Myeloid cell iron uptake pathways and paramagnetic rim formation in multiple sclerosis

In multiple sclerosis (MS), sustained inflammatory activity can be visualized by iron-sensitive magnetic resonance imaging (MRI) at the edges of chronic lesions. These paramagnetic rim lesions (PRLs) are associated with clinical worsening, although the cell type-specific and molecular pathways of iron uptake and metabolism are not well known. We studied two postmortem cohorts: an exploratory formalin-fixed paraffin-embedded (FFPE) tissue cohort of 18 controls and 24 MS cases and a confirmatory snap-frozen cohort of 6 controls and 14 MS cases. Besides myelin and non-heme iron imaging, the haptoglobin-hemoglobin scavenger receptor CD163, the iron-metabolizing markers HMOX1 and HAMP as well as immune-related markers P2RY12, CD68, C1QA and IL10 were visualized in myeloid cell (MC) subtypes at RNA and protein levels across different MS lesion areas. In addition, we studied PRLs in vivo in a cohort of 98 people with MS (pwMS) via iron-sensitive 3 T MRI and haptoglobin genotyping by PCR. CSF samples were available from 38 pwMS for soluble CD163 (sCD163) protein level measurements by ELISA. In postmortem tissues, we observed that iron uptake was linked to rim-associated C1QA-expressing MC subtypes, characterized by upregulation of CD163, HMOX1, HAMP and, conversely, downregulation of P2RY12. We found that pwMS with [Formula: see text] 4 PRLs had higher sCD163 levels in the CSF than pwMS with [Formula: see text] 3 PRLs with sCD163 correlating with the number of PRLs. The number of PRLs was associated with clinical worsening but not with age, sex or haptoglobin genotype of pwMS. However, pwMS with Hp2-1/Hp2-2 haplotypes had higher clinical disability scores than pwMS with Hp1-1. In summary, we observed upregulation of the CD163-HMOX1-HAMP axis in MC subtypes at chronic active lesion rims, suggesting haptoglobin-bound hemoglobin but not transferrin-bound iron as a critical source for MC-associated iron uptake in MS. The correlation of CSF-associated sCD163 with PRL counts in MS highlights the relevance of CD163-mediated iron uptake via haptoglobin-bound hemoglobin. Also, while Hp haplotypes had no noticeable influence on PRL counts, pwMS carriers of a Hp2 allele might have a higher risk to experience clinical worsening

Lesional Antibody Synthesis and Complement Deposition Associate With De Novo Antineuronal Antibody Synthesis After Spinal Cord Injury

BACKGROUND AND OBJECTIVES: Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane. METHODS: This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored. RESULTS: Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier. DISCUSSION: This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes

Interleukin-6 receptor blockade in treatment-refractory MOG-IgG–associated disease and neuromyelitis optica spectrum disorders

BACKGROUND AND OBJECTIVES: To evaluate the long-term safety and efficacy of tocilizumab (TCZ), a humanized anti–interleukin-6 receptor antibody in myelin oligodendrocyte glycoprotein–IgG–associated disease (MOGAD) and neuromyelitis optica spectrum disorders (NMOSD). METHODS: Annualized relapse rate (ARR), Expanded Disability Status Scale score, MRI, autoantibody titers, pain, and adverse events were retrospectively evaluated in 57 patients with MOGAD (n = 14), aquaporin-4 (AQP4)-IgG seropositive (n = 36), and seronegative NMOSD (n = 7; 12%), switched to TCZ from previous immunotherapies, particularly rituximab. RESULTS: Patients received TCZ for 23.8 months (median; interquartile range 13.0–51.1 months), with an IV dose of 8.0 mg/kg (median; range 6–12 mg/kg) every 31.6 days (mean; range 26–44 days). For MOGAD, the median ARR decreased from 1.75 (range 0.5–5) to 0 (range 0–0.9; p = 0.0011) under TCZ. A similar effect was seen for AQP4-IgG+ (ARR reduction from 1.5 [range 0–5] to 0 [range 0–4.2]; p < 0.001) and for seronegative NMOSD (from 3.0 [range 1.0–3.0] to 0.2 [range 0–2.0]; p = 0.031). During TCZ, 60% of all patients were relapse free (79% for MOGAD, 56% for AQP4-IgG+, and 43% for seronegative NMOSD). Disability follow-up indicated stabilization. MRI inflammatory activity decreased in MOGAD (p = 0.04; for the brain) and in AQP4-IgG+ NMOSD (p < 0.001; for the spinal cord). Chronic pain was unchanged. Regarding only patients treated with TCZ for at least 12 months (n = 44), ARR reductions were confirmed, including the subgroups of MOGAD (n = 11) and AQP4-IgG+ patients (n = 28). Similarly, in the group of patients treated with TCZ for at least 12 months, 59% of them were relapse free, with 73% for MOGAD, 57% for AQP4-IgG+, and 40% for patients with seronegative NMOSD. No severe or unexpected safety signals were observed. Add-on therapy showed no advantage compared with TCZ monotherapy. DISCUSSION: This study provides Class III evidence that long-term TCZ therapy is safe and reduces relapse probability in MOGAD and AQP4-IgG+ NMOSD

