MEX3B as a Potent Regulator of Inflammasome Signaling 2267250

Genome-wide CRISPR Identification of Regulators Governing the NLRP3 Inflammasome 2331537

Dysregulated NLRP3 inflammasome activation drives chronic inflammation in autoimmune and autoinflammatory diseases. Sustained NLRP3-dependent IL-1β production promotes tissue damage, yet current therapies rely on broad immunosuppression or cytokine blockade that can impair host defense and provide incomplete disease control. Although direct NLRP3 inhibitors are emerging, their utility is limited by toxicity and an incomplete understanding of inflammasome regulation. Defining genetic networks that tune inflammasome activation is therefore critical for identifying therapeutic targets that selectively limit pathological inflammation while preserving protective innate immune responses. We conducted a genome-wide CRISPR—Cas9 KO screen in human THP-1 macrophages stably expressing ASC-GFP to identify genetic regulators of NLRP3 inflammasome activation. After inflammasome activation, cells are sorted by flow cytometry based on ASC speck formation. Candidate genes were prioritized using enrichment statistics and pathway analysis. Top hits were validated using independent sgRNAs, with readouts including IL-1β ELISA and imaging-based ASC speck quantification. NF-κB phosphorylation was assessed to exclude false-positive regulators affecting inflammasome priming rather than activation. The screen robustly identified known NLRP3 pathway components, validating screen performance, and revealed multiple previously uncharacterized regulators spanning cytoskeletal dynamics, mitochondrial homeostasis, and ubiquitin signaling. Genetic perturbation of top candidates significantly altered IL-1β release without affecting NF-κB priming. Our study defines a comprehensive genetic landscape governing human NLRP3 inflammasome activation and uncovers novel regulatory nodes that decouple inflammasome assembly from upstream priming. These findings provide mechanistic insight into innate immune regulation and nominate new targets for selective therapeutic modulation of inflammasome-driven disease. n/a Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Investigating Inflammasome Responses to Pathogenic Fungi 2331094

Fungal pathogens are increasingly recognized as important activators of the innate immune system, particularly through engagement of inflammasomes. These are large protein complexes that trigger pyroptosis, an inflammatory cell death resulting in the release of IL-1 family cytokines. Although a large body of evidence demonstrates that diverse fungal species activate inflammasomes, they employ various evasion mechanisms that can limit the level of cell death and IL-1 release. Among the inflammasomes, NLRP3 has emerged as a central mediator of antifungal immunity. However, NLRP3 is not regarded as a direct pathogen sensor, but rather integrates signals related to potassium efflux, reactive oxygen species, and lysosomal damage. Given that many cytosolic sensors have defined ligands, a question that remains largely unexplored in the field of fungal innate immunity is whether specific cytosolic detection pathways exist for fungal cell wall components. Our work aims to understand the molecular mechanisms governing fungal intracellular sensing and inflammasome activation, to provide critical insights into host—fungi interactions and inform the development of therapies for the increasing emergence of invasive and inflammatory fungal diseases.   n/a Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

A Review on Structural Insights into NLRP3 and NEK7: Domain Architecture, Subcellular Localization, and Their Interaction in Inflammasome Assembly and Signaling

The dynamic and heterogeneous structure of the non-canonical inflammasome.

Inflammasomes are high-molecular-weight complexes that play integral roles in the innate immune system, triggering an inflammatory cascade to protect against cellular stresses such as pathogenic bacteria. Both canonical and non-canonical inflammasomes have been described in the literature and detailed structural studies of many components of the more complex and larger canonical versions have been reported. In contrast, corresponding structures of the non-canonical inflammasome have not emerged even though it consists of only two components: lipopolysaccharide (LPS) from Gram-negative bacteria and one of caspase-4 or caspase-5 in humans or caspase-11 in mice. Here, we determine the stoichiometry of the non-canonical inflammasome showing that it is heterogeneous, comprised of three major complexes with different numbers of LPS and caspase molecules. NMR spectroscopy studies of the N-terminal caspase activation and recruitment domain (CARD) of caspase-11, which binds LPS, establish that it is largely unstructured in the absence of lipid, with pervasive dynamics on the µs-ms timescale. Formation of this complex increases the α-helical content of the CARD, but the dynamics persist, multiple conformers are formed, and tertiary contacts are transient. The NMR results presented establish that the protease domain of caspase-11 is monomeric in isolation. As proteolysis is linked with dimerization, the protease domains are inactive in this state, but upon formation of the non-canonical inflammasome, dimerization occurs, priming the complex for rapid processing of substrates. Our study highlights the utility of NMR for studies of heterogeneous systems that are recalcitrant to crystallographic or cryo-EM analyses.

