Journal of Neuroinflammation

Microglia represent the immune component of the central nervous system (CNS) that displays dynamic responses to injury and disease. Across the developing and mature CNS, microglia emerge as immunocompetent cells that continuously survey their surroundings to maintain tissue homeostasis and respond to threats. There remains a gap in 3D in vitro models that contain microglia and can provide both developmental and mature functional hallmarks. Using a 3D neural multicellular model, cortical microtissues, derived from primary rat cortical cells, we conducted live imaging to monitor microglia dynamics from early, middle, and late stage microtissue maturation. We optimized a within-micromold imaging approach that allows for live microglia imaging without removing microtissues from their culturing environment. We confirm that microglia exhibit baseline surveillance characterized by relatively stationary somas and highly dynamic cell processes that continuously extend and retract. Following proinflammatory challenges, microglia engulf lipopolysaccharide particles, accompanied by dynamic shifts in motility patterns; and rapidly respond to laser-induced tissue damage through process extension, whole-cell displacement, and local recruitment. Lastly, we show that microtissue age in culture strongly influences both baseline and directed motility profiles. Collectively, these studies demonstrate that within a 3D microenvironment, microglia exhibit pronounced changes in morphology, surveillance area, motility, and injury response across microtissue maturation. Microtissues can serve as a valuable in vitro platform for both microglia developmental studies and investigations of brain inflammation related to CNS injuries, infections, and diseases.

BackgroundTraumatic brain injury (TBI) is increasingly recognized as a chronic condition with lasting systemic consequences. Beyond persistent neuroinflammation, long-term TBI disrupts peripheral immune homeostasis, increasing susceptibility to infection and organ dysfunction, particularly in older patients. The voltage-gated proton channel Hv1, expressed in microglia and peripheral immune cells, regulates oxidative injury via modulation of NADPH oxidase activity. Yet few animal studies extend long enough to recapitulate the lifelong trajectory of human TBI, leaving the long-term effects of Hv1 deficiency on systemic immune homeostasis unresolved. MethodsYoung adult (3-month-old) male wild-type (WT) and Hv1 knockout (Hv1KO) mice were subjected to a moderate controlled cortical impact (CCI), and survival was monitored for up to 18 months post-injury, with endpoint analyses performed at 21 months of age. After neurological behavioral assessments, spleen, lung, liver, gut, ipsilateral cortex, and blood samples were collected for flow cytometry, qPCR, NanoString nCounter Panels, and in vivo plasma transfer studies. ResultsHv1 deficiency resulted in significantly increased mortality following TBI, starting at 14 months post-injury, compared with WT/TBI mice. No significant difference in survival was observed between the two sham groups. At 18 months post-injury, Hv1KO mice exhibited significant weight loss and splenomegaly. qPCR revealed an approximately 30-fold increase of pan-bacterial 16S rRNA levels in the spleens of Hv1KO/TBI mice, but not in the lungs or liver. Furthermore, chronic TBI in the Hv1KO mice led to a compromised intestinal tight junction and mucus barrier integrity, accompanied by aberrant activation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway in the spleen. Transcriptomic profiling of the spleen, liver, and lung revealed distinct post-injury immune signatures in Hv1KO mice. In contrast, surviving Hv1KO/TBI mice showed modest behavioral resilience and a partially neuroprotective cortical transcriptomic profile. Lastly, systemic transfer of plasma from WT/TBI or Hv1KO donors into naive young adult mice altered immune responses in the spleen, lung, and brain. ConclusionsHv1 plays a critical role in maintaining peripheral immune integrity and antibacterial defense throughout the chronic course of TBI. Despite conferring modest neuroprotection through attenuation of microglial-mediated oxidative stress, Hv1 deficiency exacerbated systemic phagocyte dysfunction and significantly reduced long-term survival.

