James Bagnall

Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer’s disease in rodent models

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase-1 (COX-1) and COX-2 enzymes. The NLRP3 inflammasome is a multi-protein complex responsible for the processing of the proinflammatory cytokine interleukin-1β and is implicated in many inflammatory diseases. Here we show that several clinically approved and widely used NSAIDs of the fenamate class are effective and selective inhibitors of the NLRP3 inflammasome via inhibition of the volume-regulated anion channel in macrophages, independently of COX enzymes. Flufenamic acid and mefenamic acid are efficacious in NLRP3-dependent rodent models of inflammation in air pouch and peritoneum. We also show therapeutic effects of fenamates using a model of amyloid beta induced memory loss and a transgenic mouse model of Alzheimers disease. These data suggest that fenamate NSAIDs could be repurposed as NLRP3 inflammasome inhibitors and Alzheimers disease therapeutics.

Inflammasome-dependent IL-1β release depends upon membrane permeabilisation.

Interleukin-1β (IL-1β) is a critical regulator of the inflammatory response. IL-1β is not secreted through the conventional ER–Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1β release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1β, we have discovered that the secretion of IL-1β after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1β in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1β release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

Tight Control of Hypoxia-inducible Factor-α Transient Dynamics Is Essential for Cell Survival in Hypoxia

Background: Hypoxia inducible factor-α (HIF-α) is the main transcription factor activated in low oxygen conditions. Results: Single cell imaging reveals pulses in nuclear levels of HIF-α. Conclusion: The transient nature of the HIF-α nuclear accumulation is required to avoid cell death. Significance: The duration of HIF-α response depends on cellular oxygenation, and can encode information and dictate cell fate. Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.

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.

Modeling the dynamics of hypoxia inducible factor-1α (HIF-1α) within single cells and 3D cell culture systems.

HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells. There is increasing evidence that cell signaling pathways encode temporal information, and thus cell fate may be determined by the dynamics of protein levels. We have developed a mathematical model to describe the transient dynamics of the HIF-1α protein measured in single cells subjected to hypoxic shock. The essential characteristics of these data are modeled with a system of differential equations describing the feedback inhibition between HIF-1α and prolyl hydroxylases (PHD) oxygen sensors. Heterogeneity in the single-cell data is accounted through parameter variation in the model. We previously identified the PHD2 isoform as the main PHD sensor responsible for controlling the HIF-1α transient response, and make here testable predictions regarding HIF-1α dynamics subject to repetitive hypoxic pulses. The model is further developed to describe the dynamics of HIF-1α in cells cultured as 3D spheroids, with oxygen dynamics parameterized using experimental measurements of oxygen within spheroids. We show that the dynamics of HIF-1α and transcriptional targets of HIF-1α display a non-monotone response to the oxygen dynamics. Specifically we demonstrate that the dynamic transient behavior of HIF-1α results in differential dynamics in transcriptional targets.

Stochasticity in the miR-9/Hes1 oscillatory network can account for clonal heterogeneity in the timing of differentiation

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted. DOI: http://dx.doi.org/10.7554/eLife.16118.001

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated ex vivo using enzyme‐linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility.

Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.

Anti-inflammatory effects of infliximab in mice are independent of TNFα neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated ex vivo using enzyme‐linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

Integration of kinase and calcium signaling at the level of chromatin underlies inducible gene activation in T cells

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.