Abstract Background Monogenic autoinflammatory disorders arise from genetic defects that pathologically activate innate immunity. IRAK4, a serine/threonine kinase in the Myddosome pathway, mediates IL-1 and Toll-like receptor signaling, driving proinflammatory cytokine and type I interferon responses. While biallelic loss-of-function IRAK4 variants cause an immunodeficiency, recent reports implicate biallelic IRAK4 variants in severe neuro- and systemic autoinflammation (NASA). We investigated a child with a similar phenotype and screened unsolved autoinflammatory leukoencephalopathies in the Myelin Disorders Biorepository Project (MDBP). Methods Individuals with unexplained autoinflammatory leukoencephalopathy and no unifying molecular diagnosis were identified in the Myelin Disorders Biorepository Project (MDBP), and genome sequencing was reanalyzed to prioritize rare, protein-altering and splice-affecting variants. Candidate variants and their splicing consequences were interrogated with short-read and targeted long-read RNA sequencing, benchmarked against control PBMC and normal-tissue transcriptomes. Nonsense mediated decay of transcripts was also assessed. Clinical, genetic, and treatment data were extracted by standardized deep phenotyping, and brain MRI was reviewed in consensus by two pediatric neuroradiologists. Results We identified six patients from five unrelated families with biallelic, rare IRAK4 variants presenting with severe, persistent autoinflammation without immunodeficiency. Variants included two homozygous and three compound heterozygous changes. All patients had a concordant clinical and radiologic syndrome: episodic, waxing–waning encephalopathy with refractory seizures; neuroimaging showed transient white matter edema that evolved to gliosis, superimposed on marked calcifications and ensuing cerebral atrophy. Biomarkers indicated neuroinflammation and anemia in all cases. Median age at neurologic symptom onset was 12.96 years (IQR 9.44). Immune-suppressive therapies achieved partial benefit, but most patients had ongoing seizures, persistent neuroinflammation, and progressive disease, and without treatment, loss of life. Conclusion In these six patients, a strongly concordant clinical and radiological phenotype emerges of IRAK4 - mediated autoinflammation, expanding the phenotypic and mutational spectrum of IRAK4 -related disease. Further studies are needed to define mechanisms and optimal treatments.

Regulated cell death (RCD) plays a central role in host defenses against infection. It is also an important driver of pathology in sepsis. The proteins that regulate RCD in mice and, to a lesser degree, in humans are increasingly well understood. In other species, how RCD, especially pyroptosis, occurs in response to infection is less clear. In this review, we outline what is currently known about genes involved in RCD with a focus on pyroptosis across different species and consider the potential consequences that the genetic diversity might have on the host response to infection.

Toll-like receptors (TLRs) drive innate immunity via assembly of macromolecular signal transduction platforms [supramolecular organizing centers (SMOCs)] coordinated by adaptor proteins such as Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing interferon-β (TRIF), but whether oligomeric TRIFosomes form is unknown. Here, using cryo-electron microscopy and biophysical characterization of full-length TRIF in vitro, we show that it forms filamentous oligomers, which associate with the TRIF signaling partners receptor interacting protein 1 (RIP1) and RIP3 kinases, suggesting that oligomeric TRIFosomes could form. Endogenous TRIF, however, is predominantly monomeric in the absence of ligand, only forming TRIFosome oligomers in macrophages after stimulation of TLR4 or TLR3 when large, macromolecular signaling complexes form. TRIFosomes are fully formed 45 min after TLR3 or 60 min after TLR4 stimulation, commensurate with activation of nuclear factor κB in these cells. TLR3/4 activation triggers rapid interferon signaling prior to TRIFosome formation through monomeric TRIF, unexpectedly suggesting that a macromolecular platform of TRIF is not required to drive this signaling pathway. Collectively, these data show TRIFosome macromolecular platform formation and, unexpectedly, that TLR signaling can be SMOC-independent in addition to being SMOC-dependent.

The gut microbiome modulates immunotherapy treatment responses, and this may explain why immune checkpoint inhibitors, such as anti-PD-1, are only effective in some patients. Previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis; however, LPS from diverse bacterial species can range from immunostimulatory to inhibitory. Here, by functionally analysing faecal metagenomes from 112 patients with melanoma, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in microbiomes of clinical responders. In an implanted tumour mouse model of anti-PD-1 treatment, microbiota-derived hexa-acylated LPS was required for effective anti-tumour immune responses, and LPS-binding antibiotics and a small-molecule TLR4 antagonist abolished anti-PD-1 efficacy. Conversely, oral administration of hexa-acylated LPS to mice significantly augmented anti-PD-1-mediated anti-tumour immunity. Penta-acylated LPS did not improve anti-PD-1 efficacy in vivo and inhibited hexa-acylated LPS-induced immune activation in vitro. Microbiome hexa-acylated LPS therefore represents an accessible predictor and potential enhancer of immunotherapy responses.

Pattern Recognition Receptors (PRRs, [118]) (nomenclature as agreed by NC-IUPHAR sub-committee on Pattern Recognition Receptors, [21]) participate in the innate immune response to microbial agents, the stimulation of which leads to activation of intracellular enzymes and regulation of gene transcription. PRRs express multiple leucine-rich regions to bind a range of microbially-derived ligands, termed PAMPs or pathogen-associated molecular patterns or endogenous ligands, termed DAMPS or damage-associated molecular patterns. These include peptides, carbohydrates, peptidoglycans, lipoproteins, lipopolysaccharides, and nucleic acids. PRRs include both cell-surface and intracellular proteins. PRRs may be divided into signalling-associated members, identified here, and endocytic members, the function of which appears to be to recognise particular microbial motifs for subsequent cell attachment, internalisation and destruction. Some are involved in inflammasome formation, and modulation of IL-1β cleavage and secretion, and others in the initiation of the type I interferon response. PRRs included in the Guide To PHARMACOLOGY are:Catalytic PRRs (see links below this overview)Toll-like receptors (TLRs)Nucleotide-binding oligomerization domain, leucine-rich repeat containing receptors (NLRs, also known as NOD (Nucleotide oligomerisation domain)-like receptors)RIG-I-like receptors (RLRs)Caspase 4 and caspase 5 Non-catalytic PRRsAbsent in melanoma (AIM)-like receptors (ALRs)C-type lectin-like receptors (CLRs)Other pattern recognition receptorsAdvanced glycosylation end-product specific receptor (RAGE)

RNA editing is a posttranscriptional mechanism that targets changes in RNA transcripts to modulate innate immune responses. We report the role of astrocyte-specific, ADAR1-mediated RNA editing in neuroinflammation in Parkinson's disease (PD). We generated human induced pluripotent stem cell-derived astrocytes, neurons and cocultures and exposed them to small soluble alpha-synuclein aggregates. Oligomeric alpha-synuclein triggered an inflammatory glial state associated with Toll-like receptor activation, viral responses, and cytokine secretion. This reactive state resulted in loss of neurosupportive functions and the induction of neuronal toxicity. Notably, interferon response pathways were activated leading to up-regulation and isoform switching of the RNA deaminase enzyme, ADAR1. ADAR1 mediates A-to-I RNA editing, and increases in RNA editing were observed in inflammatory pathways in cells, as well as in postmortem human PD brain. Aberrant, or dysregulated, ADAR1 responses and RNA editing may lead to sustained inflammatory reactive states in astrocytes triggered by alpha-synuclein aggregation, and this may drive the neuroinflammatory cascade in Parkinson's.