Early adjunctive anti-inflammatory therapy could modulate the immune response and improve clinical outcomes in Herpes Simplex Virus encephalitis (HSE). However, data on the array of inflammatory mediators to target and the immunomodulatory drugs to use are limited. This study aimed to determine key cytokines associated with HSV-induced neuroinflammation, brain injury, and clinical outcomes, and the effect of dexamethasone on their blood levels. We profiled the levels of forty-eight cytokines and four brain injury biomarkers (GFAP, NfL, Tau and UCHL-1) in CSF and serum samples collected from HSE adult patients; recruited in a randomised clinical trial to receive either adjunctive dexamethasone plus intravenous aciclovir or aciclovir alone. We found that cytokines of the IL-1 superfamily (IL-1, IL-18, IL-1RA) and IL-6 were most consistently associated with neuroinflammation, brain injury and poor clinical outcomes. Spearman correlation analysis revealed positive associations between CSF concentrations of IL-1RA, IL-18, and IL-10 and serum concentrations of IL-1RA and IL-6 with the astrocytic marker, Glial fibrillary acidic protein (GFAP). Levels of CSF IL-1RA and IL-18 were associated with increased volume of cerebral oedema on MRI and were significantly raised (p < 0.05) in both patients with abnormal GCS scores (< 15) and worse outcomes (LoS <3). Moreover, the blood levels of these key mediators were unaffected by adjunctive dexamethasone treatment. Taken together, this work identified key targets for direct, targeted adjunctive anti-inflammatory and neuroprotective therapy to be taken forward in clinical trials, aiming to improve outcomes following HSE.

Microglia, the resident macrophages of the central nervous system, are recognized for their heterogeneity and integral role in brain function and diseases. In the context of high fat diet (HFD) feeding and obesity, microglia become overactive, acquiring a prevailing lipid associated microglial phenotype (also known as LAM). Yet, how microgliosis is induced and regulated remains unclear. Here we report a key role for the Complement 3a Receptor (C3aR), on HFD-induced hypothalamic gliosis and weight gain in mice. HFD consumption leads to elevated microglial expression of C3aR, which parallels widespread accumulation of reactive microglia, selectively in the hypothalamus. Conditional microglial C3aR deletion protects mice from HFD-induced hypothalamic reactive microgliosis. C3aR deletion or pharmacological antagonism opposes HFD-induced weight gain in male but not female mice. Mechanistically, we demonstrated that C3aR is essential for lipid-induced lipid droplet formation, and acquisition of a LAM molecular signature. In summary, we uncovered a previously unknown role for C3aR in the acquisition of a LAM signature driving diet-induced gliosis, identifying this receptor as a new viable therapeutic candidate for conditions associated with hypothalamic neuroinflammation.

Traumatic brain injury (TBI) initiates a secondary inflammatory cascade in which sustained microglial activation contributes to long-term neurological dysfunction. Microglial inflammatory states depend on glycolytic reprogramming, suggesting that targeted modulation of metabolic regulators may attenuate post-traumatic inflammation while preserving essential immune functions. Hexokinase-2 (HK2), a rate-limiting glycolytic enzyme, regulates inflammatory signaling and inflammasome activation in microglia in neurodegenerative contexts; however, its role in TBI remains undefined. We therefore examined whether partial suppression of microglial HK2 modulates inflammatory responses following severe TBI. HK2 was robustly induced in microglia during the sub-acute phase after injury. Pharmacological inhibition of HK2 improved motor coordination without impairing locomotion or cognitive performance and selectively reduced inflammasome-related gene expression and ASC accumulation, particularly within the hippocampal hilus. Importantly, HK2 antagonism slowed microglial proliferation while preserving efferocytic capacity. Partial genetic reduction of microglial HK2 phenocopied these molecular and behavioral effects, supporting an HK2-dependent mechanism. Together, these findings identify microglial HK2 as a therapeutically targetable regulator of inflammatory amplification after TBI. Partial modulation of this pathway attenuates secondary neuroinflammation while maintaining critical microglial functions, highlighting HK2 as a promising strategy to improve functional recovery after traumatic brain injury.

