The relation between BTK expression and iron accumulation of myeloid cells in multiple sclerosis.

Activation of Bruton's tyrosine kinase (BTK) has been shown to play a crucial role in the proinflammatory response of B cells and myeloid cells upon engagement with B cell, Fc, Toll-like receptor, and distinct chemokine receptors. Previous reports suggest BTK actively contributes to the pathogenesis of multiple sclerosis (MS). The BTK inhibitor Evobrutinib has been shown to reduce the numbers of gadolinium-enhancing lesions and relapses in relapsing-remitting MS patients. In vitro, BTK inhibition resulted in reduced phagocytic activity and modulated BTK-dependent inflammatory signaling of microglia and macrophages. Here, we investigated the protein expression of BTK and CD68 as well as iron accumulation in postmortem control (n = 10) and MS (n = 23) brain tissue, focusing on microglia and macrophages. MS cases encompassed active, chronic active, and inactive lesions. BTK+ and iron+ cells positively correlated across all regions of interests and, along with CD68, revealed highest numbers in the center of active and at the rim of chronic active lesions. We then studied the effect of BTK inhibition in the human immortalized microglia-like HMC3 cell line in vitro. In particular, we loaded HMC3 cells with iron-dextran and subsequently administered the BTK inhibitor Evobrutinib. Iron treatment alone induced a proinflammatory phenotype and increased the expression of iron importers as well as the intracellular iron storage protein ferritin light chain (FTL). BTK inhibition of iron-laden cells dampened the expression of microglia-related inflammatory genes as well as iron-importers, whereas the iron-exporter ferroportin was upregulated. Our data suggest that BTK inhibition not only dampens the proinflammatory response but also reduces iron import and storage in activated microglia and macrophages with possible implications on microglial iron accumulation in chronic active lesions in MS.

B Cell Depletion and SARS-CoV-2 Vaccine Responses in Neuroimmunologic Patients.

OBJECTIVE: The study was undertaken to assess the impact of B cell depletion on humoral and cellular immune responses to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccination in patients with various neuroimmunologic disorders on anti-CD20 therapy. This included an analysis of the T cell vaccine response to the SARS-CoV-2 Delta variant. METHODS: We investigated prospectively humoral and cellular responses to SARS-CoV-2 mRNA vaccination in 82 patients with neuroimmunologic disorders on anti-CD20 therapy and 82 age- and sex-matched healthy controls. For quantification of antibodies, the Elecsys anti-SARS-CoV-2 viral spike (S) immunoassay against the receptor-binding domain (RBD) was used. IFN-gamma enzyme-linked immunosorbent spot assays were performed to assess T cell responses against the SARS-CoV-2 Wuhan strain and the Delta variant. RESULTS: SARS-CoV-2-specific antibodies were found less frequently in patients (70% [57/82]) compared with controls (82/82 [100%], p < 0.001). In patients without detectable B cells (<1 B cell/mcl), seroconversion rates and antibody levels were lower compared to nondepleted (≥1 B cell/mcl) patients (p < 0.001). B cell levels ≥1 cell/mcl were sufficient to induce seroconversion in our cohort of anti-CD20 treated patients. In contrast to the antibody response, the T-cell response against the Wuhan strain and the Delta variant was more pronounced in frequency (p < 0.05) and magnitude (p < 0.01) in B-cell depleted compared to nondepleted patients. INTERPRETATION: Antibody responses to SARS-CoV-2 mRNA vaccinnation can be attained in patients on anti-CD20 therapy by the onset of B cell repopulation. In the absence of B cells, a strong T cell response is generated which may help to protect against severe coronavirus disease 2019 (COVID-19) in this high-risk population. ANN NEUROL 2022;91:342-352.

Bruton's Tyrosine Kinase Inhibition in the Treatment of Preclinical Models and Multiple Sclerosis.

Significant progress has been made to understand the immunopathogenesis of multiple sclerosis (MS) over recent years. Successful clinical trials with CD20-depleting monoclonal antibodies have corroborated the fundamental role of B cells in the pathogenesis of MS and reinforced the notion that cells of the B cell lineage are an attractive treatment target. Therapeutic inhibition of Bruton's tyrosine kinase (BTK), an enzyme involved in B cell and myeloid cell activation and function, is regarded as a next-generation approach that aims to attenuate both errant innate and adaptive immune functions. Moreover, brain-penetrant BTK inhibitors may impact compartmentalized inflammation and neurodegeneration within the central nervous system by targeting brain-resident B cells and microglia, respectively. Preclinical studies in animal models of MS corroborated an impact of BTK inhibition on meningeal inflammation and cortical demyelination. Notably, BTK inhibition attenuated the antigen-presenting capacity of B cells and the generation of encephalitogenic T cells. Evobrutinib, a selective oral BTK inhibitor, has been tested recently in a phase 2 study of patients with relapsing-remitting MS. The study met the primary endpoint of a significantly reduced cumulative number of Gadolinium-enhancing lesions under treatment with evobrutinib compared to placebo treatment. Thus, the results of ongoing phase 2 and 3 studies with evobrutinib, fenobrutinib, and tolebrutinib in relapsing-remitting and progressive MS are eagerly awaited. This review article introduces the physiological role of BTK, summarizes the pre-clinical and trial evidence, and addresses the potential beneficial effects of BTK inhibition in MS.

Neuropathological Variability within a Spectrum of NMDAR-Encephalitis.

OBJECTIVE: To describe the neuropathological features of N-methyl-D-aspartate receptor (NMDAR)-encephalitis in an archival autopsy cohort. METHODS: We examined four autopsies from patients with NMDAR-encephalitis; two patients were untreated, three had comorbidities: small cell lung cancer, brain post-transplant lymphoproliferative disease (PTLD), and overlapping demyelination. RESULTS: The two untreated patients had inflammatory infiltrates predominantly composed of perivascular and parenchymal CD3+ /CD8- T cells and CD79a+ B cells/plasma cells in basal ganglia, amygdala, and hippocampus with surrounding white matter. The hippocampi showed a significant decrease of NMDAR-immunoreactivity that correlated with disease severity. The patient with NMDAR-encephalitis and immunosuppression for kidney transplantation developed a brain monomorphic PTLD. Inflammatory changes were compatible with NMDAR-encephalitis. Additionally, plasma cells accumulated in the vicinity of the necrotic tumor along with macrophages and activated microglia that strongly expressed pro-inflammatory activation markers HLA-DR, CD68, and IL18. The fourth patient developed demyelinating lesions in the setting of a relapse 4 years after NMDAR-encephalitis. These lesions exhibited the hallmarks of classic multiple sclerosis with radially expanding lesions and remyelinated shadow plaques without complement or immunoglobulin deposition, compatible with a pattern I demyelination. INTERPRETATION: The topographic distribution of inflammation in patients with NMDAR-encephalitis reflects the clinical symptoms of movement disorders, abnormal behavior, and memory dysfunction with inflammation dominantly observed in basal ganglia, amygdala, and hippocampus, and loss of NMDAR-immunoreactivity correlates with disease severity. Co-occurring pathologies influence the spatial distribution, composition, and intensity of inflammation, which may modify patients' clinical presentation and outcome. ANN NEUROL 2021;90:725-737.

Longitudinal CSF Findings in Autoimmune Encephalitis-A Monocentric Cohort Study.

Autoimmune encephalitis (AIE) poses a diagnostic challenge due to its heterogeneous clinical presentation, which overlaps with various neurological and psychiatric diseases. During the diagnostic work-up, cerebrospinal fluid (CSF) is routinely obtained, allowing for differential diagnostics as well as for the determination of antibody subclasses and specificities. In this monocentric cohort study, we describe initial and serial CSF findings of 33 patients diagnosed with antibody-associated AIE (LGI1 (n=8), NMDA (n=7), CASPR2 (n=3), IgLON5 (n=3), AMPAR (n=1), GAD65/67 (n=4), Yo (n=3), Ma-1/2 (n=2), CV2 (n=2)). Routine CSF parameters of 12.1% of AIE patients were in normal ranges, while 60.6% showed elevated protein levels and 45.4% had intrathecal oligoclonal bands (OCBs). Repeated CSF analyses showed a trend towards normalization of initial pathological CSF findings, while relapses were more likely to be associated with increased cell counts and total protein levels. OCB status conversion in anti-NMDARE patients coincided with clinical improvement. In summary, we show that in routine CSF analysis at diagnosis, a considerable number of patients with AIE did not exhibit alteration in the CSF and therefore, diagnosis may be delayed if antibody testing is not performed. Moreover, OCB status in anti-NMDAR AIE patients could represent a potential prognostic biomarker, however further studies are necessary to validate these exploratory findings.