Impaired mitochondrial bioenergetics in macrophages can drive hyperinflammatory cytokine responses, but whether this may also be caused by inherited mtDNA mutations is unknown. Here, we address this question using a multi-omic approach that integrates super-resolution imaging and metabolic analyses to profile macrophages from a mouse model of mitochondrial disease arising from a heteroplasmic mutation (m.5019A>G) in the mitochondrial tRNA for alanine. These m.5019A>G macrophages exhibit defects in respiratory chain complexes and oxidative phosphorylation (OxPhos) due to decreased intra-mitochondrial translation. To adapt to this metabolic stress, mitochondrial fusion, reductive glutamine metabolism, and aerobic glycolysis are all increased. Upon inflammatory activation, type I interferon (IFN-I) release is enhanced, while the production of pro-inflammatory cytokines and oxylipins are restrained in m.5019A>G macrophages. Finally, an in vivo endotoxemia model using m.5019A>G mice reveal elevated IFN-I levels and sickness behaviour. In conclusion, our study identifies an unexpected imbalance in innate immune signalling in response to a pathogenic mtDNA mutation, with important implications for the progression of pathology in patients with mtDNA diseases. ### Competing Interest Statement The authors have declared no competing interest.

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well-structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point-in-time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70233. The Concise Guides provide expert-curated recommendations of 'Gold Standard' selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid-2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier 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), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. 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. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

Acute respiratory distress syndrome (ARDS) is present in >10% of all people admitted to critical care and is associated with severe morbidity and mortality. Despite more than half a century since its first description, no efficacious pharmacological therapies have been developed, and little progress has been made in improving clinical outcomes. Neutrophils are the principal drivers of ARDS, with their priming and subsequent aberrant downstream functions, including interleukin (IL) 1β and IL-18 secretion, central to the disease pathogenesis. The dominant pathways through which IL-1β and IL-18 are believed to be elaborated are multimeric protein structures called inflammasomes that consist of sensor proteins, adaptor proteins and an effector enzyme. The inflammasome’s initial activation depends on one of a variety of damage-associated (DAMP) or pathogen-associated (PAMP) molecular patterns. However, once activated, a common downstream inflammatory pathway is initiated regardless of the specific DAMP or PAMP involved. Several inflammasomes exist in humans. The nucleotide-binding domain leucine-rich repeat (NLR) family, pyrin domain-containing 3 (NLRP3), inflammasome is the best described in the context of ARDS and is known to be activated in both infective and sterile cases. The NLR family, caspase activation and recruitment domain-containing 4 (NLRC4) and absent in melanoma 2 (AIM2) inflammasomes have also been implicated in various ARDS settings, as have inflammasome-independent pathways. Further work is required to understand human biology as much of our knowledge is extrapolated from rodent experimental models. Experimental lung injury models have demonstrated beneficial responses to inflammasome, IL-1β and IL-18 blockade. However, findings have yet to be successfully translated into humans with ARDS, likely due to an underappreciation of the central role of the neutrophil inflammasome. A thorough understanding of inflammasome pathways is vital for critical care clinicians and researchers and for the development of beneficial therapies. In this review, we describe the central role of the inflammasome in the development of ARDS and its potential for immunomodulation, highlighting key areas for future research.

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. 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 Vβ21.3+ 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 Vβ21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Highly pathogenic avian influenza virus (HPAIV) presents a global threat to chicken livestock; chickens infected by HPAIV tend to show severe symptoms and high mortality rates. In 2022, the largest recorded outbreak of HPAIV in Europe resulted in millions of chickens being culled in the UK alone to try to prevent further spread. Unlike chickens, mallard ducks show reduced symptom severity and lower mortality rates to HPAIV infection. Research into the immune system responses of these two species shows they differ in their molecular outputs: chickens produce a pro-inflammatory response; mallards produce an anti-viral response. These differences in immune responses are thought to be in part due to chickens missing pattern recognition receptor retinoic acid-inducible gene-I (RIG-I). This project aimed to model the innate immune systems of chickens and mallard ducks to an abstracted molecular level. A literature search was conducted, and the immune systems were modelled in NetLogo as an avian innate immune response agent-based model (AIIRABM). The AIIRABM enabled examination of the relative importance of molecular differences between the chicken and mallard duck innate immune systems and produced similar differences in chicken and mallard duck molecular outputs to those observed in vitro and in vivo. Simulation experiments with the AIIRABM supported the molecular difference RIG-I as key in causing the differences in the chicken and mallard duck innate immune responses to HPAIV. The AIIRABM will be used in further research on the chicken and mallard duck immune responses to HPAIV as the baseline in an iterative modelling cycle. ### Competing Interest Statement The authors have declared no competing interest.

