RESUMO
A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.
Assuntos
Descoberta de Drogas , Doenças Inflamatórias Intestinais , Procedimentos Analíticos em Microchip , Modelos Biológicos , Células CACO-2 , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Dispositivos Lab-On-A-Chip , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic ß-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.
Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/química , Fatores de Troca do Nucleotídeo Guanina/agonistas , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , AMP Cíclico/farmacologia , Desenho de Fármacos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Ligação ProteicaRESUMO
Basal cells play a critical role in the response of the airway epithelium to injury and are recently recognized to also contribute to epithelial immunity. Antimicrobial proteins and peptides are essential effector molecules in this airway epithelial innate immunity. However, little is known about the specific role of basal cells in antimicrobial protein and peptide production and about the regulation of the ubiquitous antimicrobial protein RNase 7. In this study, we report that basal cells are the principal cell type producing RNase 7 in cultured primary bronchial epithelial cells (PBEC). Exposure of submerged cultured PBEC (primarily consisting of basal cells) to the respiratory pathogen nontypeable Haemophilus influenzae resulted in a marked increase in expression of RNase 7, although this was not observed in differentiated air-liquid interface cultured PBEC. However, transient epithelial injury in air-liquid interface-cultured PBEC induced by cigarette smoke exposure led to epidermal growth factor receptor-mediated expression of RNase 7 in remaining basal cells. The selective induction of RNase 7 in basal cells by cigarette smoke was demonstrated using confocal microscopy and by examining isolated luminal and basal cell fractions. Taken together, these findings demonstrate a phenotype-specific innate immune activity of airway epithelial basal cells, which serves as a second line of airway epithelial defense that is induced by airway epithelial injury.
Assuntos
Células Epiteliais/metabolismo , Imunidade Inata , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Ribonucleases/biossíntese , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , Receptores ErbB/metabolismo , Expressão Gênica , Haemophilus influenzae/imunologia , Humanos , Modelos Biológicos , Mucosa Respiratória/microbiologia , Ribonucleases/genética , Fumaça/efeitos adversosRESUMO
In early systemic sclerosis (Scleroderma, SSc), the vasculature is impaired. Although the exact etiology of endothelial cell damage in SSc remains unclear, it is hypothesized that endothelial to mesenchymal transition (EndoMT) plays a key role. To perform physiologically relevant angiogenic studies, we set out to develop an angiogenesis-on-a-chip platform that is suitable for assessing disease parameters that are relevant to SSc and other vasculopathies. In the model, we substituted Fetal Bovine Serum (FBS) with Human Serum without impairing the stability of the culture. We showed that 3D microvessels and angiogenic factor-induced sprouts exposed to key pro-inflammatory and pro-fibrotic cytokines (TNFα and TGFß) undergo structural alterations consisting of destructive vasculopathy (loss of small vessels). We also showed that these detrimental effects can be prevented by compound-mediated inhibition of TGFß-ALK5 signaling or addition of a TNFα neutralizing antibody to the 3D cultures. This demonstrates that our in vitro model is suitable for compound testing and identification of new drugs that can protect from microvascular destabilization or regression in disease-mimicking conditions. To support this, we demonstrated that sera obtained from SSc patients can exert an anti-angiogenic effect on the 3D vessel model, opening the doors to screening for potential SSc drugs, enabling direct patient translatability and personalization of drug treatment.
Assuntos
Escleroderma Sistêmico , Fator de Necrose Tumoral alfa , Indutores da Angiogênese , Anticorpos Neutralizantes , Humanos , Dispositivos Lab-On-A-Chip , Microvasos , Neovascularização Patológica , Soroalbumina Bovina , Fator de Crescimento Transformador betaRESUMO
Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.
Assuntos
Anti-Inflamatórios/farmacologia , Células Dendríticas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , RNA Interferente Pequeno/genética , Apresentação de Antígeno , Antígenos de Bactérias/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Humanos , Tolerância Imunológica , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Programas de Rastreamento , SimbioseRESUMO
Inflammatory bowel disease (IBD) is a complex multi-factorial disease for which physiologically relevant in vitro models are lacking. Existing models are often a compromise between biological relevance and scalability. Here, we integrated intestinal epithelial cells (IEC) derived from human intestinal organoids with monocyte-derived macrophages, in a gut-on-a-chip platform to model the human intestine and key aspects of IBD. The microfluidic culture of IEC lead to an increased polarization and differentiation state that closely resembled the expression profile of human colon in vivo. Activation of the model resulted in the polarized secretion of CXCL10, IL-8 and CCL-20 by IEC and could efficiently be prevented by TPCA-1 exposure. Importantly, upregulated gene expression by the inflammatory trigger correlated with dysregulated pathways in IBD patients. Finally, integration of activated macrophages offers a first-step towards a multi-factorial amenable IBD platform that could be scaled up to assess compound efficacy at early stages of drug development or in personalized medicine.
