Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation

Abstract

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 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.

Macrophage-specific NF-κB activation dynamics can segregate inflammatory bowel disease patients

“…Notably, only a single p65 translocation event (rather than repeated oscillation) was observed within the 3h data acquisition period of confocal imaging. This is similar to previous analyses of murine bone marrow-derived macrophages from transgenic mice in response to either Lipid A or LPS (9,10).…”

“…NF-B transcriptional activity was monitored using a lentiviral construct (B-NLSluc) that expresses firefly luciferase under the control of the classical NF-B promoter (38). For imaging, the human p65 sequence was amplified from p65-dsRedXp (32) and C-terminally fused with AmCyan protein using a previously described lentiviral vector (9). Lentivirus production was carried out as per (39).…”

“…The nuclear translocation of NF-B proteins is a highly regulated, dynamic event, characterised not only by the transport of NF-B dimers into the nucleus following stimulation, but also by shuttling between the nucleus and cytoplasm of the cell, with context-specific oscillatory frequency; such as has been observed for transcriptionally active p65 subunit dimers (4). To date, these heterogeneous dynamics have been demonstrated using mouse (5)(6)(7) and human cell-lines (8), and primary cells obtained from transgenic mice expressing fluorescent fusion proteins (9,10). NF-B activation and dysregulated cytokine production has previously been reported in various cell types in IBD patients (11,12).…”

Macrophages employ quorum licensing to regulate collective activation

“…Since the sensing step involves capturing extracellular IL-2, the feedback is confined to local regions and only some cells sense sufficient to IL-2 to proliferate 63 . Competitive cytokine uptake is involved in TNF paracrine signaling as well 64 . In the case of a synthetic genetic circuit implemented in yeast, with topological similarity to TNF intercellular feedback and that exhibited strong positive feedback and rapid degradation of the secreted cue, the proportions of cells in upper and lower modes of activation were found to depend on cell density 65 .…”

Heat shock response pathways regulate stimulus-specificity and sensitivity of NF-κB signalling to temperature stress

“…In general, we expect that the NF-κB system (or in fact the combined action of other systems, including MAP kinase (Rouse et al, 1994) or p53 tumour suppressor (Alexandrova and Marchenko, 2015)) in individual cell types might exhibit different sensitivities and recovery kinetics following temperature exposure. For example, we previously showed that TNFα stimulation in human osteosarcoma cells resulted in "all-or-nothing" NF-κB responses following HS (Kardynska et al, 2018), while tissue-level architecture might impose additional spatial constraints (Bagnall et al, 2018). We suggest that further efforts should combine dynamical modelling with cell fate, in order to better understand the relationship between temperature and NF-κB as well as cell proliferation and apoptosis in the more relevant cancer or inflammatory context.…”