NF-κB Dynamics Research Articles

Ciliary proteins specify the cell inflammatory response by tuning NFκB signalling, independently of primary cilia.

ABSTRACTComplex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation.This article has an associated First Person interview with the first author of the paper.

Decoding patterns of macrophage TNF production to model NF-κB dynamics and cell-cell communication

Abstract Immune and stromal cells communicate through cytokines to initiate intracellular signaling, gene expression, and a multifaceted immune response. Cytokine signaling coordinates the dynamics of intracellular pathways that regulate inflammation. Immune and stromal cells both produce and respond to tumor necrosis factor-alpha (TNF), mediating inflammation and enabling communication between these compartments. The inflammatory role of TNF, activating the nuclear factor-κB (NF-κB) signaling and mitogen-activated protein kinase (MAPK) pathways, is well known. However, the effect of varying the time and amplitude patterns of TNF production in neighboring immune and stromal cells has not been systematically studied. Moreover, little is known about how time and amplitude patterns of an inflammatory signal input impact the nature of inflammatory responses. To study this relationship between production and response to TNF and its role in the dynamics of TNF-dependent NF-κB and MAPK signaling, we are using a systems immunology approach. Specifically, we are experimentally determining the patterns of TNF produced by human macrophages stimulated with heat-killed bacteria and characterizing the effect of these TNF stimulation patterns in the dynamics of NF-κB and MAPK responses in human cell lines. Additionally, we will use these experimental results to calibrate our deterministic 2 feedback with competitor (D2FC) mathematical model and model the dynamics of NF-κB signaling. Findings from this research will likely apply to other cytokine and signaling pathways, contributing to understanding the integration of time varying cytokine stimuli.

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

Gene Regulatory Strategies that Decode the Duration of NFκB Dynamics Contribute to LPS- versus TNF-Specific Gene Expression.

Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and Tcell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.

HEMATOPOIETIC STEM AND PROGENITOR CELLS EXHIBIT CELL TYPE SPECIFIC NF-ΰB SIGNALING DYNAMICS UPON INFLAMMATORY STIMULATION

Cells sense external stimuli with receptors and further process these stimuli via a variety of signaling pathways consisting of complex biochemical reaction cascades. For decades, cellular signaling was measured at population average level with snapshot data, without taking into account changing signaling activity over time (e.g. signaling dynamics) and cell-to-cell heterogeneity. Recent studies however show the importance of signaling dynamics for controlling future cell fates. As a master regulator of immune responses, the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB) coordinates the expression of hundreds of target genes including its own inhibitors NFKBIA and TNFAIP3 in many cell types. While a link between Nf-κB dynamics and future cell state was shown in cell lines, its role in relevant primary cell types remains elusive. Here, we use continuous single-cell quantitative time-lapse imaging of 10 different hematopoietic cell types, including stem and early progenitor cells, from a GFPp65/H2BmCherry reporter mouse line, in custom microfluidic devices, to observe the dynamics of the Nf-κB signaling pathway activity, and investigate how those dynamics influence their fates. We demonstrate that upon an inflammatory challenge by tumor necrosis factor alpha (TNFα), different HSPCs exhibit different and cell type specific Nf-κB dynamics including sustained, transient and oscillatory behaviors. RNA expression kinetics analyses identify molecular regulators responsible for these different dynamics. Additionally, Nf-κB signaling dynamics can identify novel progenitor cell subpopulations with distinct lineage potential. These data demonstrate that signaling dynamics can identify subpopulations of traditionally described cell populations, and suggest a role of Nf-κB signaling dynamics for lineage choice and cell fate control.

ROCK2 regulates TGF-β-induced expression of CTGF and profibrotic genes via NF-κB and cytoskeleton dynamics in mesangial cells.