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. 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 Aβ 1–42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aβ 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 Aβ 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 Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

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. Recent research suggests that at least some of these assumptions should be reconsidered, especially as many of these receptors are important therapeutic targets for drug development, so understanding the mechanisms by which they signal is critical.

Summary: 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. Inhibition of cyclooxygenase by nonsteroidal anti-inflammatory drugs decreases eicosanoid, but not AA, production. It also reduces both IL-1β 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.

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. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets (e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.

AbstractImmunotherapeutic 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. The number of ASC specks increased and showed altered morphology in the blood of early-stage Parkinson’s and Alzheimer’s patients compared to controls, mimicking those found in the brain and cerebrospinal fluid. In serum samples we also measured the number of Aβ, p-tau and α-syn aggregates and formed a composite biomarker of (ASC + p-tau)/Aβ and (ASC + α-syn)/Aβ ratios that distinguished age-matched healthy controls from patients with early-stage Alzheimer’s with AUC of 92% and early-stage Parkinson’s with AUC of 97%. Our findings confirm ASC specks as a fluid candidate biomarker of inflammation for neurodegenerative diseases with blood being the main focus for further development as convenient sample for diagnostics and clinical trials.

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment, but they are only effective for a minority of patients. The gut microbiome plays a crucial role in modulating immunotherapy treatment responses, and previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis. However, LPS from diverse bacterial species have activities ranging from immunostimulatory to inhibitory. By functionally analyzing fecal metagenomes from 112 melanoma patients prior to anti-PD-1 therapy, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in the microbiomes of clinical responders. We confirmed robust activation of the NF-kB pathway by hexa-acylated LPS in vitro , and this activation was significantly inhibited by penta-acylated LPS in a dose-dependent manner. Microbiome hexa-acylated LPS may therefore represent an accessible predictor and potential enhancer of clinical anti-PD-1 immunotherapy responses. Statement of significance Functional rather than taxonomic profiling of patient gut microbiomes reveals hexa-acylated LPS as a novel biomarker of responsiveness and a targetable pathway for enhancing responses to anti-PD-1, informing future studies and current patient treatment. ### Competing Interest Statement The authors have declared no competing interest.

Changes in gene expression during bacterial infection are the combined result of altered transcription and translation, with the latter comparatively understudied. Gram-negative bacteria rapidly trigger cytokine gene transcription in macrophages through the activation of pathogen associated molecular pattern receptors, for example detection of Salmonella lipopolysaccharide (LPS) from the bacterial cell envelope by Toll-like receptor 4 (TLR4). Here, through time-resolved parallel translatomic and transcriptomic profiling, we now show temporal TLR4-specific translational upregulation of cell signalling proteins in macrophages induced by Salmonella . While transcriptional upregulation of these genes is dampened through the activity of the Salmonella SPI-1 type three secretion system, a robust translational response remains. These data reveal an important host-pathogen translational regulatory network that modifies the innate immune response of macrophages to infection.### Competing Interest StatementThe authors have declared no competing interest.

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 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. 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 Aβ 1-42 fibrils (80 seconds vs 372 seconds). The mean lifetimes of the Myddosomes was also shorter when triggered by LPS compared to sonicated Aβ 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 suggests that compared to LPS the multivalency of Aβ 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 Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.