ASC specks as a single-molecule fluid biomarker of inflammation in neurodegenerative diseases.

Immunotherapeutic strategies for Alzheimer's and Parkinson's disease would be facilitated by better measures of inflammation. Here we established an ultra-sensitive single-molecule pull-down immunoassay combined with direct stochastic optical reconstruction microscopy (dSTORM) to measure the number, size and shape of individual extracellular inflammasome ASC specks. We assayed human post-mortem brain, serum and cerebrospinal fluid of patients with Parkinson's and Alzheimer's as well as healthy elderly.

Rethinking Toll-like receptor signalling.

Since the discovery of Toll and Toll-like receptors (TLRs) in the 90s, an extensive body of research has been performed to determine how Pattern Recognition Receptors (PRRs) recognise 'ligands' and signal. The families of PRRs now include membrane and cytosolic proteins, which broadly signal by forming large protein platforms or supramolecular organising centres (SMOCs). The concept of SMOC-driven signalling has led to the development of a set of assumptions, particularly for TLRs, based on experimental data, to explain the physiological consequences of PRR activation.

A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations.

Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of 'omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell.

The delayed kinetics of Myddosome formation explains why amyloid-beta aggregates trigger Toll-like receptor 4 less efficiently than lipopolysaccharide.

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aβ) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vβ21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vβ21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children.

An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally.

Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

Elevated interleukin (IL)-1β levels, NLRP3 inflammasome activity, and systemic inflammation are hallmarks of chronic metabolic inflammatory syndromes, but the mechanistic basis for this is unclear. Here, we show that levels of plasma IL-1β are lower in fasting compared to fed subjects, while the lipid arachidonic acid (AA) is elevated. Lipid profiling of NLRP3-stimulated mouse macrophages shows enhanced AA production and an NLRP3-dependent eicosanoid signature.

Inflammasomes as regulators of mechano-immunity.

Mechano-immunity, the intersection between cellular or tissue mechanics and immune cell function, is emerging as an important factor in many inflammatory diseases. Mechano-sensing defines how cells detect mechanical changes in their environment. Mechano-response defines how cells adapt to such changes, e.

The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.

Mechanisms of type I interferon production by chicken TLR21.

The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs.

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.

NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum.

PLK1 inhibition dampens NLRP3 inflammasome-elicited response in inflammatory disease models.

Unabated activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome is linked with the pathogenesis of various inflammatory disorders. Polo-like kinase 1 (PLK1) has been widely studied for its role in mitosis. Here, using both pharmacological and genetic approaches, we demonstrate that PLK1 promoted NLRP3 inflammasome activation at cell interphase.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

Background: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection.

The Achromobacter type 3 secretion system drives pyroptosis and immunopathology via independent activation of NLRC4 and NLRP3 inflammasomes.

How the opportunistic Gram-negative pathogens of the genus Achromobacter interact with the innate immune system is poorly understood. Using three Achromobacter clinical isolates from two species, we show that the type 3 secretion system (T3SS) is required to induce cell death in human macrophages by inflammasome-dependent pyroptosis. Macrophages deficient in the inflammasome sensors NLRC4 or NLRP3 undergo pyroptosis upon bacterial internalization, but those deficient in both NLRC4 and NLRP3 do not, suggesting either sensor mediates pyroptosis in a T3SS-dependent manner.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages.

Label-free cell segmentation of diverse lymphoid tissues in 2D and 3D.

Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, segmentation of all cells imaged. Currently, achieving this is complex and requires exogenous fluorescent labels that occupy significant spectral bandwidth, increasing the duration and complexity of imaging experiments while limiting the number of channels remaining to address the study's objectives. We demonstrate that the excitation light reflected during routine confocal microscopy contains sufficient information to achieve accurate, label-free cell segmentation in 2D and 3D.

Biphasic JNK signaling reveals distinct MAP3K complexes licensing inflammasome formation and pyroptosis.

Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live-cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent process of biphasic JNK activation.

Characterization of full-length p53 aggregates and their kinetics of formation.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species.

The IRAK4 scaffold integrates TLR4-driven TRIF and MYD88 signaling pathways.

Interleukin-1 receptor-associated kinases (IRAKs) -4, -2, and -1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling.

Investigation of Host-Microbe-Parasite Interactions in an In Vitro 3D Model of the Vertebrate Gut.

In vitro models of the gut-microbiome axis are in high demand. Conventionally, intestinal monolayers grown on Transwell setups are used to test the effects of commensals/pathogens on the barrier integrity, both under homeostatic and pathophysiological conditions. While such models remain valuable for deepening the understanding of host-microbe interactions, often, they lack key biological components that mediate this intricate crosstalk.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed.

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.