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Downton P.♦, Bagnall James S.♦, England H.♦, Spiller David G.♦, Humphreys Neil E.♦, Jackson Dean A.♦, Paszek P., White Michael R.R.♦, Adamson Antony D.♦, Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression,
Frontiers in Molecular Biosciences, ISSN: 2296-889X, DOI: 10.3389/fmolb.2023.1187187, Vol.10, pp.1187187-1-15, 2023Streszczenie: 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⍺ (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 IκB⍺-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⍺ also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⍺ 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⍺ and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response. Słowa kluczowe: NF-κB, inflammation, IκB⍺, overexpression, gene expression, localisation Afiliacje autorów:
Downton P. | - | inna afiliacja | Bagnall James S. | - | inna afiliacja | England H. | - | inna afiliacja | Spiller David G. | - | inna afiliacja | Humphreys Neil E. | - | inna afiliacja | Jackson Dean A. | - | inna afiliacja | Paszek P. | - | IPPT PAN | White Michael R.R. | - | University of Manchester (GB) | Adamson Antony D. | - | inna afiliacja |
| | 140p. |
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Bagnall J.♦, Rowe W.♦, Alachkar N.♦, Roberts J.♦, England H.♦, Clark C.♦, Platt M.♦, Jackson Dean A.♦, Muldoon M.♦, Paszek P.♦, Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling,
Cell Systems, ISSN: 2405-4712, DOI: 10.1016/j.cels.2020.08.007, Vol.11, No.3, pp.300-314, 2020Streszczenie: 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-1β 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. Słowa kluczowe: cellular heterogeneity, transcriptional bursting, stochastic gene expression, toll-like receptor, single-cell transcriptomics, stochastic modeling, TNF-α, IL-1β Afiliacje autorów:
Bagnall J. | - | inna afiliacja | Rowe W. | - | inna afiliacja | Alachkar N. | - | inna afiliacja | Roberts J. | - | inna afiliacja | England H. | - | inna afiliacja | Clark C. | - | inna afiliacja | Platt M. | - | inna afiliacja | Jackson Dean A. | - | inna afiliacja | Muldoon M. | - | inna afiliacja | Paszek P. | - | inna afiliacja |
| | 200p. |
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Papoutsopoulou S.♦, Burkitt Michael D.♦, Bergey F.♦, England H.♦, Hough R.♦, Schmidt L.♦, Spiller David G.♦, White Michael H. R.R.♦, Paszek P.♦, Jackson Dean A.♦, Martins Dos Santos Vitor A. P.♦, Sellge G.♦, Pritchard D. M.♦, Campbell Barry J.♦, Müller W.♦, Probert Chris S.♦, Macrophage-Specific NF-κB Activation Dynamics Can Segregate Inflammatory Bowel Disease Patients,
Frontiers in Immunology, ISSN: 1664-3224, DOI: 10.3389/fimmu.2019.02168, Vol.10, pp.2168-1-11, 2019Streszczenie: 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. Słowa kluczowe: inflammatory bowel disease, NF-kB, macrophages, cytokines, Crohn’s disease, ulcerative colitis Afiliacje autorów:
Papoutsopoulou S. | - | inna afiliacja | Burkitt Michael D. | - | inna afiliacja | Bergey F. | - | inna afiliacja | England H. | - | inna afiliacja | Hough R. | - | inna afiliacja | Schmidt L. | - | inna afiliacja | Spiller David G. | - | inna afiliacja | White Michael H. R.R. | - | University of Manchester (GB) | Paszek P. | - | inna afiliacja | Jackson Dean A. | - | inna afiliacja | Martins Dos Santos Vitor A. P. | - | inna afiliacja | Sellge G. | - | inna afiliacja | Pritchard D. M. | - | inna afiliacja | Campbell Barry J. | - | inna afiliacja | Müller W. | - | inna afiliacja | Probert Chris S. | - | inna afiliacja |
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Bagnall J.♦, Boddington C.♦, England H.♦, Brignall R.♦, Downton P.♦, Alsoufi Z.♦, Boyd J.♦, Rowe W.♦, Bennett A.♦, Walker C.♦, Adamson A.♦, Patel Nisha M. X.♦, O’Cualain R.♦, Schmidt L.♦, Spiller David G.♦, Jackson Dean A.♦, Müller W.♦, Muldoon M.♦, White Michael R. H.R.♦, Paszek P.♦, Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation,
Science Signaling, ISSN: 1945-0877, DOI: 10.1126/scisignal.aaf3998, Vol.11, No.540, pp.1-15, 2018Streszczenie: 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. Afiliacje autorów:
Bagnall J. | - | inna afiliacja | Boddington C. | - | inna afiliacja | England H. | - | inna afiliacja | Brignall R. | - | inna afiliacja | Downton P. | - | inna afiliacja | Alsoufi Z. | - | inna afiliacja | Boyd J. | - | inna afiliacja | Rowe W. | - | inna afiliacja | Bennett A. | - | inna afiliacja | Walker C. | - | inna afiliacja | Adamson A. | - | inna afiliacja | Patel Nisha M. X. | - | inna afiliacja | O’Cualain R. | - | inna afiliacja | Schmidt L. | - | inna afiliacja | Spiller David G. | - | inna afiliacja | Jackson Dean A. | - | inna afiliacja | Müller W. | - | inna afiliacja | Muldoon M. | - | inna afiliacja | White Michael R. H.R. | - | University of Manchester (GB) | Paszek P. | - | inna afiliacja |
| | 40p. |
5. |
Brignall R.♦, Cauchy P.♦, Bevington Sarah L.♦, Gorman B.♦, Pisco Angela O.♦, Bagnall J.♦, Boddington C.♦, Rowe W.♦, England H.♦, Rich K.♦, Schmidt L.♦, Dyer Nigel P.♦, Travis Mark A.♦, Ott S.♦, Jackson Dean A.♦, Cockerill Peter N.♦, Paszek P.♦, Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells,
JOURNAL OF IMMUNOLOGY, ISSN: 0022-1767, DOI: 10.4049/jimmunol.1602033, Vol.199, No.8, pp.2652-2667, 2017Streszczenie: 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 ∼2100 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 (∼600 and ∼350, 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. Afiliacje autorów:
Brignall R. | - | inna afiliacja | Cauchy P. | - | inna afiliacja | Bevington Sarah L. | - | inna afiliacja | Gorman B. | - | inna afiliacja | Pisco Angela O. | - | inna afiliacja | Bagnall J. | - | inna afiliacja | Boddington C. | - | inna afiliacja | Rowe W. | - | inna afiliacja | England H. | - | inna afiliacja | Rich K. | - | inna afiliacja | Schmidt L. | - | inna afiliacja | Dyer Nigel P. | - | inna afiliacja | Travis Mark A. | - | inna afiliacja | Ott S. | - | inna afiliacja | Jackson Dean A. | - | inna afiliacja | Cockerill Peter N. | - | inna afiliacja | Paszek P. | - | inna afiliacja |
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