Leucine-rich glioma-inactivated antibody associated autoimmune encephalitis (LGI-1 AE) is a rare immune mediated neuroinflammatory disease resulting in seizures and cognitive impairment. LGI-1 AE can respond well to immunotherapy, however residual symptoms, particularly cognitive dysfunction may persist despite treatment. We have previously identified perturbations of peripheral innate immune cells (monocytes) in LGI-1 AE in the treated (chronic) phase of the disease. To further understand these peripheral changes, we performed a proteomic analysis of plasma from individuals with LGI-1 AE in the untreated (acute) and chronic phase of the illness. High-throughput proteomic profiling was performed on plasma samples from nine acute and 27 chronic LGI-1 AE participants and 16 age- and sex-matched controls using the SomaLogic 11k proteomic panel. Differentially expressed proteins (DEP) were identified using limma with false discovery rate (FDR) < 0.05. Bulk RNA sequencing was performed on isolated monocytes from ten of the participants with chronic LGI-1 AE and 28 controls. Differential gene analysis was performed using DESeq2 with FDR < 0.1. Pathway analysis was performed on the transcriptomic and proteomic datasets with gene set enrichment analysis using the Gene Ontology Biological Processes database and FDR < 0.05. The plasma analysis identified 215 overexpressed and 1015 suppressed proteins in the acute cohort compared with the healthy cohort. Upregulated proteins included proteins related to innate immune activation, chemoattractants for myeloid cells, components of the complement cascade as well as lymphocyte signalling and activation molecules. Significantly downregulated pathways included those related to autophagy. There were no significantly differentially expressed proteins (DEP) between the acute and chronic or chronic and control cohorts. Monocyte transcriptomic analysis revealed 18 upregulated and 170 downregulated genes, highlighting interleukin (IL)-6 and downstream IL-6 pathway signalling as key dysregulated pathways. This study identified IL-6 signalling and activation of the terminal complement cascade as potential pathophysiological immune mechanisms, highlighting opportunities for therapeutic repurposing using existing agents in LGI-1 AE. In addition, the observed suppression of autophagy- related pathways suggests a previously underappreciated mechanism that may contribute to disease pathogenesis, warranting further investigations into the role of autophagy dysregulation in CNS autoimmunity associated with LGI-1 AE.

Age-related macular degeneration (AMD) is characterized by disruption of the choriocapillaris (CC) and retinal pigment epithelium (RPE) dysfunction, leading to drusen accumulation. The CC and RPE form a tightly interdependent unit that maintains homeostasis where the CC supplies oxygen and nutrients to the RPE, while the RPE produces vascular endothelial growth factor (VEGF) to maintain the CC. Genetic studies link alternative complement pathway variants to AMD, and complement deposition on the CC increases during both aging and AMD. Macrophages express complement protein, receptors, and inhibitors, suggesting that they may be a missing link in understanding the role of complement in AMD. In support, previous groups have shown that macrophage depletion disrupts RPE-CC homeostasis, leading to AMD-like pathology, but the mechanism remains unclear. To investigate the role of macrophages in CC-RPE homeostasis, we generated Cx3cr1CreERCsf1ri-DTR and Ms4a3CreRosa26DTR mice. In Cx3cr1CreERCsf1ri-DTR mice, tamoxifen administration induced diphtheria toxin receptor (DTR) expression, allowing ablation of all macrophages (many tamoxifen injections) or long-lived, tissue-resident macrophages (tamoxifen followed by a 3-4 week wash out period). Ms4a3CreRosa26DTR mice were used to deplete monocyte-derived macrophages. Ablation of all macrophages caused decreased CC density, increased CC apoptosis, RPE disorganization, and membrane attack complex (MAC) accumulation. Tissue-resident macrophage ablation phenocopied this result while monocyte-derived macrophage ablation had no phenotype. Additionally, long-term depletion of tissue-resident macrophages led to formation of drusen-like sub-RPE deposits and retinal thinning, mimicking AMD pathology. Finally, pharmacologic depletion of all macrophages similarly reduced CC density to genetic ablation, but C3-/- mice showed an attenuated phenotype. These data demonstrate that long-lived, tissue-resident macrophages are essential for maintaining CC-RPE homeostasis while monocyte-derived macrophages are dispensable in this context. Further, ocular macrophage ablation led to MAC accumulation, while C3-/- mice were resistant to CC regression. Together, these findings suggest that tissue-resident choroidal macrophages maintain CC-RPE homeostasis partially by complement dependent mechanisms. Further, the loss of tissue-resident choroidal macrophages over time is a potential mechanism of AMD pathogenesis.

Acute provoked neonatal seizures are a major risk factor for acquired epilepsy, yet clinicians lack reliable tools to identify neonates at highest risk. Preclinical data implicate innate immune activation and neuronal injury as key drivers of epileptogenesis, suggesting blood-based biomarkers could provide mechanistic insight and prognostic utility. We sought to identify biomarkers of epileptogenesis in neonates with acute provoked seizures after brain injury using multicenter cohorts. We conducted a prospective, multi-cohort analysis across two independent studies. NSR-RISE enrolled neonates with EEG-confirmed acute provoked seizures of diverse etiologies. The HEAL trial enrolled neonates with hypoxic-ischemic encephalopathy; analyses were limited to those with seizures. Plasma proteins were quantified 48-96 h after seizure onset. Associations with acquired epilepsy by 24-months were evaluated using log-link models with robust standard errors and false-discovery rate correction (FDR < 0.05). Significant proteins were added to models including established clinical predictors (≥ 3 days of EEG seizures and abnormal neurological examination at discharge). Exploratory pathway enrichment used KEGG databases. NSR-RISE participants also underwent plasma microRNA (miRNA) sequencing with integrative pathway analyses. Among 35 neonates in NSR-RISE, 7 (20%) developed epilepsy; among 40 neonates in HEAL, 6 (15%) developed epilepsy. Across both cohorts, neonates with epilepsy had higher concentrations of the pro-inflammatory cytokine IL-1β and the neuronal injury marker UCHL1 compared to those without epilepsy. Growth hormone (GH), measured only in NSR-RISE, was decreased in neonates with epilepsy. Incorporation of biomarkers improved prognostic accuracy for epilepsy beyond clinical features alone (Area under the precision-recall curve (AUPRC) 0.30 (95%CI, 0.28-0.32) versus 0.91 (95%CI, 0.89-0.92); p < 0.001). Pathway enrichment analyses implicated innate immune signaling, including TLR/IL1/NF-κB-related and MAPK-associated IL-17 signaling. miRNA profiling identified 11 species differentially expressed between neonates with and without epilepsy, including brain-enriched miRNAs. Network analysis identified a co-expression module enriched for let-7f-5p and miR-146a-5p targeting TLR/IL1/NF-κB, MAPK, and JAK/STAT pathways. Across two cohorts, mechanistically informed biomarkers were associated with acquired epilepsy after neonatal seizures. IL-1β, UCHL1, and GH reflect inflammation, neuronal injury, and impaired trophic signaling, while circulating miRNAs provide complementary mechanistic insight. Findings support a translational biomarker panel and highlight inflammation as a biologically plausible therapeutic target.

Given the prevalence of metabolic perturbations in a variety of neurological and neurodegenerative diseases, understanding and monitoring brain metabolism is a key step in our advancement of therapies. The details of the citric acid cycle were established at the beginning of the last century but only recently have its metabolic intermediates been observed in vivo in the brain. In this study, we employed orthogonal analyses to investigate metabolic alterations in response to acute neuroinflammation in vivo, demonstrating a multi-technique approach that could be used for future studies.Hyperpolarized [1-13C] pyruvate spectroscopy revealed an early decline in pyruvate metabolism via pyruvate dehydrogenase (PDH), leading to reduced 13C-bicarbonate formation. This metabolic disruption occurred despite the absence of structural or perfusion changes on conventional MRI. Further analysis of polar metabolites in the ipsilateral hemisphere confirmed ongoing inflammatory processes. These findings highlight the potential of this dual technique approach to inform upon metabolic changes due to neuroinflammation.Combining methods to probe metabolism in invasive (metabolomics) and non-invasive (hyperpolarized MRI) manners, this represents a promising translational approach for real-time metabolic assessments in an area of the body, the brain, where studying processes such as metabolism has traditionally been challenging. This study has demonstrated the approach to monitor changes in metabolism in response to inflammation in the brain.

Alzheimer's disease (AD) disproportionately affects women, yet the biological basis of this sex bias remains unclear. Here, we identify sex-dependent interferon signaling as a contributor to this disparity. Transcriptomic profiling of postmortem AD tissue and APP/PS1 mice revealed preferential enrichment of interferon-responsive gene programs in females. In APP/PS1 mice, heightened interferon responses were associated with increased neurodegenerative features, and single-cell transcriptomic analyses identified microglia as a major cellular compartment engaging interferon responses. To test causality, we manipulated interferon signaling in vivo. Acute systemic interferon activation promoted AD-like neuropathological alterations. Genetic amplification of interferon signaling in microglia exacerbated neuroinflammatory and neurodegenerative features in APP/PS1 mice, whereas pharmacological inhibition through cGAS-STING blockade suppressed interferon responses, reduced neuropathology, and preserved cognitive performance in female APP/PS1 mice. Together, these findings identify microglial interferon signaling as a modifiable contributor to AD-associated neuropathology and suggest a neuroimmune mechanism underlying the increased vulnerability of females to the disease.

Mounting evidence implicates pathogen infections in the pathogenesis of Alzheimer's disease (AD), yet the cellular mechanisms underlying infection-induced neurodegeneration remain poorly understood. Central to this process is the dysfunction of astrocyte-neuron interactions, which are critical for maintaining neuroinflammatory balance and synaptic homeostasis. Here, we demonstrate that astrocytic protein tyrosine phosphatase 1B (PTP1B) acts as a key regulator of astrocyte reactivity during infection, leading to impaired neuroglial communications and cognitive decline. In a murine model of chronic Toxoplasma gondii (T. gondii) infection, elevated PTP1B levels in astrocytes were closely associated with neuroinflammation and cognitive impairments. Conditional deletion of astrocytic PTP1B or its pharmacological inhibition mitigated neuroinflammation, restored synaptic integrity, and rescued cognitive function. Mechanistically, astrocytic PTP1B induced the polarization of A1-like neurotoxic reactive astrocytes, enhanced glutamate-mediated excitotoxicity, and triggered neuronal senescence, collectively contributing to synaptic damage and cognitive deficits. Notably, elevated levels of PTP1B, GAFP and cellular senescence markers were observed in the serum samples from T. gondii IgG-seropositive individuals and in hippocampal transcriptomes from AD patients, underscoring the translational relevance. Together, our findings reveal that PTP1B-mediated disorder of astrocyte-neuron crosstalk represents a novel mechanism of pathogen-driven neurodegeneration.

Neonatal sevoflurane exposure in mice induces microglial activation and long-term cognitive deficits, a finding that raises significant concerns for pediatric anesthesia. The lung-brain axis, a critical pathway mediating pulmonary-central nervous system communication, is indispensable for maintaining organismal homeostasis. However, existing research on anesthetic neurotoxicity has focused predominantly on central mechanisms, with insufficient attention to the lung-a major immune organ with extensive bidirectional crosstalk with the brain. Herein, we aim to explore the lung-brain interactions underlying long-term cognitive sequelae of neonatal sevoflurane exposure. C57BL/6J mice were selected and exposed to 3% sevoflurane for 2h daily on postnatal days 6-8. Upon reaching adulthood, cognitive function and microglial activation status were evaluated. At 4 weeks post-exposure, 16S rRNA gene sequencing and metabolomic analysis were performed respectively to characterize the structure of the pulmonary microbiota and the metabolite profile. Proximity ligation assay (PLA), fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET), and co-immunoprecipitation (COIP) were employed to investigate the molecular mechanisms by which lung-derived metabolites mediate brain effects. Additionally, rescue experiments were conducted by administering the sphingosine-1-phosphate receptor modulator FTY720 and Moce to validate the aforementioned effects. Repeated neonatal sevoflurane exposure impaired adult cognitive function, induced microglial activation, and was concurrent with pulmonary microbiome dysbiosis and metabolic alterations. Notably, sphingosine-a key membrane lipid-was significantly decreased. Intratracheal administration of FTY720, a sphingosine analog, alleviated neuroinflammation and ameliorated cognitive deficits. Mechanistically, sevoflurane exposure upregulated HDAC1 and downregulated KLF4, whereas FTY720 significantly rescued these sevoflurane-induced expression aberrations, implicating the HDAC1/KLF4 axis in the regulation of neuroinflammation. Additionally, MOCE significantly alleviated neuroinflammation and ameliorated cognitive deficits. Developmental sevoflurane exposure induces microglial activation and cognitive decline via a pulmonary dysbiosis-sphingosine reduction cascade. The sphingosine-1-phosphate receptor modulator FTY720 mitigates this impairment by regulating microglial activation and neuroinflammation. These findings reveal novel mechanisms of anesthetic neurotoxicity and identify potential neuroprotective targets for pediatric anesthesia.

Stroke triggers a rapid and complex immune response that is not yet fully understood, especially within hours after an ischemic infarct. Our previous study in stroke patients revealed a significant increase in interferon-gamma (IFN-γ) immediately (hyperacute) and downstream of the ischemic ictus, within the arterial compartment. The present study investigated the source, inciting factors, and role of IFN-γ in a preclinical murine model. Stroke was produced using transient middle cerebral artery occlusion, and immune cells within the arterial vasculature distal to the occlusion (pre- and post-occlusion) were characterized using flow cytometry. Compared with the control samples, the post-occlusion samples presented an increase in IFN-γ+ and CD69+ cells, whereas no significant increase was detected in IL17+, IL4+, and CD25+ cells. Further analysis of the IFN-γ+ population revealed two novel attributes. First, interrogation of the identity of these IFN-γ+ cells revealed that the increase in IFN-γ production was largely driven by CD14+ myeloid cells in the post-occlusion sample, with negligible contributions from other canonical IFN-γ-producing cells (CD4, CD8, NK). Second, the IFN-γ+ cells exhibited two distinct clusters, an IFN-γlow and an IFN-γhi population. Further analysis revealed that the IFN-γ low population was largely composed of CD14+ myeloid cells, whereas the IFN-γhi population was dominated by CD4+ T-cells. To explore the conditions driving IFN-γ production, an in vitro ischemia model involving oxygen-glucose deprivation (OGD) was employed. Co-culturing of naïve splenocytes with OGD-treated CNS cells and OGD-derived supernatant resulted in a significant increase in IFN-γ+CD14+ cells, as compared to normoxic controls, an effect that coincided with marked loss of DAPI+ and NeuN+DAPI+ cells in mixed cortical (neuronal and glial) cultures. In summary, this study identified intra-arterial CD14+ myeloid cells as novel early sources of IFN-γ in the hyperacute phase of stroke, a role traditionally attributed to adaptive immune cells. Using in vivo and in vitro ischemia models, the findings reveal that injury-associated signals from CNS cells are sufficient to directly induce IFN-γ production in CD14+ myeloid cells, redefining early stroke immunopathology and uncovering a potential target for timely immunomodulation.

Vitamin D deficiency (VD-deficiency) has been implicated in various autoimmune diseases, including uveitis, yet its mechanistic role remains unclear. Emerging evidence suggests that gut microbial dysbiosis and metabolic disturbances may serve as critical intermediates linking VD-deficiency and autoimmune disease pathogenesis. Here, using an experimental autoimmune uveitis (EAU) model induced in B10RIII mice, we demonstrate that VD-deficiency exacerbates EAU severity, accompanied by enhanced Th1 and Th17 responses, suppressed regulatory T cells, and disruption of both intestinal and blood–retinal barrier integrity. Integrated microbiota and metabolomic profiling revealed a coordinated pattern of gut dysbiosis, characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals, together with host metabolic reprogramming in VD-deficient EAU mice. KEGG analysis highlighted disruption of vitamin-related pathways, particularly the vitamin digestion and absorption pathway. Mechanistically, VD-deficiency associated disturbances were linked to LRAT downregulation in retinal pigment epithelial (RPE) cells, accompanied by a pro-inflammatory intraocular microenvironment characterized by increased IL-6 and CXCL family chemokines expression. Gain- and loss-of-function studies in complementary in vivo and in vitro RPE-based models further demonstrated that LRAT functions as a negative regulator of retinal inflammatory responses, with LRAT knockdown enhancing, whereas LRAT overexpression suppressing, the production of IL-6, CXCL1, CXCL2, and CXCL8. Collectively, our findings suggest that VD-deficiency exacerbates autoimmune uveitis by disrupting effector–regulatory T cell balance and compromising blood–retinal barrier and intestinal integrity, accompanied by considerable alterations in gut microbiota and systemic metabolism, while identifying LRAT as a potential regulator linking VD-deficiency to retinal inflammatory amplification.