Brain development, especially developmental myelination, continues through young adulthood. Concordantly, children may be particularly vulnerable to neural-immune challenges. To investigate the consequences of major childhood immune challenges, juvenile mice were exposed to respiratory influenza (H1N1) infection. White matter-specific microglial reactivity accompanied by oligodendrocyte loss was evident until two months following infection. Mice exhibited hyperlocomotion and impaired attention, but not anxiety-like behavior, at one month following infection. Linking the oligodendroglial and behavioral deficits, genetic disruption of oligodendrocyte development at the same juvenile timepoint recapitulated this behavioral phenotype. Microglial reactivity and oligodendrocyte numbers normalized by young adulthood. However, myelin development was disrupted, with persistently decreased myelinated axon density and reduced myelin sheath thickness. Hyperlocomotion resolved, but anxiety-related behaviors emerged at two months after infection. At 6 months, anxiety resolved but cognitive deficits persisted. Elevated CSF chemokines and microglial chemokine expression prompted testing the role of the multi-chemokine receptor CCR3. CCR3 inhibition rescued these cellular and behavioral aberrations after juvenile H1N1 infection. Together, these findings underscore the potential for disruption of myelin development and lasting cognitive and neuropsychiatric sequelae following major immune challenges during the juvenile period and highlight chemokine signaling as an important therapeutic target.

BackgroundPhagocyte NADPH oxidase 2 (NOX2) is an enzyme complex responsible for reactive oxygen species (ROS) production. Chronic NOX2 activity sustains oxidative stress/damage and drives neuroinflammation following traumatic brain injury (TBI). NOX2 acts as a priming signal for NLRP3 inflammasome activation, which also plays a role in secondary injury after TBI. GSK2795039 is a small molecule brain penetrable drug that inhibits NOX2 in a NADPH competitive manner. Here, we investigated whether pharmacological inhibition of NOX2 using GSK2795039 can reduce secondary neuroinflammation after TBI, specifically via inhibition of downstream NLRP3 inflammasome activation, in both resident microglia and infiltrating myeloid cells in the injured brain. MethodsImmortalised microglial (IMG) cells or primary microglia were pre-treated with GSK2795039 (NOX2 inhibitor) or MCC950 (NLRP3 inhibitor) and stimulated with lipopolysaccharide and nigericin to induce NOX2/ROS and NLRP3 inflammasome activation. The controlled cortical impact model, pharmacokinetic analyses, multi-dimensional flow cytometry, histology and neurobehavioral assessments were used to translate in vitro findings to an experimental TBI model in adult male C57BL6/J mice. ResultsThe small molecule NOX2 inhibitor, GSK2795039, attenuated microglial NOX2 activity, thereby reducing ROS, nitrite and cytokine levels, as well as NLRP3 inflammasome components in pro-inflammatory microglia. TBI recruited NOX2/ROS/IL-1{beta}+ neutrophils and inflammatory monocytes into injured brain parenchyma with peak monocytic NOX2/ROS/IL-1{beta} production at 3 days post-injury (DPI), coincident with peak NOX2/ROS/IL-1{beta} expression in microglia. Systemic administration of GSK2795039 (100mg/kg; I.P.) starting 2 hours post-injury attenuated NOX2/IL-1{beta}+ microglial and infiltrating myeloid cell activation at 3 DPI. In addition, NOX2 inhibition reduced numbers of IL-1R+ T cells in the brain of TBI mice, indicating that myeloid-T cell crosstalk was altered by GSK2795039 treatment. Innate and adaptive neuroimmune changes were associated with improvements in motor function post-TBI. In the chronic phase through 28 DPI, pharmacological inhibition of NOX2 by GSK2795039 treatment resulted in modest improvements in neurobehavioral deficits and TBI neuropathology. ConclusionsThese preclinical studies identify the NOX2-ROS-NLRP3 inflammasome axis along with myeloid-T cell crosstalk as effective targets for TBI neuroinflammation. Our translational studies indicate that NOX2 may be a promising therapeutic target for mitigating neuroinflammation in microglia, and peripheral immune cells, following experimental TBI in mice.

