RESUMO
Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⺠(IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκBâº-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⺠also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⺠accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⺠and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.
RESUMO
The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., Fos and Cxcl10. Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.
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Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper.
Assuntos
Reposicionamento de Medicamentos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Análise de Sistemas , Animais , Células Cultivadas , Claritromicina/farmacologia , Claritromicina/uso terapêutico , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , DNA/metabolismo , Sulfato de Dextrana , Redes Reguladoras de Genes , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Lipopolissacarídeos , Luciferases/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1ß involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.
Assuntos
RNA Mensageiro/genética , Receptores Toll-Like/metabolismo , Humanos , Modelos Teóricos , Transdução de SinaisRESUMO
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.
Assuntos
Núcleo Celular/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição RelA/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Adulto , Animais , Núcleo Celular/genética , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transdução de Sinais/genética , Fator de Transcrição RelA/genéticaRESUMO
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.
Assuntos
Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Inflamação/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Células RAW 264.7 , Análise de Célula Única , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.
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Boro/química , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Células da Medula Óssea/citologia , Boro/farmacologia , Compostos de Boro/química , Compostos de Boro/metabolismo , Compostos de Boro/farmacologia , Cálcio/metabolismo , Células Cultivadas , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Conformação Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
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Cálcio/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Ionóforos de Cálcio/imunologia , Humanos , Células Jurkat , Ativação Linfocitária , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fosfotransferases/metabolismo , Receptor Cross-Talk , Transdução de Sinais , Fator de Transcrição AP-1/metabolismoRESUMO
The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1ß, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1ß. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1ß processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.
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Apoptose , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Animais , Cálcio/metabolismo , Calpaína/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , NecroseRESUMO
The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons.