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Three researchers at the Kennedy Institute have been awarded grants by Arthritis Research UK to continue the fight against various forms of arthritis.
TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation.
NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.
Formation and closure of macropinocytic cups in Dictyostelium.
Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.
Tools to Image Germplasm Dynamics During Early Zebrafish Development
During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.
CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes
Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.
Controlling Anomalous Diffusion in Lipid Membranes.
Diffusion in cell membranes is not just simple two-dimensional Brownian motion but typically depends on the timescale of the observation. The physical origins of this anomalous subdiffusion are unresolved, and model systems capable of quantitative and reproducible control of membrane diffusion have been recognized as a key experimental bottleneck. Here, we control anomalous diffusion using supported lipid bilayers containing lipids derivatized with polyethylene glycol (PEG) headgroups. Bilayers with specific excluded area fractions are formed by control of PEG lipid mole fraction. These bilayers exhibit a switch in diffusive behavior, becoming anomalous as bilayer continuity is disrupted. Using a combination of single-molecule fluorescence and interferometric imaging, we measure the anomalous behavior in this model over four orders of magnitude in time. Diffusion in these bilayers is well described by a power-law dependence of the mean-square displacement with observation time. Anomaleity in this system can be tailored by simply controlling the mole fraction of PEG lipid, producing bilayers with diffusion parameters similar to those observed for anomalous diffusion in biological membranes.
Urea-mediated anomalous diffusion in supported lipid bilayers.
Diffusion in biological membranes is seldom simply Brownian motion; instead, the rate of diffusion is dependent on the time scale of observation and so is often described as anomalous. In order to help better understand this phenomenon, model systems are needed where the anomalous diffusion of the lipid bilayer can be tuned and quantified. We recently demonstrated one such model by controlling the excluded area fraction in supported lipid bilayers (SLBs) through the incorporation of lipids derivatized with polyethylene glycol. Here, we extend this work, using urea to induce anomalous diffusion in SLBs. By tuning incubation time and urea concentration, we produce bilayers that exhibit anomalous behaviour on the same scale as that observed in biological membranes.
Which first? Surgery or biologic therapy for ileocolic Crohn's disease in the real world.
BackgroundA laparoscopic ileocolic resection could lead to a better outcome to infliximab for ileocolic Crohn's disease. The aim of this study was to explore real world clinical outcomes in biologic-naïve patients with ileocolic Crohn's disease.Research design and methodsAll patients with ileocolic Crohn's disease treated at our institution between January 2011 and December 2018 with biologics or surgical resection were included.ResultsOverall, 222 patients were included, of which 149 (67%) underwent surgery before biologic therapy. Among these, 54 patients (36%) required post-operative biologic therapy. Seventy-three patients were treated with biologics first, of which 29 (40%) subsequently required a surgical resection (p = 0.60). There were 95 patients (43%) who were successfully treated with a surgery-first approach alone. Median follow-up was 73 months (0-406). Characteristics associated on multivariable analysis with change from surgery to biologics were: gender (female) (p = 0.010), presence of obstructive symptoms (p = 0.028), and smoking (p = 0.030). Characteristics associated with changing from biologics to surgery were: isolated terminal ileum disease (p = 0.001) and the presence of obstructive symptoms (p = 0.003).ConclusionsIn our cohort, the risk of recurrent ileocolic Crohn's disease was similar whether patients were treated with a 'surgery first' or 'biologic first' approach.
Bowel urgency in ulcerative colitis: effect of baseline urgency and change in urgency in response to mirikizumab.
BACKGROUND: Mirikizumab has demonstrated efficacy in moderately to severely active ulcerative colitis. A 1-2-point change in Urgency Numeric Rating Scale (NRS) score can be meaningful for patients. In these post-hoc analyses, we evaluated the efficacy of mirikizumab compared to placebo by baseline Urgency NRS score groups (0-3, 4-6, and 7-10) and its effect on bowel urgency severity over time. METHODOLOGY: Urgency NRS was measured as a secondary outcome at baseline, week 12, and week 52. Bowel urgency improvement was assessed for patients who achieved and did not achieve multiple efficacy endpoints. Data were analyzed using Fisher's exact test with nonresponder imputation. RESULTS: At weeks 12 and 52, a significantly higher percentage of mirikizumab-treated patients achieved clinical response as well as clinical, endoscopic, and symptomatic remission compared to placebo-treated patients, regardless of baseline Urgency NRS score category (higher proportions versus placebo, delta 9%-45%). Improvement in Urgency NRS score category at weeks 12 and 52 for mirikizumab-treated patients was observed when other efficacy outcomes were achieved (13%-90%) and not achieved (12%-75%). CONCLUSIONS: A greater proportion of mirikizumab-treated patients with ulcerative colitis achieved symptomatic, clinical, and endoscopic remission endpoints compared to placebo-treated patients, regardless of baseline bowel urgency severity. After one year, bowel urgency was improved to a greater extent with mirikizumab than with placebo, even for patients who did not achieve other clinical outcomes. Small improvements in bowel urgency are associated with significant health-related quality-of-life improvements. Monitoring shifts in urgency severity over time using the Urgency NRS can aid in understanding patients' treatment outcomes. TRIAL REGISTRATION: LUCENT-1 (NCT03518086) Registered 04 May 2018 https://clinicaltrials.gov/study/NCT03518086 . LUCENT-2 (NCT03524092) Registered 10 May 2018 https://clinicaltrials.gov/study/NCT03524092 .
Development and validation of peripheral blood DNA methylation signatures to predict response to biological therapy in adults with Crohn's disease (EPIC-CD): an epigenome-wide association study.
BACKGROUND: Biological therapeutics are widely used in Crohn's disease, with evidence of efficacy from randomised trials and real-world experience. Primary non-response is a common, poorly understood problem. We aimed to assess blood methylation as a predictor of response to adalimumab, vedolizumab, or ustekinumab in patients with Crohn's disease. METHODS: This epigenome-wide association study used data from two ongoing biobanks (one from the Amsterdam University Medical Centre, University of Amsterdam, Amsterdam, Netherlands [discovery cohort] and the other from the John Radcliffe Hospital, Oxford, UK [validation cohort]) that recruited patients between Oct 1, 2009, and June 17, 2022. Adult participants (age ≥18 years) with active symptomatic and endoscopic Crohn's disease who were scheduled to start adalimumab, vedolizumab, or ustekinumab treatment were included. Patients with ongoing malignancy or serious concomitant inflammatory diseases were excluded. Treatment response was assessed after a median of 28 weeks of treatment (IQR 18-36). Response was defined as a combination of endoscopic criteria (50% or more reduction in the Simple Endoscopic Score for Crohn's Disease) with either clinical or biochemical criteria (corticosteroid-free clinical response: ≥3 point decrease in Harvey-Bradshaw Index [HBI] score or remission [HBI ≤4] and no systemic steroids at follow up; biochemical response: C-reactive protein reduction ≥50% or ≤5 mg/L and faecal calprotectin reduction ≥50% or ≤250 μg/g) compared with baseline. Epigenome-wide DNA methylation and transcriptome-wide gene expression analyses were done on whole peripheral blood leukocyte samples that were collected before the start of treatment. To identify baseline DNA methylation markers associated with response or non-response to treatment, we performed supervised machine learning through stability selected gradient boosting. In a post-hoc analysis, we compared our DNA methylation-based prediction model with clinical decision support tools (CDSTs). FINDINGS: We profiled the peripheral blood DNA methylome of 273 adults with Crohn's disease scheduled to start adalimumab, vedolizumab, or ustekinumab in the discovery (Amsterdam, n=183; 108 [59·0%] female and 75 [41·0%] male) and the validation cohort (Oxford, n=90; 46 [51·1%] female and 44 [48·9%] male). In the discovery cohort, we defined a panel of DNA methylation biomarkers that were associated with combined endoscopic and clinical or biochemical response to adalimumab (18 markers), vedolizumab (25 markers), or ustekinumab (68 markers), with an area under the curve (AUC) of 0·86 (95% CI 0·58-0·97) for adalimumab, 0·87 (0·67-0·98) for vedolizumab, and 0·89 (0·76-1·00) for ustekinumab. Validation in the Oxford cohort yielded an AUC of 0·25 (0·10-0·35) for adalimumab, 0·75 (0·65-0·85) for vedolizumab, and 0·75 (0·65-0·87) for ustekinumab. In comparison, implementing the CDSTs in the validation cohort yielded an AUC of 0·56 (0·44-0·68) for vedolizumab and 0·66 (0·54-0·77) for ustekinumab. Previous anti-TNF exposure was associated with a reduction in accuracy of the methylation models for vedolizumab (0·66 [0·55-0·73]) and ustekinumab (0·63 [0·52-0·70]) when analysed in the validation cohort. INTERPRETATION: Our findings provide evidence for the potential use of DNA methylation as a modality for personalised medicine for Crohn's disease by predicting response to vedolizumab and ustekinumab. The models were more accurate in biologically naive patients and outperform available vedolizumab and ustekinumab CDSTs. We were unable to predict response to adalimumab. The vedolizumab and ustekinumab prediction models are currently being tested in a multicentre randomised clinical trial. FUNDING: The Leona M and Harry B Helmsley Charitable Trust.
Fecal Calprotectin and C-Reactive Protein Association With Histologic and Endoscopic Endpoints in Mirikizumab-Treated Patients With Ulcerative Colitis
Lay Summary: Associations between fecal calprotectin and C-reactive protein biomarker improvement for patients with moderately to severely active ulcerative colitis treated with mirikizumab were assessed. Results indicate that biomarker analyses may provide a noninvasive monitoring strategy for use in clinical practice.
STAT3 phosphorylation in the rheumatoid arthritis immunological synapse.
Targeting the JAK/STAT pathway has emerged as a key therapeutic strategy for managing Rheumatoid Arthritis (RA). JAK inhibitors suppress cytokine-mediated signaling, including the critical IL-6/STAT3 axis, thereby effectively targeting different aspects of the pathological process. However, despite their clinical efficacy, a subset of RA patients remains refractory to JAK inhibition, underscoring the need for alternative approaches. Here, we identify a novel JAK-independent mechanism of STAT3 activation, which is triggered by the formation of the immunological synapse (IS) in naive CD4+ T cells. Our data demonstrates that LCK mediates the TCR-dependent phosphorylation of STAT3 at the IS, highlighting this pathway as a previously unrecognized hallmark of early T cell activation. Furthermore, we show that the synaptic LCK/TCR-STAT3 pathway is compromised in RA. This discovery highlights a new therapeutic target for RA beyond JAK inhibitors, offering potential avenues for treating patients resistant to current therapies.
TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells.
BACKGROUND: Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1β and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-β pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-β in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs). METHODS: TGF-β mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with normouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-β priming experiments were performed with three inhibitors of TGF-β signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-β receptor II. RESULTS: TGF-β mRNA levels were elevated in gout patients compared to healthy controls. TGF-β-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-β correlated positively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-β but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-β. CONCLUSIONS: TGF-β is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-β signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-β relation.