Profiling of microglia nodules in multiple sclerosis reveals propensity for lesion formation.

Microglia nodules (HLA-DR+ cell clusters) are associated with brain pathology. In this post-mortem study, we investigated whether they represent the first stage of multiple sclerosis (MS) lesion formation. We show that microglia nodules are associated with more severe MS pathology. Compared to microglia nodules in stroke, those in MS show enhanced expression of genes previously found upregulated in MS lesions. Furthermore, genes associated with lipid metabolism, presence of T and B cells, production of immunoglobulins and cytokines, activation of the complement cascade, and metabolic stress are upregulated in microglia nodules in MS. Compared to stroke, they more frequently phagocytose oxidized phospholipids and possess a more tubular mitochondrial network. Strikingly, in MS, some microglia nodules encapsulate partially demyelinated axons. Taken together, we propose that activation of microglia nodules in MS by cytokines and immunoglobulins, together with phagocytosis of oxidized phospholipids, may lead to a microglia phenotype prone to MS lesion formation.

Coordinated changes in the expression of Wnt pathway genes following human and rat peripheral nerve injury.

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration.

IgG Immune Complexes Break Immune Tolerance of Human Microglia.

Microglia are phagocytic cells involved in homeostasis of the brain and are key players in the pathogenesis of multiple sclerosis (MS). A hallmark of MS diagnosis is the presence of IgG Abs, which appear as oligoclonal bands in the cerebrospinal fluid. In this study, we demonstrate that myelin obtained post mortem from 8 out of 11 MS brain donors is bound by IgG Abs. Importantly, we show that IgG immune complexes strongly potentiate activation of primary human microglia by breaking their tolerance for microbial stimuli, such as LPS and Poly I:C, resulting in increased production of key proinflammatory cytokines, such as TNF and IL-1ß. We identified FcγRI and FcγRIIa as the two main responsible IgG receptors for the breaking of immune tolerance of microglia. Combined, these data indicate that IgG immune complexes potentiate inflammation by human microglia, which may play an important role in MS-associated inflammation and the formation of demyelinating lesions.

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis.

Myeloid cells contribute to inflammation and demyelination in the early stages of multiple sclerosis (MS), but it is still unclear to what extent these cells are involved in active lesion formation in progressive MS (PMS). Here, we have harnessed the power of single-cell mass cytometry (CyTOF) to compare myeloid cell phenotypes in active lesions of PMS donors with those in normal-appearing white matter from the same donors and control white matter from non-MS donors. CyTOF measurements of a total of 74 targeted proteins revealed a decreased abundance of homeostatic and TNFhi microglia, and an increase in highly phagocytic and activated microglia states in active lesions of PMS donors. Interestingly, in contrast to results obtained from studies of the inflammatory early disease stages of MS, infiltrating monocyte-derived macrophages were scarce in active lesions of PMS, suggesting fundamental differences of myeloid cell composition in advanced stages of PMS.

Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions.

Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.

Transcriptional profiling of human microglia reveals grey-white matter heterogeneity and multiple sclerosis-associated changes.

Here we report the transcriptional profile of human microglia, isolated from normal-appearing grey matter (GM) and white matter (WM) of multiple sclerosis (MS) and non-neurological control donors, to find possible early changes related to MS pathology. Microglia show a clear region-specific profile, indicated by higher expression of type-I interferon genes in GM and higher expression of NF-κB pathway genes in WM. Transcriptional changes in MS microglia also differ between GM and WM. MS WM microglia show increased lipid metabolism gene expression, which relates to MS pathology since active MS lesion-derived microglial nuclei show similar altered gene expression. Microglia from MS GM show increased expression of genes associated with glycolysis and iron homeostasis, possibly reflecting microglia reacting to iron depositions. Except for ADGRG1/GPR56, expression of homeostatic genes, such as P2RY12 and TMEM119, is unaltered in normal-appearing MS tissue, demonstrating overall preservation of microglia homeostatic functions in the initiation phase of MS.

Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables.

Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.

Gene Expression Profiling of Multiple Sclerosis Pathology Identifies Early Patterns of Demyelination Surrounding Chronic Active Lesions.

In multiple sclerosis (MS), activated microglia and infiltrating macrophages phagocytose myelin focally in (chronic) active lesions. These demyelinating sites expand in time, but at some point turn inactive into a sclerotic scar. To identify molecular mechanisms underlying lesion activity and halt, we analyzed genome-wide gene expression in rim and peri-lesional regions of chronic active and inactive MS lesions, as well as in control tissue. Gene clustering revealed patterns of gene expression specifically associated with MS and with the presumed, subsequent stages of lesion development. Next to genes involved in immune functions, we found regulation of novel genes in and around the rim of chronic active lesions, such as NPY, KANK4, NCAN, TKTL1, and ANO4. Of note, the presence of many foamy macrophages in active rims was accompanied by a congruent upregulation of genes related to lipid binding, such as MSR1, CD68, CXCL16, and OLR1, and lipid uptake, such as CHIT1, GPNMB, and CCL18. Except CCL18, these genes were already upregulated in regions around active MS lesions, showing that such lesions are indeed expanding. In vitro downregulation of the scavenger receptors MSR1 and CXCL16 reduced myelin uptake. In conclusion, this study provides the gene expression profile of different aspects of MS pathology and indicates that early demyelination, mediated by scavenger receptors, is already present in regions around active MS lesions. Genes involved in early demyelination events in regions surrounding chronic active MS lesions might be promising therapeutic targets to stop lesion expansion.