Assuntos
Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Dispositivos Lab-On-A-Chip , Macrófagos/patologia , Linhagem Celular , Células Cultivadas , Descoberta de Drogas , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Organoides/metabolismo , Organoides/patologia , TranscriptomaRESUMO
The down-regulation of the high-molecular-weight isoforms of tropomyosin (TM) is considered to be an essential event in cellular transformation. In ras-transformed fibroblasts, the suppression of TM is dependent on the activity of the Raf-1 kinase; however, the requirement for other downstream effectors of Ras, such as MEK and ERK, is less clear. In this study, we have utilized the mitogen-activated protein kinase scaffolding protein Kinase Suppressor of Ras (KSR) to further investigate the regulation of TM and to clarify the importance of MEK/ERK signaling in this process. Here, we report that overexpression of wild-type KSR1 in ras-transformed fibroblasts restores TM expression and induces cell flattening and stress fiber formation. Moreover, we find that the transcriptional activity of a TM-alpha promoter is decreased in ras-transformed cells and that the restoration of TM by KSR1 coincides with increased transcription from this promoter. Although ERK activity was suppressed in cells overexpressing KSR1, ERK inhibition alone was insufficient to upregulate TM expression. The KSR1-mediated effects on stress fiber formation and TM transcription required the activity of the ROCK kinase, because these effects could be suppressed by the ROCK inhibitor, Y27632. Overexpression of KSR1 did not directly regulate ROCK activity, but did permit the recoupling of ROCK to the actin polymerization machinery. Finally, all of the KSR1-induced effects were mediated by the C-terminal domain of KSR1 and were dependent on the KSR-MEK interaction.
Assuntos
Proteínas Quinases/metabolismo , Tropomiosina/química , Regulação para Cima , Proteínas ras/metabolismo , Células 3T3 , Adenoviridae/genética , Amidas/farmacologia , Animais , Western Blotting , Linhagem Celular Transformada , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Fosforilação , Regiões Promotoras Genéticas , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estrutura Terciária de Proteína , Piridinas/farmacologia , Transcrição Gênica , Tropomiosina/metabolismo , Quinases Associadas a rhoRESUMO
Dendritic cell-specific transcript (DC-SCRIPT) is a putative DC zinc (Zn) finger-type transcription factor described recently in humans. Here, we illustrate that DC-SCRIPT is highly conserved in evolution and report the initial characterization of the murine ortholog of DC-SCRIPT, which is also preferentially expressed in DC as shown by real-time quantitative polymerase chain reaction, and its distribution resembles that of its human counterpart. Studies undertaken in human embryonic kidney 293 cells depict its nuclear localization and reveal that the Zn finger domain of the protein is mainly responsible for nuclear import. The human and the mouse genes are located in syntenic chromosomal regions and exhibit a similar genomic organization with numerous common transcription factor-binding sites in their promoter region, including sites for many factors implicated in haematopoiesis and DC biology, such as Gfi, GATA-1, Spi-B, and c-Rel. Taken together, these data show that DC-SCRIPT is well-conserved in evolution and that the mouse homologue is more than 80% homologous to the human protein. Therefore, mouse models can be used to elucidate the function of this novel DC marker.
Assuntos
Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas Nucleares/química , Proteínas Repressoras/química , Fatores de Transcrição/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Proteínas de Transporte , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , Sequência Conservada , Proteínas de Ligação a DNA/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Dedos de Zinco/fisiologiaRESUMO
Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Fumar Cigarros/efeitos adversos , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Mucosa Respiratória/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Bacteriólise , Células Cultivadas , Humanos , Imunidade , Imunomodulação , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Mucosa Respiratória/microbiologia , Transdução de SinaisRESUMO
Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.
