NF-κB Dynamics Research Articles

Time-varying stimuli that prolong IKK activation promote nuclear remodeling and mechanistic switching of NF-κB dynamics.

Temporal properties of molecules within signaling networks, such as sub-cellular changes in protein abundance, encode information that mediate cellular responses to stimuli. How dynamic signals relay and process information is a critical gap in understanding cellular behaviors. In this work, we investigate transmission of information about changing extracellular cytokine concentrations from receptor-level supramolecular assemblies of IκB kinases (IKK) downstream to the nuclear factor κB (NF-κB) transcription factor (TF). In a custom robot-controlled microfluidic cell culture, we simultaneously measure input-output (I/O) encoding of IKK-NF-κB in dual fluorescent-reporter cells. When compared with single cytokine pulses, dose-conserving pulse trains prolong IKK assemblies and lead to disproportionately enhanced retention of nuclear NF-κB. Using particle swarm optimization, we demonstrate that a mechanistic model does not recapitulate this emergent property. By contrast, invoking mechanisms for NF-κB-dependent chromatin remodeling to the model recapitulates experiments, showing how temporal dosing that prolongs IKK assemblies facilitates switching to permissive chromatin that sequesters nuclear NF-κB. Remarkably, using simulations to resolve single-cell receptor data accurately predicts same-cell NF-κB time courses for more than 80% of our single cell trajectories. Our data and simulations therefore suggest that cell-to-cell heterogeneity in cytokine responses are predominantly due to mechanisms at the level receptor-associated protein complexes.

Actin networks modulate heterogeneous NF-κB dynamics in response to TNFα.

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in pancreatic ductal adenocardinoma (PDAC) tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single-cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single-cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.

Synergy and antagonism in the integration of BCR and CD40 signals that control B-cell proliferation.

In response to infection or vaccination, a successful antibody response must enrich high-affinity antigen-reactive B-cells through positive selection, but eliminate auto-reactive B-cells through negative selection. B-cells receive signals from the B-cell receptor (BCR) which binds the antigen, and the CD40 receptor which is stimulated by neighboring T-cells that also recognize the antigen. How BCR and CD40 signaling are integrated quantitatively to jointly determine B-cell fate decision and proliferation remains unclear. To investigate this, we developed a differential-equations-based model of the BCR and CD40 signaling networks activating NFκB. Our model accurately recapitulates the NFκB dynamics of B-cells stimulated through their BCR and CD40 receptors, correctly predicting that costimulation induces more NFκB activity. However, when linking it to established cell fate decision models of cell survival and cell cycle control, it predicted potentiated population expansion that was not observed experimentally. We found that this discrepancy was due to a time-dependent functional antagonism exacerbated by BCR-induced caspase activity that can trigger apoptosis in founder cells, unless NFκB-induced survival gene expression protects B-cells in time. Guided by model predictions, sequential co-stimulation experiments revealed how the temporal dynamics of BCR and CD40 signaling control the fate decision between negative and positive selection of B-cell clonal expansion. Our quantitative findings highlight a complex non-monotonic integration of BCR and CD40 signals that is controlled by a balance between NFκB and cell-death pathways, and suggest a mechanism for regulating the stringency of B-cell selection during an antibody response.

NFκB dynamics-dependent epigenetic changes modulate inflammatory gene expression and induce cellular senescence.

Upregulation of nuclear factor κB (NFκB) signaling is a hallmark of aging and a major cause of age-related chronic inflammation. However, its effect on cellular senescence remains unclear. Here, we show that alteration of NFκB nuclear dynamics from oscillatory to sustained by depleting a negative feedback regulator of NFκB pathway, NFκB inhibitor alpha (IκBα), in the presence of tumor necrosis factor α (TNFα) promotes cellular senescence. Sustained NFκB activity enhanced inflammatory gene expression through increased NFκB-DNA binding and slowed the cell cycle. IκBα protein was decreased under replicative or oxidative stress in vitro. Furthermore, a decrease in IκBα protein and an increase in DNA-NFκB binding at the transcription start sites of age-associated genes in aged mouse hearts suggested that nuclear NFκB dynamics may play a critical role in the progression of aging. Our study suggests that nuclear NFκB dynamics-dependent epigenetic changes regulated over time in a living system, possibly through a decrease in IκBα, enhance the expression of inflammatory genes to advance the cells to a senescent state.

Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

Macrophages sense pathogens and orchestrate specific immune responses. Stimulus specificity is thought to be achieved through combinatorial and dynamical coding by signaling pathways. While NFκB dynamics are known to encode stimulus information, dynamical coding in other signaling pathways and their combinatorial coordination remain unclear. Here, we established live-cell microscopy to investigate how NFκB and p38 dynamics interface in stimulated macrophages. Information theory and machine learning revealed that p38 dynamics distinguish cytokine TNF from pathogen-associated molecular patterns and high doses from low, but contributed little to information-rich NFκB dynamics when both pathways are considered. This suggests that immune response genes benefit from decoding immune signaling dynamics or combinatorics, but not both. We found that the heterogeneity of the two pathways is surprisingly uncorrelated. Mathematical modeling revealed potential sources of uncorrelated heterogeneity in the branched pathway network topology and predicted it to drive gene expression variability. Indeed, genes dependent on both p38 and NFκB showed high scRNAseq variability and bimodality. These results identify combinatorial signaling as a mechanism to restrict NFκB-AND-p38-responsive inflammatory cytokine expression to few cells.

Small transcriptional differences among cell clones lead to distinct NF-κB dynamics

Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at a single-cell level, even within homogeneous populations. We asked how such heterogeneity emerges for the nuclear factor κB (NF-κB). We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct NF-κB dynamics upon tumor necrosis factor ɑ (TNF-ɑ) stimulation including persistent, oscillatory, and weak activation, giving rise to differences in the transcription of its targets. By combining transcriptomics and simulations we show how less than two-fold differences in the expression levels of genes coding for key proteins of the signaling cascade and feedback system are predictive of the differences of the NF-κB dynamic response of the clones to TNF-ɑ and IL-1β. We propose that small transcriptional differences in the regulatory circuit of a transcription factor can lead to distinct signaling dynamics in cells within homogeneous cell populations and among different cell types.

Modulating the dynamics of NFκB and PI3K enhances the ensemble-level TNFR1 signaling mediated apoptotic response

Cell-to-cell variability during TNFα stimulated Tumor Necrosis Factor Receptor 1 (TNFR1) signaling can lead to single-cell level pro-survival and apoptotic responses. This variability stems from the heterogeneity in signal flow through intracellular signaling entities that regulate the balance between these two phenotypes. Using systematic Boolean dynamic modeling of a TNFR1 signaling network, we demonstrate that the signal flow path variability can be modulated to enable cells favour apoptosis. We developed a computationally efficient approach “Boolean Modeling based Prediction of Steady-state probability of Phenotype Reachability (BM-ProSPR)” to accurately predict the network’s ability to settle into different phenotypes. Model analysis juxtaposed with the experimental observations revealed that NFκB and PI3K transient responses guide the XIAP behaviour to coordinate the crucial dynamic cross-talk between the pro-survival and apoptotic arms at the single-cell level. Model predicted the experimental observations that ~31% apoptosis increase can be achieved by arresting Comp1 – IKK* activity which regulates the NFκB and PI3K dynamics. Arresting Comp1 – IKK* activity causes signal flow path re-wiring towards apoptosis without significantly compromising NFκB levels, which govern adequate cell survival. Priming an ensemble of cancerous cells with inhibitors targeting the specific interaction involving Comp1 and IKK* prior to TNFα exposure could enable driving them towards apoptosis.

NFAT and NF-κB dynamically co-regulate TCR and CAR signaling responses in human T cells

While it has been established that the responses of Tcells to antigens are combinatorially regulated by multiple signaling pathways, it remains elusive what mechanisms cells utilize to quantitatively modulate Tcell responses during pathway integration. Here, we show that two key pathways in Tcell signaling, calcium/nuclear factor of activated Tcells (NFAT) and protein kinase C (PKC)/nuclear factor κB (NF-κB), integrate through a dynamic and combinatorial strategy to fine-tune Tcell response genes. At the cis-regulatory level, the two pathways integrate through co-binding of NFAT and NF-κB to immune response genes. Pathway integration is further regulated temporally, where Tcell receptor (TCR) and chimeric antigen receptor (CAR) activation signals modulate the temporal relationships between the nuclear localization dynamics of NFAT and NF-κB. Such physical and temporal integrations together contribute to distinct modes of expression modulation for genes. Thus, the temporal relationships between regulators can be modulated to affect their co-targets during immune responses, underscoring the importance of dynamic combinatorial regulation in cellular signaling.

