[18F]F-AraG imaging reveals association between neuroinflammation and brown- and bone marrow adipose tissue.

Brown and brown-like adipose tissues have attracted significant attention for their role in metabolism and therapeutic potential in diabetes and obesity. Despite compelling evidence of an interplay between adipocytes and lymphocytes, the involvement of these tissues in immune responses remains largely unexplored. This study explicates a newfound connection between neuroinflammation and brown- and bone marrow adipose tissue. Leveraging the use of [18F]F-AraG, a mitochondrial metabolic tracer capable of tracking activated lymphocytes and adipocytes simultaneously, we demonstrate, in models of glioblastoma and multiple sclerosis, the correlation between intracerebral immune infiltration and changes in brown- and bone marrow adipose tissue. Significantly, we show initial evidence that a neuroinflammation-adipose tissue link may also exist in humans. This study proposes the concept of an intricate immuno-neuro-adipose circuit, and highlights brown- and bone marrow adipose tissue as an intermediary in the communication between the immune and nervous systems. Understanding the interconnectedness within this circuitry may lead to advancements in the treatment and management of various conditions, including cancer, neurodegenerative diseases and metabolic disorders.

Asymptomatic herpes simplex virus brain infection elicits cellular senescence phenotypes in the central nervous system of mice suffering multiple sclerosis-like disease.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease affecting the central nervous system (CNS) in animals that parallels several clinical and molecular traits of multiple sclerosis in humans. Herpes simplex virus type 1 (HSV-1) infection mainly causes cold sores and eye diseases, yet eventually, it can also reach the CNS, leading to acute encephalitis. Notably, a significant proportion of healthy individuals are likely to have asymptomatic HSV-1 brain infection with chronic brain inflammation due to persistent latent infection in neurons. Because cellular senescence is suggested as a potential factor contributing to the development of various neurodegenerative disorders, including multiple sclerosis, and viral infections may induce a premature senescence state in the CNS, potentially increasing susceptibility to such disorders, here we examine the presence of senescence-related markers in the brains and spinal cords of mice with asymptomatic HSV-1 brain infection, EAE, and both conditions. Across all scenarios, we find a significant increases of senescence biomarkers in the CNS with some differences depending on the analyzed group. Notably, some senescence biomarkers are exclusively observed in mice with the combined conditions. These results indicate that asymptomatic HSV-1 brain infection and EAE associate with a significant expression of senescence biomarkers in the CNS.

Extracellular Vesicles in Multiple Sclerosis: Their Significance in the Development and Possible Applications as Therapeutic Agents and Biomarkers.

Extracellular vesicles (EVs) are "micro-shuttles" that play a role as mediators of intercellular communication. Cells release EVs into the extracellular environment in both physiological and pathological conditions and are involved in intercellular communication, due to their ability to transfer proteins, lipids, and nucleic acids, and in the modulation of the immune system and neuroinflammation. Because EVs can penetrate the blood-brain barrier and move from the central nervous system to the peripheral circulation, and vice versa, recent studies have shown a substantial role for EVs in several neurological diseases, including multiple sclerosis (MS). MS is a demyelinating disease where the main event is caused by T and B cells triggering an autoimmune reaction against myelin constituents. Recent research has elucidate the potential involvement of extracellular vesicles (EVs) in the pathophysiology of MS, although, to date, their potential role both as agents and therapeutic targets in MS is not fully defined. We present in this review a summary and comprehensive examination of EVs' involvement in the pathophysiology of multiple sclerosis, exploring their potential applications as biomarkers and indicators of therapy response.

Oxidative Stress Markers in Multiple Sclerosis.

The pathogenesis of multiple sclerosis (MS) is not completely understood, but genetic factors, autoimmunity, inflammation, demyelination, and neurodegeneration seem to play a significant role. Data from analyses of central nervous system autopsy material from patients diagnosed with multiple sclerosis, as well as from studies in the main experimental model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), suggest the possibility of a role of oxidative stress as well. In this narrative review, we summarize the main data from studies reported on oxidative stress markers in patients diagnosed with MS and in experimental models of MS (mainly EAE), and case-control association studies on the possible association of candidate genes related to oxidative stress with risk for MS. Most studies have shown an increase in markers of oxidative stress, a decrease in antioxidant substances, or both, with cerebrospinal fluid and serum/plasma malonyl-dialdehyde being the most reliable markers. This topic requires further prospective, multicenter studies with a long-term follow-up period involving a large number of patients with MS and controls.

Digital Pathology Identifies Associations between Tissue Inflammatory Biomarkers and Multiple Sclerosis Outcomes.

BACKGROUND: Multiple sclerosis (MS) is a clinically heterogeneous disease underpinned by inflammatory, demyelinating and neurodegenerative processes, the extent of which varies between individuals and over the course of the disease. Recognising the clinicopathological features that most strongly associate with disease outcomes will inform future efforts at patient phenotyping. AIMS: We used a digital pathology workflow, involving high-resolution image acquisition of immunostained slides and opensource software for quantification, to investigate the relationship between clinical and neuropathological features in an autopsy cohort of progressive MS. METHODS: Sequential sections of frontal, cingulate and occipital cortex, thalamus, brain stem (pons) and cerebellum including dentate nucleus (n = 35 progressive MS, females = 28, males = 7; age died = 53.5 years; range 38-98 years) were immunostained for myelin (anti-MOG), neurons (anti-HuC/D) and microglia/macrophages (anti-HLA). The extent of demyelination, neurodegeneration, the presence of active and/or chronic active lesions and quantification of brain and leptomeningeal inflammation was captured by digital pathology. RESULTS: Digital analysis of tissue sections revealed the variable extent of pathology that characterises progressive MS. Microglia/macrophage activation, if found at a higher level in a single block, was typically elevated across all sampled blocks. Compartmentalised (perivascular/leptomeningeal) inflammation was associated with age-related measures of disease severity and an earlier death. CONCLUSION: Digital pathology identified prognostically important clinicopathological correlations in MS. This methodology can be used to prioritise the principal pathological processes that need to be captured by future MS biomarkers.

CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis.

Multiple sclerosis (MS) is characterized by heterogeneity in disease course and prediction of long-term outcome remains a major challenge. Here, we investigate five myeloid markers - CHIT1, CHI3L1, sTREM2, GPNMB and CCL18 - in the cerebrospinal fluid (CSF) at diagnostic lumbar puncture in a longitudinal cohort of 192 MS patients. Through mixed-effects and machine learning models, we show that CHIT1 is a robust predictor for faster disability progression. Integrative analysis of 11 CSF and 26 central nervous system (CNS) parenchyma single-cell/nucleus RNA sequencing samples reveals CHIT1 to be predominantly expressed by microglia located in active MS lesions and enriched for lipid metabolism pathways. Furthermore, we find CHIT1 expression to accompany the transition from a homeostatic towards a more activated, MS-associated cell state in microglia. Neuropathological evaluation in post-mortem tissue from 12 MS patients confirms CHIT1 production by lipid-laden phagocytes in actively demyelinating lesions, already in early disease stages. Altogether, we provide a rationale for CHIT1 as an early biomarker for faster disability progression in MS.

Association of Cortical Lesions With Regional Glutamate, GABA, N-Acetylaspartate, and Myoinositol Levels in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Cortical lesions contribute to disability in multiple sclerosis (MS), but their impact on regional neurotransmitter levels remains to be clarified. We tested the hypothesis that cortical lesions are associated with regional glutamate and gamma-aminobutyric acid (GABA) concentrations within the affected cortical region. METHODS: In this cross-sectional study, we used structural 7T MRI to segment cortical lesions and 7T proton MR-spectroscopy of the bilateral sensorimotor hand areas to quantify regional GABA, glutamate, N-acetylaspartate, and myoinositol concentrations in patients with MS (inclusion criteria: diagnosis of relapsing-remitting [RR] or secondary progressive MS [SPMS]; age 18-80 years) and age and sex-matched healthy controls. Data were collected at a single center between August 2018 and September 2020. Linear mixed-effects models were used to test for associations between metabolite concentrations and cortical lesion volumes within the same MR-spectroscopy voxel. RESULTS: Forty-seven patients with MS (34 RRMS, 13 SPMS; 45.1 ± 12.5 years; 31 women) and 23 healthy controls (44.4 ± 13 years, 15 women) were studied. In patients, higher regional glutamate and lower regional GABA concentrations were associated with larger cortical lesion volume within the MR-spectroscopy voxel [glutamate: 0.61 (95% CI 0.19-1.03) log(mm3), p = 0.005, GABA: -0.71 (-1.24 to -0.18) log(mm3), p = 0.01]. In addition, lower N-acetylaspartate levels [-0.37 (-0.67 to -0.07) log(mm3), p = 0.016] and higher myoinositol levels [0.48 (0.03-0.93) log(mm3), p = 0.037] were associated with a larger regional cortical lesion volume. Furthermore, glutamate concentrations were reduced in patients with SPMS compared with healthy participants [-0.75 (-1.3 to -0.19) mM, p = 0.005] and patients with RRMS [-0.55 (-1.07 to -0.02) mM, p = 0.04]. N-acetylaspartate levels were lower in both patients with RRMS [-0.81 (-1.39 to -0.24) mM, p = 0.003] and SPMS [-1.31 (-2.07 to -0.54) mM, p < 0.001] when compared with healthy controls. Creatine-normalized N-acetylaspartate levels were associated with performance in the 9-hole peg test of the contralateral hand [-0.004 (-0.007 to -0.002) log(s), p = 0.002], and reduced mean creatine-normalized glutamate was associated with increased Expanded Disability Status Scale (R = -0.39, p = 0.02). DISCUSSION: Cortical lesions are associated with local increases in glutamate and a reduction in GABA concentration within the lesional or perilesional tissue. Further studies are needed to investigate the causal relationship between cortical lesions and changes in neurotransmitter concentrations.

Experimental Autoimmune Encephalomyelitis Influences GH-Axis in Female Rats.

Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.

Neuroinflammation: From Molecular Basis to Therapy.

Neuroinflammatory conditions in the central nervous system (CNS) are implicated in the pathogenesis of several neuroimmune disorders such as acquired demyelinating syndromes, autoimmune encephalopathies, acute or chronic bacterial and viral CNS infections as well as multiple sclerosis (MS) [...].

Amniotic fluid stem cell-derived extracellular vesicles educate type 2 conventional dendritic cells to rescue autoimmune disorders in a multiple sclerosis mouse model.

Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.

Microglia and Dendritic Cells as a Source of IL-6 in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease of central nervous system (CNS) characterized by the myelin sheath destruction and compromised nerve signal transmission. Understanding molecular mechanisms driving MS development is critical due to its early onset, chronic course, and therapeutic approaches based only on symptomatic treatment. Cytokines are known to play a pivotal role in the MS pathogenesis with interleukin-6 (IL-6) being one of the key mediators. This study investigates contribution of IL-6 produced by microglia and dendritic cells to the development of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS. Mice with conditional inactivation of IL-6 in the CX3CR1+ cells, including microglia, or CD11c+ dendritic cells, displayed less severe symptoms as compared to their wild-type counterparts. Mice with microglial IL-6 deletion exhibited an elevated proportion of regulatory T cells and reduced percentage of pathogenic IFNγ-producing CD4+ T cells, accompanied by the decrease in pro-inflammatory monocytes in the CNS at the peak of EAE. At the same time, deletion of IL-6 from microglia resulted in the increase of CCR6+ T cells and GM-CSF-producing T cells. Conversely, mice with IL-6 deficiency in the dendritic cells showed not only the previously described increase in the proportion of regulatory T cells and decrease in the proportion of TH17 cells, but also reduction in the production of GM-CSF and IFNγ in the secondary lymphoid organs. In summary, IL-6 functions during EAE depend on both the source and localization of immune response: the microglial IL-6 exerts both pathogenic and protective functions specifically in the CNS, whereas the dendritic cell-derived IL-6, in addition to being critically involved in the balance of regulatory T cells and TH17 cells, may stimulate production of cytokines associated with pathogenic functions of T cells.

N-butyl-ß-carboline-3-carboxylate (ß-CCB) systemic administration promotes remyelination in the cuprizone demyelinating model in mice.

Demyelination is generated in several nervous system illnesses. Developing strategies for effective clinical treatments requires the discovery of promyelinating drugs. Increased GABAergic signaling through γ-aminobutyric acid type A receptor (GABAAR) activation in oligodendrocytes has been proposed as a promyelinating condition. GABAAR expressed in oligodendroglia is strongly potentiated by n-butyl-ß-carboline-3-carboxylate (ß-CCB) compared to that in neurons. Here, mice were subjected to 0.3% cuprizone (CPZ) added in the food to induce central nervous system demyelination, a well-known model for multiple sclerosis. Then ß-CCB (1 mg/Kg) was systemically administered to analyze the remyelination status in white and gray matter areas. Myelin content was evaluated using Black-Gold II (BGII) staining, immunofluorescence (IF), and magnetic resonance imaging (MRI). Evidence indicates that ß-CCB treatment of CPZ-demyelinated animals promoted remyelination in several white matter structures, such as the fimbria, corpus callosum, internal capsule, and cerebellar peduncles. Moreover, using IF, it was observed that CPZ intake induced an increase in NG2+ and a decrease in CC1+ cell populations, alterations that were importantly retrieved by ß-CCB treatment. Thus, the promyelinating character of ß-CCB was confirmed in a generalized demyelination model, strengthening the idea that it has clinical potential as a therapeutic drug.

Fluorinated apelin-13 mediates neuroprotective effects in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease leading to demyelination and axonal loss. Current treatments are immunomodulatory or immunosuppressive drugs acting on the inflammatory component. However, these treatments do not adequately address the crucial aspect of neuroprotection. Recently, an association between an altered balance of adipokines and MS has been proposed as both a risk factor for developing MS and a chronic disease aggravating factor. Specifically, a decrease of apelin plasma levels in MS patients compared to controls correlates with the number of relapses and disease severity. Here we report a dramatic downregulation of apelin levels in the CNS of EAE mice which is also detected in MS patients brain samples compared to controls. Exploiting innovative design and synthesis techniques, we engineered a novel fluorinated apelin-13 peptide characterized by enhanced plasmatic stability compared to its native counterpart. With this peptide, we assessed the potential therapeutic benefits of apelin preventive supplementation in the EAE mouse model. We show that the fluorinated Apelin-13 peptide ameliorates EAE clinical score and preserves myelin content in the EAE MOG model recapitulating the progressive form of disease. These results combined with ex-vivo experiments in brain organotypic slices and in vitro studies in neurons and primary microglia and macrophages suggest that apelin has neuroprotective effects and influences the microglia/macrophages function.

IL-6 Inhibition as a Therapeutic Target in Aged Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) onset at an advanced age is associated with a higher risk of developing progressive forms and a greater accumulation of disability for which there are currently no effective disease-modifying treatments. Immunosenescence is associated with the production of the senescence-associated secretory phenotype (SASP), with IL-6 being one of the most prominent cytokines. IL-6 is a determinant for the development of autoimmunity and neuroinflammation and is involved in the pathogenesis of MS. Herein, we aimed to preclinically test the therapeutic inhibition of IL-6 signaling in experimental autoimmune encephalomyelitis (EAE) as a potential age-specific treatment for elderly MS patients. Young and aged mice were immunized with myelin oligodendrocyte protein (MOG)35-55 and examined daily for neurological signs. Mice were randomized and treated with anti-IL-6 antibody. Inflammatory infiltration was evaluated in the spinal cord and the peripheral immune response was studied. The blockade of IL-6 signaling did not improve the clinical course of EAE in an aging context. However, IL-6 inhibition was associated with an increase in the peripheral immunosuppressive response as follows: a higher frequency of CD4 T cells producing IL-10, and increased frequency of inhibitory immune check points PD-1 and Tim-3 on CD4+ T cells and Lag-3 and Tim-3 on CD8+ T cells. Our results open the window to further studies aimed to adjust the anti-IL-6 treatment conditions to tailor an effective age-specific therapy for elderly MS patients.

Pregnant Women with Multiple Sclerosis: An Overview of Gene Expression and Molecular Interaction Using Bioinformatics Analysis.

Multiple sclerosis (MS) is a common disease in young women of reproductive age, characterized by demyelination of the central nervous system (CNS). Understanding how genes related to MS are expressed during pregnancy can provide insights into the potential mechanisms by which pregnancy affects the course of this disease. This review article presents evidence-based studies on these patients' gene expression patterns. In addition, it constructs interaction networks using bioinformatics tools, such as STRING and KEGG pathways, to understand the molecular role of each of these genes. Bioinformatics research identified 25 genes and 21 signaling pathways, which allows us to understand pregnancy patients' genetic and biological phenomena and formulate new questions about MS during pregnancy.

Fibulin-2 is an extracellular matrix inhibitor of oligodendrocytes relevant to multiple sclerosis.

Impairment of oligodendrocytes and myelin contributes to neurological disorders including multiple sclerosis (MS), stroke, and Alzheimer's disease. Regeneration of myelin (remyelination) decreases the vulnerability of demyelinated axons, but this repair process commonly fails with disease progression. A contributor to inefficient remyelination is the altered extracellular matrix (ECM) in lesions, which remains to be better defined. We have identified fibulin-2 (FBLN2) as a highly upregulated ECM component in lesions of MS and stroke and in proteome databases of Alzheimer's disease and traumatic brain injury. Focusing on MS, the inhibitory role of FBLN2 was suggested in the experimental autoimmune encephalomyelitis (EAE) model, in which genetic FBLN2 deficiency improved behavioral recovery by promoting the maturation of oligodendrocytes and enhancing remyelination. Mechanistically, when oligodendrocyte progenitors were cultured in differentiation medium, FBLN2 impeded their maturation into oligodendrocytes by engaging the Notch pathway, leading to cell death. Adeno-associated virus deletion of FBLN2 in astrocytes improved oligodendrocyte numbers and functional recovery in EAE and generated new myelin profiles after lysolecithin-induced demyelination. Collectively, our findings implicate FBLN2 as a hitherto unrecognized injury-elevated ECM, and a therapeutic target, that impairs oligodendrocyte maturation and myelin repair.

The neuropathobiology of multiple sclerosis.

Chronic low-grade inflammation and neuronal deregulation are two components of a smoldering disease activity that drives the progression of disability in people with multiple sclerosis (MS). Although several therapies exist to dampen the acute inflammation that drives MS relapses, therapeutic options to halt chronic disability progression are a major unmet clinical need. The development of such therapies is hindered by our limited understanding of the neuron-intrinsic determinants of resilience or vulnerability to inflammation. In this Review, we provide a neuron-centric overview of recent advances in deciphering neuronal response patterns that drive the pathology of MS. We describe the inflammatory CNS environment that initiates neurotoxicity by imposing ion imbalance, excitotoxicity and oxidative stress, and by direct neuro-immune interactions, which collectively lead to mitochondrial dysfunction and epigenetic dysregulation. The neuronal demise is further amplified by breakdown of neuronal transport, accumulation of cytosolic proteins and activation of cell death pathways. Continuous neuronal damage perpetuates CNS inflammation by activating surrounding glia cells and by directly exerting toxicity on neighbouring neurons. Further, we explore strategies to overcome neuronal deregulation in MS and compile a selection of neuronal actuators shown to impact neurodegeneration in preclinical studies. We conclude by discussing the therapeutic potential of targeting such neuronal actuators in MS, including some that have already been tested in interventional clinical trials.

The interplay between mitochondrial dysfunction and NLRP3 inflammasome in multiple sclerosis: Therapeutic implications and animal model studies.

Multiple sclerosis (MS) is a complex autoimmune disorder that impacts the central nervous system (CNS), resulting in inflammation, demyelination, and neurodegeneration. The NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome, a multiprotein complex of the innate immune system, serves an essential role in the pathogenesis of MS by regulating the production of pro-inflammatory cytokines (IL-1ß & IL-18) and the induction of pyroptotic cell death. Mitochondrial dysfunction is one of the main potential factors that can trigger NLRP3 inflammasome activation and lead to inflammation and axonal damage in MS. This highlights the importance of understanding how mitochondrial dynamics modulate NLRP3 inflammasome activity and contribute to the inflammatory and neurodegenerative features of MS. The lack of a comprehensive understanding of the pathogenesis of MS and the urge for the introduction of new therapeutic strategies led us to review the therapeutic potential of targeting the interplay between mitochondrial dysfunction and the NLRP3 inflammasome in MS. This paper also evaluates the natural and synthetic compounds that can improve mitochondrial function and/or inhibit the NLRP3 inflammasome, thereby providing neuroprotection. Moreover, it summarizes the evidence from animal models of MS that demonstrate the beneficial effects of these compounds on reducing inflammation, demyelination, and neurodegeneration. Finally, this review advocates for a deeper investigation into the molecular crosstalk between mitochondrial dynamics and the NLRP3 inflammasome as a means to refine therapeutic targets for MS.

