Neurofilament Light Chain Serum Levels Mirror Age and Disability in Secondary Progressive Multiple Sclerosis: A Cross-Sectional Study.

OBJECTIVES: To assess neurofilament light chain serum (sNfL) levels in patients with secondary progressive multiple sclerosis (SP-MS). METHODS: Using a single molecule array, we analyzed sNfL levels in a cross-sectional cohort study of 153 patients with SP-MS hospitalized for rehabilitation in a clinic specialized in the care for patients with multiple sclerosis (MS). In addition, we investigated the correlation of disease activity with sNfL levels in 36 patients with relapsing-remitting MS (RR-MS). RESULTS: Mean sNfL levels in patients with SP-MS were consistently elevated when compared with age-matched controls and patients with RR-MS. In SP-MS, age dependency of sNfL levels was pronounced, whereas patients with RR-MS younger than 41 years without recent disease activity were not distinguishable from age-matched healthy controls. In a multivariate analysis, clinical disability was a risk factor for elevated sNfL levels in SP-MS, whereas no correlation with comorbidities, such as cardiovascular disease, diabetes mellitus, smoking status, or vitamin D serum levels, could be detected. DISCUSSION: These findings highlight that measurement of sNfL levels represents a useful tool to assess the extent of neuroaxonal damage as a surrogate for clinical progression in patients with SP-MS, when age and disease activity as major confounders are taken into account.

An Animal Model for Chronic Meningeal Inflammation and Inflammatory Demyelination of the Cerebral Cortex.

Modeling chronic cortical demyelination allows the study of long-lasting pathological changes observed in multiple sclerosis such as failure of remyelination, chronically disturbed functions of oligodendrocytes, neurons and astrocytes, brain atrophy and cognitive impairments. We aimed at generating an animal model for studying the consequences of chronic cortical demyelination and meningeal inflammation. To induce long-lasting cortical demyelination and chronic meningeal inflammation, we immunized female Lewis rats against myelin oligodendrocyte glycoprotein (MOG) and injected lentiviruses for continuing overexpression of the cytokines TNFα and IFNγ in the cortical brain parenchyma. Immunization with MOG and overexpression of TNFα and IFNγ led to widespread subpial demyelination and meningeal inflammation that were stable for at least 10 weeks. We demonstrate here that immunization with MOG is necessary for acute as well as chronic cortical demyelination. In addition, long-lasting overexpression of TNFα and IFNγ in the brain parenchyma is sufficient to induce chronic meningeal inflammation. Our model simulates key features of chronic cortical demyelination and inflammation, reminiscent of human multiple sclerosis pathology. This will allow molecular, cellular and functional investigations for a better understanding of the adaptation mechanisms of the cerebral cortex in multiple sclerosis.

Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2-/- mice using CRISPR/Cas9-mediated genome editing. Rnaset2-/- mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2-/- mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies.

High class filtering facepiece (FFP) are fundamental and effective in protection of emergency health care workers: an observational cohort study in a German community.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious airborne virus inducing pandemic coronavirus disease 2019 (COVID-19). This is most relevant for medical staff working under harmful conditions in emergencies often dealing with patients and an undefined SARS-CoV-2 status. We aimed to measure the effect of high-class filtering facepieces (FFP) in emergency medical service (EMS) staff by analyzing seroprevalence and history of positive polymerase chain reaction (PCR) for SARS-CoV-2. METHOD: This observational cohort study included workers in EMS, who were compared with hospital staff (HS) and staff, which was not directly involved in patient care (NPC). All direct patient contacts of EMS workers were protected by FFP2/N95 (filtering face piece protection class 2/non-oil-based particulates filter efficiency 95%) masks, whereas HS was protected by FFP2/N95 exclusively when a patient had a proven or suspected SARS-CoV-2 infection. NPC was not protected by higher FFP. The seroprevalence of SARS-CoV-2 antibodies was analyzed by immunoassay by end of 12/2020 together with the history of a positive PCR. In addition, a self-assessment was performed regarding the quantity of SARS-CoV-2 positive contacts, about flu symptoms and personal belief of previous COVID-19 infections. RESULTS: The period in which contact to SARS-CoV-2 positive patients has been possible was 10 months (March to December 2020)-with 54,681 patient contacts documented for EMS-either emergencies (n = 33,241) or transportation services (n = 21,440). Seven hundred-thirty (n = 730) participants were included into the study (n = EMS: 325, HS: 322 and NPC: 83). The analysis of the survey showed that the exposure to patients with an unknown and consecutive positive SARS-CoV-2 result was significantly higher for EMS when compared to HS (EMS 55% vs. HS 30%, p = 0.01). The incidence of a SARS-CoV-2 infection in our cohort was 1.2% (EMS), 2.2% (HS) and 2.4% (NPC) within the three groups (ns) and lowest in EMS. Furthermore, the belief of previous COVID-19 was significant higher in EMS (19% vs. 10%), CONCLUSION: The consistent use of FFP2/N95 in EMS is able to prevent work-related SARS-CoV-2 infections in emergency situations. The significance of physical airway protection in exposed medical staff is still relevant especially under the aspect of new viral variants and unclear effectiveness of new vaccines.

SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

Targeted metabolomics revealed changes in phospholipids during the development of neuroinflammation in Abcd1tm1Kds mice and X-linked adrenoleukodystrophy patients.

X-linked adrenoleukodystrophy (X-ALD) is the most common leukodystrophy. Despite intensive research in recent years, it remains unclear, what drives the different clinical disease courses. Due to this missing pathophysiological link, therapy for the childhood cerebral disease course of X-ALD (CCALD) remains symptomatic; the allogenic hematopoietic stem cell transplantation or hematopoietic stem-cell gene therapy is an option for early disease stages. The inclusion of dried blood spot (DBS) C26:0-lysophosphatidylcholine to newborn screening in an increasing number of countries is leading to an increasing number of X-ALD patients diagnosed at risk for CCALD. Current follow-up in asymptomatic boys with X-ALD requires repetitive cerebral MRIs under sedation. A reliable and easily accessible biomarker that predicts CCALD would therefore be of great value. Here we report the application of targeted metabolomics by AbsoluteIDQ p180-Kit from Biocrates to search for suitable biomarkers in X-ALD. LysoPC a C20:3 and lysoPC a C20:4 were identified as metabolites that indicate neuroinflammation after induction of experimental autoimmune encephalitis in the serum of Abcd1tm1Kds mice. Analysis of serum from X-ALD patients also revealed different concentrations of these lipids at different disease stages. Further studies in a larger cohort of X-ALD patient sera are needed to prove the diagnostic value of these lipids for use as early biomarkers for neuroinflammation in CCALD patients.

Blood-brain barrier resealing in neuromyelitis optica occurs independently of astrocyte regeneration.

Approximately 80% of neuromyelitis optica spectrum disorder (NMOSD) patients harbor serum anti-aquaporin-4 autoantibodies targeting astrocytes in the CNS. Crucial for NMOSD lesion initiation is disruption of the blood-brain barrier (BBB), which allows the entrance of Abs and serum complement into the CNS and which is a target for new NMOSD therapies. Astrocytes have important functions in BBB maintenance; however, the influence of their loss and the role of immune cell infiltration on BBB permeability in NMOSD have not yet been investigated. Using an experimental model of targeted NMOSD lesions in rats, we demonstrate that astrocyte destruction coincides with a transient disruption of the BBB and a selective loss of occludin from tight junctions. It is noteworthy that BBB integrity is reestablished before astrocytes repopulate. Rather than persistent astrocyte loss, polymorphonuclear leukocytes (PMNs) are the main mediators of BBB disruption, and their depletion preserves BBB integrity and prevents astrocyte loss. Inhibition of PMN chemoattraction, activation, and proteolytic function reduces lesion size. In summary, our data support a crucial role for PMNs in BBB disruption and NMOSD lesion development, rendering their recruitment and activation promising therapeutic targets.

Pre-treatment with the viral Toll-like receptor 3 agonist poly(I:C) modulates innate immunity and protects neutropenic mice infected intracerebrally with Escherichia coli.

BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.

B cell depletion can be effective in multiple sclerosis but failed in a patient with advanced childhood cerebral X-linked adrenoleukodystrophy.

Rituximab exerts its clinical efficacy by its specific pattern of depletion of CD20+ B lymphocytes and it has been demonstrated that rituximab is an effective treatment for relapsing remitting multiple sclerosis. X-linked adrenoleukodystrophy (X-ALD), the most common monogenetic neuroinflammatory disorder, shares substantial overlap with multiple sclerosis in the neuropathological changes found in brain tissues in advanced stages of the disease. While there is no effective therapy for these patients, we hypothesized that rituximab might be effective in arresting the neuroinflammatory process. Our detailed clinical, imaging and immunological data revealed that rituximab is not effective in advanced stages of X-ALD and consequently should not be applied for compassionate use in these patients.

Microglia damage precedes major myelin breakdown in X-linked adrenoleukodystrophy and metachromatic leukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) are two relatively common examples of hereditary demyelinating diseases caused by a dysfunction of peroxisomal or lysosomal lipid degradation. In both conditions, accumulation of nondegraded lipids leads to the destruction of cerebral white matter. Because of their high lipid content, oligodendrocytes are considered key to the pathophysiology of these leukodystrophies. However, the response to allogeneic stem cell transplantation points to the relevance of cells related to the hematopoietic lineage. In the present study, we aimed to better characterize the pathogenetic role of microglia in the above-mentioned diseases. Applying recently established microglia markers to human autopsy cases of X-ALD and MLD we were able to delineate distinct lesion stages in evolving demyelinating lesions. The immune-phenotype of microglia was altered already early in lesion evolution, and microglia loss preceded full-blown myelin degeneration both in X-ALD and MLD. DNA fragmentation indicating phagocyte death was observed in areas showing microglia loss. The morphology and dynamics of phagocyte decay differed between the diseases and between lesion stages, hinting at distinct pathways of programmed cell death. In summary, the present study shows an early and severe damage to microglia in the pathogenesis of X-ALD and MLD. This hints at a central pathophysiologic role of these cells in the diseases and provides evidence for an ongoing transfer of toxic substrates primarily enriched in myelinating cells to microglia.

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.

Laquinimod, a prototypic quinoline-3-carboxamide and aryl hydrocarbon receptor agonist, utilizes a CD155-mediated natural killer/dendritic cell interaction to suppress CNS autoimmunity.

BACKGROUND: Quinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1). METHODS: We explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed. RESULTS: We demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing. CONCLUSIONS: This study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.

Maintenance of high proteolipid protein level in adult central nervous system myelin is required to preserve the integrity of myelin and axons.

Proteolipid protein (PLP) is the most abundant integral membrane protein in central nervous system (CNS) myelin. Expression of the Plp-gene in oligodendrocytes is not essential for the biosynthesis of myelin membranes but required to prevent axonal pathology. This raises the question whether the exceptionally high level of PLP in myelin is required later in life, or whether high-level PLP expression becomes dispensable once myelin has been assembled. Both models require a better understanding of the turnover of PLP in myelin in vivo. Thus, we generated and characterized a novel line of tamoxifen-inducible Plp-mutant mice that allowed us to determine the rate of PLP turnover after developmental myelination has been completed, and to assess the possible impact of gradually decreasing amounts of PLP for myelin and axonal integrity. We found that 6 months after targeting the Plp-gene the abundance of PLP in CNS myelin was about halved, probably reflecting that myelin is slowly turned over in the adult brain. Importantly, this reduction by 50% was sufficient to cause the entire spectrum of neuropathological changes previously associated with the developmental lack of PLP, including myelin outfoldings, lamellae splittings, and axonal spheroids. In comparison to axonopathy and gliosis, the infiltration of cytotoxic T-cells was temporally delayed, suggesting a corresponding chronology also in the genetic disorders of PLP-deficiency. High-level abundance of PLP in myelin throughout adult life emerges as a requirement for the preservation of white matter integrity.

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood-brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.

Differential contribution of immune effector mechanisms to cortical demyelination in multiple sclerosis.

Cortical demyelination is a widely recognized hallmark of multiple sclerosis (MS) and correlate of disease progression and cognitive decline. The pathomechanisms initiating and driving gray matter damage are only incompletely understood. Here, we determined the infiltrating leukocyte subpopulations in 26 cortical demyelinated lesions of biopsied MS patients and assessed their contribution to cortical lesion formation in a newly developed mouse model. We find that conformation-specific anti-myelin antibodies contribute to cortical demyelination even in the absence of the classical complement pathway. T cells and natural killer cells are relevant for intracortical type 2 but dispensable for subpial type 3 lesions, whereas CCR2+ monocytes are required for both. Depleting CCR2+ monocytes in marmoset monkeys with experimental autoimmune encephalomyelitis using a novel humanized CCR2 targeting antibody translates into significantly less cortical demyelination and disease severity. We conclude that biologics depleting CCR2+ monocytes might be attractive candidates for preventing cortical lesion formation and ameliorating disease progression in MS.

The Early Adaptive Immune Response in the Pathophysiological Process of Pneumococcal Meningitis.

BACKGROUND: The adaptive immune system has been considered to play a minimal role in the early host response during bacterial meningitis. METHODS: We investigated the progression and outcome of pneumococcal meningitis in Rag1-/- mice lacking functional B and T cells by assessing overall and symptom-free survival, bacteriological and histological studies, as well as flow cytometry and measurements of proinflammatory mediators. RESULTS: The intracerebral injection of S. pneumoniae D39 induced the recruitment of B and T cells (CD4+, γδ and natural killer) into the brain of wild-type mice. Mice with no functional B and T cells developed clinical symptoms and succumbed to the infection earlier than the wild-type group. In the CNS, Rag1-/- mice showed lower levels of interleukin 1ß, reduced microglial proliferation, and impaired granulocyte recruitment with an earlier spread of pneumococci into the bloodstream, compared with wild-type mice. Lack of B and T cells resulted in a severe impairment of bacterial clearance in blood, spleen, and liver and an exaggerated systemic inflammatory response. CONCLUSIONS: B and T cells are important effector cells delaying the spread of pneumococci from the brain to the systemic circulation and shaping the immune response, thereby prolonging the survival of the host in the absence of antibiotic treatment.

A New Targeted Model of Experimental Autoimmune Encephalomyelitis in the Common Marmoset.

Multiple sclerosis (MS) is the most common cause for sustained disability in young adults, yet treatment options remain very limited. Although numerous therapeutic approaches have been effective in rodent models of experimental autoimmune encephalomyelitis (EAE), only few proved to be beneficial in patients with MS. Hence, there is a strong need for more predictive animal models. Within the past decade, EAE in the common marmoset evolved as a potent, alternative model for MS, with immunological and pathological features resembling more closely the human disease. However, an often very rapid and severe disease course hampers its implementation for systematic testing of new treatment strategies. We here developed a new focal model of EAE in the common marmoset, induced by myelin oligodendrocyte glycoprotein (MOG) immunization and stereotactic injections of proinflammatory cytokines. At the injection site of cytokines, confluent inflammatory demyelinating lesions developed that strongly resembled human MS lesions. In a proof-of-principle treatment study with the immunomodulatory compound laquinimod, we demonstrate that targeted EAE in marmosets provides a promising and valid tool for preclinical experimental treatment trials in MS research.

Remyelination After Cuprizone-Induced Demyelination Is Accelerated in Juvenile Mice.

Remyelination capacity decreases with age in adult mice, but data comparing remyelination capacity after toxic demyelination in developing mice versus adult mice are not available. We treated 3-week-old and adult C57BL/6 mice with cuprizone for 1 to 5 weeks and studied demyelination/remyelination and cellular reactions in the corpus callosum and motor cortex by histology, immunohistochemistry, and electron microscopy. We compared results between the 2 treated groups and age-matched controls. In juvenile mice, significant demyelination was detectable in the corpus callosum on Week 2 and in the motor cortex on Week 5. Oligodendrocyte loss, microglial activation, and acute axonal damage peaked on Week 2. Increased numbers of oligodendrocyte precursor cells were evident on Week 1, and remyelination was detectable on Week 3. Juvenile mice showed more rapid demyelination than adult mice, which may be related to greater vulnerability of oligodendrocytes, lower myelin content, or dose-dependent cuprizone effects. Earlier activation of microglia and proliferation of oligodendrocyte precursor cells probably contributed to accelerated remyelination and less pronounced axonal damage. Our data indicate that oligodendroglial regeneration and remyelination are enhanced in the maturing rodent brain compared with the young-adult rodent brain.