RESUMEN
The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.
Asunto(s)
Núcleo Celular/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Factor de Transcripción ReIA/inmunología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Adulto , Animales , Núcleo Celular/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Femenino , Humanos , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Transducción de Señal/genética , Factor de Transcripción ReIA/genéticaRESUMEN
Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.
Asunto(s)
Inflamación/inmunología , Activación de Macrófagos , Macrófagos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Células RAW 264.7 , Análisis de la Célula Individual , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at â¼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (â¼600 and â¼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation.
Asunto(s)
Calcio/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Ionóforos de Calcio/inmunología , Humanos , Células Jurkat , Activación de Linfocitos , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosfotransferasas/metabolismo , Receptor Cross-Talk , Transducción de Señal , Factor de Transcripción AP-1/metabolismoRESUMEN
Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.
Asunto(s)
Interleucina-1beta/farmacología , Inhibidor NF-kappaB alfa/genética , FN-kappa B/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular Tumoral , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Inhibidor NF-kappaB alfa/inmunología , FN-kappa B/inmunología , Neuronas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunología , Proteína Fluorescente RojaRESUMEN
Leptin regulates energy balance by suppressing appetite and increasing energy expenditure through actions in the hypothalamus. Recently we demonstrated that the effects of leptin are, at least in part, mediated by the release of interleukin (IL)-1beta in the brain. Microglia constitute the major source of IL-1beta in the brain but it is not known whether these cells express leptin receptors, or respond to leptin to produce IL-1beta. Using RT-PCR and immunocytochemistry, we demonstrate that primary rat microglial cells express the short (non-signalling) and long (signalling) isoforms of the leptin receptors (Ob-R)s. Immunoassays performed on cell medium collected 24 h after leptin treatment (0.01-10 microg/mL) demonstrated a dose-dependent production and release of IL-1beta and its endogenously occurring receptor antagonist IL-1RA. In addition leptin-induced IL-1beta release occurs via a signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. Western blot analysis demonstrated that leptin induced the synthesis of pro-IL-1beta in microglial cells and the release of mature 17 kDa isoform into the culture medium. Leptin-induced IL-1beta release was neither inhibited by the pan-caspase inhibitor BOC-D-FMK, nor by the caspase 1 inhibitor Ac-YVAD-CHO indicating that IL-1 cleavage is independent of caspase activity. These results confirm our earlier observations in vivo and demonstrate that microglia are an important source of IL-1beta in the brain in response to leptin.