RESUMO
Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.
Assuntos
Asma/imunologia , Comunicação Celular/imunologia , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Proliferação de Células/fisiologia , Conexinas/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Sistema Respiratório/imunologiaRESUMO
Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.
Assuntos
Cálcio/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Técnicas de Cultura de Células , Endocitose/efeitos dos fármacos , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica , Técnicas de Genotipagem , Immunoblotting , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/genéticaRESUMO
BACKGROUND: Clinical phenotype information greatly facilitates genetic diagnostic interpretations pipelines in disease. While post-hoc extraction using natural language processing on unstructured clinical notes continues to improve, there is a need to improve point-of-care collection of patient phenotypes. Therefore, we developed "PheNominal", a point-of-care web application, embedded within Epic electronic health record (EHR) workflows, to permit capture of standardized phenotype data. METHODS: Using bi-directional web services available within commercial EHRs, we developed a lightweight web application that allows users to rapidly browse and identify relevant terms from the Human Phenotype Ontology (HPO). Selected terms are saved discretely within the patient's EHR, permitting reuse both in clinical notes as well as in downstream diagnostic and research pipelines. RESULTS: In the 16 months since implementation, PheNominal was used to capture discrete phenotype data for over 1500 individuals and 11,000 HPO terms during clinic and inpatient encounters for a genetic diagnostic consultation service within a quaternary-care pediatric academic medical center. An average of 7 HPO terms were captured per patient. Compared to a manual workflow, the average time to enter terms for a patient was reduced from 15 to 5 min per patient, and there were fewer annotation errors. CONCLUSIONS: Modern EHRs support integration of external applications using application programming interfaces. We describe a practical application of these interfaces to facilitate deep phenotype capture in a discrete, structured format within a busy clinical workflow. Future versions will include a vendor-agnostic implementation using FHIR. We describe pilot efforts to integrate structured phenotyping through controlled dictionaries into diagnostic and research pipelines, reducing manual effort for phenotype documentation and reducing errors in data entry.
Assuntos
Registros Eletrônicos de Saúde , Sistemas Automatizados de Assistência Junto ao Leito , Criança , Documentação , Humanos , Processamento de Linguagem Natural , SoftwareRESUMO
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.
Assuntos
Tecido Adiposo , Antígenos de Diferenciação , Metabolismo Energético , Macrófagos , Obesidade , Fosfolipídeos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant "renin recruitment" as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.
Assuntos
Conexinas , Renina , Trifosfato de Adenosina , Animais , Pressão Sanguínea , Conexinas/genética , Feminino , Homeostase , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genéticaRESUMO
RATIONALE: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. OBJECTIVE: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. METHODS AND RESULTS: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. CONCLUSIONS: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.
Assuntos
Anti-Hipertensivos/farmacologia , Conexinas/antagonistas & inibidores , Diuréticos/farmacologia , Hipertensão/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Espironolactona/farmacologia , Animais , Anti-Hipertensivos/uso terapêutico , Arteríolas/efeitos dos fármacos , Conexinas/metabolismo , Diuréticos/uso terapêutico , Células HEK293 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Espironolactona/uso terapêuticoRESUMO
TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.
Assuntos
Cloridrato de Fingolimode , Canais de Cátion TRPM , Cloridrato de Fingolimode/farmacologia , Ciclopentanos , FosforilaçãoRESUMO
OBJECTIVE: Prompt ascertainment is crucial for the management of hyperammonemic infants. Because these patients are rare and recognition of hyperammonemia is often delayed, we designed and implemented an electronic medical record (EMR)-based tool to assist physicians in the detection of hyperammonemia. METHODS: We retrospectively evaluated the hospitalizations of prior hyperammonemic infants to identify codable elements that could trigger an EMR-based warning. An alert was designed and implemented and its utilization was prospectively analyzed. RESULTS: Blood gas studies were obtained universally and early in the retrospectively evaluated infants (x¯=26h before ammonia level). Prompting physicians to evaluate ammonia after ordering blood gas studies would have accelerated the initial ammonia order in 89% of retrospective cases. The alert has activated 184 times over the first six months of operation leading to 63 laboratory evaluations and detection of one hyperammonemic infant. CONCLUSION: Implementation of an EMR-based warning system can improve surveillance for hyperammonemia in a susceptible population.
Assuntos
Amônia/metabolismo , Registros Eletrônicos de Saúde , Hiperamonemia/diagnóstico , Ureia/metabolismo , Monitoramento Epidemiológico , Feminino , Humanos , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Lactente , Recém-Nascido , Masculino , Estudos RetrospectivosRESUMO
OBJECTIVE: State agencies have developed reporting systems of safety events that include events related to health information technology (HIT). These data come from hospital reporting systems where staff submit safety reports and nurses, in the role of safety managers, review, and code events. Safety managers may have varying degrees of experience with identifying events related to HIT. Our objective was to review events potentially involving HIT and compare those with what was reported to the state. METHODS: We performed a structured review of 1 year of safety events from an academic pediatric healthcare system. We reviewed the free-text description of each event and applied a classification scheme derived from the AHRQ Health IT Hazard Manager and compared the results with events reported to the state as involving HIT. RESULTS: Of 33,218 safety events for a 1-year period, 1247 included key words related to HIT and/or were indicated by safety managers as involving HIT. Of the 1247 events, the structured review identified 769 as involving HIT. In comparison, safety managers only identified 194 of the 769 events (25%) as involving HIT. Most events, 353 (46%), not identified by safety managers were documentation issues. Of the 1247 events, the structured review identified 478 as not involving HIT while safety managers identified and reported 81 of these 478 events (17%) as involving HIT. CONCLUSIONS: The current process of reporting safety events lacks standardization in identifying health technology contributions to safety events, which may minimize the effectiveness of safety initiatives.
Assuntos
Documentação , Informática Médica , Criança , Humanos , Centros de Atenção Terciária , Relatório de Pesquisa , Atenção à SaúdeRESUMO
TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine 1-phosphate (S1P) receptors. In this study, we tested non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo , for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, an effect that is predominantly but not solely mediated by TRPM7. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo . Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting. These inhibitors exhibit potent anti-inflammatory properties that are mediated by TRPM7 and likely other molecular targets that remain to be identified.
RESUMO
Mitochondrial dysfunction is linked to age-associated inflammation or inflammaging, but underlying mechanisms are not understood. Analyses of 700 human blood transcriptomes revealed clear signs of age-associated low-grade inflammation. Among changes in mitochondrial components, we found that the expression of mitochondrial calcium uniporter (MCU) and its regulatory subunit MICU1, genes central to mitochondrial Ca2+ (mCa2+) signaling, correlated inversely with age. Indeed, mCa2+ uptake capacity of mouse macrophages decreased significantly with age. We show that in both human and mouse macrophages, reduced mCa2+ uptake amplifies cytosolic Ca2+ oscillations and potentiates downstream nuclear factor kappa B activation, which is central to inflammation. Our findings pinpoint the mitochondrial calcium uniporter complex as a keystone molecular apparatus that links age-related changes in mitochondrial physiology to systemic macrophage-mediated age-associated inflammation. The findings raise the exciting possibility that restoring mCa2+ uptake capacity in tissue-resident macrophages may decrease inflammaging of specific organs and alleviate age-associated conditions such as neurodegenerative and cardiometabolic diseases.
Assuntos
Cálcio , Proteínas de Transporte da Membrana Mitocondrial , Camundongos , Animais , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Cálcio/metabolismo , Mitocôndrias/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Proteínas de Ligação ao Cálcio/genéticaRESUMO
Efficient clearance of apoptotic cells by phagocytosis, also known as efferocytosis, is fundamental to developmental biology, organ physiology, and immunology. Macrophages use multiple mechanisms to detect and engulf apoptotic cells, but the signaling pathways that regulate the digestion of the apoptotic cell cargo, such as the dynamic Ca2+ signals, are poorly understood. Using an siRNA screen, we identify TRPM7 as a Ca2+-conducting ion channel essential for phagosome maturation during efferocytosis. Trpm7-targeted macrophages fail to fully acidify or digest their phagosomal cargo in the absence of TRPM7. Through perforated patch electrophysiology, we demonstrate that TRPM7 mediates a pH-activated cationic current necessary to sustain phagosomal acidification. Using mice expressing a genetically-encoded Ca2+ sensor, we observe that phagosome maturation requires peri-phagosomal Ca2+-signals dependent on TRPM7. Overall, we reveal TRPM7 as a central regulator of phagosome maturation during macrophage efferocytosis.
Assuntos
Sinalização do Cálcio , Fagocitose , Canais de Cátion TRPM , Animais , Macrófagos/metabolismo , Camundongos , Fagocitose/fisiologia , Fagossomos/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Acetyl-CoA carboxylase (ACC) regulates lipid synthesis; however, its role in inflammatory regulation in macrophages remains unclear. We generated mice that are deficient in both ACC isoforms in myeloid cells. ACC deficiency altered the lipidomic, transcriptomic, and bioenergetic profile of bone marrow-derived macrophages, resulting in a blunted response to proinflammatory stimulation. In response to lipopolysaccharide (LPS), ACC is required for the early metabolic switch to glycolysis and remodeling of the macrophage lipidome. ACC deficiency also resulted in impaired macrophage innate immune functions, including bacterial clearance. Myeloid-specific deletion or pharmacological inhibition of ACC in mice attenuated LPS-induced expression of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß, while pharmacological inhibition of ACC increased susceptibility to bacterial peritonitis in wild-type mice. Together, we identify a critical role for ACC in metabolic regulation of the innate immune response in macrophages, and thus a clinically relevant, unexpected consequence of pharmacological ACC inhibition.
Assuntos
Acetil-CoA Carboxilase , Glucose , Animais , Camundongos , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Camundongos Knockout , Macrófagos/metabolismo , Inflamação/metabolismoRESUMO
Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.
Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Tecido Adiposo Marrom/metabolismo , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Termogênese/fisiologia , Adipócitos Marrons/metabolismo , Adiponectina/metabolismo , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Baixa , Conexinas/genética , Fluordesoxiglucose F18 , Resistência à Insulina , Lipólise , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Obesidade/metabolismo , Transdução de Sinais , Termogênese/genética , TranscriptomaRESUMO
Herpes simplex virus-1 (HSV-1) establishes a latent infection in neurons and periodically reactivates to cause disease. The stimuli that trigger HSV-1 reactivation have not been fully elucidated. We demonstrate HSV-1 reactivation from latently infected mouse neurons induced by forskolin requires neuronal excitation. Stimuli that directly induce neurons to become hyperexcitable also induced HSV-1 reactivation. Forskolin-induced reactivation was dependent on the neuronal pathway of DLK/JNK activation and included an initial wave of viral gene expression that was independent of histone demethylase activity and linked to histone phosphorylation. IL-1ß is released under conditions of stress, fever and UV exposure of the epidermis; all known triggers of clinical HSV reactivation. We found that IL-1ß induced histone phosphorylation and increased the excitation in sympathetic neurons. Importantly, IL-1ß triggered HSV-1 reactivation, which was dependent on DLK and neuronal excitability. Thus, HSV-1 co-opts an innate immune pathway resulting from IL-1 stimulation of neurons to induce reactivation.
Assuntos
Herpesvirus Humano 1/fisiologia , Interleucina-1beta/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Neurônios/virologia , Ativação Viral/fisiologia , Animais , Herpes Simples/imunologia , Herpes Simples/metabolismo , Camundongos , Latência Viral/fisiologiaRESUMO
The thymic development of regulatory T (Treg) cells, crucial suppressors of the responses of effector T (Teff) cells, is governed by the transcription factor FOXP3. Despite the clinical importance of Treg cells, there is a dearth of druggable molecular targets capable of increasing their numbers in vivo. We found that inhibiting the function of the TRPM7 chanzyme (ion channel and enzyme) potentiated the thymic development of Treg cells in mice and led to a substantially higher frequency of functional Treg cells in the periphery. In addition, TRPM7-deficient mice were resistant to T cell-driven hepatitis. Deletion of Trpm7 and inhibition of TRPM7 channel activity by the FDA-approved drug FTY720 increased the sensitivity of T cells to the cytokine interleukin-2 (IL-2) through a positive feed-forward loop involving increased expression of the IL-2 receptor α-subunit and activation of the transcriptional regulator STAT5. Enhanced IL-2 signaling increased the expression of Foxp3 in thymocytes and promoted thymic Treg (tTreg) cell development. Thus, these data indicate that inhibiting TRPM7 activity increases Treg cell numbers, suggesting that it may be a therapeutic target to promote immune tolerance.
Assuntos
Interleucina-2/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Canais de Cátion TRPM/imunologia , Timo/imunologia , Animais , Feminino , Deleção de Genes , Interleucina-2/genética , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética , Canais de Cátion TRPM/genética , Timo/crescimento & desenvolvimentoRESUMO
Phagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca2+ (phase 1), and by concurrently activating the mitochondrial Ca2+ (mCa2+) uniporter (MCU), they trigger a rapid influx of Ca2+ into the mitochondria (phase 2). mCa2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa2+ signaling, Mcu-/- macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca2+ signaling is an electrometabolic switch to fuel phagosome killing.
Assuntos
Cálcio/metabolismo , Candida albicans/patogenicidade , Mitocôndrias/metabolismo , Fagossomos/metabolismo , Animais , Camundongos , Transdução de SinaisRESUMO
Insulin secretion from ß-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of ß-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of ß-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.
Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the ß-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when ß-cells become exhausted from increased insulin demand and stop producing insulin. ß-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing ß-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy ß-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.
Assuntos
Cálcio/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Colesterol/metabolismo , Citocinas/farmacologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Exocitose/efeitos dos fármacos , Humanos , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células PC12 , Palmitatos/farmacologia , Ratos , Proteínas SNARE/metabolismo , Via Secretória , Esfingomielinas/metabolismo , Sinaptotagminas/metabolismoRESUMO
Duty hour monitoring is required in accredited training programs, however trainee self-reporting is onerous and vulnerable to bias. The objectives of this study were to use an automated, validated algorithm to measure duty hour violations of pediatric trainees over a full academic year and compare to self-reported violations. Duty hour violations calculated from electronic health record (EHR) logs varied significantly by trainee role and rotation. Block-by-block differences show 36.8% (222/603) of resident-blocks with more EHR-defined violations (EDV) compared to self-reported violations (SRV), demonstrating systematic under-reporting of duty hour violations. Automated duty hour tracking could provide real-time, objective assessment of the trainee work environment, allowing program directors and accrediting organizations to design and test interventions focused on improving educational quality.