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 (Aß) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aß 1-42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aß fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aß fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aß fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ 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 Vß21.3+ 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. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

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. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

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

Elevated interleukin (IL)-1ß 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-1ß 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. Inhibition of cyclooxygenase by nonsteroidal anti-inflammatory drugs decreases eicosanoid, but not AA, production. It also reduces both IL-1ß and IL-18 production in response to NLRP3 activation. AA inhibits NLRP3 inflammasome activity in human and mouse macrophages. Mechanistically, AA inhibits phospholipase C activity to reduce JNK1 stimulation and hence NLRP3 activity. These data show that AA is an important physiological regulator of the NLRP3 inflammasome and explains why fasting reduces systemic inflammation and also suggests a mechanism to explain how nonsteroidal anti-inflammatory drugs work.

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.g. form synapses, signal or migrate. Inflammasomes are intracellular immune sensors that detect changes in tissue and cell homoeostasis during infection or injury. We and others recently found that mechano-sensing of tissue topology (swollen tissue), topography (presence and distribution of foreign solid implant) or biomechanics (stiffness), alters inflammasome activity. Once activated, inflammasomes induce the secretion of inflammatory cytokines, but also change cellular mechanical properties, which influence how cells move, change their shape, and interact with other cells. When overactive, inflammasomes lead to chronic inflammation. This clearly places inflammasomes as important players in mechano-immunity. Here, we discuss a model whereby inflammasomes integrate pathogen- and tissue-injury signals, with changes in tissue mechanics, to shape the downstream inflammatory responses and allow cell and tissue mechano-adaptation. We will review the emerging evidence that supports this model.

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. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-κB and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-κB and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species.

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.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16180. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

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. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.

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. Using an unbiased proximity-dependent biotin identification (Bio-ID) screen for the PLK1 interactome in macrophages, we show an enhanced proximal association of NLRP3 with PLK1 upon NLRP3 inflammasome activation. We further confirmed the interaction between PLK1 and NLRP3 and identified the interacting domains. Mechanistically, we show that PLK1 orchestrated the microtubule-organizing center (MTOC) structure and NLRP3 subcellular positioning upon inflammasome activation. Treatment with a selective PLK1 kinase inhibitor suppressed IL-1ß production in in vivo inflammatory models, including LPS-induced endotoxemia and monosodium urate-induced peritonitis in mice. Our results uncover a role of PLK1 in regulating NLRP3 inflammasome activation during interphase and identify pharmacological inhibition of PLK1 as a potential therapeutic strategy for inflammatory diseases with excessive NLRP3 inflammasome activation.

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. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

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. Detailed analysis of the intracellular trafficking of one isolate indicates that the intracellular bacteria reside in a late phagolysosome. Using an intranasal mouse infection model, we observe that Achromobacter damages lung structure and causes severe illness, contingent on a functional T3SS. Together, we demonstrate that Achromobacter species can survive phagocytosis by promoting macrophage cell death and inflammation by redundant mechanisms of pyroptosis induction in a T3SS-dependent manner.

Naturally-occurring serotype 3 Streptococcus pneumoniae strains that lack functional pneumolysin and autolysin have attenuated virulence but induce localized protective immune responses.

Streptococcus pneumoniae is an important cause of fatal pneumonia in humans. These bacteria express virulence factors, such as the toxins pneumolysin and autolysin, that drive host inflammatory responses. In this study we confirm loss of pneumolysin and autolysin function in a group of clonal pneumococci that have a chromosomal deletion resulting in a pneumolysin-autolysin fusion gene Δ(lytA'-ply')593. The Δ(lytA'-ply')593 pneumococci strains naturally occur in horses and infection is associated with mild clinical signs. Here we use immortalized and primary macrophage in vitro models, which include pattern recognition receptor knock-out cells, and a murine acute pneumonia model to show that a Δ(lytA'-ply')593 strain induces cytokine production by cultured macrophages, however, unlike the serotype-matched ply+lytA+ strain, it induces less tumour necrosis factor α (TNFα) and no interleukin-1ß production. The TNFα induced by the Δ(lytA'-ply')593 strain requires MyD88 but, in contrast to the ply+lytA+ strain, is not reduced in cells lacking TLR2, 4 or 9. In comparison to the ply+lytA+ strain in a mouse model of acute pneumonia, infection with the Δ(lytA'-ply')593 strain resulted in less severe lung pathology, comparable levels of interleukin-1α, but minimal release of other pro-inflammatory cytokines, including interferon-γ, interleukin-6 and TNFα. These results suggest a mechanism by which a naturally occurring Δ(lytA'-ply')593 mutant strain of S. pneumoniae that resides in a non-human host has reduced inflammatory and invasive capacity compared to a human S. pneumoniae strain. These data probably explain the relatively mild clinical disease in response to S. pneumoniae infection seen in horses in comparison to humans.

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

Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, in situ 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. This is achieved using a simple convolutional neural network trained to predict the probability that reflected light pixels belong to either nucleus, cytoskeleton, or background classifications. We demonstrate the approach across diverse lymphoid tissues and provide video tutorials demonstrating deployment in Python and MATLAB or via standalone software for Windows.

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. We find that this biphasic post-trigger JNK signaling initially facilitates the mitochondrial reactive oxygen species generation needed to support core inflammasome formation, then supports the gasdermin-mediated cell permeation required for release of active IL-1ß from human macrophages. We further identify and characterize a xanthine oxidase-ROS activated MAP3K5/JNK2 substrate licensing complex as a novel regulator of the GSDMD mobilization which precedes pyroptosis. We show that inhibitors targeting this MAP3K5 cascade alleviate morbidity in mouse models of colitis and dampen both augmented IL-1ß release and cell permeation in monocytes derived from patients with gain-of-function inflammasomopathies.

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. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.

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. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-ß (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 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. Here, a 3D in vitro model of the vertebrate intestinal epithelium, interfaced with immune cells surviving in culture for over 3 weeks, is developed and applied to proof-of-concept studies of host-microbe interactions. More specifically, the establishment of stable host-microbe cocultures is described and functional and morphological changes in the intestinal barrier induced by the presence of commensal bacteria are shown. Finally, evidence is provided that the 3D vertebrate gut models can be used as platforms to test host-microbe-parasite interactions. Exposure of gut-immune-bacteria cocultures to helminth "excretory/secretory products" induces in vivo-like up-/down-regulation of certain cytokines. These findings support the robustness of the modular in vitro cell systems for investigating the dynamics of host-microbe crosstalk and pave the way toward new approaches for systems biology studies of pathogens that cannot be maintained in vitro, including parasitic helminths.

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 3ß 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. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

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. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

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.