Tonic TNF conditioning of macrophages safeguards stimulus‐specific inflammatory responses

Tumor necrosis factor (TNF) is a key inflammatory cytokine that warns recipient cells of a nearby infection or tissue damage. Acute exposure to TNF activates characteristic oscillatory dynamics of the transcription factor NFκB and induces a characteristic gene expression program; these are distinct from the responses of cells directly exposed to pathogen‐associated molecular patterns (PAMPs). Here, we report that tonic TNF exposure is critical for safeguarding TNF's specific functions. In the absence of tonic TNF conditioning, acute exposure to TNF causes (i) NFκB signaling dynamics that are less oscillatory and more like PAMP‐responsive NFκB dynamics, (ii) immune gene expression that is more similar to the Pam3CSK4 response program, and (iii) broader epigenomic reprogramming that is characteristic of PAMP‐responsive changes. We show that the absence of tonic TNF signaling effects subtle changes to TNF receptor availability and dynamics such that enhanced pathway activity results in non‐oscillatory NFκB. Our results reveal tonic TNF as a key tissue determinant of the specific cellular responses to acute paracrine TNF exposure, and their distinction from responses to direct exposure to PAMPs.

Double knock-in reporter mice: a novel tool to study age-associated NF-κB dynamics in primary microglia

Abstract Microglia are immune sentinels in the brain that are capable of orchestrating potent inflammatory responses in aging and neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. NF-κB signaling pathway is commonly recognized as a significant regulator of inflammation and aging. Mapping the spatiotemporal complexity of NF-κB signaling is crucial to understand its impact and function in vivo, but the lack of tools to directly monitor NF-κB protein components has hindered such efforts. Our lab has generated reporter mice with the endogenous RelA (p65) and c-Rel labeled with distinct fluorescent proteins and a double knock-in line with both labeled subunits. To understand how aging affects microglial function, we isolated primary microglia from young and old animals. We cultured them in vitro and validated by P2RY12 +, CD11b +and CD45 int. Interestingly, we observed two different microglial populations, one motile, and the other forming clusters. Microglia from aged animals showed a higher prevalence of motile, free-roaming microglia. We also observed these distinct microglial populations in brain slices from the mice ex-vivo. Live-cell imaging of NF-kB dynamics in primary microglia from young and old mice revealed a shift towards c-Rel in old brains. Further, we stained for Ki67 as a proliferation marker to functionally characterize these microglia populations. We observed only the free-roaming microglia to be proliferative in nature. Furthermore, we are investigating the senescence pattern between the two populations to better understand the molecular mechanisms associated with age-dependent and inflammation-derived changes in young and old brains.

Spatiotemporal NF-κB dynamics encodes the position, amplitude, and duration of local immune inputs.

Infected cells communicate through secreted signaling molecules like cytokines, which carry information about pathogens. How differences in cytokine secretion affect inflammatory signaling over space and how responding cells decode information from propagating cytokines are not understood. By computationally and experimentally studying NF-κB dynamics in cocultures of signal-sending cells (macrophages) and signal-receiving cells (fibroblasts), we find that cytokine signals are transmitted by wave-like propagation of NF-κB activity and create well-defined activation zones in responding cells. NF-κB dynamics in responding cells can simultaneously encode information about cytokine dose, duration, and distance to the cytokine source. Spatially resolved transcriptional analysis reveals that responding cells transmit local cytokine information to distance-specific proinflammatory gene expression patterns, creating “gene expression zones.” Despite single-cell variability, the size and duration of the signaling zone are tightly controlled by the macrophage secretion profile. Our results highlight how macrophages tune cytokine secretion to control signal transmission distance and how inflammatory signaling interprets these signals in space and time.

NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli.

Many scenarios in cellular communication require cells to interpret multiple dynamic signals. It is unclear how exposure to inflammatory stimuli alters transcriptional responses to subsequent stimulus. Using high-throughput microfluidic live-cell analysis, we systematically profile the NF-κB response to different signal sequences in single cells. We find that NF-κB dynamics store the short-term history of received signals: depending on the prior pathogenic or cytokine signal, the NF-κB response to subsequent stimuli varies from no response to full activation. Using information theory, we reveal that these stimulus-dependent changes in the NF-κB response encode and reflect information about the identity and dose of the prior stimulus. Small-molecule inhibition, computational modeling, and gene expression profiling show that this encoding is driven by stimulus-dependent engagement of negative feedback modules. These results provide a model for how signal transduction networks process sequences of inflammatory stimuli to coordinate cellular responses in complex dynamic environments.

Insights on the NF-κB System Using Live Cell Imaging: Recent Developments and Future Perspectives.

The transcription factor family of nuclear factor kappa B (NF-κB) proteins is widely recognized as a key player in inflammation and the immune responses, where it plays a fundamental role in translating external inflammatory cues into precise transcriptional programs, including the timely expression of a wide variety of cytokines/chemokines. Live cell imaging in single cells showed approximately 15 years ago that the canonical activation of NF-κB upon stimulus is very dynamic, including oscillations of its nuclear localization with a period close to 1.5 hours. This observation has triggered a fruitful interdisciplinary research line that has provided novel insights on the NF-κB system: how its heterogeneous response differs between cell types but also within homogeneous populations; how NF-κB dynamics translate external cues into intracellular signals and how NF-κB dynamics affects gene expression. Here we review the main features of this live cell imaging approach to the study of NF-κB, highlighting the key findings, the existing gaps of knowledge and hinting towards some of the potential future steps of this thriving research field.

6-OR: Vascular Endothelial ROCK2 Deficiency Promotes Browning and Burning of White Adipose Tissue

The accumulation of visceral fat is associated with multiple coronary risk factors such as glucose intolerance, dyslipidemia, and hypertension. We have previously demonstrated that Rho/Rho-associated protein kinase (ROCK) signaling is activated in animal models of diabetes and is involved in the pathogenesis of vascular complications. Specifically, ROCK is implicated in the molecular basis of inflammation- and ischemia-induced tissue damage through the actions on NF-κB dynamics. These findings support the hypothesis that ROCK signaling could be one of the important therapeutic targets for preventing cardiorenal events. However, isoform-specific roles of ROCKs have not been clarified yet. In this study, we aimed to determine the tissue-intrinsic roles of ROCK2 in the setting of obesity. To this end, we generated vascular endothelium-specific ROCK2 knockout mice (ER2KO) by crossing ROCK2 floxed mice with VE-Cadherin-Cre mice. ER2KO mice were fertile and had the same lifespan as wild type mice. Notably, ER2KO gained weight at a slower rate than wild type mice under high-fat diet feeding. Fat composition, as assessed by magnetic resonance imaging, was significantly lower in ER2KO, even though ER2KO displayed increased food intake. The gene expression analysis revealed increased expression of PPARα, a browning-inducing factor, and its downstream components, CIDEA, IL-4, and IL-10, in white adipose tissue obtained from ER2KO mice. ER2KO mice also showed an elevation in body temperature after fasting, suggesting that endothelial ROCK2 may negatively regulate fat browning. In summary, vascular endothelial ROCK2 negatively regulates fat accumulation through the mechanism of browning inhibition. Disclosure Y. Takeda: None. K. Matoba: None. R. Ukichi: None. Y. Nagai: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker's Bureau; Abbott Diabetes, Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic, Merck & Co., Inc., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Holdings Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Terumo Corporation. Funding a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, the MSD Life Science Foundation, the Takeda Science Foundation, the Suzuken Memorial Foundation, Sanofi KK, Tanabe Pharma, and Takeda Pharmaceutical., Kazunori Utsunomiya has received research support from Terumo, Novo Nordisk Pharma, Taisho Pharmaceutical, Böehringer Ingelheim, Kyowa Hakko Kirin, Sumitomo Dainippon Pharma, and OPharmaceutical as, Rimei Nishimura has received speaker honoraria from Astellas Pharma, Nippon Boehringer Ingelheim, Eli Lilly Japan KK, Kissei Pharmaceutical, Medtronic Japan, MSD, Novartis Pharma KK, Novo Nordisk Phar.