CNS-resident immune cells are uniquely adapted to their microenvironment; however, the extent of their regional specialisation remains unclear. We combined morphometric and transcriptomic profiling of microglia across the healthy adult mouse CNS, including the olfactory bulbs, cortex, hippocampus, cerebellum and retina, to define their regional and sub-regional heterogeneity. Bulk RNA-sequencing revealed region-specific signatures, with retinal microglia showing the most divergent transcriptomes, and genes related to antigen presentation, phagocytosis and chemokine signalling among the top differentially expressed genes. Single-cell RNA sequencing identified predominantly homeostatic microglia across all examined regions, alongside smaller clusters of interferon-responsive, chemokine-enriched, apolipoprotein-enriched and proliferative microglia. Apolipoprotein-enriched microglia were restricted to the olfactory bulbs, whereas interferon-responsive microglia were most abundant in the retina. Single-cell profiling of human retinal microglia confirmed clusters enriched for interferon-stimulated genes. Together, this study reveals previously unrecognised microglial heterogeneity within the healthy brain and eye and provides a comparison of microglia transcriptomes across different neuroanatomical regions of the CNS.

AimsCLN2 disease is a fatal inherited childhood neurodegenerative disorder. Although a disease-modifying therapy now exists, a fundamental lack of understanding of disease pathogenesis has hampered development of more effective therapies. To better understand the cellular pathophysiology of CLN2 disease, we investigated the nature and progression of neuropathological and neurological changes in the recently generated Cln2R207X mouse. MethodsWe have detailed microglial activation, astrogliosis, cytokine and chemokine expression, and neuron loss across the forebrain and spinal cords of Cln2R207X mice, along with quantitative gait analysis. We also performed long-term electroencephalography (EEG) recordings to characterize seizure activity, a clinically-relevant phenotype yet to be defined in any CLN2 disease model. ResultsHistology revealed early localized microglial activation months before neuron loss in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. These pathological changes were more pronounced and occurred in the cortex before the thalamus or spinal cord. There were early-onset and progressive changes in the expression of specific chemokines and cytokines including IL-33, IP-10, and MIP-1. Gait analysis revealed impaired performance only at disease end stage. EEG recordings revealed robust and progressive epileptiform activity from disease mid-stage including spontaneous seizures, which were accompanied by a profound loss of cortical GABAergic interneurons. ConclusionsOur data reveal novel phenotypes in Cln2R207X mice that differ markedly in their timing and progression through the CNS from other NCL mouse models. Our findings provide new insights on CLN2 disease pathogenesis and clinically-relevant readouts for future therapeutic studies.

Intronic G4C2 hexanucleotide repeat expansions (HRE) of C9orf72 are the most common cause of familial variants of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). G4C2 HREs in C9orf72 undergo non-canonical repeat-associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain. Previous work has demonstrated the profound effects of a poly(GR) model of C9orf72 FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease. Here, using an established mouse model of C9orf72 FTD/ALS we investigate the contributions of the nod-like receptor pyrin-containing 3 (NLRP3) inflammasome in the pathogenesis of FTD/ALS. We find that inflammasome-mediated neuroinflammation is increased with microglial activation, cleavage of caspase-1, production of IL-1{beta} and upregulation of Cxcl10 in the brain of C9orf72 FTD/ALS mice. Excitingly, we find that genetic ablation of Nlrp3 significantly improved survival, protected behavioral deficits and prevented neurodegeneration suggesting a novel mechanism involving HRE-mediated induction of innate immunity. The findings provide experimental evidence of the integral role of HRE in inflammasome-mediated innate immunity in the C9orf72 variant of FTD/ALS pathogenesis and suggest the NLRP3 inflammasome as a therapeutic target.

The novel coronavirus SARS-CoV-2 has caused significant global morbidity and mortality and continues to burden patients with persisting neurological dysfunction. COVID-19 survivors develop debilitating symptoms to include neuro-psychological dysfunction, termed "Long COVID", which can cause significant reduction of quality of life. Despite vigorous model development, the possible cause of these symptoms and the underlying pathophysiology of this devastating disease remains elusive. Mouse adapted (MA10) SARS-CoV-2 is a novel mouse-based model of COVID-19 which simulates the clinical symptoms of respiratory distress associated with SARS-CoV-2 infection in mice. In this study, we evaluated the long-term effects of MA10 infection on brain pathology and neuroinflammation. 10-week and 1-year old female BALB/cAnNHsd mice were infected intranasally with 104 plaque-forming units (PFU) and 103 PFU of SARS-CoV-2 MA10, respectively, and the brain was examined 60 days post-infection (dpi). Immunohistochemical analysis showed a decrease in the neuronal nuclear protein NeuN and an increase in Iba-1 positive amoeboid microglia in the hippocampus after MA10 infection, indicating long-term neurological changes in a brain area which is critical for long-term memory consolidation and processing. Importantly, these changes were seen in 40-50% of infected mice, which correlates to prevalence of LC seen clinically. Our data shows for the first time that MA10 infection induces neuropathological outcomes several weeks after infection at similar rates of observed clinical prevalence of "Long COVID". These observations strengthen the MA10 model as a viable model for study of the long-term effects of SARS-CoV-2 in humans. Establishing the viability of this model is a key step towards the rapid development of novel therapeutic strategies to ameliorate neuroinflammation and restore brain function in those suffering from the persistent cognitive dysfunction of "Long-COVID".

BackgroundTraumatic brain injury (TBI) initiates a rapidly evolving neuroinflammatory response; however, the temporal relationship between early innate immune activation, T cell polarization, and neurobehavioural recovery remains poorly understood. Here, we hypothesize that interleukin-1{beta} (IL-1{beta}) is a critical upstream mediator that polarizes T cells towards pro-inflammatory and cytotoxic effector functions following TBI. MethodsUsing a controlled cortical impact model in adult male C57BL/6J mice, we mapped post-injury immune dynamics and investigated whether targeting key innate inflammatory compartments influenced subsequent T cell programming and neurological outcomes. We conducted longitudinal immune profiling by multiparameter spectral flow cytometry and quantitative polymerase chain reaction up to 10 days post-injury. Antibody-based immune depletion strategies were used to investigate neutrophil and monocyte contributions to the post-traumatic T cell response, while pharmacological inhibition of NLRP3 inflammasome by MCC950 treatment was used to investigate the contribution of IL-1{beta}. ResultsTBI elicited a structured early innate immune response, marked by rapid chemokine induction, followed by temporally distinct infiltration of neutrophils, monocytes, and dendritic cells. Neutrophils and monocytes were the predominant early IL-1{beta}-producing infiltrating populations. This was followed by a delayed adaptive phase characterized by sustained recruitment of T cell subsets (CD4+, CD8+, {gamma}{delta}+), alongside dynamic effector cytokine production (IL-17, IFN-{gamma}). Neutrophil depletion altered the early myeloid composition but did not result in durable improvements in T cell effector responses or neurobehavioral outcomes. Depletion of CCR2-dependent inflammatory monocytes reduced acute monocyte accumulation and attenuated early downstream T cell responses; however, these effects were not sustained and only resulted in modest neurobehavioural benefits. In contrast, inhibition of the NLRP3 inflammasome suppressed microglial IL-1{beta} production, without significantly altering leukocyte recruitment or subacute T cell effector phenotypes. These phenotypic changes were associated with improvements in motor and cognitive function recovery. ConclusionWe show that early monocyte IL-1{beta} signalling actively regulates downstream T cell infiltration and effector function after TBI. In addition, inhibition of NLRP3 inflammasome after TBI attenuates microglial IL-1{beta}-associated immune activation and results in behavioural improvement despite ongoing leukocyte recruitment, indicating that targeting the nature and cellular source of IL-1{beta} signalling can dissociate immune cell burden from neurological outcomes. Collectively, our findings identify myeloid IL-1{beta}-linked pathways as a viable bridge between innate and adaptive immunity post-TBI, and underscore cellular compensation as a critical design consideration for next-generation immunotherapies.

