Karianne G. Schuurman

Phenotyping primary human microglia: Tight regulation of LPS responsiveness

Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b+CD45dim phenotype and a smaller size compared with CD11b+CD45high macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL‐4 resulted in the induction of HLA‐DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL‐4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease.

Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease

The ubiquitin-proteasome system is the main regulated intracellular proteolytic pathway. Increasing evidence implicates impairment of this system in the pathogenesis of diseases with ubiquitin-positive pathology. A mutant ubiquitin, UBB+1, accumulates in the pathological hallmarks of tauopathies, including Alzheimers disease, polyglutamine diseases, liver disease and muscle disease and serves as an endogenous reporter for proteasomal dysfunction in these diseases. UBB+1 is a substrate for proteasomal degradation, however it can also inhibit the proteasome. Here, we show that UBB+1 properties shift from substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB+1 expression system. At low expression levels, UBB+1 was efficiently degraded by the proteasome. At high levels, the proteasome failed to degrade UBB+1, causing its accumulation, which subsequently induced a reversible functional impairment of the ubiquitin-proteasome system. Also in brain slice cultures, UBB+1 accumulation and concomitant proteasome inhibition was only induced at high expression levels. Our findings show that by varying UBB+1 expression levels, the dual proteasome substrate and inhibitory properties can be optimally used to serve as a research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease.

Expression of Vitamin D Receptor and Metabolizing Enzymes in Multiple Sclerosis—Affected Brain Tissue

Vitamin D deficiency has been implicated as a risk factor for multiple sclerosis (MS), but how vitamin D metabolism affects MS pathophysiology is not understood. We studied the expression of vitamin D receptor (VDR) and related enzymes, including 1,25(OH)(2)D-24-hydroxylase (24-OHase; CYP24A1) and 25(OH)D-1α-hydroxylase (CYP27B1), in CNS tissues of 39 MS patients and 20 controls and in primary human glial cells in vitro. In control and MS normal-appearing white matter (NAWM), nuclear VDR immunostaining was observed in oligodendrocyte-like cells, human leukocyte antigen (HLA)-positive microglia, and glial fibrillary acidic protein-positive astrocytes. There was a 2-fold increase in VDR transcripts in MS NAWM versus control white matter (p = 0.03). In chronic active MS lesions, HLA-positive microglia/macrophages showed nuclear VDR staining; astrocytes showed nuclear and cytoplasmic VDR staining. Staining for 24-OHase was restricted to astrocytes.VDR and CYP27B1 mRNA expressions were increased in active MS lesions versus NAWM (p < 0.01, p = 0.04, respectively). In primary human astrocytes in vitro, the active form of vitamin D, 1,25(OH)(2)D(3), induced upregulation of VDR and CYP24A1. Tumor necrosis factor and interferon-γ upregulated CYP27B1 mRNA in primary human microglia and astrocytes. Increased VDR expression in MS NAWM and inflammatory cytokine-induced amplified expression of VDR and CYP27B1 in chronic active MS lesions suggest increased sensitivity to vitamin D in NAWM and a possible endogenous role for vitamin D metabolism in the suppression of active MS lesions.

Gender differences in multiple sclerosis : induction of estrogen signaling in male and progesterone signaling in female lesions

The basis of gender differences in the prevalence and clinical progression of multiple sclerosis (MS) is not understood. Here, we identify gender-specific responses in steroid synthesis and signaling in the brains of MS patients as possible contributors to these differences. We investigated gene expression changes in these pathways and of inflammatory cytokines in MS lesions and normal-appearing white matter (NAWM) of male and female patients (n=21) and control NAWM (n=14) using quantitative polymerase chain reaction (25 MS lesions, 21 MS NAWM, and 14 control NAWM) and immunohistochemistry (3-4 sections per group). In MS lesions in males, there was local upregulation of aromatase (an enzyme involved in estrogen biosynthesis), estrogen receptor-β (ERβ), and tumor necrosis factor (TNF) mRNA; whereas in females, there was local upregulation of 3β-hydroxysteroid-dehydrogenase, a progesterone synthetic enzyme, and of progesterone receptor. Astrocytes in the rim and center of MS lesions were found to be the primary source of steroidogenic enzyme and receptor expression. Aromatase and ERα mRNA levels were positively correlated with that of TNF in primary cultures of human microglia and astrocytes; TNF caused increased ERα, suggesting that inflammatory signals stimulate estrogen signaling in this cell type. Together, these findings suggest that there are gender differences in the CNS of MS patients that may affect lesion pathogenesis, that is, in males, estrogen synthesis and signaling are induced; whereas in females, progestogen synthesis and signaling are induced. These differences may represent contributing factors to gender differences in the prevalence and course of MS.

Selective Upregulation of Scavenger Receptors in and Around Demyelinating Areas in Multiple Sclerosis

Autoantibodies and complement opsonization have been implicated in the process of demyelination in the major human CNS demyelinating disease multiple sclerosis (MS), but scavenger receptors (SRs) may also play pathogenetic roles. We characterized SR mRNA and protein expression in postmortem brain tissue from 13 MS patients in relation to active demyelination. CD68, chemokine (C-X-C motif) ligand 16 (CXCL16), class A macrophage SR (SR-AI/II), LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1), FcγRIII, and LRP-1 (low-density lipoprotein receptor-related protein 1) mRNA were upregulated in the rims of chronic active MS lesions. CD68 and CXCL16 mRNA were also upregulated around chronic active MS lesions. By immunohistochemistry, CD68, CXCL16, and SR-AI/II were expressed by foamy macrophages in the rim and by ramified microglia around chronic active MS lesions. CXCL16 and SR-AI/II were also expressed by astrocytes in MS lesions and by primary human microglia and astrocytes in vitro. These data suggest that SRs are involved in myelin uptake in MS, and that upregulation of CD68, CXCL16, and SR-AI/II is one of the initial events in microglia as they initiate myelin phagocytosis. As demyelination continues, additional upregulation of LOX-1, FcγRIII, and LRP-1 may facilitate this process.

Characteristics of differentiated CD8(+) and CD4 (+) T cells present in the human brain.

Immune surveillance of the central nervous system (CNS) by T cells is important to keep CNS-trophic viruses in a latent state, yet our knowledge of the characteristics of CNS-populating T cells is incomplete. We performed a comprehensive, multi-color flow-cytometric analysis of isolated T cells from paired corpus callosum (CC) and peripheral blood (PB) samples of 20 brain donors. Compared to PB, CC T cells, which were mostly located in the perivascular space and sporadically in the parenchyma, were enriched for cells expressing CD8. Both CD4+ and CD8+ T cells in the CC had a late-differentiated phenotype, as indicated by lack of expression of CD27 and CD28. The CC contained high numbers of T cells expressing chemokine receptor CX3CR1 and CXCR3 that allow for homing to inflamed endothelium and tissue, but hardly cells expressing the lymph node-homing receptor CCR7. Despite the late-differentiated phenotype, CC T cells had high expression of the IL-7 receptor α-chain CD127 and did not contain the neurotoxic cytolytic enzymes perforin, granzyme A, and granzyme B. We postulate that CNS T cells make up a population of tissue-adapted differentiated cells, which use CX3CR1 and CXCR3 to home into the perivascular space, use IL-7 for maintenance, and lack immediate cytolytic activity, thereby preventing immunopathology in response to low or non-specific stimuli. The presence of these cells in this tightly regulated environment likely enables a fast response to local threats. Our results will enable future detailed exploration of T-cell subsets in the brain involved in neurological diseases.

Microglia in normal appearing white matter of multiple sclerosis are alerted but immunosuppressed

Little is known about the functional phenotype of microglia in normal appearing white matter (NAWM) of multiple sclerosis (MS), although it may hold valuable clues about mechanisms for lesion development. Therefore, we studied microglia from NAWM obtained post‐mortem from controls (n = 25) and MS patients (n = 21) for their phenotype ex vivo and their immune responsiveness in vitro, using a microglia isolation method that omits culture and adherence. By flow cytometry, microglia in MS NAWM displayed elevated CD45 levels and increased size and granularity but were distinct from autologous choroid plexus macrophages by absent or low expression of additional markers, in particular CD206. Flow cytometric analysis of microglia from NAWM of three controls and four MS patients showed alterations in levels of Fc‐gamma receptors in MS. In primary microglia from a bigger sample of subjects, analysis of Fc‐gamma receptors by quantitative PCR indicated a significant increase in mRNA levels of the inhibitory CD32b isoform in MS NAWM. Despite their changed activation status, microglia from MS NAWM were unresponsive to lipopolysaccharide in vitro. Notably, culture with dexamethasone led to an impaired induction of the inflammation‐limiting cytokine CCL18 in microglia from MS NAWM compared with those from control NAWM. Together, these data demonstrate that microglia in MS NAWM are in an alerted state, but display features of immunosuppression. Thus, the activation status of microglia in NAWM of MS patients likely reflects a response to ongoing neuroinflammation, which coincides with upregulation of immunoregulatory molecules to prevent full activation and damage to the vulnerable milieu. GLIA 2013;61:1848–1861

Staining of HLA-DR, Iba1 and CD68 in human microglia reveals partially overlapping expression depending on cellular morphology and pathology

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimers disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.

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.