Assuntos
Anticorpos , Animais , Humanos , Terminologia como AssuntoRESUMO
Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and are characterized by a plethora of functions that facilitate tumor outgrowth. Mechanisms of macrophage attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced interleukin (IL-10) production, are ill-defined. We aimed to identify signaling pathways that contribute to the generation of TAM-like macrophages using an adenoviral RNAi-based approach. Primary human monocyte-derived macrophages were stimulated with apoptotic tumor cell supernatants (ACM) to induce a TAM-like phenotype, characterized by secretion of IL-10, IL-6, IL-8 but repression of IL-12. For the high-throughput screen, macrophages were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect(®) library of Galapagos BV, which aims at identifying druggable targets. We identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of targets regulating both IL-10 and IL-6. Validation of five targets within the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors in human primary macrophages. Among those, interleukin 4 receptor-α and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion by ACM-stimulated macrophages. Our approach characterizes cellular functions of transfection-resistant, highly plastic and versatile cells and identifies novel targets involved in the generation of a TAM-like phenotype in human macrophages.
Assuntos
Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Neoplasias/imunologia , RNA Interferente Pequeno/genética , Adenoviridae/genética , Antígenos de Neoplasias/imunologia , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-10/genética , Interleucina-6/genética , Terapia de Alvo Molecular , Cultura Primária de Células , Receptor CB2 de Canabinoide/metabolismo , Receptores de Superfície Celular/metabolismo , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Pathogenic mechanisms involved in fibrosis of various organs share many common features. Myofibroblasts are thought to play a major role in fibrosis through excessive deposition of extracellular matrix during wound healing processes. Myofibroblasts are observed in fibrotic lesions, and whereas these derive from the hepatic stellate cells in liver, in lung they appear to originate from fibroblasts. The source of these fibroblasts has been the object of numerous studies over the recent years and points towards multiple sources. First of all, resident fibroblasts are thought to differentiate into the more contractile myofibroblasts, secreting many extracellular matrix proteins. Secondly, the epithelial to mesenchymal transition (EMT) of epithelial cells may also account for increased numbers of fibroblasts, though in vivo evidence in patient tissue is still scarce. Thirdly, the enigmatic fibrocytes, stemming from the bone marrow, may also account for increasing numbers of fibroblasts in fibrotic lesions. These pathogenic processes are further augmented by the generation of so-called alternatively activated macrophages, which have direct and indirect effects on myofibroblast accumulation and collagen deposition. TGFß, which is produced predominantly by macrophages, plays a central role in all these processes by inducing EMT, driving differentiation of fibrocytes, and differentiation towards myofibroblasts. This review describes the potential origins and roles of these fibrotic cells in the lung and discusses models to study these cells in vitro. These models offer innovative approaches in target and drug discovery, aiming to uncover novel therapeutic targets that regulate the profibrotic phenotype of these cells.
Assuntos
Modelos Biológicos , Miofibroblastos/patologia , Fibrose Pulmonar/fisiopatologia , Animais , Diferenciação Celular/fisiologia , Colágeno/metabolismo , Desenho de Fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Macrófagos/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/fisiologiaRESUMO
Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
Assuntos
Padronização Corporal , Biologia do Desenvolvimento/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Automação , Biologia Computacional , Biblioteca Gênica , Humanos , Sistema Imunitário , Inflamação , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
Dendritic cells (DC) compose a heterogeneous population of cells that hold a leading role in initiating and directing immune responses. Although their function in recognizing, capturing, and presenting Ags is well defined, the molecular mechanisms that control their differentiation and immune functions are still largely unknown. In this study, we report the isolation and characterization of DC-SCRIPT, a novel protein encoded by an 8-kb mRNA that is preferentially expressed in DC. DC-SCRIPT is expressed in multiple DC subsets in vivo, including myeloid DC, plasmacytoid DC, and Langerhans cells. At the protein level, DC-SCRIPT consists of a proline-rich region, 11 C2H2-type zinc fingers, and an acidic region. Localization studies reveal that DC-SCRIPT resides in the nucleus and that nuclear localization is critically dependent on the zinc fingers. The protein displays no transcriptional activation properties according to assorted transactivation assays, but interacts with the corepressor C-terminal binding protein 1. Taken together, our results show that we have isolated a novel DC marker that could be involved in transcriptional repression. In contrast to other DC molecules, DC-SCRIPT identifies all DC subsets tested to date.