Disease-specific molecular events in cortical multiple sclerosis lesions

Cortical lesions constitute an important part of multiple sclerosis pathology. Although inflammation appears to play a role in their formation, the mechanisms leading to demyelination and neurodegeneration are poorly understood. We aimed to identify some of these mechanisms by combining gene expression studies with neuropathological analysis. In our study, we showed that the combination of inflammation, plaque-like primary demyelination and neurodegeneration in the cortex is specific for multiple sclerosis and is not seen in other chronic inflammatory diseases mediated by CD8-positive T cells (Rasmussen’s encephalitis), B cells (B cell lymphoma) or complex chronic inflammation (tuberculous meningitis, luetic meningitis or chronic purulent meningitis). In addition, we performed genome-wide microarray analysis comparing micro-dissected active cortical multiple sclerosis lesions with those of tuberculous meningitis (inflammatory control), Alzheimer’s disease (neurodegenerative control) and with cortices of age-matched controls. More than 80% of the identified multiple sclerosis-specific genes were related to T cell-mediated inflammation, microglia activation, oxidative injury, DNA damage and repair, remyelination and regenerative processes. Finally, we confirmed by immunohistochemistry that oxidative damage in cortical multiple sclerosis lesions is associated with oligodendrocyte and neuronal injury, the latter also affecting axons and dendrites. Our study provides new insights into the complex mechanisms of neurodegeneration and regeneration in the cortex of patients with multiple sclerosis

Inflammatory mechanisms and their relation to demyelination and neurodegeneration in Multiple Sclerosis