Traumatic brain injury (TBI) induces both neuroinflammation and gut microbiome dysbiosis, yet the influence of antibiotics (ABX) on TBI-related neuropathology remains unclear. We administered a broad-spectrum oral ABX regimen to deplete the gut microbiome in single and repeated TBI mouse models. In male mice, ABX treatment significantly reduced neuroinflammation and neurodegeneration post-TBI, with no effects observed in uninjured controls. ABX also altered microbiome composition and decreased serum and fecal short-chain fatty acid levels, while intestinal damage and dysbiosis were further exacerbated by TBI severity. Notably, germ-free male mice exhibited heightened neuroinflammation and larger lesion volumes following TBI, underscoring the microbiomes essential role in recovery. Metagenomic analyses revealed Parasutterella excrementihominis and Lactobacillus johnsonii as potential ABX-resistant taxa post-injury. These findings suggest that short-term ABX treatment may attenuate TBI-induced neuroinflammation by reshaping the gut microbiome, offering directions for microbiome-targeted therapies in TBI.

Parkinsons disease (PD) is a common age-related neurodegenerative disorder characterized by the aggregation of -synuclein (SYN) building up intraneuronal inclusions termed Lewy pathology. Mounting evidence suggests that neuron-released SYN aggregates could be central to microglial activation, which in turn mounts and orchestrates neuroinflammatory processes potentially harmful to neurons. Therefore, understanding the mechanisms that drive microglial cell activation, polarization and function in PD might have important therapeutic implications. Here, using primary microglia, we investigated the inflammatory potential of pure SYN fibrils derived from PD patients. We further explored and characterized microglial cell responses to a chronic-type inflammatory stimulation combining PD patient-derived SYN fibrils (FPD), Tumor necrosis factor- (TNF) and prostaglandin E2 (PGE2) (TPFPD). We showed that FPD hold stronger inflammatory potency than pure SYN fibrils generated de novo. When combined with TNF and PGE2, FPD polarizes microglia toward a particular functional phenotype departing from FPD-treated cells and featuring lower inflammatory cytokine and higher glutamate release. Whereas metabolomic studies showed that TPFPD-exposed microglia were closely related to classically activated M1 proinflammatory cells, notably with similar tricarboxylic acid cycle disruption, transcriptomic analysis revealed that TPFPD-activated microglia assume a unique molecular signature highlighting upregulation of genes involved in glutathione and iron metabolisms. In particular, TPFPD-specific upregulation of Slc7a11 (which encodes the cystine-glutamate antiporter xCT) was consistent with the increased glutamate response and cytotoxic activity of these cells toward midbrain dopaminergic neurons in vitro. Together, these data further extend the structure-pathological relationship of SYN fibrillar polymorphs to their innate immune properties and demonstrate that PD-derived SYN fibrils, TNF and PGE2 act in concert to drive microglial cell activation toward a specific and highly neurotoxic chronic-type inflammatory phenotype characterized by robust glutamate release and iron retention.

HIV-associated neurocognitive disorders (HAND) have become a major clinical concern, particularly among the aging HIV-1-seropositive population, which is generally characterized by persistent viral reservoirs and a lower level of chronic inflammation. NLRP3 inflammasome activation exhibits its unique role in the progression of many chronic inflammatory diseases. Furthermore, pyroptosis, an inflammatory form of programmed cell death, has been implicated in numerous neurological diseases. However, the mechanisms linking EcoHIV infection, microglial pyroptosis, and NLRP3 inflammasome activation remain incompletely understood. In this study, EcoHIV was retro-orbitally injected into C57BL/6J wild-type mice and analyzed at 14-, 30-, 60-, and 90-days post-infection to establish a NeuroHIV model. Additionally, in vitro, BV2 microglial cell line was infected with EcoHIV and treated with MCC950, an inhibitor of the NLRP3 inflammasome, for three days. Pyroptosis marker GSDMD, NLRP3 inflammasome components, Caspase-1 (a marker of inflammasome activation), HLA-DR (an immune activation marker), Programmed-death 1 (PD-1, an immune checkpoint molecule), and Ki67 (a cellular proliferation marker) were assessed by immunofluorescence staining. Results showed that EcoHIV-infected mice showed a peak in NLRP3 expression at 14 days post-infection, compared with controls, followed by a modest decline at 30 days, while GSDMD expression increased progressively across 14 and 30 days. These findings demonstrate dynamic changes in microglial pyroptosis and NLRP3 inflammasome activation over the course of EcoHIV infection. In vitro, EcoHIV-infected BV2 cells exhibited significantly increased EcoHIV-eGFP fluorescence compared with controls, confirming the utility of BV2 cells as an in vitro model of microglial EcoHIV infection. Expression levels of GSDMD and NLRP3 were elevated following infection, indicating enhanced pyroptosis and neuroinflammation. Treatment with MCC950 significantly reduced the expression of GSDMD, NLRP3, HLA-DR, PD-1, and Ki67, suggesting that inhibition of NLRP3 inflammasome activity suppresses both pyroptosis and microglial activation and proliferation. Together, elucidating the interplay between microglial pyroptosis and NLRP3 inflammasome activation may provide new insights into the pathogenesis and potential therapeutic strategies for NeuroHIV in the aging HIV-1-seropositive population.