NF-κB Rel subunit exchange on a physiological timescale.

The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

Stochastic pooling of IKK/NEMO complexes describes information transmission in the NF-KB pathway in response to IL-1 and TNF stimulation.

Abstract A myriad of inflammatory cytokines regulates signaling pathways for normal cellular physiology. The IKK complex is an integration hub for extracellular cytokines that govern NF-κB signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here we track dynamics of IKK complexes and nuclear NF-κB to identify upstream signaling features that determine same-cell responses. Quantitative imaging experiments and computational modeling of single complexes reveals that their size, number, and timing relays cytokine-specific information with feedback control that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables parsimonious and cytokine-specific signaling. We argue that emergent properties of stochastic pooling motif are general principles of receptor signaling, and we present new modules of regulation of NF-kB signaling in response to cytokine stimulation.

Time will tell: The temporal code of immune threats

Activation of NF-κB is a common downstream consequence of inflammatory stimulation, yet it achieves stimulus-specific transcriptional responses. In this issue of Immunity, Adelaja etal. use single-cell imaging and computational approaches to understand temporal features of NF-κB dynamics that transmit information about immune threats.

Decoding the temporal nature of brain GR activity in the NF\u03baB signal transition leading to depressive-like behavior

The fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

An incoherent feedforward loop interprets NFκB/RelA dynamics to determine TNF‐induced necroptosis decisions

Balancing cell death is essential to maintain healthy tissue homeostasis and prevent disease. Tumor necrosis factor (TNF) not only activates nuclear factor κB (NFκB), which coordinates the cellular response to inflammation, but may also trigger necroptosis, a pro‐inflammatory form of cell death. Whether TNF‐induced NFκB affects the fate decision to undergo TNF‐induced necroptosis is unclear. Live‐cell microscopy and model‐aided analysis of death kinetics identified a molecular circuit that interprets TNF‐induced NFκB/RelA dynamics to control necroptosis decisions. Inducible expression of TNFAIP3/A20 forms an incoherent feedforward loop to interfere with the RIPK3‐containing necrosome complex and protect a fraction of cells from transient, but not long‐term TNF exposure. Furthermore, dysregulated NFκB dynamics often associated with disease diminish TNF‐induced necroptosis. Our results suggest that TNF's dual roles in either coordinating cellular responses to inflammation, or further amplifying inflammation are determined by a dynamic NFκB‐A20‐RIPK3 circuit, that could be targeted to treat inflammation and cancer.

A Quantitative Modular Modeling Approach Reveals the Effects of Different A20 Feedback Implementations for the NF-kB Signaling Dynamics.

Signaling pathways involve complex molecular interactions and are controled by non-linear regulatory mechanisms. If details of regulatory mechanisms are not fully elucidated, they can be implemented by different, equally reasonable mathematical representations in computational models. The study presented here focusses on NF-κB signaling, which is regulated by negative feedbacks via IκBα and A20. A20 inhibits NF-κB activation indirectly through interference with proteins that transduce the signal from the TNF receptor complex to activate the IκB kinase (IKK) complex. A number of pathway models has been developed implementing the A20 effect in different ways. We here focus on the question how different A20 feedback implementations impact the dynamics of NF-κB. To this end, we develop a modular modeling approach that allows combining previously published A20 modules with a common pathway core module. The resulting models are fitted to a published comprehensive experimental data set and therefore show quantitatively comparable NF-κB dynamics. Based on defined measures for the initial and long-term behavior we analyze the effects of a wide range of changes in the A20 feedback strength, the IκBα feedback strength and the TNFα stimulation strength on NF-κB dynamics. This shows similarities between the models but also model-specific differences. In particular, the A20 feedback strength and the TNFα stimulation strength affect initial and long-term NF-κB concentrations differently in the analyzed models. We validated our model predictions experimentally by varying TNFα concentrations applied to HeLa cells. These time course data indicate that only one of the A20 feedback models appropriately describes the impact of A20 on the NF-κB dynamics in this cell type.