Mikroglia repräsentieren die mononukleären Phagozyten des ZNS und spielen in der Homöostase des ZNS im Gesunden sowie im Kranken eine Rolle. Mikroglia/Makrophagen detektieren pathologische Geschehnisse und akkumulieren um inflammatorische oder vaskuläre Läsionen. Janus-artige Effekte werden ihnen im Rahmen von Pathologien zugeschrieben. Bis zum heutigen Zeitpunkt weiß man wenig über den Anteil und die Polarisation von Mikroglia und peripheren Makrophagen während der Entwicklung einer Läsion. In den letzten Jahren konnten mittels RNA-Sequenzierung weitreichende Erkenntnisse bzgl. Homöostase und Aktivierung von Mikroglia gewonnen werden. Diese führten zur Definition geeigneter Marker mittels derer die Eigenschaften von Mikroglia erforscht werden können. In einem ersten Ansatz zielten wir darauf ab die Reaktion von Mikroglia und Makrophagen auf Läsionen hingehend ihrer inflammatorischen und vaskulären Genese zu charakterisieren. Die Studien wurden ausgeführt mittels immunohistochemischen Techniken auf in Paraffin eingebetteten Autopsien, welche im Zentrum für Hirnforschung gesammelt wurden. Zusätzlich untersuchten wir die Aktivierung von Mikroglia in Reaktion auf Neurodegeneration. Wir konnten zeigen, dass in frühen Stadien der inflammatorischen und ischämischen Läsionen ein substantieller Anteil aus dem Mikrogliapool rekrutiert wird, während bei weiterer Läsionsprogression der Hauptanteil der Phagozyten von peripheren Makrophagen stammt. Der homöostatische Marker war unterschiedlich zwischen den Läsionen reguliert. Während in MS Läsionen der Marker komplett herunterreguliert wurde, war bei ischämischen Läsionen eine niedrige Expression vorhanden. Im Gegensatz zu Versuchstieren war bei humanen Kontrollhirnen ein partieller Verlust des Markers zu sehen. Die Polarisation zwischen den Krankheitsentitäten war sehr ähnlich. In den Anfangsstadien war eine starke Aufregulation des pro-inflammatorischen Profils sichtbar, welche später in einen intermediären Phänotyp, Co-Expression von pro- und antiinflammatorischen Markern, überging. Des Weiteren konnten wir zeigen, dass Neurodegeneration zu einem Verlust des homöostatischen Markers führt. Obwohl über 100 Risikovarianten mit MS assoziiert wurden, kann die erbliche Komponente nur teilweise erklärt werden. Wir rekrutierten deswegen familiäre MS Fälle um Hochrisikovarianten identifizieren zu können. Anschließend testeten wir gefundene Varianten mittels Immunhistochemie auf humanen Autopsien. Durch Sequenzierung einer von MS betroffenen consanguinen Familie konnten wir eine seltene Variante im TPP2 Gen identifizieren und weiter zeigen, dass TPP2 in Makrophagen und Mikroglia in MS Läsionen aufreguliert wird. Zusammenfassend konnten wir wichtige Erkenntnisse zu den Eigenschaften von Mikroglia sowohl im Gesunden als auch bei pathologischen Ereignissen beisteuern, welche in Zukunft bei der Planung von klinischen und experimentellen Studien einbezogen werden können. Zusätzlich konnten wir mittels Next Generation Sequencing eine mögliche Hochrisikovariante für MS identifizieren, welche eventuell eine wichtige Rolle in der generellen Pathologie der MS spielt.Microglia represent the resident mononuclear phagocytes and are implicated in CNS homeostasis in health and disease. Microglia/macrophages are sensors for pathological CNS events and accumulate at sites of inflammatory or vascular lesions and are hypothesized to have a Janus face role in the disease process both detrimental and beneficial. However, knowledge about the polarization and contribution of microglia and peripheral macrophages during lesion formation is sparse in humans due to the absence of suitable markers. In the last couple of years, RNA sequencing revealed major insights into microglial homeostasis and activation and thereby elucidated targetable proteins for studying microglia properties. In a first approach, we aimed to provide an in-depth characterization of the microglia and macrophage response in brain lesions of inflammatory and vascular diseases of the human CNS. These studies were performed by immunohistochemistry on paraffin-embedded autopsy brains collected at Center for Brain Research. Additionally, we intended to study microglia activation with regard to neurodegeneration. In the process of lesion formation, we could demonstrate that in the early stages of ischemic and inflammatory lesions, a substantial number of phagocytes was derived from the microglia pool while during further lesion maturation the majority of phagocytes derive from recruited macrophages from the periphery. The homeostatic microglia marker P2RY12 was differentially regulated being completely lost in multiple sclerosis lesions while low expression was observed in ischemic lesions. In contrast to rodents, the homeostatic profile was already partially lost in human control patients. Lesion polarization was similar in these disease entities showing a pro-inflammatory profile in the early stages and transition to an intermediate phenotype (co-expressing pro and anti-inflammatory markers) over lesion maturation. Additionally, we could demonstrate that neurodegeneration leads to a loss of the homeostatic phenotype. Although over 100 common polymorphisms have been associated with multiple sclerosis, the heritability can only be partly explained. Therefore, we aimed to identify high risk variants by recruiting familial cases of MS. Once identified variants were further studied by immunohistochemistry on human autopsy brains. By sequencing of a consanguine family suffering from MS, we were able to identify a rare variant in the TPP2 gene and could further show that TPP2 is upregulated in microglia and macrophages in active MS lesions. In conclusion, our findings add valuable information on microglia participation in health and during pathological events in the CNS and will be considered in future clinical and experimental trials. Additionally, by next-generation sequencing, we provide a possible high-risk gene variant for multiple sclerosis, coding for a protein expressed in microglia/macrophages in active lesions, which might play a possible role in the general disease process.submitted by Tobias ZrvavyMedizinische Universität Wien, Dissertation, 2017OeBB(VLID)236661

Exome-sequence analyses of four multi-incident multiple sclerosis families

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the Central Nervous System (CNS). Currently, it is estimated that 30&ndash;40% of the phenotypic variability of MS can be explained by genetic factors. However, low susceptibility variants identified through Genome Wide Association Study (GWAS) were calculated to explain about 50% of the heritability. Whether familial high-risk variants also contribute to heritability is a subject of controversy. In the last few years, several familial variants have been nominated, but none of them have been unequivocally confirmed. One reason for this may be that genetic heterogeneity and reduced penetrance are hindering detection. Sequencing a large number of MS families is needed to answer this question. In this study, we performed whole exome sequencing in four multi-case families, of which at least three affected individuals per family were analyzed. We identified a total of 138 rare variants segregating with disease in each of the families. Although no single variant showed convincing evidence for disease causation, some genes seemed particularly interesting based on their biological function. The main aim of this study was to provide a complete list of all rare segregating variants to provide the possibility for other researchers to cross-check familial candidate genes in an unbiased manner

Immunosenescence in Neurological Diseases—Is There Enough Evidence?

The aging of the immune system has recently attracted a lot of attention. Immune senescence describes changes that the immune system undergoes over time. The importance of immune senescence in neurological diseases is increasingly discussed. For this review, we considered studies that investigated cellular changes in the aging immune system and in neurological disease. Twenty-six studies were included in our analysis (for the following diseases: multiple sclerosis, stroke, Parkinson’s disease, and dementia). The studies differed considerably in terms of the patient groups included and the cell types studied. Evidence for immunosenescence in neurological diseases is currently very limited. Prospective studies in well-defined patient groups with appropriate control groups, as well as comprehensive methodology and reporting, are essential prerequisites to generate clear insights into immunosenescence in neurological diseases

Brain Pathology / Dominant role of microglial and macrophage innate immune responses in human ischemic infarcts

Inflammatory mechanisms, involving granulocytes, Tcells, Bcells, macrophages and activated microglia, have been suggested to play a pathogenic role in experimental models of stroke and may be targets for therapeutic intervention. However, knowledge on the inflammatory response in human stroke lesions is limited. Here, we performed a quantitative study on the inflammatory reaction in human ischemic infarct lesions. We found increased numbers of Tlymphocytes, mainly CD8+ cells, but not of Blymphocytes. Their number was very low in comparison to that seen in inflammatory diseases of the central nervous system and they did not show signs of activation. Polymorphonuclear leukocytes were present in meninges and less prominently in the perivascular space in early lesions, but their infiltration into the lesioned tissue was sparse with the exception of a single case. Microglia were lost in the necrotic core of fresh lesions, their number was increased in the surrounding penumbra, apparently due to proliferation. Using TMEM119 as a marker for the resident microglia pool, macrophages in lesions were in part derived from the original microglia pool, depending on the lesion stage. Most microglia and macrophages revealed a proinflammatory activation pattern, expressing molecules involved in phagocytosis, oxidative injury, antigen presentation and iron metabolism and had partially lost the expression of P2RY12, an antigen expressed on homeostatic (“resting”) microglia in rodents. At later lesion stages, the majority of macrophages showed intermediate activation patterns, expressing proinflammatory and antiinflammatory markers. Microglia in the normal white matter of controls and stroke patients were already partly activated toward a proinflammatory phenotype. Our data suggest that the direct contribution of lymphocytes and granulocytes to active tissue injury in human ischemic infarct lesions is limited and that stroke therapy that targets proinflammatory microglia and macrophage activation may be effective.(VLID)480011

Microglia pre-activation and neurodegeneration precipitate neuroinflammation without exacerbating tissue injury in experimental autoimmune encephalomyelitis

Abstract Human inflammatory or neurodegenerative diseases, such as progressive multiple sclerosis (MS), occur on a background of age-related microglia activation and iron accumulation as well as pre-existing neurodegeneration. Most experimental models for CNS diseases, however, are induced in rodents, which are naturally characterized by a homeostatic microglia phenotype, low cellular iron load and absence of neurodegeneration. Here, we show that naïve LEWzizi rats – Lewis rats with a zitter rat background – show a spontaneous phenotype partly mimicking the changes seen in human aging and particularly in the normal-appearing white and grey matter of patients with progressive MS. Using this model system, we further aimed to investigate (i) whether the acute monophasic MS model experimental autoimmune encephalomyelitis (EAE) transforms into chronic progressive disease and (ii) whether EAE-induced neuroinflammation and tissue damage aggravate on the LEWzizi background. We found that the pre-existing LEWzizi-specific pathology precipitated EAE-related neuroinflammation into forebrain areas, which are devoid of EAE lesions in normal Lewis rats. However, EAE-related tissue damage was neither modified by the LEWzizi-specific pathology nor did EAE-induced neuroinflammation modify the LEWzizi-related pathological process. Our data indicate that the interaction between pre-activated microglia and CD4+ autoreactive T cells during the induction and propagation of tissue damage in the CNS is limited