Immune checkpoint inhibitor (ICI) combinations that block cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) signaling have revolutionized cancer care but also exert a range of immune-related adverse events (irAE) in various tissues, including the brain. Our understanding of the mechanisms of irAE in the brain is still evolving, and we recently demonstrated that ICI (blockade of CTLA-4 and PD-1) perturbs hippocampal-dependent memory function by derailing neuro-immune homeostasis and compromising synaptic integrity. However, the spatial patterns and the cell-type-specific molecular mechanisms underlying ICI-related brain dysfunction remain not well-defined. To address this gap, we performed spatial transcriptomic profiling of the hippocampal region using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to map gene expression at single-cell resolution. By integrating spatial single-cell data with bulk RNA-seq, we define the distribution of microglia, astrocytes, synaptic, and neuroinflammatory markers, and determine how ICI reshapes hippocampal cellular composition in a syngeneic murine melanoma model. MERFISH revealed upregulation of microglial, astrocytic, oligodendrocytic, and T cell markers post-ICI treatment, revealing unique pathways driving neuroinflammation, synaptic function, and cellular signaling. Furthermore, immunofluorescence analysis of postmortem brains from patients treated with ICI corroborates our findings of ICI-related immune activation of microglia. Finally, using a conditional deletion model, we show that T cells are indispensable for ICI-driven microglial activation. Altogether, our study provides a high-resolution spatial framework for understanding irAEs in brain function and a T cell-microglia crosstalk axis as a driving mechanism of dysregulated neuro-immune homeostasis during ICI.

BackgroundHypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal mortality and neurodevelopmental impairments. Following brain hypoxia-ischemia (HI), microglia face substantial metabolic stress; and upon phagocytosis, they become overloaded with lipids derived from engulfed dead neurons and myelin debris. It is unclear how microglia respond to and process the lipid cargo, and whether lipid accumulation may affect microglia function following neonatal HI. MethodsThe postnatal day 10 mice were subjected to HI using the Vannucci model. Lipid droplets (LD) were assessed by histology and immunofluorescent staining. Single-nucleus RNA sequencing (snRNA-seq) was performed using brain tissue from HI-injured and sham-operated mice at 72 hours after HI. LD-accumulating microglia (LDAM) were identified by a specific LD marker gene perilipin 2 (Plin2). Differential gene expression was analyzed between Plin2-positive and Plin2-negative microglia after HI. Human HIE brain sections were also examined for LD accumulation. The dynamic changes of PLIN2-expressing microglia and infiltrating monocyte-derived macrophages (MDM) at 24 hours, 72 hours and 7 days after HI were compared using flow cytometry. In addition, mouse BV2 microglia were subjected to oxygen-glucose deprivation (OGD) to study phagocytosis and cytokine expression. ResultsLipid droplets accumulated primarily in microglia after HI in neonatal mice and in human HIE brain. LD were not found in astrocytes or neurons. Plin2-expressing LDAM emerged as new microglia clusters after HI. Compared with microglia without LD, LDAM showed a distinct transcriptional profile with upregulation of genes linked to microglial activation, enhanced cholesterol and lipid processing, and a shift towards phagocytic and pro-inflammatory state. Blocking LD biogenesis reduced elevated phagocytosis and IL-1{beta} expression in BV2 cells following OGD. ConclusionOur study revealed that microglia accumulate lipid droplets as part of their metabolic responses to HI in the neonatal brain. Microglial lipid droplet formation is associated with a pro-inflammatory phenotype at early stage after HI, and increased phagocytosis in vitro. The lipid metabolic changes may regulate microglial function and influence HI outcomes.

