Biology subjects

Allan, S. M.

Publications and source records attributed to Allan, S. M..

18 recordsLinked to original sources

bioRxiv2026

HTRA1 deficiency in COL4A1 mutant hiPSC-derived astrocytes, a convergent mechanism of cerebral small vessel disease

Cerebral small vessel disease (cSVD) is a major contributor to stroke and cognitive decline, ultimately leading to vascular dementia (VaD). Genetic factors play a key role in disease susceptibility and progression, and variants in COL4A1 cause one of the most common forms of genetic cSVD. COL4A1 encodes the 1 chain of collagen type IV which is the major structural component of the basement membrane, a specialised extracellular matrix (ECM) structure, in the vasculature. In addition to this vascular basement membrane (vBM), in the central nervous system (CNS), the neurovascular unit (NVU) also has a unique parenchymal basement membrane (pBM) that is largely produced by astrocytes and forms a critical interface anchoring astrocyte end-feet to vascular cells. Together, these BMs play essential roles in regulating blood-brain barrier (BBB) function. However, the role of the pBM in cSVD has been relatively less investigated compared to the vBM. The lack of relevant human disease models that faithfully recapitulate the pBM, makes it difficult to dissect pBM-related cell-cell and cell-matrix interactions specific to cSVD, hindering the identification of effective therapeutic targets. In this study, we hypothesised that astrocyte-mediated ECM remodelling contributes to BBB dysfunction in COL4A1-associated cSVD. To investigate this, human induced pluripotent stem cells (iPSCs) derived from a patient carrying the COL4A1G755Rvariant and its isogenic control line were differentiated into astrocytes and brain microvascular endothelial cells (BMECs). Comparing to isogenic controls, the COL4A1G755Rastrocytes significantly reduced the expression of ECM-related genes and increased glutamate uptake. ECM preparations from COL4A1G755R astrocytes significantly damaged the tight junction (TJ) structure formed by control iPSC-BMECs and failed to rescue the TJ integrity in COL4A1G755R BMECs. The secretome from COL4A1G755Rastrocytes exacerbated the ECM defects in COL4A1G755R BMECs. Most importantly, COL4A1G755R astrocytes exhibited reduced expression of HTRA1, a serine protease that regulates both ECM turnover and homeostasis, and increased TGF-{beta} signalling. Functional rescue by recombinant human HTRA1 protein rescues TJ defects in COL4A1G755R BMECs, and normalized TGF-{beta} signalling and glutamate uptake in COL4A1G755R astrocytes. Together, these findings define a previously unrecognised astrocyte-driven pBM mechanism in COL4A1-associated cSVD and highlight HTRA1 in ECM remodelling as a therapeutic target for cSVD.

neuroscience↗

bioRxiv2026

Blood-brain barrier dysfunction predicts cognitive trajectory after ischemic stroke

Ischemic stroke doubles the risk of dementia.1-4 Stroke severity and location affect cognition early,5,6 but late dementia risk is not related to infarct characteristics, nor is it reduced by preventing additional strokes,3,6,7 and its mechanism is unknown. We identified a plasma proteomic signature of chronic stroke that was consistent with blood-brain barrier (BBB) dysfunction, including a 58% decrease in plasma levels of platelet-derived growth factor B and downregulation of its pathway compared to healthy controls. During 2 years of follow-up, the stroke-specific proteome was accentuated in stroke survivors who subsequently declined in the processing speed/executive function cognitive domain. To test BBB function, we performed dynamic contrast-enhanced MRI 6-9 months after stroke in an additional cohort and found 1.7-fold higher whole brain BBB leakage compared to controls. Finally, we compared autopsy tissue from people with infarcts and dementia at death to those with infarcts and no dementia. Those who died with dementia had dramatic loss of vascular mural cell coverage compared to those without dementia (median 0.7% vs. 27%). Thus, our proteomic, functional, and structural data implicate chronic BBB dysfunction in cognitive decline late after stroke, revealing potential proteomic and imaging biomarkers and, importantly, a novel target for intervention.

neuroscience↗

bioRxiv2026

ABERRANT HIPPOCAMPAL NEUROGENESIS IS A CONSERVED RESPONSE TO STROKE IN MICE: A MULTI-CENTER MULTIMODEL STUDY

BackgroundAdult hippocampal neurogenesis is markedly altered after cerebral ischemia. Although stroke increases the production of newborn neurons, many of these cells display aberrant morphological and positional features that impair their functional integration and contribute to long-term cognitive decline. Given the clinical heterogeneity of ischemic stroke and the persistent translational failures of preclinical approaches relying on single-model studies it remains unknown whether post-stroke neurogenic alterations are conserved across different experimental paradigms. This study aimed to define common and model-specific features of hippocampal neurogenesis across complementary focal ischemia models. MethodsWe performed a multi-center, multimodel analysis within the STROKE-IMPaCT consortium using permanent and transient middle cerebral artery occlusion (MCAO) paradigms (MCAO via ligation or cauterization under normoxic (dMCAO) or hypoxic conditions (dMCAO+Hypoxia); and filament-based tMCAO across six international sites. Brains from adult C57BL/6J mice were collected 3 days, 7 days, or 2 months after ischemia, sham, or naive conditions. Hippocampal cell proliferation (Ki67) and neuroblast density (DCX) were quantified, and the morphological maturation of newborn neurons was evaluated using high-resolution analyses of dendritic architecture and somatodendritic polarity. All analyses were performed blind to experimental group. ResultsAcross all stroke models, ischemia induced a robust bilateral increase in hippocampal cell proliferation, most pronounced at 3 days and still elevated at 7 days, with levels returning to baseline by 2 months. Neuroblast density was similarly increased at 7 days, particularly in the ipsilateral hippocampus, but normalized by 2 months. Despite recovery in cell number, long-term morphological analysis revealed a consistent reduction in apical dendrite length and a higher proportion of neurons exhibiting aberrant features including ectopic localization, multipolar or inverted polarity, and abnormal lateral growth across all models. These abnormalities were observed both when pooling data across sites and when analyzing each model or center individually. ConclusionsIschemia induces an early, transient increase in hippocampal neurogenesis across diverse stroke paradigms, but the newborn neurons generated after stroke consistently display maladaptive morphological features. These cross-model, cross-site abnormalities indicate that aberrant hippocampal neurogenesis represents a robust hallmark of post-stroke pathology within the investigated species, independent of ischemia type or surgical approach, despite known differences in the spatial distribution of primary injury across experimental stroke models. Our findings support the concept that maladaptive neurogenesis may contribute to chronic post-stroke cognitive impairment and underscore the need to consider the quality not only the quantity of newborn neurons when developing therapeutic strategies.

animal behavior and cognition↗

bioRxiv2025

Specific deletion of interleukin-1 beta in microglia improves acute outcome and modulates neurogenesis after ischemic stroke

Interleukin-1 (IL-1) signaling is a major driver of post-ischemic neuroinflammation, yet the cell- and isoform-specific roles of the two major IL-1 receptor type 1 agonists, IL-1 and IL-1{beta}, remain incompletely defined in the context of stroke. Microglia rapidly express IL-1 after cerebral ischemia, whereas IL-1{beta} expression is delayed and restricted to a small subset of microglia and infiltrating immune cells. Here, we investigated for the first time the specific contribution of microglial-derived IL-1{beta} to acute injury and post-stroke neurorepair after transient middle cerebral artery occlusion in male and female mice, through microglial-specific tamoxifen-inducible Cre-loxP-mediated recombination. Deletion of microglial IL-1{beta} improved acute neurological outcome, reduced neutrophil accumulation in the ischemic brain and dampened systemic inflammatory cytokines. These effects were most evident during the acute phase and in female in mice. In contrast, long-term functional recovery was largely unaffected. However, microglial IL-1{beta} deletion differentially regulated post-stroke neurogenesis, enhancing subventricular zone neurogenic responses and ectopic neuroblast migration while limiting hippocampal neurogenesis. Together, these findings identify microglial IL-1{beta} as a key amplifier of early inflammatory injury after stroke, exerting region-specific effects on neurogenic niches, and highlight distinct, non-redundant roles for microglial IL-1 isoforms in ischemic brain injury and repair.

neuroscience↗

bioRxiv2025

Assessing post-stroke cognition in pre-clinical models: lessons and recommendations from a multi-center study

Cognitive decline is a significant long-term consequence of stroke and has no available treatments. To aid in therapy development, we sought to achieve robust detection of cognitive performance after stroke in a multi-site design. Ischemic stroke was induced in adult and middle-aged male C57BL/6J mice utilizing three well-established models: distal middle cerebral artery occlusion (dMCAO), dMCAO with hypoxia and transient MCAO. Cognitive outcomes were assessed via Novel Object Recognition (NOR) and Barnes Maze (BM) tests prior to surgery, and during sub-acute (1-2 weeks) and chronic (8 weeks) phases post-stroke. Histology and immunostaining were used to assess infarct size, tissue damage and neuronal loss, and plasma neurofilament light was quantified. We did not detect a reliable cognitive deficit after stroke using NOR but saw a promising signal from BM (single site tested only). Overall, our study highlights the often-encountered challenges in detecting post-stroke cognitive impairment within the pre-clinical stroke community, as well as a number of complexities in the design and execution of pre-clinical stroke cognition studies, particularly as applied to a multi-site structure. We provide recommendations and suggest important aspects of stroke cognition studies to consider in the future, whether operating as an individual lab or a multi-site group.

animal behavior and cognition↗

bioRxiv2025

Graphene Micro-transistor Arrays Reveal Perfusion-Dependent Electrophysiological and Haemodynamic Signatures of Cortical Spreading Depolarizations in Ischaemic Stroke

Cortical spreading depolarizations (CSDs) are large-scale disruptions of neuronal homeostasis that contribute to secondary injury in ischaemic stroke. In healthy cortex, CSDs induce vasodilation to meet metabolic demand, whereas in ischaemic tissue, they can provoke vasoconstriction and sustained hypoperfusion, exacerbating damage. We present a multimodal neurotechnology platform integrating flexible, transparent graphene solution-gated field-effect transistor (gSGFET) arrays with laser speckle contrast imaging (LSCI), enabling simultaneous DC-coupled electrophysiological recordings and cerebral blood flow imaging with high spatiotemporal fidelity. gSGFETs enable stable, distortion-free recording of infraslow potentials essential for resolving CSD waveforms in vivo. In two murine stroke models, we show that CSD waveform duration and morphology scale with local perfusion, delineating electrophysiological subtypes predictive of tissue viability and haemodynamic response. In metabolically compromised cortex, CSDs exhibit double peaks or negative ultraslow components that result in vasoconstriction, contrasting with narrow, monophasic waveforms eliciting vasodilation in healthy regions. Systemic ketamine shortens CSD duration and converts vasoconstriction to vasodilation in perfusion-deficit tissue, revealing a mechanism for its neuroprotective action. This platform enables high-fidelity investigation of brain-blood flow interactions in vivo.

neuroscience↗

bioRxiv2025

Prominent white matter related abnormalities associated with diverse molecular alterations in a mouse model of Col4a1 cerebral small vessel disease.

White matter abnormalities are a hallmark of cerebral small vessel disease (cSVD) and are closely linked to cognitive decline and dementia. However, despite their clinical importance, underlying mechanisms remain poorly understood. Collagen IV, encoded by genes COL4A1/COL4A2, is a major component of the basement membrane, a specialised extracellular matrix (ECM) structure. Mutations in these genes cause a genetic form of cSVD. We tested the hypothesis that ECM defects caused by a Col4a1 mutation lead to white matter pathology using an established mouse model of cSVD (Col4a1+/Svc). Behavioural testing with magnetic resonance diffusion tensor imaging, pathology and ultrastructural investigations of white matter were studied. The studies revealed that Col4a1+/Svc mice have cognitive impairments, reduced myelinating oligodendrocyte pools, axonal myelination defects, and altered white matter structural integrity. Proteomic analysis, of isolated white matter from Col4a1+/Svcmice, identified extensive changes to ECM composition and endoplasmic reticulum (ER) biology including ER stress induction. We also demonstrated that targeting protein folding to promote collagen secretion and reduce ER stress, increased myelinating oligodendrocytes and axon-glial integrity in Col4a1+/Svc mice. These data provide novel insight into the pathomolecular mechanisms of white matter abnormalities in cSVD and identify a modifiable pathway as a putative therapeutic target for cSVD.

neuroscience↗

bioRxiv2024

Cell volume tunes macrophage innate inflammatory responses through promoting type I interferon signalling.

Macrophages are key effectors in co-ordinating inflammatory and immune responses to threats to the host. How macrophages decipher diverse danger signals to tailor inflammatory responses remains an unanswered question. Cell volume control is critical for normal cellular function. Disturbances in extracellular and intracellular homeostasis induce changes in cell volume, but the impact of disruptions in cell volume in controlling macrophage inflammatory responses is poorly understood. Here, we discover that macrophages use cell volume control as a bona fide danger sensing mechanism to promote and augment inflammatory responses. Using macrophages deficient in the volume regulated anion channel (VRAC), which lack cell volume control under hypo-osmotic conditions, we show that disruptions in cell volume are sensed by macrophages to drive a large transcriptomic response and induction of inflammation. Cell volume disruption, particularly loss of cell volume control, induces type I interferon signalling through a DNA- and STING-dependent mechanism, but independent of cGAS and 23cGAMP transport. Further, we found that cell volume changes synergise with diverse pathogen-mediated signalling to augment type I interferon responses and exacerbate the cytokine storm in a mouse model of hyperinflammation. Our findings highlight cell volume as an important regulator in shaping inflammatory responses, adding to our understanding of how macrophages sense complex danger signals and threats.

immunology↗

bioRxiv2024

Cerebral ischaemic stroke results in altered mucosal IgA responses and host-commensal microbiota interactions.

Stroke is a devastating neurological event with a high risk of mortality, but also results in long-term sequalae in survivors that extend beyond the central nervous system. Notably, these include gastrointestinal dysfunction and alterations in the commensal microbiota in both patients and mouse models, which have been suggested to contribute to secondary infection and poor outcome following stroke. Strikingly changes in commensal microbial community composition occur rapidly in both humans and animal models following stroke and correlate with disease severity. Despite these observations the underpinning mechanisms that drive alterations in the microbiota post-stroke remain poorly understood. The gastrointestinal tract is home to a complex network of tissue-resident immune cells that act constitutively to maintain microbial community and prevent bacterial-driven inflammation. Here we demonstrate that mice subjected to ischaemic stroke exhibit alterations in the intestinal immune system, most notably in antibody secreting B cells and the production of Immunoglobulin A (IgA) - a major effector response against commensal microbes. Mice lacking secretory IgA binding to commensal bacteria exhibit a partial reversion of stroke-induced changes in microbiota composition. Notably we also report increases in B cell and IgA-producing plasma cell frequencies in the brain and meninges following stroke. Together these findings demonstrate stroke is associated with perturbations in antibody producing immune responses both in mucosal tissues and the CNS following stroke, which in part explain stroke-induced changes in the intestinal microbiota. A mechanistic understanding of the immunological basis of stroke-associated pathologies in the periphery may open new avenues to manage the secondary complications and long-term prognosis of patients suffering from neurological disease.

immunology↗

bioRxiv2024

Cerebrovascular Health Mediates Processing Speed Change Through Anterior White Matter Alterations: A UK Biobank Study

Cerebrovascular disease is associated with an increased likelihood of developing dementia. While cardiovascular risk factors are modifiable and may reduce the risk of later-life cognitive dysfunction, the relationship between cerebrovascular risk factors, brain integrity and cognition remains poorly characterised. Using a large UK Biobank sample of predominantly middle-aged adults, without neurological disease, our structural equation mediation models showed that poor cerebrovascular health, indicated by the presence of cerebrovascular risk factors, was associated with slowed processing speed. This effect was best explained by anterior white matter microstructure changes, rather than posterior changes. Effects were also significantly reduced when considering other forms of cognition, demonstrating both regional- and cognitive-specificity of our effects. Critically, our findings also demonstrate that including measures of risk factor duration may be particularly important for improving estimations of cerebrovascular burden. In summary, our study demonstrates the specific impact of early cerebrovascular burden on brain structure and cognitive function, highlighting the necessary next steps for improving cerebrovascular burden quantification and improving clinical predictions.

neuroscience↗

bioRxiv2024

The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-{gamma}. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.

immunology↗

bioRxiv2024

Selective deletion of interleukin-1 alpha in microglia regulates neuronal activity and neurorepair processes after experimental ischemic stroke.

Inflammation is a key contributor to stroke pathogenesis and exacerbates brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. However, the distinct roles of the two major IL-1 receptor type 1 agonists, IL-1 and IL-1{beta}, and the specific role of IL-1 in ischemic stroke remain largely unknown. Here we show that IL-1 and IL-1{beta} have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1 expression (4 h) and delayed IL-1{beta} expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined for the first time the specific role of microglial-derived IL-1 in experimental permanent and transient ischemic stroke through microglial-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1 deletion did not influence acute brain damage, cerebral blood flow, IL-1{beta} expression, neutrophil infiltration, microglial nor endothelial activation after ischemic stroke. However, microglial IL-1 knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside long-term impaired functional recovery. Our study identifies for the first time a critical role for microglial IL-1 on neurorepair and functional recovery after stroke, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop more effective therapies.

neuroscience↗

bioRxiv2023

A novel human iPSC model of COL4A1/A2 small vessel disease unveils a key pathogenic role of matrix metalloproteinases in extracellular matrix abnormalities

Abstract/SummaryCerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodelling and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cells tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMP) and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD. HighlightsO_LIA novel human iPSC-derived model of genetic SVD due to collagen IV (COL4A1/A2) mutations is described C_LIO_LIMural cells expressing COL4A1/A2 mutations have prominent ECM abnormalities as seen in patients and mouse models and contribute to endothelial cells defects C_LIO_LIECM and endothelial cells abnormalities can be rescued by MMP inhibition in the COL4A1/A2 model C_LI

cell biology↗

bioRxiv2022

Targeting neuronal homeostasis to prevent seizures

Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.

neuroscience↗

bioRxiv2021

Treatment with IgM-enriched intravenous immunoglobulins (IgM-IVIg) enhances clearance of stroke-associated bacterial lung infection

Post-stroke infection is a common complication of stroke that is associated with increased mortality and morbidity. We previously found that experimental stroke induces an ablation of multiple sub-populations of B cells and reduced levels of IgM antibody that coincide with the development of spontaneous bacterial pneumonia. Reduced circulating IgM concentrations were also observed in acute stroke patients. The loss of IgM antibody after stroke could be an important determinant of infection susceptibility and highlights this pathway as an important target for intervention. We treated mice with a low (replacement), dose of IgM-enriched intravenous immunoglobulin (IgM-IVIg) prior to and 24 h after experimental stroke induced by middle cerebral artery occlusion (MCAO) or sham surgery, then recovered mice for 2 d or 5 d. The effect of treatment on lung bacterial burden, lung pathology, brain infarct volume, antibody levels and both lung and systemic cellular and cytokine immune profiles was determined. Treatment with IgM-IVIg enhanced bacterial clearance from the lung after MCAO and improved pathology but did not impact infarct volume. IgM-IVIg treatment induced immunomodulatory effects systemically including rescue of splenic plasma B cell numbers and endogenous mouse IgM and IgA circulating immunoglobulin concentrations that were reduced by MCAO, and treatment also reduced concentrations of pro-inflammatory cytokines in the lung. The effects of MCAO and IgM-IVIg treatment on the immune system were tissue specific as no impact on B cells or mouse immunoglobulins were found within the lung. However, the presence of human immunoglobulins from the IgM-IVIg treatment led to increased total lung immunoglobulin concentration. IgM-IVIg treatment did not increase the number of lung mononuclear phagocytes or directly modulate macrophage phagocytic capacity but enhanced their capability to phagocytose Staphylococcus aureus bioparticles in vitro by increasing opsonisation. Low dose IgM-IVIg contributes to increased clearance of spontaneous lung bacteria after MCAO likely via increasing availability of antibody in the lung to enhance phagocytic activity. Immunomodulatory effects of IgM-IVIg treatment, including reduced pro-inflammatory cytokine production, may also contribute to reduced levels of damage in the lung after MCAO. IgM-IVIg shows promise as an antibacterial and immunomodulatory agent to use in the treatment of post-stroke infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/472965v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c8684forg.highwire.dtl.DTLVardef@384688org.highwire.dtl.DTLVardef@9eb7f7org.highwire.dtl.DTLVardef@3fe58e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Itaconate and fumarate derivatives exert a dual inhibitory effect on canonical NLRP3 activation in macrophages and microglia

The NLRP3 inflammasome is a multi-protein complex that regulates the protease caspase-1 and subsequent interleukin (IL)-1{beta} release from cells of the innate immune system, or microglia in the brain, in response to infection or injury. Derivatives of the metabolites itaconate and fumarate, dimethyl itaconate (DMI), 4-octyl itaconate (4OI) and dimethyl fumarate (DMF), limit both expression of IL-1{beta}, and IL-1{beta} release following NLRP3 inflammasome activation. However, the direct effects of these metabolite derivatives on NLRP3 inflammasome responses in macrophages and microglia require further investigation. Using murine bone marrow-derived macrophages, mixed glia and organotypic hippocampal slice cultures (OHSCs), we demonstrate that DMI and 4OI pre-treatment limited IL-1{beta}, IL-6 and tumor necrosis factor production in response to lipopolysaccharide (LPS) priming, as well as inhibiting subsequent NLRP3 inflammasome activation. DMI, 4OI, DMF and monomethyl fumarate (MMF), another fumarate derivative, also directly inhibited biochemical markers of NLRP3 activation in LPS-primed macrophages, mixed glia and OHSCs, including ASC speck formation, caspase-1 activation, gasdermin D cleavage and IL-1{beta} release. Finally, DMF, an approved treatment for multiple sclerosis, as well as DMI, 4OI and MMF, inhibited NLRP3 activation in macrophages in response to the phospholipid lysophosphatidylcholine, which is used to induce demyelination, suggesting a possible mechanism of action for DMF in multiple sclerosis through NLRP3 inhibition. Together, these findings reveal the importance of immunometabolic regulation for both the priming and activation steps of NLRP3 activation in macrophages and microglia. Furthermore, we highlight itaconate and fumarate derivatives as a potential therapeutic option in NLRP3-driven diseases, including in the brain. Summary statementWe show that itaconate and fumarate derivatives inhibit both the priming and activation steps of NLRP3 inflammasome responses in macrophages and microglia, revealing the importance of immunometabolic NLRP3 regulation.

immunology↗

bioRxiv2019

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence

AimBlockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. MethodsWe assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the SNS was determined by measuring noradrenaline, a major SNS mediator. ResultsThere were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. ConclusionThese data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations, and is therefore unlikely to further aggravate stroke-associated infection susceptibility through reduced availability of these key anti-microbial mediators.

immunology↗

bioRxiv2019

Selective Liposomal Transport Through Blood Brain Barrier Disruption in Ischaemic Stroke Reveals Two Distinct Therapeutic Opportunities

The development of new therapies for stroke continues to face repeated translational failures. Brain endothelial cells form paracellular and transcellular barriers to many blood-borne therapies and the development of efficient delivery strategies is highly warranted. Here, in a mouse model of stroke, we show selective recruitment of clinically used liposomes into the ischaemic brain that correlates with biphasic blood brain barrier (BBB) breakdown. Intravenous administration of liposomes into mice exposed to transient middle cerebral artery occlusion took place at early (0.5h and 4h) and delayed (24h and 48h) timepoints, covering different phases of BBB disruption after stroke. Using a combination of in vivo real-time imaging and histological analysis we show that selective liposomal brain accumulation coincides with biphasic enhancement in transcellular transport followed by a delayed impairment to the paracellular barrier. This process precedes neurological damage in the acute phase and maintains long-term liposomal co-localisation within the neurovascular unit, which could have great potential for neuroprotection. Levels of liposomal uptake by glial cells are similarly selectively enhanced in the ischaemic region late after experimental stroke (2-3 days), highlighting their potential for blocking delayed inflammatory responses or shifting the polarization of microglia/macrophages towards brain repair.\n\nThese findings demonstrate the capability of liposomes to maximise selective translocation into the brain after stroke and identify for the first time two windows for therapeutic manipulation. This emphasizes the benefits of selective drug delivery for efficient tailoring of new stroke treatments.

neuroscience↗