MARCO is an IFN-restricted immunometabolic decoy LPS receptor 2308885

Cytosolic sensing of bacterial lipopolysaccharide (LPS) is mediated by caspase-11 (caspase-4/5 in humans), which triggers non-canonical inflammasome activation. This leads to gasdermin D cleavage, pyroptosis, and release of pro-inflammatory cytokines. Severe gram-negative bacterial infections cause endotoxic septic shock, with systemic inflammation, organ failure, and potential death. Bacterial derived LPS gains access to the cytosol through extracellular vesicles and CD14 receptor mediated endocytosis. However, how this process is controlled during infection to maintain homeostasis is unknown. We performed an unbiased proteomic screen with biotinylated LPS and identified MARCO as an LPS-binding protein by mass spectrometry. Using macrophages from transgenic mice, we defined the mechanism by which MARCO facilitates LPS clearance to limit activation of the non-canonical inflammasome. MARCO is a scavenger receptor expressed on tissue resident macrophages (TRMs) and is known to promote clearance of pathogens and apoptotic debris. However, there is a lack of genetic studies and a paucity in our understanding of the functional role MARCO plays in innate immunity and how MARCO expression is regulated on TRMs. Here, we show that activation of the non-canonical inflammasome is preceded by a priming step by which type I interferon (IFN) suppresses MARCO expression on TRMs to allow for optimal recognition of LPS and activation of caspase-11. Remarkably, the metabolite itaconate and transcription factor NRF2 mediate MARCO expression. IFN signaling inhibits NRF2 stabilization, reducing surface MARCO levels. In vivo, MARCO-deficient mice show increased sensitivity to LPS-induced septic shock due to enhanced caspase-11 and non-canonical inflammasome activation. IFN-mediated suppression of MARCO expression is a previously unknown innate checkpoint that facilitates the recognition of LPS by caspase-11, activation of the non-canonical inflammasome, and progression of septic shock. n/a Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Sublethal disulfide stress facilitates TLR2-dependent NLRP3 inflammasome activation and pyroptosis in leukemia cells

Inflammasomes are innate immune signaling platforms that activate caspase-1, which in turn cleaves gasdermin D (GSDMD) into its mature form, leading to pyroptosis, an inflammatory form of regulated necrosis. It has been reported that bacterial lipopeptides (BLPs) trigger pyroptosis through NLRP7 inflammasome activation. However, the underlying recognition mechanism remains entirely unclear, and the concept of BLP sensing by NLRP7 has not been well established. In this study, we sought to elucidate the mechanism by which BLPs induce inflammasome activation and pyroptosis in human acute myeloid leukemia cell lines. Through comprehensive analyses, we demonstrate that BLPs induce GSDMD-dependent pyroptosis not via the NLRP7 inflammasome, but through the NLRP3 inflammasome without the need for additional stimuli. Addition of KCl to the culture medium significantly suppressed BLP-induced pyroptosis, suggesting the involvement of K + efflux in inflammasome activation. However, BLPs alone are generally thought to be insufficient to directly induce K + efflux, and K + channel inhibitors had minimal effects, prompting us to hypothesize that K + efflux might result from sublethal membrane damage associated with a distinct cell death mechanism. Among several cell death inhibitors tested, reducing agents, inhibitors of disulfidptosis, which is necrotic cell death caused by excessive disulfide bonding in the actin cytoskeleton, markedly attenuated BLP-induced K + efflux and pyroptosis. Consistent with this, BLP stimulation led to NADPH depletion and increased disulfide bond formation in β-actin, hallmarks of disulfidptosis. These findings suggest that disulfide stress contributes to NLRP3 inflammasome activation and pyroptosis triggered by BLPs in leukemia cells. To our knowledge, this is the first report linking disulfidptosis-associated disulfide stress to innate immune signaling, providing a conceptual framework for exploiting pyroptosis induction in leukemia cells.