RESUMEN
The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.
Asunto(s)
Factores de Restricción Antivirales , Cromatina , Animales , Ratones , Cromatina/genética , Motivos de Nucleótidos , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Interferones/metabolismoRESUMEN
IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5'-GAGA-3' and 5'-GACA-3', were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Factor 3 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Animales , Línea Celular , Cromatina/genética , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Humanos , Aprendizaje Automático , Ratones , FN-kappa B/genética , Motivos de Nucleótidos/genética , Análisis de Componente Principal , Elementos de Respuesta/genética , Factores de Transcripción/genéticaRESUMEN
Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , Receptor alfa de Estrógeno/genética , Estrógenos/genética , MicroARNs/genética , Biomarcadores de Tumor/genética , Cadherinas/genética , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Endodesoxirribonucleasas/genética , Transición Epitelial-Mesenquimal/genética , Estradiol/metabolismo , Estrógenos/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Neoplasias/genética , ARN Mensajero/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Ovarian cancer is one of the most common cancer types in women characterized by a high mortality rate due to lack of early diagnosis. Circulating miRNAs besides being important regulators of cancer development could be potential biomarkers to aid diagnosis. We performed the circulating miRNA expression analysis in plasma samples obtained from ovarian cancer patients stratified into FIGO I, FIGO III, and FIGO IV stages and from healthy females using the NanoString quantitative assay. Forty-five miRNAs were differentially expressed, out of these 17 miRNAs showed significantly different expression between controls and patients, 28 were expressed only in patients, among them 19 were expressed only in FIGO I patients. Differentially expressed miRNAs were ranked by the network-based analysis to assess their importance. Target genes of the differentially expressed miRNAs were identified then functional annotation of the target genes by the GO and KEGG-based enrichment analysis was carried out. A general and an ovary-specific protein-protein interaction network was constructed from target genes. Results of our network and the functional enrichment analysis suggest that besides HSP90AA1, MYC, SP1, BRCA1, RB1, CFTR, STAT3, E2F1, ERBB2, EZH2, and MET genes, additional genes which are enriched in cell cycle regulation, FOXO, TP53, PI-3AKT, AMPK, TGFß, ERBB signaling pathways and in the regulation of gene expression, proliferation, cellular response to hypoxia, and negative regulation of the apoptotic process, the GO terms have central importance in ovarian cancer development. The aberrantly expressed miRNAs might be considered as potential biomarkers for the diagnosis of ovarian cancer after validation of these results in a larger cohort of ovarian cancer patients.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , MicroARN Circulante/genética , Perfilación de la Expresión Génica/métodos , Neoplasias Ováricas/genética , Plasma/química , Adulto , Anciano , Carcinoma Epitelial de Ovario/patología , Estudios de Casos y Controles , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Persona de Mediana Edad , Anotación de Secuencia Molecular , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Mapas de Interacción de ProteínasRESUMEN
Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (MΦ), and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)α, IFNγ, and Toll-like receptor (TLR)4 activate pro-inflammatory gene expression and are pro-atherogenic. Gene expression regulation of many pro-inflammatory genes has shown to rely on Signal Integration (SI) between IFNs and TLR4 through combinatorial actions of the Signal Transducer and Activator of Transcription (STAT)1 complexes ISGF3 and γ-activated factor (GAF), and Nuclear Factor-κB (NFκB). Thus, IFN pre-treatment ("priming") followed by LPS stimulation leads to enhanced transcriptional responses as compared to the individual stimuli. To characterize the mechanism of priming-induced IFNα + LPS- and IFNγ + LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, MΦ, and DC in response to IFNα, IFNγ, and/or LPS. Thus, we identified IFNα + LPS or IFNγ + LPS induced genes commonly expressed in these cell types that bound STAT1 and p65 at comparable γ-activated sequence (GAS), Interferon-stimulated response element (ISRE), or NFκB sites in promoter proximal and distal regions. Comparison of the relatively high number of overlapping ISRE sites in these genes unraveled a novel role of ISGF3 and possibly STAT1/IRF9 in IFNγ responses. In addition, similar STAT1-p65 co-binding modes were detected for IFNα + LPS and IFNγ + LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NFκB or ISRE/NFκB composite sites already upon IFNα or IFNγ treatment. This STAT1-p65 co-binding significantly increased after subsequent LPS exposure and correlated with histone acetylation, PolII recruitment, and amplified target gene transcription in a STAT1-p65 co-bound dependent manner. Thus, co-binding of STAT1-containing transcription factor complexes and NFκB, activated by IFN-I or IFN-II together with LPS, provides a platform for robust transcriptional activation of pro-inflammatory genes. Moreover, our data offer an explanation for the comparable effects of IFNα or IFNγ priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.
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Regulación de la Expresión Génica , Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Ontología de Genes , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Transcripción GenéticaRESUMEN
The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Macrófagos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Biología Computacional , Regulación de la Expresión Génica/fisiología , Genoma , Aprendizaje Automático , Ratones , Ratones Endogámicos C57BL , Unión Proteica/fisiología , Coloración y Etiquetado/métodos , Activación Transcripcional/fisiologíaRESUMEN
The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.
Asunto(s)
Trastornos de la Memoria/genética , Receptores X Retinoide/metabolismo , Tretinoina/análogos & derivados , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptores X Retinoide/química , Receptores X Retinoide/genética , Proteínas Celulares de Unión al Retinol/genética , Proteínas Celulares de Unión al Retinol/metabolismo , Tretinoina/metabolismoRESUMEN
Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-ß stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Interferón beta/inmunología , Receptor Toll-Like 3/inmunología , Animales , Reactividad Cruzada/inmunología , Humanos , Interferón beta/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/virología , Poli I-C/inmunología , Receptor de Interferón alfa y beta/inmunología , Rhinovirus/inmunología , Virus Sendai/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
All-trans retinoic acid (ATRA) has a key role in dendritic cells (DCs) and affects T cell subtype specification and gut homing. However, the identity of the permissive cell types and the required steps of conversion of vitamin A to biologically active ATRA bringing about retinoic acid receptor-regulated signaling remains elusive. Here we present that only a subset of murine and human DCs express the necessary enzymes, including RDH10, RALDH2, and transporter cellular retinoic acid binding protein (CRABP)2, to produce ATRA and efficient signaling. These permissive cell types include CD103(+) DCs, granulocyte-macrophage colony-stimulating factor, and interleukin-4-treated bone marrow-derived murine DCs and human monocyte-derived DCs (mo-DCs). Importantly, in addition to RDH10 and RALDH2, CRABP2 also appears to be regulated by the fatty acid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and colocalize in human gut-associated lymphoid tissue DCs. In our model of human mo-DCs, all three proteins (RDH10, RALDH2, and CRABP2) appeared to be required for ATRA production induced by activation of PPARγ and therefore form a linear pathway. This now functionally validated PPARγ-regulated ATRA producing and signaling axis equips the cells with the capacity to convert precursors to active retinoids in response to receptor-activating fatty acids and is potentially amenable to intervention in diseases involving or affecting mucosal immunity.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Células Dendríticas/metabolismo , PPAR gamma/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinal-Deshidrogenasa/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Oxidorreductasas de Alcohol/deficiencia , Oxidorreductasas de Alcohol/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Células Dendríticas/citología , Células Dendríticas/enzimología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Intestinos/citología , Masculino , Ratones , Monocitos/citología , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transporte de Proteínas , Receptores de Ácido Retinoico/deficiencia , Receptores de Ácido Retinoico/genética , Retinal-Deshidrogenasa/deficiencia , Retinal-Deshidrogenasa/genética , Transglutaminasas/metabolismoRESUMEN
IL-2 is the primary growth factor for promoting survival and proliferation of activated T cells that occurs following engagement of the Janus Kinase (JAK)1-3/and Signal Transducer and Activator of Transcription (STAT) 5 signaling pathway. STAT5 has two isoforms: STAT5A and STAT5B (commonly referred to as STAT5) which, in T cells, play redundant roles transcribing cell cycle and survival genes. As such, inhibition of STAT5 by a variety of mechanisms can rapidly induce apoptosis in certain lymphoid tumor cells, suggesting that it and its target genes represent therapeutic targets to control certain lymphoid diseases. To search for these molecules we aligned IL-2 regulated genes detected by Affymetrix gene