Wild-type 8- to 12-week-old C57BL/6 male mice (n = 47) and apolipoprotein E-deficient (ApoE-/-) mice (n = 20) were used in two models to demonstrate the effects of RvD1 on AAA growth. In the topical elastase AAA model, wild-type mice were divided into three groups: a deactivated elastase control group, in which sham surgery was performed using deactivated elastase and mice were intravenously injected with phosphate-buffered saline (PBS) once a day until harvest; an elastase group, in which active elastase was used to induce AAA and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which AAA was induced and mice were injected with RvD1 daily until harvest. In the angiotensin II (Ang II)-induced AAA model, ApoE-/- mice were fed a high-fat diet and implanted with osmotic infusion pumps containing Ang II (1000 ng/kg/min). The Ang II model was divided into two groups: an Ang II control group, in which Ang II was delivered and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which Ang II was delivered and mice were injected with RvD1 daily until harvest. On postoperative day 3, day 14, or day 28, aortic and blood samples were collected for Western blot, histology, cytokine array, enzyme-linked immunosorbent assay, and gelatin zymography after aortic diameter measurement. RESULTS: The day 14 RvD1-treated group demonstrated 42% reduced AAA diameter compared with the elastase group (P < .001). On postoperative day 3, the RvD1-treated group showed decreased levels of NETosis markers citrullinated histone H3 (P = .04) and neutrophil elastase (P = .002) compared with the elastase group. Among important cytokines involved in AAA formation, interleukin (IL) 1ß was downregulated (P = .02) whereas IL-10, a protective cytokine, was upregulated (P = .01) in the RvD1-treated group. Active matrix metalloproteinase 2 also decreased in the RvD1-treated group (P = .03). The RvD1-treated group in the Ang II AAA model, a second model, demonstrated reduced AAA diameter compared with the Ang II control group on day 28 (P < .046). The RvD1-treated group showed decreased levels of citrullinated histone H3 on day 3 (P = .002). Cytokines interferon γ, IL-1ß, C-X-C motif chemokine ligand 10, monocyte chemotactic protein 1, and regulated on activation, normal T cell expressed and secreted (RANTES) were all decreased on day 28 (P < .05). CONCLUSIONS: RvD1-mediated inhibition of NETosis may represent a future medical treatment for the attenuation of AAA growth.
Asunto(s)
Antiinflamatorios/farmacología , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Ácidos Docosahexaenoicos/farmacología , Trampas Extracelulares/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Citrulinación , Citocinas/metabolismo , Modelos Animales de Enfermedad , Trampas Extracelulares/metabolismo , Histonas/metabolismo , Mediadores de Inflamación/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Neutrófilos/metabolismo , Neutrófilos/patología , Elastasa Pancreática , Remodelación Vascular/efectos de los fármacosRESUMEN
OBJECTIVE: B-cell depletion therapy is widely used for treatment of cancers and autoimmune diseases. B cells are abundant in abdominal aortic aneurysms (AAA); however, it is unknown whether B-cell depletion therapy affects AAA growth. Using experimental models of murine AAA, we aim to examine the effect of B-cell depletion on AAA formation. APPROACH AND RESULTS: Wild-type or apolipoprotein E-knockout mice were treated with mouse monoclonal anti-CD20 or control antibodies and subjected to an elastase perfusion or angiotensin II infusion model to induce AAA, respectively. Anti-CD20 antibody treatment significantly depleted B1 and B2 cells, and strikingly suppressed AAA growth in both models. B-cell depletion resulted in lower circulating IgM levels, but did not affect the levels of IgG or cytokine/chemokine levels. Although the total number of leukocyte remained unchanged in elastase-perfused aortas after anti-CD20 antibody treatment, the number of B-cell subtypes was significantly lower. Interestingly, plasmacytoid dendritic cells expressing the immunomodulatory enzyme indole 2,3-dioxygenase were detected in the aortas of B-cell-depleted mice. In accordance with an increase in indole 2,3-dioxygenase+ plasmacytoid dendritic cells, the number of regulatory T cells was higher, whereas the expression of proinflammatory genes was lower in aortas of B-cell-depleted mice. In a coculture model, the presence of B cells significantly lowered the number of indole 2,3-dioxygenase+ plasmacytoid dendritic cells without affecting total plasmacytoid dendritic cell number. CONCLUSIONS: The present results demonstrate that B-cell depletion protects mice from experimental AAA formation and promotes emergence of an immunosuppressive environment in aorta.
Asunto(s)
Anticuerpos/farmacología , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Linfocitos B/efectos de los fármacos , Depleción Linfocítica/métodos , Angiotensina II , Animales , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Aorta Abdominal/inmunología , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores/sangre , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo , Citocinas/sangre , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Predisposición Genética a la Enfermedad , Inmunoglobulina M/sangre , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Mediadores de Inflamación/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Elastasa Pancreática , Fenotipo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Recent reports of rupture in patients with abdominal aortic aneurysm (AAA) receiving B-cell depletion therapy highlight the importance of understanding the role of B cells (B1 and B2 subsets) in the development of AAA. We hypothesized that B2 cells aggravate experimental aneurysm formation. The IHC staining revealed infiltration of B cells in the aorta of wild-type (C57BL/6) mice at day 7 after elastase perfusion and persisted through day 21. Quantification of immune cell types using flow cytometry at day 14 showed significantly greater infiltration of mononuclear cells, including B cells (B2: 93% of total B cells) and T cells in elastase-perfused aortas compared with saline-perfused or normal aortas. muMT (mature B-cell deficient) mice were prone to AAA formation similar to wild-type mice in two different experimental AAA models. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AAA formation (102.0% ± 7.3% versus 75.2% ± 5.5%; P < 0.05) with concomitant increase in the splenic regulatory T cell (0.24% ± 0.03% versus 0.92% ± 0.23%; P < 0.05) and decrease in aortic infiltration of mononuclear cells. Our data suggest that B2 cells constitute the largest population of B cells in experimental AAA. Furthermore, B2 cells, in the absence of other B-cell subsets, increase splenic regulatory T-cell population and suppress AAA formation.
Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Linfocitos B/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/metabolismoRESUMEN
OBJECTIVE: The impact of leukotriene production by the 5-lipoxygenase (5-LO) pathway in the pathophysiology of abdominal aortic aneurysms (AAAs) has been debated. Moreover, a clear mechanism through which 5-LO influences AAA remains unclear. APPROACH AND RESULTS: Aneurysm formation was attenuated in 5-LO(-/-) mice, and in lethally irradiated wild-type mice reconstituted with 5-LO(-/-) bone marrow in an elastase perfusion model. Pharmacological inhibition of 5-LO-attenuated aneurysm formation in both aortic elastase perfused wild-type and angiotensin II-treated LDLr(-/-) (low-density lipoprotein receptor) mice, with resultant preservation of elastin and fewer 5-LO and MMP9 (matrix metalloproteinase)-producing cells. Separately, analysis of wild-type mice 7 days after elastase perfusion showed that 5-LO inhibition was associated with reduced polymorphonuclear leukocyte infiltration to the aortic wall. Importantly, 5-LO inhibition initiated 3 days after elastase perfusion in wild-type mice arrested progression of small AAA. Human AAA and control aorta corroborated these elastin and 5-LO expression patterns. CONCLUSIONS: Inhibition of 5-LO by pharmacological or genetic approaches attenuates aneurysm formation and prevents fragmentation of the medial layer in 2 unique AAA models. Administration of 5-LO inhibitor in small AAA slows progression of AAA. Targeted interruption of the 5-LO pathway is a potential treatment strategy in AAA.
Asunto(s)
Aneurisma de la Aorta Abdominal/enzimología , Araquidonato 5-Lipooxigenasa/metabolismo , Anciano , Angiotensina II/metabolismo , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/enzimología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/etiología , Aneurisma de la Aorta Abdominal/patología , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Infiltración Neutrófila , Elastasa Pancreática/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal , Quimera por Trasplante/metabolismoRESUMEN
Activation of the adenosine 2A receptor (A2AR) reduces inflammation in models of acute injury but contribution in development of chronic abdominal aortic aneurysms (AAAs) is unknown. Elastase perfusion to induce AAA formation in A2AR-knockout (A2ARKO) and C57BL6/J wild-type (WT) mice resulted in nearly 100% larger aneurysms in A2ARKO compared to WT at d 14 (P<0.05), with evidence of greater elastin fragmentation, more immune cell infiltration, and increased matrix metallatoproteinase (MMP) 9 expression (P<0.05). Separately, exogenous A2AR antagonism in elastase-perfused WT mice also resulted in larger aneurysms (P<0.05), while A2AR agonism limited aortic dilatation (P<0.05). Activated Thy-1.2(+) T lymphocytes from WT mice treated in vitro with A2AR antagonist increased cytokine production, and treatment with A2AR agonist decreased cytokine production (P<0.05 for all). Primary activated CD4(+) T lymphocytes from A2ARKO mice exhibited greater chemotaxis (P<0.05). A2AR antagonist increased chemotaxis of activated CD4(+) cells from WT mice in vitro, and A2AR agonist reduced this effect (P<0.05). A2AR activation attenuates AAA formation partly by inhibiting immune cell recruitment and reducing elastin fragmentation. These findings support augmenting A2AR signaling as a putative target for limiting aneurysm formation.
Asunto(s)
Aneurisma de la Aorta Abdominal/metabolismo , Receptores de Adenosina A2/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/patología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Elastasa Pancreática/administración & dosificación , Fenetilaminas/farmacología , Fenotipo , Receptores de Adenosina A2/deficiencia , Receptores de Adenosina A2/genética , Triazinas/farmacología , Triazoles/farmacologíaRESUMEN
B cell-activating factor (BAFF) is a critical TNF-family cytokine that regulates homeostasis and peripheral tolerance of B2 cells. BAFF overproduction promotes autoantibody generation and autoimmune diseases. During obesity, BAFF is predominantly produced by white adipose tissue (WAT), and IgG autoantibodies against adipocytes are identified in the WAT of obese humans. However, it remains to be determined if the autoantibodies formed during obesity affect WAT remodeling and systemic insulin resistance. Here, we show that IgG autoantibodies are generated in high-fat diet (HFD)-induced obese mice that bind to apoptotic adipocytes and promote their phagocytosis by macrophages. Next, using murine models of obesity in which the gonadal WAT undergoes remodeling, we found that BAFF neutralization depleted IgG autoantibodies, increased the number of dead adipocytes, and exacerbated WAT inflammation and insulin resistance. RNA sequencing of the stromal vascular fraction from the WAT revealed decreased expression of immunoglobulin light-chain and heavy-chain variable genes suggesting a decreased repertoire of B cells after BAFF neutralization. Further, the B cell activation and the phagocytosis pathways were impaired in the WAT of BAFF-neutralized mice. In vitro, plasma IgG fractions from BAFF-neutralized mice reduced the phagocytic clearance of apoptotic adipocytes. Altogether, our study suggests that IgG autoantibodies developed during obesity, at least in part, dampens exacerbated WAT inflammation and systemic insulin resistance.
Asunto(s)
Adipocitos , Autoanticuerpos , Factor Activador de Células B , Inmunoglobulina G , Resistencia a la Insulina , Obesidad , Fagocitosis , Animales , Resistencia a la Insulina/inmunología , Factor Activador de Células B/metabolismo , Factor Activador de Células B/inmunología , Ratones , Obesidad/inmunología , Obesidad/metabolismo , Autoanticuerpos/inmunología , Adipocitos/inmunología , Adipocitos/metabolismo , Fagocitosis/inmunología , Inmunoglobulina G/inmunología , Masculino , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Apoptosis/inmunología , Tejido Adiposo Blanco/inmunología , Tejido Adiposo Blanco/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Animales de EnfermedadRESUMEN
RATIONALE: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. OBJECTIVE: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. METHODS AND RESULTS: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2(-/-) mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR(-/-)) mice. CONCLUSIONS: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.
Asunto(s)
Aterosclerosis/metabolismo , Inmunofenotipificación , Macrófagos/fisiología , Factor 2 Relacionado con NF-E2/fisiología , Fosfolípidos/fisiología , Animales , Aterosclerosis/genética , Células Cultivadas , Femenino , Macrófagos/clasificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/biosíntesis , Factor 2 Relacionado con NF-E2/deficiencia , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción , Fosfolípidos/metabolismoRESUMEN
TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, gene regulation, growth, apoptosis, cellular senescence, DNA repair, drug resistance, malignant transformation, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene TP53 is mutated frequently (50-75%) in PDAC. Different types of TP53 mutations have been observed including gain of function (GOF) point mutations and various deletions of the TP53 gene resulting in lack of the protein expression. Most PDACs have point mutations at the KRAS gene which result in constitutive activation of KRas and multiple downstream signaling pathways. It has been difficult to develop specific KRas inhibitors and/or methods that result in recovery of functional TP53 activity. To further elucidate the roles of TP53 in drug-resistance of pancreatic cancer cells, we introduced wild-type (WT) TP53 or a control vector into two different PDAC cell lines. Introduction of WT-TP53 increased the sensitivity of the cells to multiple chemotherapeutic drugs, signal transduction inhibitors, drugs and nutraceuticals and influenced key metabolic properties of the cells. Therefore, TP53 is a key molecule which is critical in drug sensitivity and metabolism of PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Proliferación Celular , Suplementos Dietéticos , Receptores ErbB/genética , Mutación con Ganancia de Función , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor , Neoplasias PancreáticasRESUMEN
Glycogen synthase kinase-3 (GSK-3) is a regulator of signaling pathways. KRas is frequently mutated in pancreatic cancers. The growth of certain pancreatic cancers is KRas-dependent and can be suppressed by GSK-3 inhibitors, documenting a link between KRas and GSK-3. To further elucidate the roles of GSK-3ß in drug-resistance, we transfected KRas-dependent MIA-PaCa-2 pancreatic cells with wild-type (WT) and kinase-dead (KD) forms of GSK-3ß. Transfection of MIA-PaCa-2 cells with WT-GSK-3ß increased their resistance to various chemotherapeutic drugs and certain small molecule inhibitors. Transfection of cells with KD-GSK-3ß often increased therapeutic sensitivity. An exception was observed with cells transfected with WT-GSK-3ß and sensitivity to the BCL2/BCLXL ABT737 inhibitor. WT-GSK-3ß reduced glycolytic capacity of the cells but did not affect the basal glycolysis and mitochondrial respiration. KD-GSK-3ß decreased both basal glycolysis and glycolytic capacity and reduced mitochondrial respiration in MIA-PaCa-2 cells. As a comparison, the effects of GSK-3 on MCF-7 breast cancer cells, which have mutant PIK3CA, were examined. KD-GSK-3ß increased the resistance of MCF-7 cells to chemotherapeutic drugs and certain signal transduction inhibitors. Thus, altering the levels of GSK-3ß can have dramatic effects on sensitivity to drugs and signal transduction inhibitors which may be influenced by the background of the tumor.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Suplementos Dietéticos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Adenilato Quinasa/metabolismo , Antineoplásicos/farmacología , Berberina/farmacología , Berberina/uso terapéutico , Compuestos de Bifenilo/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Diabetes Mellitus/tratamiento farmacológico , Progresión de la Enfermedad , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Femenino , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Glucólisis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Células MCF-7 , Malaria/tratamiento farmacológico , Metformina/farmacología , Metformina/uso terapéutico , Metástasis de la Neoplasia , Nitrofenoles/farmacología , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Tiadiazoles/farmacología , Tiadiazoles/uso terapéutico , Ensayo de Tumor de Célula Madre , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismo , GemcitabinaRESUMEN
OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of ß-adrenergic receptor (ßAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by ß3AR stimulation via a novel mechanism that involves Gßγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of ß3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective ß3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gßγ-dependent Panx1 channel activation is involved in ß3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of ß3AR activation may have therapeutic implications.
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Tejido Adiposo Pardo/metabolismo , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Termogénesis/fisiología , Adipocitos Marrones/metabolismo , Adiponectina/metabolismo , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/metabolismo , Animales , Frío , Conexinas/genética , Fluorodesoxiglucosa F18 , Resistencia a la Insulina , Lipólisis , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Obesidad/metabolismo , Transducción de Señal , Termogénesis/genética , TranscriptomaRESUMEN
Large-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has insufficient volume for neutron diffraction. The thin physical dimension has been identified as corresponding to the b cell edge and the X-ray structure identified a solvent-mediated crystal contact adjacent to position Glu81 that was hypothesized to limit efficient crystal growth in this dimension. In this report, a series of mutations at this crystal contact designed to both reduce side-chain entropy and replace the solvent-mediated interface with direct side-chain contacts are reported. The results suggest that improved crystal growth is achieved upon the introduction of direct crystal contacts, while little improvement is observed with side-chain entropy-reducing mutations alone.
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Cristalización/métodos , Factor 1 de Crecimiento de Fibroblastos/química , Ingeniería de Proteínas/métodos , Solventes/química , Cristalografía por Rayos X , Factor 1 de Crecimiento de Fibroblastos/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Difracción de Neutrones , Conformación Proteica , Difracción de Rayos XRESUMEN
OBJECTIVE: Macrophages control tissue homeostasis and inflammation by sensing and responding to environmental cues. However, the metabolic adaptation of macrophages to oxidative tissue damage and its translation into inflammatory mechanisms remains enigmatic. METHODS: Here we identify the critical regulatory pathways that are induced by endogenous oxidation-derived DAMPs (oxidized phospholipids, OxPL) in vitro, leading to formation of a unique redox-regulatory metabolic phenotype (Mox), which is strikingly different from conventional classical or alternative macrophage activation. RESULTS: Unexpectedly, metabolomic analyses demonstrated that Mox heavily rely on glucose metabolism and the pentose phosphate pathway (PPP) to support GSH production and Nrf2-dependent antioxidant gene expression. While the metabolic adaptation of macrophages to OxPL involved transient suppression of aerobic glycolysis, it also led to upregulation of inflammatory gene expression. In contrast to classically activated (M1) macrophages, Hif1α mediated expression of OxPL-induced Glut1 and VEGF but was dispensable for Il1ß expression. Mechanistically, we show that OxPL suppress mitochondrial respiration via TLR2-dependent ceramide production, redirecting TCA metabolites to GSH synthesis. Finally, we identify spleen tyrosine kinase (Syk) as a critical downstream signaling mediator that translates OxPL-induced effects into ceramide production and inflammatory gene regulation. CONCLUSIONS: Together, these data demonstrate the metabolic and bioenergetic requirements that enable macrophages to translate tissue oxidation status into either antioxidant or inflammatory responses via sensing OxPL. Targeting dysregulated redox homeostasis in macrophages could therefore lead to novel therapies to treat chronic inflammation.
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Ceramidas/metabolismo , Homeostasis , Macrófagos/metabolismo , Estrés Oxidativo , Quinasa Syk/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Células Cultivadas , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glutatión/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Vía de Pentosa Fosfato , Transducción de Señal , Quinasa Syk/genética , Receptor Toll-Like 2/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The 6-kDa early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10), expressed from the region of deletion-1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in virulence. In this study, we explored the thermodynamic and biochemical changes associated with the formation of the 1 : 1 heterodimeric complex between ESAT-6 and CFP-10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 x 10(7) M(-1) and -9.95 kcal.mol(-1), respectively. Strikingly, the thermal unfolding of the ESAT-6-CFP-10 heterodimeric complex was completely reversible, with a T(m) of 53.4 degrees C and DeltaH of 69 kcal.mol(-1). Mixing of ESAT-6 and CFP-10 at any temperature below the T(m) of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT-6 and CFP-10. Enhanced biochemical stability of the complex was indicated by protection of ESAT-6 and an N-terminal fragment of CFP-10 from proteolysis with trypsin. However, the flexible C-terminal of CFP-10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT-6, but not CFP-10 and the complex, showed an increase in alpha-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT-6, CFP-10 and/or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT-6 family proteins and for screening ESAT-6 and CFP-10 mutants.
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Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Estructura Cuaternaria de Proteína , Antígenos Bacterianos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calorimetría , Dicroismo Circular , Dimerización , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Complejos Multiproteicos , Mycobacterium tuberculosis/genética , Resonancia Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , TermodinámicaRESUMEN
OBJECTIVE: B lymphocytes are generally considered to be activators of the immune response; however, recent findings have shown that a subtype of B lymphocytes, regulatory B lymphocytes, play a role in attenuating the immune response. Bronchiolitis obliterans remains the major limitation to modern-day lung transplantation. The role of regulatory B lymphocytes in bronchiolitis obliterans has not been elucidated. We hypothesized that regulatory B lymphocytes play a role in the attenuation of bronchiolitis obliterans. METHODS: We performed a standard heterotopic tracheal transplant model. Tracheas from Balb/c mice were transplanted into C57BL/6 recipients. Rapamycin treatment and dimethyl sulfoxide control groups were each treated for the first 14 days after the transplant. Tracheas were collected on days 7, 14, and 28 post-transplantation. Luminal obliteration was evaluated by hematoxylin-eosin staining and Picrosirius red staining. Immune cell infiltration and characteristics, and secretion of interleukin-10 and transforming growth factor-ß1 were accessed by immunohistochemistry. Cytokines and transforming growth factor-ß1 were measured using the Luminex assay (Bio-Rad, Hercules, Calif). RESULTS: The results revealed that intraperitoneal injection of rapamycin for 14 days after tracheal transplantation significantly reduced luminal obliteration on day 28 when compared with the dimethyl sulfoxide control group (97.78% ± 3.63% vs 3.02% ± 2.14%, P < .001). Rapamycin treatment markedly induced regulatory B lymphocytes (B220(+)IgM(+)IgG(-)IL-10(+)TGF-ß1(+)) cells when compared with dimethyl sulfoxide controls. Rapamycin treatment inhibited interleukin-1ß, 6, 13, and 17 on days 7 and 14. Rapamycin also greatly increased interleukin-10 and transforming growth factor-ß1 production in B cells and regulatory T lymphocytes infiltration on day 28. CONCLUSIONS: Mammalian target of rapamycin inhibition decreases the development of bronchiolitis obliterans via inhibition of proinflammatory cytokines and increasing regulatory B lymphocytes cell infiltration, which subsequently produces anti-inflammatory cytokines and upregulates regulatory T lymphocyte cells.
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Antiinflamatorios/farmacología , Linfocitos B/efectos de los fármacos , Bronquiolitis Obliterante/prevención & control , Quimiotaxis de Leucocito/efectos de los fármacos , Sirolimus/farmacología , Tráquea/efectos de los fármacos , Tráquea/trasplante , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/inmunología , Bronquiolitis Obliterante/metabolismo , Bronquiolitis Obliterante/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factores de Tiempo , Tráquea/inmunología , Tráquea/metabolismo , Tráquea/patologíaRESUMEN
OBJECTIVE: Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. METHODS: We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. RESULTS: Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. CONCLUSIONS: We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.