Human regulatory memory B cells defined by expression of TIM-1 and TIGIT are dysfunctional in multiple sclerosis.

Background: Regulatory B cells (Bregs) play a pivotal role in suppressing immune responses, yet there is still a lack of cell surface markers that can rigorously identify them. In mouse models for multiple sclerosis (MS), TIM-1 or TIGIT expression on B cells is required for maintaining self-tolerance and regulating autoimmunity to the central nervous system. Here we investigated the activities of human memory B cells that differentially express TIM-1 and TIGIT to determine their potential regulatory function in healthy donors and patients with relapsing-remitting (RR) MS. Methods: FACS-sorted TIM-1+/-TIGIT+/- memory B (memB) cells co-cultured with allogenic CD4+ T cells were analyzed for proliferation and induction of inflammatory markers using flow cytometry and cytokine quantification, to determine Th1/Th17 cell differentiation. Transcriptional differences were assessed by SMARTSeq2 RNA sequencing analysis. Results: TIM-1-TIGIT- double negative (DN) memB cells strongly induce T cell proliferation and pro-inflammatory cytokine expression. The TIM-1+ memB cells enabled low levels of CD4+ T cell activation and gave rise to T cells that co-express IL-10 with IFNγ and IL-17A or FoxP3. T cells cultured with the TIM-1+TIGIT+ double positive (DP) memB cells exhibited reduced proliferation and IFNγ, IL-17A, TNFα, and GM-CSF expression, and exhibited strong regulation in Breg suppression assays. The functional activity suggests the DP memB cells are a bonafide Breg population. However, MS DP memB cells were less inhibitory than HC DP memB cells. A retrospective longitudinal study of anti-CD20 treated patients found that post-treatment DP memB cell frequency and absolute number were associated with response to therapy. Transcriptomic analyses indicated that the dysfunctional MS-derived DP memB/Breg population exhibited increased expression of genes associated with T cell activation and survival (CD80, ZNF10, PIK3CA), and had distinct gene expression compared to the TIGIT+ or TIM-1+ memB cells. Conclusion: These findings demonstrate that TIM-1/TIGIT expressing memory B cell subsets have distinct functionalities. Co-expression of TIM-1 and TIGIT defines a regulatory memory B cell subset that is functionally impaired in MS.

Role of ferroptosis in neuroimmunity and neurodegeneration in multiple sclerosis revealed by multi-omics data.

Previous studies have found that ferroptosis plays an important role in a variety of neurological diseases. However, the precise role of ferroptosis in the multiple sclerosis patients remains uncertain. We defined and validated a computational metric of ferroptosis levels. The ferroptosis scores were computed using the AUCell method, which reflects the enrichment scores of ferroptosis-related genes through gene ranking. The reliability of the ferroptosis score was assessed using various methods, involving cells induced to undergo ferroptosis by six different ferroptosis inducers. Through a comprehensive approach integrating snRNA-seq, spatial transcriptomics, and spatial proteomics data, we explored the role of ferroptosis in multiple sclerosis. Our findings revealed that among seven sampling regions of different white matter lesions, the edges of active lesions exhibited the highest ferroptosis score, which was associated with activation of the phagocyte system. Remyelination lesions exhibit the lowest ferroptosis score. In the cortex, ferroptosis score were elevated in neurons, relevant to a variety of neurodegenerative disease-related pathways. Spatial transcriptomics demonstrated a significant co-localization among ferroptosis score, neurodegeneration and microglia, which was verified by spatial proteomics. Furthermore, we established a diagnostic model of multiple sclerosis based on 24 ferroptosis-related genes in the peripheral blood. Ferroptosis might exhibits a dual role in the context of multiple sclerosis, relevant to both neuroimmunity and neurodegeneration, thereby presenting a promising and novel therapeutic target. Ferroptosis-related genes in the blood that could potentially serve as diagnostic and prognostic markers for multiple sclerosis.