Abstract Multiple sclerosis (MS) is an immune-mediated demyelinating disease, with progressive neurodegeneration that is refractory to current therapies. Here, we identify microglia aging as a major factor responsible for age-related loss of beneficial demyelinating and remyelinating brain functions. Brain transcriptomics and in situ spatial gene transcription analysis revealed significant oligodendrocyte loss in both young and aged mice during cuprizone-induced experimental demyelination, but impaired microglial activation in aged mice. Age-related defects in microglial activation were associated with reduced clearance of dead myelin and accumulation of lipid droplets, and impaired remyelination, implying exhaustion of microglial function in the aged mouse brain. Transcriptomic analysis of human brain samples from MS donors with matched disease course and severity and with chronic active and inactive lesions, validated that microglial activation is strongly reduced with increasing age. To investigate microglial responses after repeated demyelinating insults and their direct impact on myelin integrity, we established an experimental model of repeated demyelinating episodes in young and aged mice to recapitulate MS features. Notably, aged mice developed a progressive neuroinflammatory response following sequential demyelinating episodes, in contrast to the alternating cycles of demyelination and remyelination reminiscent of relapsing-remitting MS that were observed in young mice. Microglia depletion and repopulation using a CSF1R antagonist recovered demyelination-remyelination capacity in aged mice. The results indicate that microglia aging is a major determinant in the pathogenesis of progressive MS, and that microglia replacement represents a promising therapeutic approach.

OBJECTIVE:The neurodegenerative processes driving the build-up of disability in progressive multiple sclerosis (P-MS) have not been fully elucidated. Recent data link cellular senescence (CS) to neurodegeneration. We investigated for evidence of CS in P-MS and sought to determine its pattern. METHODS:We used 53BP1, p16, and lipofuscin as markers of CS in white matter lesions (WMLs), normal appearing white matter (NAWM), normal appearing cortical gray matter (NAGM), control white matter (CWM), and control gray matter (CGM) on autopsy material from patient with P-MS and healthy controls. Senescence-associated secretory phenotype (SASP) factors were quantified in cerebrospinal fluid (CSF). RESULTS:P16+ cell counts were significantly increased in WMLs and GMLs, compared with NAWM, CWM, NAGM, and CGM and lipofuscin+ cells were significantly increased in WMLs, compared with NAWM and CWM, indicating more abundant CS in demyelinated lesions. The 53BP1+ cells in WMLs were significantly increased compared with NAWM and CWM. The 53BP1+ and p16+ cells were found significantly more abundant in acute active WMLs and GMLs, compared with chronic inactive lesions. Co-localization studies showed evidence of CS in neurons, astrocytes, oligodendrocytes, microglia, and macrophages. Among the quantified CSF SASP factors, IL-6, MIF, and MIP1a levels correlated with 53BP1+ cell counts in NAGM, whereas IL-10 levels correlated with p16+ cell counts in NAWM. P16+ cell counts in WMLs exhibited an inverse correlation with time to requiring a wheelchair and with age at death. INTERPRETATION:Our data indicates that CS primarily affects actively demyelinating gray and WMLs. A higher senescent cell load in P-MS is associated with faster disability progression and death. ANN NEUROL 2025;97:1074-1087.

IntroductionViruses, particularly Epstein–Barr virus (EBV), are strongly implicated in multiple sclerosis (MS) pathogenesis, yet reliable biomarkers of active viral replication remain limited. Double-stranded RNA (dsRNA) represents a hallmark of viral replication and may serve as a measurable indicator of viral infection.MethodsWe developed a sandwich ELISA to quantify dsRNA levels in matched plasma and cerebrospinal fluid (CSF) samples from 70 treatment-naïve MS patients at first clinical onset and in plasma from 26 sex- and age-matched healthy controls. We additionally assessed plasma and CSF antiviral cytokine concentrations and designed an indirect ELISA to measure anti-dsRNA antibody levels using poly(I:C) as the target antigen.ResultsPlasma dsRNA levels were significantly elevated in MS patients compared to controls and positively correlated with antiviral cytokines, including GM-CSF, IFN-λ1, IFN-λ2/3, IFN-γ, IFN-α2, and IL-12p70. CSF samples exhibited increased IP-10 and IL-8 levels, and the single patient with detectable CSF dsRNA showed among the highest concentrations of both cytokines. A subset of ten patients (14%) with serological evidence of atypical EBV reactivation (EBNA1 IgG+/IgM+) had higher plasma dsRNA and antiviral cytokine levels than the remaining patients. Anti-dsRNA IgM, but not IgG or IgA, correlated positively with plasma dsRNA in both MS patients and controls, yet anti-dsRNA IgM levels were significantly reduced in MS compared to controls.DiscussionOur findings identify increased plasma viral dsRNA coupled with reduced anti-dsRNA IgM antibody levels as potential biomarkers for a subpopulation of early MS patients and indicate a dysregulated anti-viral immune response.

Humanized immune system (HIS) mice are powerful tools for studying human immune system function and dysfunction and developing human-specific immunotherapeutics. The availability of sophisticated super immunodeficient mouse strains has allowed immune system humanization using transplants of human peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells. HIS mice are used extensively in immune-oncology, while there are fewer studies in autoimmunity, especially multiple sclerosis (MS). Using the protocol described here, we generated HIS mice that show key features of MS not represented in other widely used MS models [1]. Severely immunodeficient NOD.Cg-B2mem1Tac Prkdcscid Il2rgtm1Sug /JicTac (B2m-NOG) mice, which lack murine B, T, and NK cells and murine major histocompatibility class I molecules and have defective innate immune responses, were transplanted with PBMC from HLA-DRB1-genotyped MS patients and healthy donors. Mice were successfully engrafted with hCD4 and hCD8 T and B lymphocytes and developed both spontaneous and experimental autoimmune encephalomyelitis (EAE)-enhanced T-cell lesions in the central nervous system. B-cell engraftment was highest in mice receiving cells from MS patients with serological evidence for Epstein-Barr virus (EBV) reactivation. This humanized MS model shows advantages over EAE, particularly spontaneous hCD8 T-cell lesions in the brain and spinal cord, mixed hCD8/hCD4 T-cell lesions in EAE-immunized mice, and more severe lesions in mice engrafted with PBMC from MS donors carrying the DRB1*15 MS susceptibility allele compared to DRB1*15-positive healthy and DRB1*13-positive MS donors. MS HIS mice represent simple and rapid tools for investigating human immunopathology and the efficacy of therapeutics at a personalized level. Key features • Humanization of severely immunodeficient B2m-NOG mice with human PBMC. • Engraftment analysis of human immune system in mice using multicolor flow cytometry. • Animal familiarization and handling techniques. • Epstein-Barr virus latency evaluation in human plasma. • Ex vivo characterization of engrafted T-cell cytokine responses.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, with autoimmune and neurodegenerative components, recently linked with Epstein-Barr virus (EBV) infection. To investigate immunological alterations associated with EBV reactivation (EBV-R) in MS we analyzed immune cell profiles in matched peripheral blood (PB) and cerebrospinal fluid (CSF) samples from treatment-naive MS patients, in relation to plasma EBV serology markers. In our Hellenic MS cohort, 12 of 33 patients (39 %) exhibited serological evidence of EBV-R (VCA IgG+ with VCA IgA+, VCA IgM+, or EA(D) IgG+), and 7 of these 12 also tested positive for plasma BZLF1 mRNA, indicative of lytic infection. EBV-R patients showed reduced CD19+ B cells in PB and equal proportions in CSF compared to patients with latent EBV infection. Conversely, EBV-R patients showed increased cytotoxic CD56dim NK cells and CD14+ monocytes in PB, while cytotoxic CD56dim NK cells remained absent in the CSF, regardless of EBV status. Proportions of regulatory CD56bright NK cells were reduced in both PB and CSF during EBV-R. Our results reveal that MS patients with serological evidence of EBV-R show altered immune responses, with reduced B cell proportions in the PB in the presence of expanded cytotoxic NK cells, and unaffected B cell proportions in CSF in the absence of cytotoxic NK cell surveillance.

Background Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. Methods We used constitutive MφIKKβΚΟ mice, in which depletion of IKKβ, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚβKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG 35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. Results Global depletion of IKKβ from myeloid cells in MφIKKβΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKβ only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKβ, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKβ resulted in enhanced late long-term potentiation in EAE. Conclusions IKKβ-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKβ deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKβ on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Comparative immune profiling was performed in peripheral blood and CSF samples of 33 newly diagnosed, treatment-naive MS patients and 6 healthy controls by flow cytometry. We focused on basic immune cell markers CD45, CD66b, CD14, CD19, CD56, CD3, CD4, and CD8. Results in blood revealed elevated monocyte counts in RRMS/CIS versus PPMS, and increased neutrophil-to-lymphocyte ratio (NLR) during relapse. A higher blood CD56bright/CD56dim NK cell ratio was observed at the first MS clinical episode. These results highlight the importance of NLR and NK cell ratios in distinguishing MS subtypes, providing valuable insights for early disease prognosis and management.

Microglia are strongly implicated in demyelinating neurodegenerative diseases with increasing evidence for roles in protection and healing, but the mechanisms that control CNS remyelination are poorly understood. Here, we show that microglia-specific deletion of tumor necrosis factor receptor 1 (TNFR1) and pharmacological inhibition of soluble TNF (solTNF) or downstream interleukin-1 receptor (IL-1R) allow maturation of highly activated disease-associated microglia with increased size and myelin phagocytosis capacity that accelerate cortical remyelination and motor recovery. Single-cell transcriptomic analysis of cortex at disease onset reveals that solTNF inhibition enhances reparative IL-10-responsive while preventing damaging IL-1-related signatures of disease-associated microglia. Longitudinal brain transcriptome analysis through disease reveals earlier recovery upon therapeutic loss of microglia TNFR1. The functional relevance of microglia inflammatory polarization pathways for disease is validated in vivo. Furthermore, disease-state microglia producing downstream IL-1/IL-18/caspase-11 targets are identified in human demyelinating lesions. Overall, redirecting disease microglia polarization by targeting cytokines is a potential approach for improving CNS repair in demyelinating disorders.

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein–Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.

Autoimmune diseases affecting the CNS not only overcome immune privilege mechanisms that protect neural tissues but also peripheral immune tolerance mechanisms towards self. Together with antigen-specific T cells, myeloid cells are main effector cells in CNS autoimmune diseases such as multiple sclerosis, but the relative contributions of blood-derived monocytes and the tissue resident macrophages to pathology and repair is incompletely understood. Through the study of oxidized mannan-conjugated myelin oligodendrocyte glycoprotein 35-55 (OM-MOG), we show that peripheral maturation of Ly6C hi CCR2 + monocytes to Ly6C hi MHCII + PD-L1 + cells is sufficient to reverse spinal cord inflammation and demyelination in MOG-induced autoimmune encephalomyelitis. Soluble intradermal OM-MOG drains directly to the skin draining lymph node to be sequestered by subcapsular sinus macrophages, activates Ly6C hi CCR2 + monocytes to produce MHC class II and PD-L1, prevents immune cell trafficking to spinal cord, and reverses established lesions. We previously showed that protection by OM-peptides is antigen specific. Here, using a neutralizing anti-PD-L1 antibody in vivo and dendritic cell-specific Pdl1 knockout mice, we further demonstrate that PD-L1 in non-dendritic cells is essential for the therapeutic effects of OM-MOG. These results show that maturation of circulating Ly6C hi CCR2 + monocytes by OM-myelin peptides represents a novel mechanism of immune tolerance that reverses autoimmune encephalomyelitis.

Microglia are key players in Multiple Sclerosis (MS), expressing many susceptibility genes for this disease. They constantly survey the brain microenvironment, but the precise functional relationships between microglia and pathological processes remain unknown. We performed a detailed assessment of microglial dynamics in three distinct grey matter regions in a cuprizone-induced demyelination model. We found that microglial activation preceded detectable demyelination and showed regional specificities, such as prominent phagocytic activity in cortical layer 5 and early hypertrophic morphology in hippocampal CA1. Demyelination happened earliest in cortical layer 5, although was more complete in CA1. In cortical layer 2/3, microglial activation and demyelination were less pronounced but microglia became hyper-ramified with slower process movement during remyelination, thereby maintaining local brain surveillance. Profiling of microglia using specific morphological and motility parameters revealed region-specific heterogeneity of microglial responses in the grey matter that might serve as sensitive indicators of progression in CNS demyelinating diseases.

Background: In the context of the coronavirus disease 2019 (COVID-19) pandemic, the constant needs of people with multiple sclerosis (PwMS) and their caregivers were urgently highlighted. Aim: The present study aims to capture the effects of the COVID-19 pandemic in several aspects of the quality of life of PwMS, in perception and behavior to COVID-19 and multiple sclerosis (MS), as well as concerning healthcare, working conditions, and the willingness toward COVID-19 vaccination. Methods: This study is an initiative of the Hellenic Academy of Neuroimmunology (HEL.A.NI.) and it has been included in the MS Data Alliance (MSDA) Catalog, which can be accessed after creating an account on https://msda.emif-catalogue.eu/login. Two online questionnaires were administered: (i) impact of the COVID-19 pandemic on the quality of life, behavior, and healthcare of PwMS (Questionnaire A) and (ii) vaccination against COVID-19 (Questionnaire B). People with MS were invited to participate by the Hellenic Federation of Persons with Multiple Sclerosis (HFoPwMS). Results: Three-hundred-ninety PwMS responded to Questionnaire A, whereas 176 PwMS provided answers for Questionnaire B. Older age, longer disease duration, and higher MS-related disability were associated with the increased perceived sensitivity toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as well as the increased perceived severity of COVID-19 upon potential infection. A significant proportion of PwMS experienced restricted access to MS-related health professionals, disease-modifying therapy (DMT) prescription, and/or to MS-related laboratory examination due to the pandemic. Subgroups of PwMS reported exacerbated symptoms (i.e., chronic MS-related symptoms, fatigue and/or worsening of pre-existing fatigue, and sexual dysfunction and or/worsening of pre-existing sexual dysfunction). Overall, the majority of the participants reported either a strong willingness to get vaccinated against COVID-19 or a likeliness to undergo vaccination. Being aware of the HEL.A.NI. recommendations regarding COVID-19 vaccination for PwMS were reported to increase the willingness of the participants to receive the vaccine. Conclusions: Our results highlight the necessity of scientific and patient organizations in taking joint action to increase awareness on health-related issues during the pandemic and to provide accurate and up-to-date guidance for PwMS. Online information and communications technology (ICT) tools for polling public belief and behavior may prove valuable as means of retaining active routes of communication between stakeholders.

BACKGROUNDDuring inflammatory demyelination TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF, whereas TNFR2 mediates beneficial effects of transmembrane TNF through oligodendrocytes, microglia, and possibly other cell types. This model supports use of selective inhibitors of soluble TNF/TNFR1 as antinflammatory drugs for CNS disease. A potential obstacle is the neuroprotective effect of soluble TNF pretreatment described in cultured neurons, but the in vivo relevance is unknown. METHODSTo address this question we generated mice with neuron-specific depletion of TNFR1, TNFR2 or IKKβ and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of soluble TNF (and therefore TNFR1) neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wildtype and TNF and TNF receptor knockout cells and measuring NMDA excitotoxicity in vitro .RESULTSNeither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid excitotoxicity model in mice, and limited hippocampal neuron death. The neuroprotective effects of neuronal TNFR2 observed in vivo were further investigated in vitro . Here as expected, pretreatment of astrocyte-neuron co-cultures with soluble TNF protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte transmembrane TNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. CONCLUSIONSThese results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously-described neuroprotective effects of soluble TNF (and therefore TNFR1) against glutamate excitotoxicity in vitro are indirect, and mediated by astrocyte transmembrane TNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of soluble TNF/TNFR1 with maintenance of TNFR2 function would have anti-inflammatory and neuroprotective properties required for the safe treatment of CNS disease.