expression microarrays with the STAT5 cistrome identified by chip-on-ChIP analysis in an IL-2-dependent human leukemia cell line, Kit225. Select overlapping genes were then validated using qRT(2)PCR medium-throughput arrays in human PHA-activated PBMCs. Of 19 putative genes, one key regulator of T cell receptor signaling, PDE4B, was identified as a novel target, which was readily up-regulated at the protein level (3 h) in IL-2 stimulated, activated human PBMCs. Surprisingly, only purified CD8+ primary T-cells expressed PDE4B, but not CD4+ cells. Moreover, PDE4B was found to be highly expressed in CD4+ lymphoid cancer cells, which suggests that it may represent a physiological role unique to the CD8+ and lymphoid cancer cells and thus might represent a target for pharmaceutical intervention for certain lymphoid diseases.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Interleucina-2/fisiología , Linfoma/metabolismo , Monocitos/metabolismo , Factor de Transcripción STAT5/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Linfoma/patología , Reacción en Cadena de la PolimerasaRESUMEN
Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.
RESUMEN
A key issue in the immune system is to generate specific cell types, often with opposing activities. The mechanisms of differentiation and subtype specification of immune cells such as macrophages and dendritic cells are critical to understand the regulatory principles and logic of the immune system. In addition to cytokines and pathogens, it is increasingly appreciated that lipid signaling also has a key role in differentiation and subtype specification. In this review we explore how intracellular lipid signaling via a set of transcription factors regulates cellular differentiation, subtype specification, and immune as well as metabolic homeostasis. We introduce macrophages and dendritic cells and then we focus on a group of transcription factors, nuclear receptors, which regulate gene expression upon receiving lipid signals. The receptors we cover are the ones with a recognized physiological function in these cell types and ones which heterodimerize with the retinoid X receptor. These are as follows: the receptor for a metabolite of vitamin A, retinoic acid: retinoic acid receptor (RAR), the vitamin D receptor (VDR), the fatty acid receptor: peroxisome proliferator-activated receptor γ (PPARγ), the oxysterol receptor liver X receptor (LXR), and their obligate heterodimeric partner, the retinoid X receptor (RXR). We discuss how they can get activated and how ligand is generated and eliminated in these cell types. We also explore how activation of a particular target gene contributes to biological functions and how the regulation of individual target genes adds up to the coordination of gene networks. It appears that RXR heterodimeric nuclear receptors provide these cells with a coordinated and interrelated network of transcriptional regulators for interpreting the lipid milieu and the metabolic changes to bring about gene expression changes leading to subtype and functional specification. We also show that these networks are implicated in various immune diseases and are amenable to therapeutic exploitation.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Metabolismo de los Lípidos/inmunología , Macrófagos/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Animales , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , PPAR gamma/inmunología , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Vitamina D/farmacologíaRESUMEN
It is well established that dendritic cells (DCs) take up, process, and present lipid Ags in complex with CD1d molecules to invariant NKT cells. The lipid-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), has previously been shown to regulate CD1d expression in human monocyte-derived DCs, providing a link between lipid metabolism and lipid Ag presentation. We report that PPARγ regulates the expression of a lysosomal protease, cathepsin D (CatD), in human monocyte-derived DCs. Inhibition of CatD specifically reduced the expansion of invariant NKT cells and furthermore resulted in decreased maturation of saposins, a group of lipid transfer proteins required for lysosomal lipid Ag processing and loading. These results reveal a novel mechanism of lipid Ag presentation and identify CatD as a key component of this machinery and firmly place PPARγ as the transcriptional regulator linking lipid metabolism and lipid Ag processing.
