Biology subjects

Hoyle, C.

Publications and source records attributed to Hoyle, C..

7 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↗

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↗

bioRxiv2021

NLRP3 activation in response to disrupted endocytic traffic

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3. One-Sentence SummaryNLRP3 senses disruptions in endosome trafficking to trigger the formation of an inflammasome complex and initiate an inflammatory response.

immunology↗

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↗