Rachel A. Gottschalk

Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced NF-κB Signaling

Dueling feedback loops set the threshold for mounting an effective innate immune response to infection. Rewiring NF-κB Signaling The bacterial product lipopolysaccharide (LPS) stimulates nuclear factor κB (NF-κB) signaling, which results in the production of proinflammatory cytokines, such as tumor necrosis factor–α (TNF-α), as part of the immune response. NF-κB target genes also include those encoding proteins that inhibit NF-κB signaling through negative feedback loops. By simultaneously studying the dynamics of the nuclear translocation of the NF-κB subunit RelA and the activity of a Tnf-driven reporter in a mouse macrophage cell line, Sung et al. found that the gene encoding RelA was also a target of NF-κB. Synthesis of RelA occurred only at higher concentrations of LPS and constituted a positive feedback loop that dominated over existing negative feedback mechanisms. Genes expressed in response to a high concentration of LPS were enriched for those involved in innate immune responses. Together, these data suggest that the RelA-mediated positive feedback loop enables macrophages to mount an effective immune response only above a critical concentration of LPS. A fundamental goal in biology is to gain a quantitative understanding of how appropriate cell responses are achieved amid conflicting signals that work in parallel. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor κB (NF-κB) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela, which encodes the RelA (p65) NF-κB subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-κB signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We suggest that this switching in the relative dominance of feedback loops (“feedback dominance switching”) may be a general mechanism in immune cells to integrate opposing feedback on a key transcriptional regulator and to set a response threshold for the host.

Network representations of immune system complexity

The mammalian immune system is a dynamic multiscale system composed of a hierarchically organized set of molecular, cellular, and organismal networks that act in concert to promote effective host defense. These networks range from those involving gene regulatory and protein–protein interactions underlying intracellular signaling pathways and single‐cell responses to increasingly complex networks of in vivo cellular interaction, positioning, and migration that determine the overall immune response of an organism. Immunity is thus not the product of simple signaling events but rather nonlinear behaviors arising from dynamic, feedback‐regulated interactions among many components. One of the major goals of systems immunology is to quantitatively measure these complex multiscale spatial and temporal interactions, permitting development of computational models that can be used to predict responses to perturbation. Recent technological advances permit collection of comprehensive datasets at multiple molecular and cellular levels, while advances in network biology support representation of the relationships of components at each level as physical or functional interaction networks. The latter facilitate effective visualization of patterns and recognition of emergent properties arising from the many interactions of genes, molecules, and cells of the immune system. We illustrate the power of integrating ‘omics’ and network modeling approaches for unbiased reconstruction of signaling and transcriptional networks with a focus on applications involving the innate immune system. We further discuss future possibilities for reconstruction of increasingly complex cellular‐ and organism‐level networks and development of sophisticated computational tools for prediction of emergent immune behavior arising from the concerted action of these networks. WIREs Syst Biol Med 2015, 7:13–38. doi: 10.1002/wsbm.1288

Anti-Inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action

&NA; Despite the widespread use of glucocorticoids (GCs), their anti‐inflammatory effects are not understood mechanistically. Numerous investigations have examined the effects of glucocorticoid receptor (GR) activation prior to inflammatory challenges. However, clinical situations are emulated by a GC intervention initiated in the midst of rampant inflammatory responses. To characterize the effects of a late GC treatment, we profiled macrophage transcriptional and chromatinscapes with Dexamethasone (Dex) treatment before or after stimulation by lipopolysaccharide (LPS). The late activation of GR had a similar gene‐expression profile as from GR pre‐activation, while ameliorating the disruption of metabolic genes. Chromatin occupancy of GR was not predictive of Dex‐regulated gene expression, contradicting the “trans‐repression by tethering” model. Rather, GR activation resulted in genome‐wide blockade of NF‐&kgr;B interaction with chromatin and directly induced inhibitors of NF‐&kgr;B and AP‐1. Our investigation using GC treatments with clinically relevant timing highlights mechanisms underlying GR actions for modulating the “inflamed epigenome.” Graphical Abstract Figure. No caption available. HighlightsWe delineated the effects of glucocorticoids given after an inflammatory signalGR inhibits chromatin occupancy, but not nuclear residence, of NF‐&kgr;BGR binding has modest effects on chromatin accessibility in macrophagesGR activates negative regulators of NF‐&kgr;B and AP‐1 in resting or LPS‐induced cells &NA; Although glucocorticoids are widely used anti‐inflammatory drugs, relevant mechanisms are unclear. Oh et al. monitored the epigenomic landscape of macrophages and found that the gene‐inducing activity of GR is crucial for boosting inhibitors of inflammatory factors. This cautions the idea that GR ligands selectively promoting trans‐repression should improve therapeutic outcome.

Distinct NF-κB and MAPK Activation Thresholds Uncouple Steady-State Microbe Sensing from Anti-pathogen Inflammatory Responses

The innate immune system distinguishes low-level homeostatic microbial stimuli from those of invasive pathogens, yet we lack understanding of how qualitatively similar microbial products yield context-specific macrophage functional responses. Using quantitative approaches, we found that NF-κB and MAPK signaling was activated at different concentrations of a stimulatory TLR4 ligand in both mouse and human macrophages. Above a threshold of ligand, MAPK were activated in a switch-like manner, facilitating production of inflammatory mediators. At ligand concentrations below this threshold, NF-κB signaling occurred, promoting expression of a restricted set of genes and macrophage priming. Among TLR-induced genes, we observed an inverse correlation between MAPK dependence and ligand sensitivity, highlighting the role of this signaling dichotomy in partitioning innate responses downstream of a single receptor. Our study reveals an evolutionarily conserved innate immune response system in which danger discrimination is enforced by distinct thresholds for NF-κB and MAPK activation, which provide sequential barriers to inflammatory mediator production.

Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα 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.

Proteome and secretome analysis reveals differential post-transcriptional regulation of Toll-like receptor responses

The innate immune system is the organisms first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level.

Response to Comment on “Expression of Helios in Peripherally Induced Foxp3+ Regulatory T Cells”

We recently reported in the The Journal of Immunology that Helios is expressed in peripherally induced T regulatory cells (Treg), precluding the use of this transcription factor as a marker of thymically-derived natural Treg ([1][1]). We noted differences in the optimal conditions for Helios and

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1−/− macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/− mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.