Biology subjects

Lopez-Castejon, G.

Publications and source records attributed to Lopez-Castejon, G..

8 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

Pyroptosis leads to loss of centrosomal integrity in macrophages

NLRP3 forms a multiprotein inflammasome complex to initiate the inflammatory response when macrophages sense infection or tissue damage, which leads to caspase-1 activation and maturation and release of the inflammatory cytokines interleukin-1{beta} (IL-1{beta}) and IL-18, and Gasdermin-D (GSDMD) mediated pyroptosis. NLRP3 inflammasome activity must be controlled as unregulated and chronic inflammation underlies inflammatory and autoimmune diseases. Several findings uncovered that NLRP3 inflammasome activity is under the regulation of centrosome localized proteins such as NEK7 and HDAC6, however, whether the centrosome composition or structure is altered during the inflammasome activation is not known. Our data show that levels of the centrosomal scaffold protein pericentrin (PCNT) are reduced upon NLRP3 inflammasome activation via different activators in human and murine macrophages. PCNT loss occurs in the presence of membrane stabilizer punicalagin, suggesting this is not a consequence of membrane rupture. We found that PCNT loss is dependent on NLRP3 and active caspases as MCC950 and pan caspase inhibitor ZVAD prevent its degradation. Moreover, caspase-1 and GSDMD are both required for this NLRP3-mediated PCNT loss because absence of caspase-1 or GSDMD triggers an alternative regulation of PCNT via its cleavage by caspase-3 in response to nigericin stimulation. PCNT degradation occurs in response to nigericin, but also other NLRP3 activators including lysomotropic agent L-Leucyl-L-Leucine methyl ester (LLOMe) and hypotonicity. Our work reveals that the NLRP3 inflammasome activation affects centrosome composition and structure which may deepen our understandings of how activated NLRP3 inflammasomes are involved in the pathogenesis of inflammatory diseases.

immunology↗

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↗

bioRxiv2020

Priming Is Dispensable For NLRP3 Inflammasome Activation In Human Monocytes

Interleukin (IL)-1 family of cytokines modulate immune responses during infection and inflammation. IL-18 and IL-1{beta} are members of the IL-1 family, which contribute to inflammatory conditions such as rheumatoid arthritis and Alzheimers disease. IL-18 and IL-1{beta} are produced as inactive precursors that are activated by large macromolecular complexes called inflammasomes upon sensing damage or pathogenic signals. Canonical NLRP3 inflammasome activation is regarded to require a priming step that causes NLRP3 and IL-1{beta} gene upregulation, and also NLRP3 post-translational licencing. A subsequent activation step leads to the assembly of the inflammasome and the cleavage of pro-IL-18 and pro-IL-1{beta} by caspase-1 into their mature forms, allowing their release. Here we show that in primary human monocytes, the initial priming step is dispensable to form an active NLRP3 inflammasome. We found that, in the absence of priming, the NLRP3 activator nigericin caused the processing and release of constitutively expressed IL-18. Another IL-1 family member, IL-37, is constitutively cleaved but the release of its mature form was mediated by inflammasome activation, also in the absence of a priming step. This NLRP3 activation was characterised by ASC oligomerisation as well as caspase-1 and GSDMD cleavage and was blocked by the NLRP3 inhibitor MCC950 and in NLRP3 deficient cells. IL-18 and IL-37 release were impaired in GSDMD deficient THP-1s, suggesting that pyroptosis is required for release of these cytokines. This work highlights the readiness of the NLRP3 inflammasome to assemble in the absence of priming and hence contribute to sterile inflammatory processes in health and disease. Significance StatementThe NLRP3 inflammasome is a driver of inflammation through the processing of Interleukins (IL)-1{beta} and IL-18. Human monocytes coordinate the innate immune response through inflammasome activation following exposure to pathogens and damage signals. We currently think of NLRP3 activation as a 2 step process: priming (NLRP3 gene upregulation and post-translational licencing) and assembly. Here we show that the priming step is dispensable for NLRP3 inflammasome activation in human monocytes. The second signal alone is sufficient for caspase-1 activation, leading to cell death and the release of the constitutively expressed IL-18 and mIL-37. This reveals that in human monocytes, the NLRP3 inflammasome is already licenced and able to quickly assemble to mount an inflammatory response.

immunology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