Asunto(s)
Presentación de Antígeno/inmunología , Catepsina D/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Lipoproteínas/metabolismo , PPAR gamma/fisiología , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Catepsina D/biosíntesis , Catepsina D/fisiología , Células Cultivadas , Técnicas de Cocultivo , Humanos , Metabolismo de los Lípidos/inmunología , Lipoproteínas/inmunología , Lisosomas/enzimología , Lisosomas/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Células T Asesinas Naturales/enzimología , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Saposinas/metabolismo , Saposinas/fisiología , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression. OBJECTIVES: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment. METHOD: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction. RESULTS: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV(1). CONCLUSION: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression.
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Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Adulto , Anciano , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Volumen Espiratorio Forzado , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.
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Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Macrófagos/metabolismo , PPAR gamma/metabolismo , Factor de Transcripción STAT6/metabolismo , Animales , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-4/metabolismo , Ratones , Regiones Promotoras GenéticasRESUMEN
Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor's activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner.
Asunto(s)
Diferenciación Celular/genética , Células Dendríticas/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Monocitos/citología , Receptores de Ácido Retinoico/metabolismo , Benzoatos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Receptores X del Hígado , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Ácidos Nicotínicos/farmacología , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/metabolismo , PPAR delta/agonistas , PPAR delta/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Fenotipo , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Receptores de Ácido Retinoico/agonistas , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Tetrahidronaftalenos/farmacología , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Factor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.
Asunto(s)
Deficiencia del Factor XIII/genética , Deficiencia del Factor XIII/inmunología , Factor XIIIa/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Simulación por Computador , Deficiencia del Factor XIII/metabolismo , Factor XIIIa/genética , Factor XIIIa/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Redes Reguladoras de Genes/inmunología , Humanos , Interleucina-4/inmunología , Interleucina-4/metabolismo , Activación de Macrófagos/genética , Macrófagos/inmunología , Macrófagos/patología , Análisis por Micromatrices , Cicatrización de Heridas/genéticaRESUMEN
Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-alpha, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-kappaB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Mediadores de Inflamación/fisiología , Receptores Nucleares Huérfanos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/patología , Humanos , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos/inmunología , Receptores X del Hígado , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/fisiología , Receptores Nucleares Huérfanos/fisiología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Activation of vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D(3) (1,25-vitD) reprograms dendritic cells (DC) to become tolerogenic. Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Underpinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation. We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs. In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes, impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22, the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated 1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the previously characterized tolerogenic state.
Asunto(s)
Calcitriol/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/genética , Transcripción Genética/inmunología , Vitaminas/inmunología , Western Blotting , Calcitriol/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Quimiocina CCL22/inmunología , Quimiocina CCL22/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Tolerancia Inmunológica/inmunología , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores de Calcitriol/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genética , Vitaminas/metabolismoRESUMEN
The lipid activated transcription factor, PPARgamma appears to have multiple functions in the immune system. There are several cell types expressing the receptor, most prominently antigen presenting cells, such as macrophages and dendritic cells. The receptor's activation leads to primary transcriptional activation of many, mostly lipid metabolism-related genes. However, gene regulation also occurs on immunity and inflammation-related genes. Key questions are: in what way lipid metabolism and immune regulation are connected and how activation and/or repression of gene expression may modulate inflammatory and anti-inflammatory responses and in what way can these be utilized in therapy. Here we provide a cell type and disease centric review on the role of this lipid activated transcription factor in the various cells of the immune system it is expressed in, and in some major inflammatory diseases such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis.