Biology subjects

Green, J. P.

Publications and source records attributed to Green, J. P..

12 recordsLinked to original sources

bioRxiv2026

In vivo cellular localisation and nanoscale organisation of NLRP3 inflammasomes

The NLRP3 inflammasome is a critical regulator of inflammation, yet the localisation, organisation, and cellular sources of endogenous NLRP3 inflammasomes remain incompletely understood. Here, we generated NLRP3-mScarlet-I endogenous reporter mice enabling visualisation of NLRP3 at physiological levels in primary cells and in vivo. We show that activated NLRP3 associated with PI4P-positive membranes from multiple organelles, supporting a model where diverse membrane platforms act as a scaffold to nucleate inflammasome assembly. Super-resolution imaging revealed that NLRP3 and ASC occupy distinct nanoscale architectures within the inflammasome, with NLRP3 displaying marked structural heterogeneity and stimulus-dependent organisation. Unexpectedly, circulating monocytes and neutrophils, rather than tissue-resident populations, emerged as the dominant NLRP3-expressing cells in vivo which rapidly infiltrated tissues following systemic inflammation, highlighting an underappreciated cellular source of rapid inflammasome-driven responses. These findings reveal previously unrecognised insights into inflammasome organisation and localisation, establishing a powerful resource for investigating endogenous NLRP3 biology in health and disease.

immunology↗

bioRxiv2025

Inflammatory responses following CRISPR modification of the nuclear localisation sequence in endogenous interleukin-1α

Interleukin (IL)-1 is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1{beta}, pro-IL-1 is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1 is proposed to regulate its secretion. To-date, studies on the nuclear role of pro-IL-1 have used overexpression systems. Here, we generated a mouse where the endogenous Il1a gene was edited with CRISPR to disrupt the NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1 RNA expression levels in response to LPS but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1 in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1 release were detected, potentially due to increased levels of pro-IL-1. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1 nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1 function.

immunology↗

bioRxiv2025

Lysosome positioning affects NLRP3 inflammasome activation

The NLRP3 inflammasome, an important multi-molecular protein complex driving inflammation, is activated by multiple cellular stresses, including a loss of organelle homeostasis. Studies indicate that lysosome disruption can play a role in NLRP3 inflammasome activation, but whether lysosome positioning impacts NLRP3 inflammasome activation is currently unknown. Using HeLa and macrophage cell models, the effect of repositioning lysosomes to either the cell periphery or the perinuclear region on inflammasome activation was studied. Lysosome positioning to the cell periphery increased NLRP3 inflammasome activation in response to nigericin. This effect correlated with a change in the subcellular localisation of the inflammasome complex and an increase in its size. As macrophages at sites of tissue inflammation are exposed to environments known to influence lysosome position, these data help us understand how subcellular organelle reorganisation can regulate inflammation in response to inflammatory stimuli.

immunology↗

bioRxiv2024

Cell volume tunes macrophage innate inflammatory responses through promoting type I interferon signalling.

Macrophages are key effectors in co-ordinating inflammatory and immune responses to threats to the host. How macrophages decipher diverse danger signals to tailor inflammatory responses remains an unanswered question. Cell volume control is critical for normal cellular function. Disturbances in extracellular and intracellular homeostasis induce changes in cell volume, but the impact of disruptions in cell volume in controlling macrophage inflammatory responses is poorly understood. Here, we discover that macrophages use cell volume control as a bona fide danger sensing mechanism to promote and augment inflammatory responses. Using macrophages deficient in the volume regulated anion channel (VRAC), which lack cell volume control under hypo-osmotic conditions, we show that disruptions in cell volume are sensed by macrophages to drive a large transcriptomic response and induction of inflammation. Cell volume disruption, particularly loss of cell volume control, induces type I interferon signalling through a DNA- and STING-dependent mechanism, but independent of cGAS and 23cGAMP transport. Further, we found that cell volume changes synergise with diverse pathogen-mediated signalling to augment type I interferon responses and exacerbate the cytokine storm in a mouse model of hyperinflammation. Our findings highlight cell volume as an important regulator in shaping inflammatory responses, adding to our understanding of how macrophages sense complex danger signals and threats.

immunology↗

bioRxiv2024

The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-{gamma}. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.

immunology↗

bioRxiv2023

Brazilin is a Natural Product Inhibitor of the NLRP3 Inflammasome

Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modelling, we predict that brazilin can adopt a favourable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small-molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds.

molecular biology↗

bioRxiv2023

Proximity labelling and evolutionary evidence reveal insights into IL-1α nuclear networks and function

Interleukin (IL)-1 is a suggested dual-function cytokine that diverged from IL-1{beta} in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of IL-1, including a nuclear localisation sequence (NLS) and histone acetyl transferase (HAT)-binding domains. Why evolution modified pro-IL-1s subcellular location and protein interactome, and how this shaped IL-1s intracellular role, is unknown. TurboID proximity labelling with pro-IL-1 suggested a nuclear role for pro-IL-1 that involved interaction with HATs, including EP300. We also identified and validated inactivating mutations in the pro-IL-1 NLS of multiple mammalian species. However, HAT-binding domains were also conserved in species that had lost pro-IL-1 nuclear localisation. Together, these data suggest that HAT binding and nuclear localisation occurred together, and that while some species lost the NLS in their pro-IL-1, HAT binding was maintained. The NLS was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the NLS confers some important biological outcome.

immunology↗

bioRxiv2023

Stroke induces early recurrent vascular events by inflammasome-dependent atherosclerotic plaque rupture

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA from the ischemic tissue. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterio-arterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischemic events in atherosclerotic patients. The detailed mechanisms uncovered here provide so far clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.

immunology↗

bioRxiv2022

Discovery of an AIM2 inflammasome inhibitor for the treatment of DNA-driven inflammatory disease.

Inflammation driven by DNA sensors is now understood to be central to disease pathogenesis. Here we describe new inhibitors of pathogenic DNA sensing, primarily of the inflammasome forming sensor AIM2. Molecular modelling and biochemistry has revealed potent inhibitors of AIM2 that work by binding competitively to the DNA binding site. Though less potent, these AIM2 inhibitors, 4-sulfonic calixarenes, also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against pathogenic DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2 dependent post-stroke T cell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combatting post-stroke immunosuppression. By extension, we propose a broad utility against DNA driven inflammation in disease. Finally, we reveal that the ancient drug suramin, by virtue of its structural similarities, is an excellent inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an ever increasing clinical need.

immunology↗

bioRxiv2022

Inclusive fitness forces of selection in an age-structured population

Current evolutionary theories of senescence predict that the force of selection on survival will decline from maturity to zero at the age of last reproduction, and the force of selection on reproduction will decline monotonically from birth. These predictions rest upon the assumption that individuals within a population do not interact with one another. This assumption, however, is violated in social species, where an individuals survival and/or reproduction may shape the fitness of other group members. In such species, it is inclusive fitness that natural selection optimises. Yet, it remains unclear how the forces of selection on survival and reproduction might be modified when inclusive fitness, rather than population growth rate, is considered the appropriate metric for fitness. Here, we derive inclusive fitness forces of selection for hypothetical populations of social species. We show that selection on survival is not always constant before maturity, and can remain above zero in post-reproductive age classes, contrary to conventional models of senescence. We also show how the trajectory of the force of selection on reproduction does not always decline monotonically from birth, as predicted by classical theory, but instead depends on the balance of benefits to direct fitness and costs to indirect fitness. Our theoretical framework provides the unique opportunity to expand our understanding of senescence across social species, with important implications to species with variable life histories.

evolutionary biology↗

bioRxiv2021

Itaconate and fumarate derivatives exert a dual inhibitory effect on canonical NLRP3 activation in macrophages and microglia

The NLRP3 inflammasome is a multi-protein complex that regulates the protease caspase-1 and subsequent interleukin (IL)-1{beta} release from cells of the innate immune system, or microglia in the brain, in response to infection or injury. Derivatives of the metabolites itaconate and fumarate, dimethyl itaconate (DMI), 4-octyl itaconate (4OI) and dimethyl fumarate (DMF), limit both expression of IL-1{beta}, and IL-1{beta} release following NLRP3 inflammasome activation. However, the direct effects of these metabolite derivatives on NLRP3 inflammasome responses in macrophages and microglia require further investigation. Using murine bone marrow-derived macrophages, mixed glia and organotypic hippocampal slice cultures (OHSCs), we demonstrate that DMI and 4OI pre-treatment limited IL-1{beta}, IL-6 and tumor necrosis factor production in response to lipopolysaccharide (LPS) priming, as well as inhibiting subsequent NLRP3 inflammasome activation. DMI, 4OI, DMF and monomethyl fumarate (MMF), another fumarate derivative, also directly inhibited biochemical markers of NLRP3 activation in LPS-primed macrophages, mixed glia and OHSCs, including ASC speck formation, caspase-1 activation, gasdermin D cleavage and IL-1{beta} release. Finally, DMF, an approved treatment for multiple sclerosis, as well as DMI, 4OI and MMF, inhibited NLRP3 activation in macrophages in response to the phospholipid lysophosphatidylcholine, which is used to induce demyelination, suggesting a possible mechanism of action for DMF in multiple sclerosis through NLRP3 inhibition. Together, these findings reveal the importance of immunometabolic regulation for both the priming and activation steps of NLRP3 activation in macrophages and microglia. Furthermore, we highlight itaconate and fumarate derivatives as a potential therapeutic option in NLRP3-driven diseases, including in the brain. Summary statementWe show that itaconate and fumarate derivatives inhibit both the priming and activation steps of NLRP3 inflammasome responses in macrophages and microglia, revealing the importance of immunometabolic NLRP3 regulation.

immunology↗

bioRxiv2020

LRRC8A regulates hypotonicity-induced NLRP3 inflammasome activation

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1{beta} and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered a therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but the identity of which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of Cl- channels in the regulation of NLRP3 activation. Specifically we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to Cl- channel inhibitors, suggesting there are additional and specific Cl- sensing and regulating mechanisms controlling NLRP3.

immunology↗