RESUMO
High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1ß secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.
Assuntos
Medula Óssea , Células Dendríticas , Animais , Camundongos , Antivirais , Medula Óssea/imunologia , Diferenciação Celular , Células Dendríticas/classificação , Células Dendríticas/imunologiaRESUMO
BACKGROUND & AIMS: Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. METHODS: We developed an inducible, EC cell-specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell-depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell-depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). RESULTS: Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. CONCLUSIONS: The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.
Assuntos
Esvaziamento Gástrico/genética , Trânsito Gastrointestinal/genética , Serotonina/deficiência , Animais , Linhagem Celular , Células Enterocromafins/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Triptofano Hidroxilase/metabolismoRESUMO
BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic ß cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. METHODS: We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of ß cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Krüppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus/prevenção & controle , Esvaziamento Gástrico , Trânsito Gastrointestinal , Gastroparesia/prevenção & controle , Células Secretoras de Insulina/metabolismo , Células Intersticiais de Cajal/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Feminino , Gastroparesia/genética , Gastroparesia/metabolismo , Gastroparesia/fisiopatologia , Células HEK293 , Humanos , Células Secretoras de Insulina/patologia , Células Intersticiais de Cajal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Adulto JovemRESUMO
Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα+ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα+ cells. ADAMDEC1 protein was mainly released from PDGFRα+ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα+ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45- PDGFRα+ cells in DSS-induced colitis mice, with only minimal expression in CD45+ CD64+ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα+ cells, but not in CD64+ macrophages found in human colonic mucosal tissue affected by Crohn's disease. In summary, PDGFRα+ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn's disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn's disease.
Assuntos
Proteínas ADAM , Colite , Doença de Crohn , Doenças Inflamatórias Intestinais , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Animais , Biomarcadores , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colo/citologia , Colo/metabolismo , Doença de Crohn/metabolismo , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.
Assuntos
Interferon Tipo I , Neoplasias , Humanos , Monócitos , Neoplasias/radioterapia , Diferenciação Celular , Interferon Tipo I/farmacologia , Linfócitos do Interstício Tumoral , Microambiente TumoralRESUMO
Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self-antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen-associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.
Assuntos
Encéfalo/imunologia , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Envelhecimento/imunologia , Envelhecimento/patologia , Animais , Encéfalo/citologia , Encéfalo/patologia , Doenças do Sistema Nervoso Central/imunologia , Doenças do Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Macrófagos/patologia , Macrófagos/fisiologia , Microglia/patologia , Microglia/fisiologiaRESUMO
Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP(+) cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNgamma or IL-4 into the CNS. The administration of IFNgamma increased the number of EYFP(+)bDC but did not recruit peripheral DC into the CNS. IFNgamma, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNgamma-activated EYFP(+)bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNgamma, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Encéfalo/citologia , Células Dendríticas/imunologia , Interferon gama/farmacologia , Animais , Proteínas de Bactérias/metabolismo , Antígeno CD11c/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Epitopos/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/administração & dosagem , Interleucina-4/farmacologia , Proteínas Luminescentes/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Tolerância a Radiação/efeitos dos fármacos , Receptores CCR7/metabolismoRESUMO
Abdominal pain continues to be a major challenge and unmet need in clinical practice. Normalization of bidirectional gut-brain signaling has generated much interest as a therapeutic approach to treat chronic abdominal pain. Vagal nerve stimulation (VNS) is emerging as a potential non-pharmacologic strategy for the treatment of abdominal pain. In this review paper, we will summarize the etiologies of chronic pain in gastrointestinal disorders and discuss the rational for VNS as a therapeutic approach to chronic abdominal pain, with particular emphasis in the gammaCore stimulator which allows for noninvasive VNS.
Assuntos
Dor Abdominal/terapia , Sensibilização do Sistema Nervoso Central , Dor Crônica/terapia , Gastroenteropatias/complicações , Estimulação do Nervo Vago , Dor Abdominal/etiologia , Dor Crônica/etiologia , Humanos , Estimulação do Nervo Vago/instrumentação , Estimulação do Nervo Vago/métodosRESUMO
INTRODUCTION: It is unclear how immune perturbations may influence the pathogenesis of idiopathic gastroparesis, a prevalent functional disorder of the stomach which lacks animal models. Several studies have noted altered immune characteristics in the deep gastric muscle layer associated with gastroparesis, but data are lacking for the mucosal layer, which is endoscopically accessible. We hypothesized that immune dysregulation is present in the gastroduodenal mucosa in idiopathic gastroparesis and that specific immune profiles are associated with gastroparesis clinical parameters. METHODS: In this cross-sectional prospective case-control study, routine endoscopic biopsies were used for comprehensive immune profiling by flow cytometry, multicytokine array, and gene expression in 3 segments of the stomach and the duodenal bulb. Associations of immune endpoints with clinical parameters of gastroparesis were also explored. RESULTS: The gastric mucosa displayed large regional variation of distinct immune profiles. Furthermore, several-fold increases in innate and adaptive immune cells were found in gastroparesis. Various immune cell types showed positive correlations with duration of disease, proton pump inhibitor dosing, and delayed gastric emptying. DISCUSSION: This initial observational study showed immune compartmentalization of the human stomach mucosa and significant immune dysregulation at the level of leukocyte infiltration in idiopathic gastroparesis patients that extends to the duodenum. Select immune cells, such as macrophages, may correlate with clinicopathological traits of gastroparesis. This work supports further mucosal studies to advance our understanding of gastroparesis pathophysiology.
Assuntos
Mucosa Gástrica/imunologia , Gastroparesia/imunologia , Imunidade Adaptativa , Adolescente , Adulto , Idoso , Antígenos CD8 , Estudos de Casos e Controles , Estudos Transversais , Citocinas/sangue , Duodeno/imunologia , Feminino , Esvaziamento Gástrico , Gastroparesia/fisiopatologia , Expressão Gênica , Humanos , Imunidade Inata , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Linfócitos T/imunologia , Adulto JovemRESUMO
Glucocorticoids are potent regulators of inflammation exerting permissive, stimulatory, and suppressive effects. Glucocorticoid access to intracellular receptors is regulated by the activity of two distinct enzymes known as 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) Type 1 and Type 2, which catalyze the activation or deactivation of glucocorticoids. Although expression of these enzymes in major organ systems and their roles in the metabolic effects of glucocorticoids have been described, their role in the inflammatory response has only recently started to be addressed. In this report, we have studied the expression and activity of 11 beta HSD Type 1 and Type 2 in microglia cells. Microglia, the brain's resident macrophages, initiate and orchestrate CNS inflammatory responses. Importantly, activated microglia are implicated in most neurodegenerative conditions, making them key subjects of study. We found that microglia expressed 11 beta HSD-1, but not 11 beta HSD-2, both in ex vivo FACS-sorted adult cells and in vitro primary cultures. 11 beta HSD-1 expression was increased in LPS-activated microglia. Moreover, 11 beta HSD-1 catalyzed the metabolic conversion of 11-dehydro-corticosterone into corticosterone (CORT), which potently reduced cytokine production in activated microglia. We propose that 11 beta HSD-1 may provide microglia with an intrinsic mechanism to autoregulate and inhibit proinflammatory mediator production through CORT formation.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Microglia/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/imunologia , Células Cultivadas , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Citocinas/metabolismo , Glucocorticoides/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/imunologiaRESUMO
The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.
Assuntos
Isquemia Encefálica/imunologia , Encéfalo/imunologia , Células Dendríticas/imunologia , Acidente Vascular Cerebral/imunologia , Análise de Variância , Animais , Antígeno CD11c/imunologia , Citometria de Fluxo , Genes MHC da Classe II/imunologia , Imuno-Histoquímica , Leucócitos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
Minimizing variability in collection and processing of human blood samples for research remains a challenge. Delaying plasma or serum isolation after phlebotomy (processing delay) can cause perturbations of numerous analytes. Thus, a comprehensive understanding of how processing delay affects major endpoints used in human immunology research is necessary. Therefore, we studied how processing delay affects commonly measured cytokines and immune cell populations. We hypothesized that short-term time delays inherent to human research in serum and plasma processing impact commonly studied immunological analytes. Blood from healthy donors was subjected to processing delays commonly encountered in sample collection, and then assayed by 62-plex Luminex panel, 40-parameter mass cytometry panel, and 540,000 transcript expression microarray. Variance for immunological analytes was estimated using each individual's baseline as a control. In general, short-term processing delay led to small changes in plasma and serum cytokines (range - 10.8 to 43.5%), markers and frequencies of peripheral blood mononuclear cell phenotypes (range 0.19 to 3.54 fold), and whole blood gene expression (stable for > 20 K genes)-with several exceptions described herein. Importantly, we built an open-access web application allowing investigators to estimate the degree of variance expected from processing delay for measurements of interest based on the data reported here.
Assuntos
Citocinas/sangue , Leucócitos Mononucleares/citologia , Flebotomia/métodos , Manejo de Espécimes/métodos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Temperatura , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Gastroparesis, a chronic motility disorder characterized by delayed gastric emptying, abdominal pain, nausea, and vomiting, remains largely unexplained. Medical therapy is limited, reflecting the complex physiology of gastric sensorimotor function. Vagus nerve stimulation is an attractive therapeutic modality for gastroparesis, but prior methods required invasive surgery. In this open-label pilot study, we aimed to assess the benefit of non-invasive vagal nerve stimulation in patients with mild to moderate idiopathic gastroparesis. METHODS: Patients self-administered the gammaCore vagal nerve stimulator for 4 weeks. The gastroparesis cardinal symptom index daily diary (GCSI-dd) was assessed during a two-week run-in period, ≥4 weeks of therapy, and 4 weeks after therapy was completed. Gastric emptying and autonomic function testing were also performed. The primary endpoint was an absolute reduction in CGSI-dd of 0.75 after nVNS. RESULTS: There was a total improvement in symptom scores (2.56 ± 0.76 to 1.87 ± 1.05; P = .01), with 6/15 (40%) participants meeting our primary endpoint. Therapy was associated with a reduction in gastric emptying (T1/2 155 vs 129 minutes; P = .053, CI -0.4 to 45). Therapy did not correct autonomic function abnormalities, but was associated with modulation of reflex parasympathetic activity. CONCLUSIONS: Short-term non-invasive vagal nerve stimulation led to improved cardinal symptoms and accelerated gastric emptying in a subset of patients with idiopathic gastroparesis. Responders had more severe gastric delay at baseline and clinical improvement correlated with duration of therapy, but not with improvements in gastric emptying. Larger randomized sham-controlled trials of greater duration are needed to confirm the results of this pilot study.
Assuntos
Gastroparesia/terapia , Estimulação do Nervo Vago/métodos , Adulto , Feminino , Esvaziamento Gástrico/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
In the CNS, steroid hormones play a major role in the maintenance of brain homeostasis and it's response to injury. Since activated microglia are the pivotal immune cell involved in neurodegeneration, we investigated the possibility that microglia provide a discrete source for the metabolism of active steroid hormones. Using RT-PCR, our results showed that mouse microglia expressed mRNA for 17beta-hydroxysteroid dehydrogenase type 1 and steroid 5alpha-reductase type 1, which are involved in the metabolism of androgens and estrogens. Microglia also expressed the peripheral benzodiazepine receptor and steroid acute regulatory protein; however, the enzymes required for de novo formation of progesterone and DHEA from cholesterol were not expressed. To test the function of these enzymes, primary microglia cultures were incubated with steroid precursors, DHEA and AD. Microglia preferentially produced delta-5 androgens (Adiol) from DHEA and 5alpha-reduced androgens from AD. Adiol behaved as an effective estrogen receptor agonist in neuronal cells. Activation of microglia with pro-inflammatory factors, LPS and INFgamma did not affect the enzymatic properties of these proteins. However, PBR ligands reduced TNFalpha production signifying an immunomodulatory role for PBR. Collectively, our results suggest that microglia utilize steroid-converting enzymes and related proteins to influence inflammation and neurodegeneration within microenvironments of the brain.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Encéfalo/citologia , Encéfalo/enzimologia , Microglia/enzimologia , Esteroides/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Androstenodiol/metabolismo , Androstenodiol/farmacologia , Animais , Sequência de Bases , Encéfalo/metabolismo , Primers do DNA/genética , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/agonistas , Receptores de GABA/genética , Receptores de GABA/metabolismoRESUMO
The well-established neuroprotective effect of dehydroepiandrosterone (DHEA) has been attributed to its metabolism in the brain to provide estrogens known to be neuroprotective and to enhance memory and learning in humans and animals. However, our previous work showed that the conversion of DHEA to 4-androstenedione (AD), the precursor of estrone (E(1)) and estradiol (E(2)), is very low in several different types of neural cells, and that the main product is 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). In this study, we found that microglia are an exception and produce mainly 5-androstene-3beta,17beta-diol (Delta(5)-Adiol), a C(19) steroid with estrogen-like activity from DHEA. Virtually, no other products, including testosterone (T) were detected by TLC or HPLC in incubations of (3)H-labeled DHEA with the BV2 microglial cell line. Microglia are important brain cells that are thought to play a house-keeping role during the steady state, and that are crucial to the brain's immune reaction to injury and the healing process. Our findings suggest that the microglia-produced Delta(5)-Adiol might have a role in modulating estrogen-sensitive neuroplastic events in the brain, in the absence of adequate local synthesis of estrone and estradiol.