The small GTPase Rho and its effector Rho kinase (ROCK) are involved in the pathogenesis of diabetic kidney disease. Rho kinase has two isoforms: ROCK1 and ROCK2. However, it remains unclear which is mainly involved in the progression of diabetic glomerulosclerosis and the regulation of profibrotic mediators. Glomeruli isolated from type 2 diabetic db/db mice demonstrated increased gene expression of transforming growth factor (TGF)-β and its downstream profibrotic mediators. Chemical inhibition of ROCK suppressed the expression of profibrotic mediators in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated ROCK functions through the phosphorylation of JNK and Erk and the nuclear translocation of NF-κB via actin dynamics. Knockdown by siRNA against ROCK1 and ROCK2 showed that ROCK2 but not ROCK1 controls this fibrotic machinery. Further in vivo experiments showed that ROCK2 activity in the renal cortex of db/db mice was elevated compared with control db/m mice. Importantly, oral administration of ROCK2 inhibitor attenuated renal ROCK2 activity, albuminuria, and glomerular fibrosis in db/db mice. These observations indicate that ROCK2 is a key player in the development of diabetic renal injury. Glomerular ROCK2 may be a potential therapeutic target for the treatment of diabetic kidney disease.

Controlling Nuclear NF-κB Dynamics by β-TrCP-Insights from a Computational Model.

The canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway regulates central processes in mammalian cells and plays a fundamental role in the regulation of inflammation and immunity. Aberrant regulation of the activation of the transcription factor NF-κB is associated with severe diseases such as inflammatory bowel disease and arthritis. In the canonical pathway, the inhibitor IκB suppresses NF-κB’s transcriptional activity. NF-κB becomes active upon the degradation of IκB, a process that is, in turn, regulated by the β-transducin repeat-containing protein (β-TrCP). β-TrCP has therefore been proposed as a promising pharmacological target in the development of novel therapeutic approaches to control NF-κB’s activity in diseases. This study explores the extent to which β-TrCP affects the dynamics of nuclear NF-κB using a computational model of canonical NF-κB signaling. The analysis predicts that β-TrCP influences the steady-state concentration of nuclear NF-κB, as well as changes characteristic dynamic properties of nuclear NF-κB, such as fold-change and the duration of its response to pathway stimulation. The results suggest that the modulation of β-TrCP has a high potential to regulate the transcriptional activity of NF-κB.

NF-κB Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration.

The nuclear factor-κB (NF-κB) signaling pathway is one of the best understood immune-related pathways thanks to almost four decades of intense research. NF-κB signaling is activated by numerous discrete stimuli and is a master regulator of the inflammatory response to pathogens and cancerous cells, as well as a key regulator of autoimmune diseases. In this regard, the role of NF-κB signaling in immunity is not unlike that of the macrophage. The dynamics by which NF-κB proteins shuttle between the cytoplasm and the nucleus to initiate transcription have been studied rigorously in fibroblasts and other non-hematopoietic cells, but many questions remain as to how current models of NF-κB signaling and dynamics can be translated to innate immune cells such as macrophages. In this review, we will present recent research on the dynamics of NF-κB signaling and focus especially on how these dynamics vary in different cell types, while discussing why these characteristics may be important. We will end by looking ahead to how new techniques and technologies should allow us to analyze these signaling processes with greater clarity, bringing us closer to a more complete understanding of inflammatory transcription factor dynamics and how different cellular contexts might allow for appropriate control of innate immune responses.

Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-κB Signaling Dynamics in Single Host Cells.

During an infection, immune cells must identify the specific level of threat posed by a given bacterial input in order to generate an appropriate response. Given that they use a general non-self-recognition system, known as Toll-like receptors (TLRs), to detect bacteria, it remains unclear how they transmit information about a particular threat. To determine whether host cells can use signaling dynamics to transmit contextual information about a bacterial stimulus, we use live-cell imaging to make simultaneous quantitative measurements of host MAPK and NF-κB signaling, two key pathways downstream of TLRs, and bacterial infection and load. This combined, single-cell approach reveals that NF-κB and MAPK signaling dynamics are sufficient to discriminate between (1) pathogen-associated molecular patterns (PAMPs) versus bacteria, (2) extracellular versus intracellular bacteria, (3) pathogenic versus non-pathogenic bacteria, and (4) the presence or absence of features indicating an active intracellular bacterial infection, such as replication and effector secretion.

Signal Distortion: How Intracellular Pathogens Alter Host Cell Fate by Modulating NF-κB Dynamics.