Microglia play a critical role in synapse remodeling and neuroinflammation, both of which are dysregulated in Alzheimers disease (AD). However, most in vitro models rely on neonatal or immortalized microglia, limiting their relevance to adult pathophysiological context. Here, we present a compartmentalized microfluidic co-culture platform that enables spatially controlled interactions between primary cortical neurons and adult microglia from wild-type (WT) and APP-transgenic mice. This system allows precise functional analysis of microglia-synapse interactions under defined inflammatory conditions. Upon lipopolysaccharide (LPS) stimulation, APP microglia exhibited exaggerated morphological activation, elevated IL-1{beta} secretion, and selectively increased engulfment of synaptic material. In contrast, phagocytosis of non-specific substrates such as pHrodo Zymosan remained unchanged, suggesting a substrate-specific enhancement of microglial phagocytic activity. Blocking the complement receptor CD11b abolished the LPS-induced increase in synaptic uptake, confirming the role of complement-dependent pathways. Transcriptomic profiling revealed robust inflammatory responses in both genotypes, with selectively heightened expression of proinflammatory genes in APP microglia, consistent with a primed immune phenotype. Importantly, increased synaptic uptake occurred without measurable loss of global synaptic connectivity, highlighting the specificity and sensitivity of the system to detect microglial functional changes. This model captures genotype-dependent microglial reactivity (revealing phenotypes not fully captured by transcriptomic rofiling) and provides a physiologically relevant, tractable in vitro platform for dissecting microglial contributions to synaptic pathology in neurodegenerative disease.

Despite suppressive antiretroviral therapy (ART), HIV persists in the central nervous system (CNS) and contributes to HIV-associated neurocognitive disorder (HAND), but cell type-specific effects remain poorly defined. Using fluorescence-activated nuclei sorting of postmortem brain tissue of aviremic and viremic deceased people with HIV (DPWH) and HIV-negative individuals, we quantified the size of the HIV CNS reservoir and transcriptional alterations. Microglia were identified as the dominant CNS reservoir, harboring 103-10 HIV DNA copies per million cells by ddPCR-LTR assay in both aviremic and viremic DPWH. Bulk RNA-sequencing revealed immune pathway upregulation specifically in microglia, and downregulation of synaptic and homeostatic pathways across cell-types in viremic compared to aviremic individuals. ART partially mitigated microglial transcriptional dysregulation, but transcriptional profiles did not restore profiles to HIV-negative levels. Notably, persistent microglial infection was associated with transcriptional changes in other cell-types, underscoring microglia as a key therapeutical target for CNS-directed HIV cure strategies.

Brain tuberculosis remains associated with high mortality, and many survivors exhibit cognitive impairments. Progress in understanding the disease is hindered by the lack of human models. In this study, human neural organoids were infected, revealing that a subpopulation of neural progenitor cells (NPCs) is directly infected by apoptotic cell receptors expressed by NPCs, mediating bacterial uptake. Phagocytosed bacteria were localized in late endosomes, lysosomes, and the cytoplasm. Cytoplasmic bacteria frequently formed cords, indicating limited control of bacterial expansion. Immunostaining demonstrated that infected NPCs produce a type I interferon (IFN) response, corroborated by increased expression of type I IFN and IFN-regulated genes detected by RNA sequencing. Pathways related to innate immune response, cell death, and proliferation were also activated following Mycobacterium tuberculosis (Mtb) uptake by NPCs. The addition of color-coded microglia and monocytes to 3D neural organoids and NPCs revealed cross-infection of NPCs and other phagocytes by Mtb, suggesting a mechanism by which NPCs may access the bacteria. Infection of NPCs resulted in increased cell death, inhibition of neural differentiation, and reduced proliferation, effects that were partially mitigated by anti-IFN treatment. Differentiated neurons were not infected. These findings indicate that brain organoids and NPC-based in vitro platforms provide a novel approach for studying brain tuberculosis. Decreased NPC function may contribute to brain tuberculosis-induced cognitive disease.