Macrophages coordinate inflammatory and immune responses to threats, yet how they interpret diverse danger signals to tailor inflammation remains unclear. Disturbances in extracellular and intracellular homeostasis alter cell volume, but the consequences for macrophage inflammatory responses are poorly understood. We demonstrate that macrophages use cell volume control as a danger-sensing mechanism to promote and augment inflammation. Using volume-regulated anion channel (VRAC)-deficient macrophages, which lack cell volume control under hypo-osmotic conditions, we show that cell volume disruptions drive transcriptomic reprogramming and induction of inflammation. Cell volume disruption induced type I interferon signaling through a DNA- and TBK1-dependent mechanism, but independent of cGAS and 2 ' 3 '-cGAMP transport. VRAC deficiency enhanced macrophage antiviral responses to influenza infection. Cell volume changes synergized with diverse pathogen-associated molecular pattern-mediated signaling to augment type I interferon responses and exacerbate the cytokine storm in mouse models of hyperinflammation. Our findings highlight cell volume as an important regulator in shaping inflammatory responses, expanding our understanding of how macrophages sense complex danger signals.

Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse strain in which the endogenous Il1a gene was edited using CRISPR to disrupt the NLS, yielding a mutated NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to lipopolysaccharide (LPS) but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.

Recent advances in Mendelian genomics reveal the importance of variant-level characterization of allelic disorders. Non-muscle actin isoforms, encoded by the genes ACTB and ACTG1, are the most abundant intracellular proteins, but historically, they are often regarded as merely being “housekeeping” molecules. Here, we illuminate the extraordinary clinical heterogeneity and complex pathobiology of genetic non-muscle actinopathies. To do this, we combine human genomics studies with molecular biology. Strikingly, variants in ACTB and ACTG1 isoforms generate at least eight distinct clinical disorders. A subset of disease-associated missense variants causes dysregulated actin polymerization-depolymerization and neuronal migration defects. In contrast, nonsense, frameshift, and missense variants enhancing protein degradation cause milder phenotypes or are benign. These results emphasize the essential functional aspects of the non-muscle actin isoforms. Critically, they additionally constitute a template for the personalized genetic variant-level-driven management of the pleiotropic allelic single-gene disorders.

Abstract The NLRP3 inflammasome is a critical regulator of inflammation, yet the localisation, organisation, and cellular sources of endogenous NLRP3 inflammasomes remain incompletely understood. Here, we generated NLRP3-mScarlet-I endogenous reporter mice enabling visualisation of NLRP3 at physiological levels in primary cells and in vivo . We show that activated NLRP3 associated with PI4P-positive membranes from multiple organelles, supporting a model where diverse membrane platforms act as a scaffold to nucleate inflammasome assembly. Super-resolution imaging revealed that NLRP3 and ASC occupy distinct nanoscale architectures within the inflammasome, with NLRP3 displaying marked structural heterogeneity and stimulus-dependent organisation. Unexpectedly, circulating monocytes and neutrophils, rather than tissue-resident populations, emerged as the dominant NLRP3-expressing cells in vivo which rapidly infiltrated tissues following systemic inflammation, highlighting an underappreciated cellular source of rapid inflammasome-driven responses. These findings reveal previously unrecognised insights into inflammasome organisation and localisation, establishing a powerful resource for investigating endogenous NLRP3 biology in health and disease.

Abstract Intracerebral haemorrhage (ICH) is a severe form of stroke with high morbidity and mortality rates. For survivors, acute haematoma expansion strongly determines neurological outcome. Although blood pressure reduction is widely investigated as a strategy to limit haematoma growth, the haemodynamic mechanisms regulating haemorrhage development remain poorly understood. Zebrafish provide a tractable in vivo model to study cerebrovascular biology and spontaneous ICH, yet the contribution of vascular regulation to haemorrhage onset and expansion has not been explored in this species. Here, we investigated whether pharmacological modulation of vascular dilation influences ICH development in zebrafish larvae. We first characterised vascular changes during the developmental window in which spontaneous ICH occurs and observed increased heart rate and progressive reductions in arterial diameter between 2 and 3 days post-fertilisation, suggesting increased vascular resistance. We then tested whether vasoconstriction promotes haemorrhage using angiotensin II, which induced systemic and cerebrovascular vasoconstriction but did not increase ICH incidence or haematoma size in two independent ICH models. In contrast, pharmacological vasodilation using sodium nitroprusside or isoproterenol significantly reduced haematoma size in a high-incidence model of atorvastatin-induced ICH. Live imaging of cerebral blood flow revealed that vasodilation was associated with the confinement of red blood cells around affected vessels rather than dispersing into the brain ventricles. Together, these findings indicate that vascular dilation modulates haemorrhage progression in zebrafish ICH and establish this model as a platform to investigate haemodynamic mechanisms regulating haematoma expansion.

The antiviral enzyme cholesterol 25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25HC), which modulates cholesterol metabolism during infection, have been associated with vascular pathology. Viral infections have been linked to intracerebral haemorrhage (ICH) risk, but the molecular mechanisms leading to ICH via antiviral responses remain unknown. We hypothesised that the CH25H/25HC pathway impacts neuroendothelial integrity in the context of infection-associated ICH. Using a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein-induced zebrafish ICH model and foetal human SARS-CoV-2-associated cortical tissue containing microbleeds, we identified upregulation of CH25H in infection-associated cerebral haemorrhage. Using zebrafish models and human brain endothelial cells, we asked whether 25HC promotes neurovascular dysfunction by modulating cholesterol metabolism. We found that 25HC and pharmacological inhibition of cholesterol synthesis had an additive effect to exacerbate brain bleeding in zebrafish and in vitro neuroendothelial dysfunction. 25HC-induced dysfunction was also rescued by cholesterol supplementation in vitro. These results demonstrate that 25HC can dysregulate brain endothelial function by remodelling cholesterol metabolism. We propose that CH25H/25HC plays an important role in the pathophysiology of brain vessel dysfunction associated with infection and cholesterol dysregulation in the context of ICH.

Pneumonia is a common infection in people suffering with Alzheimer’s disease, leading to delirium, critical illness or severe neurological decline, which may be due to an amplified response of the blood-brain barrier (BBB) to peripheral insult. We assess the response of the BBB to repeated Streptococcus pneumoniae lung infection in rat model of Alzheimer’s disease (TgF344-AD), at 13- and 18-months old, using dynamic contrast-enhanced (DCE) MRI and filter exchange imaging. Higher BBB water exchange rate is initially detected in infected TgF344-AD rats. BBB water exchange rates correlated with hippocampus aquaporin-4 water channel expression in infected animals. We detected no differences in BBB permeability to gadolinium contrast agent measured by DCE-MRI, confirmed by staining for tight junction proteins, occludin and claudin-5. These findings provide insight into the mechanisms of how peripheral inflammation impacts the BBB.

Stroke is a devastating neurological event with a high risk of mortality that results in long-term sequalae that extend beyond the central nervous system. Notably these include gastrointestinal dysfunction and altered composition of the commensal microbiota in both patients and mouse models, which have been suggested to contribute to secondary infection and poor clinical outcomes following stroke. Strikingly, changes in commensal microbial community composition occur rapidly following stroke and correlate with disease severity. Despite these observations, the underpinning mechanisms that drive perturbation of the microbiota post-stroke remain poorly understood. The gastrointestinal tract is home to a complex network of tissue-resident immune cells that maintain homeostatic interactions with commensal microbes and prevent bacterial-driven inflammation. Here we demonstrate mice subjected to ischaemic stroke exhibit alterations in the intestinal immune system, most notably in class switched germinal centre B cells and the production of Immunoglobulin A (IgA) - a major effector response against commensal microbes. Mice lacking secretory antibodies, including IgA, exhibited a partial reversion of stroke-induced changes in microbiota composition. Together these findings demonstrate stroke is associated with dysregulation of antibody producing immune responses, which may in part explain changes in the intestinal microbiota. A mechanistic understanding of the immunological basis of strokeassociated pathologies in the periphery may open new avenues to manage the secondary complications and long-term prognosis of patients suffering from neurological disease.

Viral infection and hypocholesterolaemia are two independent risk factors for intracerebral haemorrhage (ICH), but the molecular mechanisms leading to vascular rupture via these risk factors remain unknown. We hypothesised that the enzyme cholesterol 25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25HC), which modulates cholesterol metabolism during infection, may offer a mechanistic link between cholesterol dysregulation and infection during neurovascular dysfunction. We identified an upregulation of CH25H in infection-associated cerebral haemorrhage, in a SARS-CoV-2-induced zebrafish ICH model and foetal human SARS-CoV-2-associated cortical microbleeds. Using human brain endothelial cells and zebrafish ICH models, we show that 25HC promotes endothelial dysfunction and exacerbates brain bleeding. These effects involved cholesterol metabolism modulation, as cholesterol supplementation rescued these effects, while 25HC and statin treatments interacted to exacerbate dysfunction. We propose that the CH25H/25HC pathway represents an important component in the pathophysiology of brain vessel dysfunction associated with infection and cholesterol dysregulation in the context of ICH.### Competing Interest StatementThe authors have declared no competing interest.

Non-disruption alterations to the blood-brain barrier (BBB) can be difficult to detect and therefore require highly sensitive tools for reliable measurement. Here, we apply a BBB filter exchange imaging (BBB-FEXI) technique to assess the rat brain in response to mild Streptococcus pneumoniae lung infection. We observe a significant 78 ± 39 % increase in BBB water permeability during infection. Higher water exchange measures were associated with higher levels of vascular inflammation, while BBB tight junction proteins remained unchanged. The expression of aquaporin-4 water channel was 38% higher in infected animals, which may drive the increase in water exchange during infection.

Frontotemporal dementia and amyotrophic lateral sclerosis are neurodegenerative diseases with considerable clinical, genetic and pathological overlap. The most common cause of both diseases is a hexanucleotide repeat expansion in C9orf72. The expansion is translated to produce five toxic dipeptides, which aggregate in patient brain. Neuroinflammation is a feature of frontotemporal dementia and amyotrophic lateral sclerosis; however, its causes are unknown. The nod-like receptor family, pyrin domain-containing 3 inflammasome is implicated in several other neurodegenerative diseases as a driver of damaging inflammation. The inflammasome is a multi-protein complex which forms in immune cells in response to tissue damage, pathogens or aggregating proteins. Inflammasome activation is observed in models of other neurodegenerative diseases such as Alzheimer's disease, and inflammasome inhibition rescues cognitive decline in rodent models of Alzheimer's disease. Here, we show that a dipeptide arising from the C9orf72 expansion, poly-glycine-arginine, activated the inflammasome in microglia and macrophages, leading to secretion of the pro-inflammatory cytokine, interleukin-1β. Poly-glycine-arginine also activated the inflammasome in organotypic hippocampal slice cultures, and immunofluorescence imaging demonstrated formation of inflammasome specks in response to poly-glycine-arginine. Several clinically available anti-inflammatory drugs rescued poly-glycine-arginine-induced inflammasome activation. These data suggest that C9orf72 dipeptides contribute to the neuroinflammation observed in patients, and highlight the inflammasome as a potential therapeutic target for frontotemporal dementia and amyotrophic lateral sclerosis.

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)-γ. 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.

Background: Cytoskeletal non-muscle actin isoforms are the most abundant intracellular proteins and extensively interact with other molecules. Biological consequences and genotype-phenotype correlations of the variants in genes encoding these isoforms, ACTB and ACTG1, are not delineated. Methods: Clinical data analysis from 290 individuals with pathogenic ACTB/ACTG1 variants; characterization of patient cells, mutant proteins, patient-derived iPSC-based models and mutant mice. Results: We show that ACTB and ACTG1 variants have distinct clinical profiles. ACTB nonsense, frameshift and missense variants that lead to rapid protein degradation result in milder phenotypes. Heterozygous Actb knockout causes altered neuronal cell morphology and abnormal expression of actin-related genes in newborn mouse brains. Truncating ACTG1 variants are likely to be non-pathogenic, but chromosomal deletions encompassing ACTG1 and flanking genes may result in susceptibility to neurodevelopmental phenotypes. Subsets of disease-causing ACTB missense variants (MVs) result in more severe Type 1 Baraitser-Winter Cerebrofrontofacial (BWCFF1) or Deafness Dystonia syndromes. Pathogenic ACTG1 MVs cause BWCFF2 or isolated hearing loss. These amino acid substitutions are associated with dramatically dysregulated actin polymerization and depolymerization dynamics and, in induced pluripotent stem cells (iPSC) derived models, lead to neuronal migration defects. A significant subset of MVs result in disorders that cannot yet be classified into recognizable groups. Conclusions: ACTB or ACTG1 variants and result in minimum eight mechanistically diverse non-muscle actinopathies. These results will improve their diagnosis and management, and pave the way for new treatment strategies. This study reflects the scale of collaborative clinical studies and multi-modal mechanistic studies required to dissect rare allelic disorders. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement NDD received grant support from Deutsche Forschungsgemeischaft (DFG) (DI 2170/3.1 and DI 2170/5.1) and Else-Kroener-Fresenius Stiftung (2020_EKES.04). NDD and DJM are supported through the European Union Horizon 2020 research and innovation program under the EJP RD COFUND EJP N 825575 with support from the German Federal Ministry of Education and Research under Grant Agreements 01GM1922A and 01GM1922B, respectively. DJM acknowledges grant support from: Deutsche Forschungsgemeinschaft (DFG) (MA1081/23.1, MA1081/28.1), the Cluster of Excellence RESIST (EXC 2155; DFG Project ID: 39087428.B11). ASW acknowledges grant support from: Medical Research Council project grant MR/T016809/1; Medical Research Council-National Institute for Health and Care Research rare disease research platform MR/Y008340/1; and Kidneys for Life pump priming grant 2017. SB acknowledges grant support from the NIHR Manchester Biomedical Research Centre (NIHR203308). SB, ASW, and AT acknowledge support from the Davies family for grant support in the form of Marsh Studentship to the University of Manchester. SB and SC acknowledge support from the Great Ormond Street Hospital Charity research grant V4621. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB the TU Dresden and Ethics committee/IRB of the Central Manchester gave ethical approval for this work (EK-127032017, BO-EK-341062021 and (02/CM/238). All patients ID were created specifically for this manuscript and are not known to anyone outside of teh research group. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data sets supporting the results of this article are available in the supplementary material or could be requested from the corresponding authors.

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 2’3’cGAMP 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. ### Competing Interest Statement The authors have declared no competing interest.

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. ### Competing Interest Statement The authors have declared no competing interest.

Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modeling, we predict that brazilin can adopt a favorable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small -molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds.

Blood–brain barrier (BBB) dysfunction occurs in many brain diseases, and there is increasing evidence to suggest that it is an early process in dementia which may be exacerbated by peripheral infection. Filter-exchange imaging (FEXI) is an MRI technique for measuring trans-membrane water exchange. FEXI data is typically analysed using the apparent exchange rate (AXR) model, yielding estimates of the AXR. Crusher gradients are commonly used to remove unwanted coherence pathways arising from longitudinal storage pulses during the mixing period. We first demonstrate that when using thin slices, as is needed for imaging the rodent brain, crusher gradients result in underestimation of the AXR. To address this, we propose an extended crusher-compensated exchange rate (CCXR) model to account for diffusion-weighting introduced by the crusher gradients, which is able to recover ground truth values of BBB water exchange ( k in ) in simulated data. When applied to the rat brain, k in estimates obtained using the CCXR model were 3.10 s −1 and 3.49 s −1 compared to AXR estimates of 1.24 s −1 and 0.49 s −1 for slice thicknesses of 4.0 mm and 2.5 mm respectively. We then validated our approach using a clinically relevant Streptococcus pneumoniae lung infection. We observed a significant 70 ± 10% increase in BBB water exchange in rats during active infection ( k in = 3.78 ± 0.42 s −1 ) compared to before infection ( k in = 2.72 ± 0.30 s −1 ; p = 0.02). The BBB water exchange rate during infection was associated with higher levels of plasma von Willebrand factor (VWF), a marker of acute vascular inflammation. We also observed 42% higher expression of perivascular aquaporin-4 (AQP4) in infected animals compared to non-infected controls, while levels of tight junction proteins remain consistent between groups. In summary, we propose a modelling approach for FEXI data which removes the bias in estimated water-exchange rates associated with the use of crusher gradients. Using this approach, we demonstrate the impact of peripheral infection on BBB water exchange, which appears to be mediated by endothelial dysfunction and associated with an increase in perivascular AQP4.

Intracerebral haemorrhage (ICH) is the deadliest form of stroke, but current treatment options are limited, meaning ICH survivors are often left with life-changing disabilities. The significant unmet clinical need and socioeconomic burden of ICH mean novel regenerative medicine approaches are gaining interest. To facilitate the regeneration of the ICH lesion, injectable biomimetic hydrogels are proposed as both scaffolds for endogenous repair and delivery platforms for pro-regenerative therapies. In this paper, the objective was to explore whether injection of a novel self-assembling peptide hydrogel (SAPH) Alpha2 was feasible, safe and could stimulate brain tissue regeneration, in a collagenase-induced ICH model in rats. Alpha2 was administered intracerebrally at 7 days post ICH and functional outcome measures, histological markers of damage and repair and RNA-sequencing were investigated for up to 8 weeks. The hydrogel Alpha2 was safe, well-tolerated and was retained in the lesion for several weeks, where it allowed infiltration of host cells. The hydrogel had a largely neutral effect on functional outcomes and expression of angiogenic and neurogenic markers but led to increased numbers of proliferating cells. RNAseq and pathway analysis showed that ICH altered genes related to inflammatory and phagocytic pathways, and these changes were also observed after administration of hydrogel. Overall, the results show that the novel hydrogel was safe when injected intracerebrally and had no negative effects on functional outcomes but increased cell proliferation. To elicit a regenerative effect, future studies could use a functionalised hydrogel or combine it with an adjunct therapy.