Acute and non-resolving inflammation associate with oxidative injury after human spinal cord injury.

Traumatic spinal cord injury is a devastating insult followed by progressive cord atrophy and neurodegeneration. Dysregulated or non-resolving inflammatory processes can disturb neuronal homeostasis and drive neurodegeneration. Here, we provide an in-depth characterization of innate and adaptive inflammatory responses as well as oxidative tissue injury in human traumatic spinal cord injury lesions compared to non-traumatic control cords. In the lesion core, microglia were rapidly lost while intermediate (co-expressing pro- as well as anti-inflammatory molecules) blood-borne macrophages dominated. In contrast, in the surrounding rim, TMEM119+ microglia numbers were maintained through local proliferation and demonstrated a predominantly pro-inflammatory phenotype. Lymphocyte numbers were low and mainly consisted of CD8+ T cells. Only in a subpopulation of patients, CD138+/IgG+ plasma cells were detected, which could serve as candidate cellular sources for a developing humoral immunity. Oxidative neuronal cell body and axonal injury was visualized by intracellular accumulation of amyloid precursor protein (APP) and oxidized phospholipids (e06) and occurred early within the lesion core and declined over time. In contrast, within the surrounding rim, pronounced APP+/e06+ axon-dendritic injury of neurons was detected, which remained significantly elevated up to months/years, thus providing mechanistic evidence for ongoing neuronal damage long after initial trauma. Dynamic and sustained neurotoxicity after human spinal cord injury might be a substantial contributor to (i) an impaired response to rehabilitation; (ii) overall failure of recovery; or (iii) late loss of recovered function (neuro-worsening/degeneration).

Iron accumulation in the choroid plexus, ependymal cells and CNS parenchyma in a rat strain with low-grade haemolysis of fragile macrocytic red blood cells.

Iron accumulation in the CNS is associated with many neurological diseases via amplification of inflammation and neurodegeneration. However, experimental studies on iron overload are challenging, since rodents hardly accumulate brain iron in contrast to humans. Here, we studied LEWzizi rats, which present with elevated CNS iron loads, aiming to characterise choroid plexus, ependymal, CSF and CNS parenchymal iron loads in conjunction with altered blood iron parameters and, thus, signifying non-classical entry sites for iron into the CNS. Non-haem iron in formalin-fixed paraffin-embedded tissue was detected via DAB-enhanced Turnbull Blue stainings. CSF iron levels were determined via atomic absorption spectroscopy. Ferroportin and aquaporin-1 expression was visualised using immunohistochemistry. The analysis of red blood cell indices and serum/plasma parameters was based on automated measurements; the fragility of red blood cells was manually determined by the osmotic challenge. Compared with wild-type animals, LEWzizi rats showed strongly increased iron accumulation in choroid plexus epithelial cells as well as in ependymal cells of the ventricle lining. Concurrently, red blood cell macrocytosis, low-grade haemolysis and significant haemoglobin liberation from red blood cells were apparent in the peripheral blood of LEWzizi rats. Interestingly, elevated iron accumulation was also evident in kidney proximal tubules, which share similarities with the blood-CSF barrier. Our data underscore the importance of iron gateways into the CNS other than the classical route across microvessels in the CNS parenchyma. Our findings of pronounced choroid plexus iron overload in conjunction with peripheral iron overload and increased RBC fragility in LEWzizi rats may be seminal for future studies of human diseases, in which similar constellations are found.

Immunology of COVID-19 and disease-modifying therapies: The good, the bad and the unknown.

OBJECTIVE: The outbreak of the SARS-CoV-2 pandemic, caused by a previously unknown infectious agent, posed unprecedented challenges to healthcare systems and unmasked their vulnerability and limitations worldwide. Patients with long-term immunomodulatory/suppressive therapies, as well as their physicians, were and are concerned about balancing the risk of infection and effects of disease-modifying therapy. Over the last few months, knowledge regarding SARS-CoV-2 has been growing tremendously, and the first experiences of infections in patients with multiple sclerosis (MS) have been reported. METHODS: This review summarizes the currently still limited knowledge about SARS-CoV-2 immunology and the commonly agreed modes of action of approved drugs in immune-mediated diseases of the central nervous system (MS and neuromyelitis optica spectrum disorder). Specifically, we discuss whether immunosuppressive/immunomodulatory drugs may increase the risk of SARS-CoV-2 infection and, conversely, may decrease the severity of a COVID-19 disease course. RESULTS: At present, it can be recommended in general that none of those therapies with a definite indication needs to be stopped per se. A possibly increased risk of infection for most medications is accompanied by the possibility to reduce the severity of COVID-19. CONCLUSIONS: Despite the knowledge gain over the last few months, current evidence remains limited, and, thus, further clinical vigilance and systematic documentation is essential.

Plasmalogens, platelet-activating factor and beyond - Ether lipids in signaling and neurodegeneration.

Glycerol-based ether lipids including ether phospholipids form a specialized branch of lipids that in mammals require peroxisomes for their biosynthesis. They are major components of biological membranes and one particular subgroup, the plasmalogens, is widely regarded as a cellular antioxidant. Their vast potential to influence signal transduction pathways is less well known. Here, we summarize the literature showing associations with essential signaling cascades for a wide variety of ether lipids, including platelet-activating factor, alkylglycerols, ether-linked lysophosphatidic acid and plasmalogen-derived polyunsaturated fatty acids. The available experimental evidence demonstrates links to several common players like protein kinase C, peroxisome proliferator-activated receptors or mitogen-activated protein kinases. Furthermore, ether lipid levels have repeatedly been connected to some of the most abundant neurological diseases, particularly Alzheimer's disease and more recently also neurodevelopmental disorders like autism. Thus, we critically discuss the potential role of these compounds in the etiology and pathophysiology of these diseases with an emphasis on signaling processes. Finally, we review the emerging interest in plasmalogens as treatment target in neurological diseases, assessing available data and highlighting future perspectives. Although many aspects of ether lipid involvement in cellular signaling identified in vitro still have to be confirmed in vivo, the compiled data show many intriguing properties and contributions of these lipids to health and disease that will trigger further research.

Vaccination in Multiple Sclerosis: Friend or Foe?

Multiple sclerosis (MS) is a debilitating disease of the central nervous systems (CNS). Disease-modifying treatments (including immunosuppressive treatments) have shown positive effects on the disease course, but are associated with systemic consequences on the immune system and may increase the risk of infections and alter vaccine efficiency. Therefore, vaccination of MS patients is of major interest. Over the last years, vaccine hesitancy has steadily grown especially in Western countries, partly due to fear of sequelae arising from vaccination, especially neurological disorders. The interaction of vaccination and MS has been discussed for decades. In this review, we highlight the immunology of vaccination, provide a review of literature and discuss the clinical consideration of MS, vaccination and immunosuppression. In conclusion, there is consensus that MS cannot be caused by vaccines, neither by inactivated nor by live vaccines. However, particular attention should be paid to two aspects: First, in immunocompromised patients, live vaccines may lead to a stronger immune reaction with signs of the disease against which the patients have been vaccinated, albeit in weakened form. Second, protection provided by vaccination should be controlled in patients who have been vaccinated while receiving immunomodulatory or immunosuppressive treatment. In conclusion, there is evidence that systemic infections can worsen MS, thus vaccination will lower the risk of relapses by reducing the risk of infections. Therefore, vaccination should be in general recommended to MS patients.

PECAM-1 Stabilizes Blood-Brain Barrier Integrity and Favors Paracellular T-Cell Diapedesis Across the Blood-Brain Barrier During Neuroinflammation.

Breakdown of the blood-brain barrier (BBB) and increased immune cell trafficking into the central nervous system (CNS) are hallmarks of the pathogenesis of multiple sclerosis (MS). Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is expressed on cells of the vascular compartment and regulates vascular integrity and immune cell trafficking. Involvement of PECAM-1 in MS pathogenesis has been suggested by the detection of increased levels of soluble PECAM-1 (sPECAM-1) in the serum and CSF of MS patients. Here, we report profound upregulation of cell-bound PECAM-1 in initial (pre-phagocytic) white matter as well as active cortical gray matter MS lesions. Using a human in vitro BBB model we observed that PECAM-1 is not essential for the transmigration of human CD4+ T-cell subsets (Th1, Th1*, Th2, and Th17) across the BBB. Employing an additional in vitro BBB model based on primary mouse brain microvascular endothelial cells (pMBMECs) we show that the lack of endothelial PECAM-1 impairs BBB properties as shown by reduced transendothelial electrical resistance (TEER) and increases permeability for small molecular tracers. Investigating T-cell migration across the BBB under physiological flow by in vitro live cell imaging revealed that absence of PECAM-1 in pMBMECs did not influence arrest, polarization, and crawling of effector/memory CD4+ T cells on the pMBMECs. Absence of endothelial PECAM-1 also did not affect the number of T cells able to cross the pMBMEC monolayer under flow, but surprisingly favored transcellular over paracellular T-cell diapedesis. Taken together, our data demonstrate that PECAM-1 is critically involved in regulating BBB permeability and although not required for T-cell diapedesis itself, its presence or absence influences the cellular route of T-cell diapedesis across the BBB. Upregulated expression of cell-bound PECAM-1 in human MS lesions may thus reflect vascular repair mechanisms aiming to restore BBB integrity and paracellular T-cell migration across the BBB as it occurs during CNS immune surveillance.

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

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.

Microglial nodules provide the environment for pathogenic T cells in human encephalitis.

Microglia nodule formation is a common feature in inflammatory brain diseases mediated by T lymphocytes such as viral and paraneoplastic encephalitis, multiple sclerosis, and Rasmussen encephalitis (RE). However, its role has not been fully understood yet. We hypothesized that, in RE, microglial nodules provide an environment for the initiation of the later dominating T-cell cytotoxicity. In RE stage 0, small primary microglia nodules could be identified in the absence of T cells. These primary nodules showed inflammasome activation and endosomal Toll-like receptor upregulation. In stage 1, T cells migrate into the parenchyma and intermingle with microglial cells, thereby forming secondary nodules in which neurons are destroyed. Whole-genome transcriptome analysis at this point showed upregulation of several inflammatory pathways including interferon signaling and major histocompatibility complex-I signaling. Inflammatory profiles, like the ones observed in RE, could be induced upon TLR3 stimulation in neonatal microglial cell cultures. Taken together, our results point towards activation of endosomal TLRs, resulting in increased interferon signaling, inflammasome activation, and chemokine upregulation as early steps in RE pathogenesis. This activity sets the scene for subsequent infiltration of T cells and destruction of neurons. Similar to RE, this microglial microenvironment might be a crucial step in other T-cell-mediated inflammatory brain diseases.

Acute microglia ablation induces neurodegeneration in the somatosensory system.

Previous studies have reported that microglia depletion leads to impairment of synapse formation and these cells rapidly repopulate from CNS progenitors. However, the impact of microglia depletion and repopulation in the long-term state of the CNS environment has not been characterized. Here, we report that acute and synchronous microglia depletion and subsequent repopulation induces gray matter microgliosis, neuronal death in the somatosensory cortex and ataxia-like behavior. We find a type 1 interferon inflammatory signature in degenerating somatosensory cortex from microglia-depleted mice. Transcriptomic and mass cytometry analysis of repopulated microglia demonstrates an interferon regulatory factor 7-driven activation state. Minocycline and anti-IFNAR1 antibody treatment attenuate the CNS type 1 interferon-driven inflammation, restore microglia homeostasis and reduce ataxic behavior. Neither microglia depletion nor repopulation impact neuropathology or T-cell responses during experimental autoimmune encephalomyelitis. Together, we found that acute microglia ablation induces a type 1 interferon activation state of gray matter microglia associated with acute neurodegeneration.

Neuroinflammatory responses in experimental and human stroke lesions.

Neuroinflammation has been suggested as an attractive treatment target in stroke, since it offers a broader therapeutic window in comparison to currently established thrombolytic approaches. Inflammatory cells of both the innate and the adaptive immune system have been identified in experimental as well as human stroke lesions. In animal models, various therapeutic strategies targeting neuroinflammation have shown beneficial effects, however, translation to human disease has so far been disappointing. Comparisons of the numerous experimental findings with until now rather limited human data reveal that despite similarities in the core signature of the inflammatory reaction, human stroke lacks clearly definable temporal and spatial sequences of lesion maturation that can be generalized and applied for a large patient population. Thus, in comparison with well-timed animal models with a clear succession of pathogenic mechanisms, precise and stage-dependent targeting of neuroinflammation in human stroke patients will be difficult to achieve. In addition, the contribution of different components of the innate and adaptive immune system quantitatively varies between humans and experimental animals and even between different modes of ischemia induction in animal models. Finally, human stroke lesions develop on a basis of age-dependent microglia pre-activation, which may have major impact on the further activation and functional differentiation of these cells. Knowledge about neuroinflammation in stroke patients, however, is currently limited and it has to be determined in future studies to what extent different causes of stroke and different co-morbidities in the patients result in distinct patterns of inflammation.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Loss of 'homeostatic' microglia and patterns of their activation in active multiple sclerosis.

Microglia and macrophages accumulate at the sites of active demyelination and neurodegeneration in the multiple sclerosis brain and are thought to play a central role in the disease process. We used recently described markers to characterize the origin and functional states of microglia/macrophages in acute, relapsing and progressive multiple sclerosis. We found microglia activation in normal white matter of controls and that the degree of activation increased with age. This microglia activation was more pronounced in the normal-appearing white matter of patients in comparison to controls and increased with disease duration. In contrast to controls, the normal-appearing white matter of patients with multiple sclerosis showed a significant reduction of P2RY12, a marker expressed in homeostatic microglia in rodents, which was completely lost in active and slowly expanding lesions. Early stages of demyelination and neurodegeneration in active lesions contained microglia with a pro-inflammatory phenotype, which expressed molecules involved in phagocytosis, oxidative injury, antigen presentation and T cell co-stimulation. In later stages, the microglia and macrophages in active lesions changed to a phenotype that was intermediate between pro- and anti-inflammatory activation. In inactive lesions, the density of microglia/macrophages was significantly reduced and microglia in part converted to a P2RY12+ phenotype. Analysis of TMEM119, which is expressed on microglia but not on recruited macrophages, demonstrated that on average 45% of the macrophage-like cells in active lesions were derived from the resident microglia pool. Our study demonstrates the loss of the homeostatic microglial signature in active multiple sclerosis with restoration associated with disease inactivity.

Cell type-specific Nrf2 expression in multiple sclerosis lesions.

Oxidative injury appears to play a major role in the propagation of demyelination and neurodegeneration in multiple sclerosis (MS). It has been suggested that endogenous anti-oxidant defense mechanisms within MS lesions are insufficient to prevent spreading of damage. Thus, current therapeutic approaches (e.g., fumarate treatment) target to up-regulate the expression of a key regulator of anti-oxidative defense, the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In this study, we show that Nrf2 is already strongly up-regulated in active MS lesions. Nuclear Nrf2 expression was particularly observed in oligodendrocytes and its functional activity is indicated by the expression of one of its downstream targets (heme oxygenase 1) in the same cells. In contrast, only a minor number of Nrf2-positive neurons were detected, even in highly inflammatory cortical lesions presenting with extensive oxidative injury. Overall, the most pronounced Nrf2 expression was found in degenerating cells, which showed signs of apoptotic or necrotic cell death. Via whole-genome microarray analyses of MS lesions, we observed a differential expression of numerous Nrf2-responsive genes, also involved in the defense against oxidative stress, predominantly in areas of initial myelin destruction within actively demyelinating white matter lesions. Furthermore, the expression patterns of Nrf2-induced genes differed between the white matter and cortical gray matter. Our study shows that in the MS brain, Nrf2 expression varies in different cell types and is associated with active demyelination in the lesions.

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models.

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Thymic stromal lymphopoietin is expressed in the intact central nervous system and upregulated in the myelin-degenerative central nervous system.

Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine expressed at barrier surfaces of the skin, gut, nose, lung, and the maternal/fetal interphase. At these sites, it is important for the generation and maintenance of non-inflammatory, tissue-resident dendritic cell responses. We show here that TSLP is also expressed in the central nervous system (CNS) where it is produced by choroid plexus epithelial cells and astrocytes in the spinal cord. Under conditions of low-grade myelin degeneration, the numbers of TSLP-expressing astrocytes increase, and microglia express transcripts for the functional TSLP receptor dimer indicating that these cells are targets for TSLP in the myelin-degenerative CNS.