Assuntos
Androstenodiol/metabolismo , Desidroepiandrosterona/metabolismo , Estrogênios/metabolismo , Microglia/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Estrogen receptors alpha and beta (ERalpha and ERbeta) are expressed in the cerebellum throughout development and in the adult suggesting an important role of 17-beta-estradiol (E2) in this brain structure. In the present study, we have characterized the functionality of estrogen receptors (ERs) expressed in the immature cerebellar granule cell line E(t)C.1 by transfecting such cells with a luciferase reporter gene (ERE-Luc) coupled to an estrogen response element promoter. The induction of luciferase activity in E(t)C.1 cells by E2 and ER-subtype selective agonists was compared in normal cells and in cells overexpressing human ERalpha or ERbeta (hERalpha or hERbeta). E2-mediated transcription of the reporter gene was blocked by the ER antagonist ICI 182,780 (ICI), demonstrating the presence of functional native ERs. The selective agonist for ERalpha (PPT) showed a reduced response in luciferase induction compared to E2. Moreover, the ERbeta agonist (DPN) was unable to induce luciferase activity. E2-induced ERE-Luc transcription was not increased by overexpression of hERalpha. In contrast, hERbeta overexpression reduced the efficacy of E2 and abolished ERalpha-selective agonist activity. The ERbeta-specific agonist did not induce gene reporter activity unless hERbeta was overexpressed in the cells, suggesting that the endogenous ERbeta in E(t)C.1 cells is transcriptionally inactive. ICI inhibition of E2 responses was not affected by overexpression of the human ERs. The data suggest that ERalpha plays a predominant role in E2-mediated transcription in E(t)C.1 cells. Our data are discussed in view of other reports alluding to the complexity and cell-type specificity of E2-mediated transcription.
Assuntos
Cerebelo/citologia , Células-Tronco Embrionárias/citologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Neurônios/citologia , Animais , Cerebelo/embriologia , Cerebelo/metabolismo , Células Clonais , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Mouse cerebellar development occurs at late embryonic stages and through the first few weeks of postnatal life. Hormones such as 17-beta-estradiol (E2) have been implicated in cerebellar development, through the expression of E2 receptors (ER). However, the role of E2 in the development and function of cerebellar neurons has yet to be fully elucidated. To gain insight into E2's actions on the developing cerebellum, we characterized a cloned neuronal cell line, E(t)C.1, derived from late embryonic cerebellum for its neural properties and responsiveness to E2. Our results revealed that E(t)C.1 cells express markers characteristic of neural progenitor cells such as Nestin, Musashi, and Doublecortin (DCX), and of the granule cell lineage such as Math1 and Zipro1. The ER alpha and beta (ERalpha and ERbeta) were also identified in this cell line. Functionality of ERs was verified using an Estrogen Response Element (ERE)-Luciferase reporter plasmid. E2 modulated ERalpha, FMRP, and IL-6, which were expressed in these cells. However, E2 did not induce changes in neural proteins nor induce maturation of E(t)C.1 cells. CREB and ERK(1/2) protein kinases were not modulated by E2 either. Interestingly, E(t)C.1 expressed active p450 Aromatase (P450arom), which was confirmed by the aromatization of androstenedione (AD) to E2 and other estrogen metabolites. Collectively, our results show that the E(t)C.1 cell line may serve as a model to study early development of cerebellar progenitor granule cells, and their responsiveness to E2.
Assuntos
Cerebelo/citologia , Células-Tronco Embrionárias/citologia , Estradiol/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Receptores de Estrogênio/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Cerebelo/embriologia , Cerebelo/metabolismo , Células Clonais , Proteínas de Ligação a DNA/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Células-Tronco Embrionárias/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismoRESUMO
Studies to elucidate the role of dehydroepiandrosterone (DHEA) metabolism in neuroprotection have compared its relative 7-hydroxylation against estrogen formation by way of 4-androstenedione (AD) in various rodent brain cell lines. In all cases, the 7alpha- and 7beta-hydroxy epimers of DHEA were found to be the dominant products with one notable exception. BV2 mouse microglia were virtually unable to hydroxylate DHEA at C-7 and converted AD to a major unknown metabolite not observed with mouse BHc hippocampal cells. In this paper, we describe the identification of this compound based on its physical properties and analysis by TLC and HPLC. Its identity as 3beta-hydroxy-4-androstene-17-one, the Delta(4)-isomer of DHEA, was confirmed by mass spectrometry (LC/MS), as well as by reverse isotope dilution analysis involving co-crystallization with the synthetic steroid. Possible mechanisms for the formation of this isomer of DHEA by BV2 microglia are proposed, together with that of other C-19 steroids detected which include testosterone (T), 5alpha-dihydrotestosterone and 5alpha-androstanedione.
Assuntos
Androstenodiona/metabolismo , Encéfalo/metabolismo , Desidroepiandrosterona/metabolismo , Microglia/metabolismo , Adjuvantes Imunológicos/metabolismo , Animais , Cromatografia Líquida , Desidroepiandrosterona/análogos & derivados , Espectrometria de Massas , Camundongos , EsteroidesRESUMO
The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.