By uncovering complex dynamics in the expression or localization of transcriptional regulators in single cells that were otherwise hidden at the population level, live cell imaging has transformed our understanding of how cells sense and orchestrate appropriate responses to changes in their internal state or extracellular environment. This has proved particularly true for the nuclear factor-kappaB (NF-κB) family of transcription factors, key regulators of the inflammatory response and innate immune function, which are capable of encoding information about the mode and intensity of stimuli in the dynamics of NF-κB nuclear accumulation and loss. While live cell imaging continues to serve as a useful tool in ongoing efforts to characterize the feedbacks that shape these dynamics and to connect dynamics to downstream gene expression, it is also proving invaluable for recent studies that seek to determine how intracellular pathogens subvert NF-κB signaling to survive and replicate within host cells by providing quantitative information about the pathogen and changes in NF-κB activity during different stages of an infection. Here, we provide a brief overview of NF-κB signaling in innate immune cells and review recent literature that uses live imaging to investigate the mechanisms by which bacterial and yeast pathogens modulate NF-κB in a variety of different host cell types to evade destruction or maintain the viability of an intracellular growth niche.

Temperature regulates NF-κB dynamics and function through timing of A20 transcription

NF-κB signaling plays a pivotal role in control of the inflammatory response. We investigated how the dynamics and function of NF-κB were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling suggested that this temperature sensitivity might be due to an A20-dependent mechanism, and A20 silencing removed the sensitivity to increased temperature. The timing of the early response of a key set of NF-κB target genes showed strong temperature dependence. The cytokine-induced expression of many (but not all) later genes was insensitive to temperature change (suggesting that they might be functionally temperature-compensated). Moreover, a set of temperature- and TNFα-regulated genes were implicated in NF-κB cross-talk with key cell-fate-controlling pathways. In conclusion, NF-κB dynamics and target gene expression are modulated by temperature and can accurately transmit multidimensional information to control inflammation.

Impact of intracellular ionic strength on dimer binding in the NF-kB Inducing kinase.

Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-κB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly.

NF-κB Dynamics Discriminate between TNF Doses in Single Cells.

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune theircytokine responses beyond the decision to activate.

Lipopolysaccharide-induced NF-\u03baB nuclear translocation is primarily dependent on MyD88, but TNF\u03b1 expression requires TRIF and MyD88

TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.

The Single-Cell Yin and Yang of Live Imaging and Transcriptomics

A new approach to monitoring both signaling over time and a global gene expression profile from the same cell establishes a functional role for NF-κB dynamics in transcription.

Measuring Signaling and RNA-Seq in the Same Cell Links Gene Expression to Dynamic Patterns of NF-κB Activation

Signaling proteins display remarkable cell-to-cell heterogeneity in their dynamic responses to stimuli, but the consequences of this heterogeneity remain largely unknown. For instance, the contribution of the dynamics of the innate immune transcription factor nuclear factor κB (NF-κB) to gene expression output is disputed. Here we explore these questions by integrating live-cell imaging approaches with single-cell sequencing technologies. We used this approach to measure both the dynamics of lipopolysaccharide-induced NF-κB activation and the global transcriptional response in the same individual cell.Our results identify multiple, distinct cytokine expression patterns that are correlated with NF-κB activation dynamics, establishing a functional role for NF-κB dynamics in determining cellular phenotypes. Applications of this approach to other model systems and single-cell sequencing technologies have significant potential for discovery, as it is now possible to trace cellular behavior from the initial stimulus, through the signaling pathways, down to genome-wide changes in gene expression, all inside of a single cell.

Transcriptional Regulation by Locking and Unlocking the Intrinsic Disorder in NfκB

Intrinsic disorder plays a crucial role in the NFκB/IκB/DNA genetic regulatory network. Past studies on intrinsic disorder in the NFκB-IκB interaction have been focusing on IκB, the inhibitor of NFκB, while the dynamics of NFκB itself remains little understood. Previous coarse-grained energy landscape models and hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments showed intrinsic disorder in the N-terminal DNA-binding domain (NTD) of NFκB which becomes “locked” upon binding of DNA or IκB.

Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast

ABSTRACTThe eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization – a proxy for NF-κB activity – oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-κB was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-κB nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-α or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-κB target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression of SOCS3, encoding a “suppressor of cytokine signaling,” was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-κB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-κB target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

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-1β 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.

High-Content Quantification of Single-Cell Immune Dynamics.

SummaryCells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-κB, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages.