Amy M. Smith

The human side of microglia

Despite increasing evidence of major differences between rodent and human immune and neurological function, relatively few biomedical studies are performed with human cells. This review takes the example of neuroimmunology research and the microglia cell type to illustrate the emerging differences between rodent and human research findings. Microglia are involved in disease states and normal aging processes of the adult human brain. Although rodent microglia are often used in studies investigating microglial function, there are important differences between rodent microglia and their human counterparts. To maximise the relevance of our basic research to the clinical setting, it is necessary to integrate more human-based research into current biomedical research practise and we discuss practical steps towards this aim.

M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia

BackgroundMicroglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation.MethodsUsing biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function.ResultsWe found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aβ1-42 peptide.ConclusionsWe show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit.

The Transcription Factor PU.1 is Critical for Viability and Function of Human Brain Microglia

Microglia are the predominant resident immune cells of the brain and can assume a range of phenotypes. They are critical for normal brain development and function but can also contribute to many disease processes. Although they are widely studied, the transcriptional control of microglial phenotype and activation requires further research. PU.1 is a key myeloid transcription factor expressed by peripheral macrophages and rodent microglia. In this article, we report the presence of PU.1 specifically in microglia of the adult human brain and we examine its functional role in primary human microglia. Using siRNA, we achieved substantial PU.1 protein knock‐down in vitro. By assessing a range of characteristic microglial proteins we found decreased viability of adult human microglia with reduced PU.1 protein expression. This observation was confirmed with PU.1 antisense DNA oligonucleotides. An important function of microglia is to clear debris by phagocytosis. We assessed the impact of loss of PU.1 on microglial phagocytosis and show that PU.1 siRNA reduces the ability of adult human microglia to phagocytose amyloid‐beta1‐42 peptide. These results show that PU.1 controls human microglial viability and function and suggest PU.1 as a molecular target for manipulation of human microglial phenotype.

Valproic acid induces microglial dysfunction, not apoptosis, in human glial cultures.

Valproic acid (VPA) is widely used for the treatment of mood disorders and epilepsy, but its mechanism of action is unclear. In vivo and in vitro studies using rodent models have demonstrated that VPA has both neuroprotective and neurotrophic effects. These beneficial effects are, in part, through modulation of glial cell function. Recently, we and others have shown that VPA selectively induces caspase-3 mediated apoptosis in rodent microglial cells. However, the effect of VPA on human microglia has not been tested. In this study, using microglia derived from adult human brains, we demonstrate that VPA does not induce microglial apoptosis as determined by the absence of caspase-3 cleavage. However, VPA does partially decrease the expression of the microglial markers PU.1 and CD45, as well as dramatically reducing microglial phagocytosis. Due to the many roles of microglia in the brain, these VPA-induced alterations in microglial phenotype could potentially have major effects on physiological and pathological actions of these cells.

Adult Human Glia, Pericytes and Meningeal Fibroblasts Respond Similarly to IFNy but Not to TGFβ1 or M-CSF

The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ1) did not inhibit this increase in HLA, nor did TGFβ1 affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ1, but not affected by M-CSF. In contrast, TGFβ1 did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ1 and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation.

Isolation of highly enriched primary human microglia for functional studies.

Microglia, the resident macrophages of the central nervous system play vital roles in brain homeostasis through clearance of pathogenic material. Microglia are also implicated in neurological disorders through uncontrolled activation and inflammatory responses. To date, the vast majority of microglial studies have been performed using rodent models. Human microglia differ from rodent counterparts in several aspects including their response to pharmacological substances and their inflammatory secretions. Such differences highlight the need for studies on primary adult human brain microglia and methods to isolate them are therefore required. Our procedure generates microglial cultures of >95% purity from both biopsy and autopsy human brain tissue using a very simple media-based culture procedure that takes advantage of the adherent properties of these cells. Microglia obtained in this manner can be utilised for research within a week. Isolated microglia demonstrate phagocytic ability and respond to inflammatory stimuli and their purity makes them suitable for numerous other forms of in vitro studies, including secretome and transcriptome analysis. Furthermore, this protocol allows for the simultaneous isolation of neural precursor cells during the microglial isolation procedure. As human brain tissue is such a precious and valuable resource the simultaneous isolation of multiple cell types is highly beneficial.

Isolation and Culture of Adult Human Microglia Within Mixed Glial Cultures for Functional Experimentation and High-Content Analysis

Microglia are thought to be involved in diseases of the adult human brain as well as normal aging processes. While neonatal and rodent microglia are often used in studies investigating microglial function, there are important differences between rodent microglia and their adult human counterparts. Human brain tissue provides a unique and valuable tool for microglial cell and molecular biology. Routine protocols can now enable use of this culture method in many laboratories. Detailed protocols and advice for culture of human brain microglia are provided here. We demonstrate the protocol for culturing human adult microglia within a mixed glial culture and use a phagocytosis assay as an example of the functional studies possible with these cells as well as a high-content analysis method of quantification.

Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles.

Parkinsons disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.

Validated Method for the Screening and Quantification of Baclofen, Gabapentin and Pregabalin in Human Post-Mortem Whole Blood Using Protein Precipitation and Liquid Chromatography–Tandem Mass Spectrometry

There has been a rapid increase in the number of prescriptions for baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) in the UK since their introduction to therapy. Recent studies across the European Union and USA have shown the illicit abuse potential of these drugs and deaths have been observed. A simple, reliable and fully validated method was developed for the screening and quantification of BLF, GBP and PGL in human post-mortem (PM) blood. The analytes and their deuterated analogs as internal standard were extracted from blood using a single addition acetonitrile protein precipitation reaction followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous confirmation and quantification. The assay was linear from 0.05 to 1.00 µg/mL for BLF and 0.5 to 50.0 µg/mL for GBP and PGL, respectively with r2 > 0.999 (n = 9) for all analytes. Intra-day and inter-day imprecisions (n = 80) were calculated using one-way ANOVA; no significant difference (P > 0.99) was observed for all analytes over 8 non-consecutive days. The average recovery for all analytes was >98.9%. The limits of detection and quantification were both 0.05 µg/mL for BLF, and 0.5 µg/mL for GBP and PGL. The method was highly selective with no interference from endogenous compounds or from 54 drugs commonly encountered in PM toxicology. To prove method applicability, 17 PM blood samples submitted for analysis were successfully analyzed. The concentration range observed in PM blood for BLF was 0.08-102.00 µg/mL (median = 0.25 µg/mL), for GBP 1.0-134.0 µg/mL (median = 49.0 µg/mL) and 2.0-540.0 µg/mL (median = 42.0 µg/mL) for PGL.

PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia

BackgroundMicroglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses.MethodsTo investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD.ResultsBioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ.ConclusionsCollectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose.