Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases.

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.

Investigating the NLRP3 inflammasome and its regulator miR-223-3p in multiple sclerosis and experimental demyelination.

Innate immune signaling pathways are essential mediators of inflammation and repair following myelin injury. Inflammasome activation has recently been implicated as a driver of myelin injury in multiple sclerosis (MS) and its animal models, although the regulation and contributions of inflammasome activation in the demyelinated central nervous system (CNS) are not completely understood. Herein, we investigated the NLRP3 (NBD-, LRR- and pyrin domain-containing protein 3) inflammasome and its endogenous regulator microRNA-223-3p within the demyelinated CNS in both MS and an animal model of focal demyelination. We observed that NLRP3 inflammasome components and microRNA-223-3p were upregulated at sites of myelin injury within activated macrophages and microglia. Both microRNA-223-3p and a small-molecule NLRP3 inhibitor, MCC950, suppressed inflammasome activation in macrophages and microglia in vitro; compared with microglia, macrophages were more prone to inflammasome activation in vitro. Finally, systemic delivery of MCC950 to mice following lysolecithin-induced demyelination resulted in a significant reduction in axonal injury within demyelinated lesions. In conclusion, we demonstrate that NLRP3 inflammasome activity by macrophages and microglia is a critical component of the inflammatory microenvironment following demyelination and represents a potential therapeutic target for inflammatory-mediated demyelinating diseases, including MS. Cover Image for this issue: https://doi.org/10.1111/jnc.15422.

TAAR1 Expression in Human Macrophages and Brain Tissue: A Potential Novel Facet of MS Neuroinflammation.

TAAR1 is a neuroregulator with emerging evidence suggesting a role in immunomodulation. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. Here, we investigate TAAR1 expression in human primary monocytes, peripherally-derived macrophages, and MS brain tissue. RT-qPCR was used to assess TAAR1 levels in MS monocytes. Using a previously validated anti-human TAAR1 antibody and fluorescence microscopy, TAAR1 protein was visualized in lipopolysaccharide-stimulated or basal human macrophages, as well as macrophage/microglia populations surrounding, bordering, and within a mixed active/inactive MS lesion. In vivo, TAAR1 mRNA expression was significantly lower in MS monocytes compared to age- and sex-matched healthy controls. In vitro, TAAR1 protein showed a predominant nuclear localization in quiescent/control macrophages with a shift to a diffuse intracellular distribution following lipopolysaccharide-induced activation. In brain tissue, TAAR1 protein was predominantly expressed in macrophages/microglia within the border region of mixed active/inactive MS lesions. Considering that TAAR1-mediated anti-inflammatory effects have been previously reported, decreased mRNA in MS patients suggests possible pathophysiologic relevance. A shift in TAAR1 localization following pro-inflammatory activation suggests its function is altered in pro-inflammatory states, while TAAR1-expressing macrophages/microglia bordering an MS lesion supports TAAR1 as a novel pharmacological target in cells directly implicated in MS neuroinflammation.

Interleukin-1 receptor antagonist: An exploratory plasma biomarker that correlates with disability and provides pathophysiological insights in relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disorder. Interleukin-1 receptor antagonist (IL-1RA) is an endogenous soluble antagonist of the IL-1 receptor and blocks the pro-inflammatory effects of IL-1ß known to contribute to MS pathology. The objectives of this study were to determine whether IL-1RA is associated with disability in MS and how this correlates with neurofilament light (NfL) levels in cerebrospinal fluid (CSF). METHODS: Peripheral blood and CSF were collected from consenting MS patients. Patient demographic and clinical variables, including past relapse activity, were also collected. Circulating levels of IL-1RA, IL-18, and IL-1ß were measured in plasma; IL-1RA and NfL were measured in the CSF via Bio-plex multiplex immunoassay kits and ELISA, respectively. IL-1RA expression was investigated in vitro using primary human macrophages and microglia, and in situ using post-mortem MS tissue. RESULTS: Following a multiple regression analysis, IL-1RA levels in plasma correlated with expanded disability status scale score independent of all other variables. In a separate cohort, CSF IL-1RA significantly correlated with NfL. In vitro, induction of the NLRP3 inflammasome, a pathological hallmark within MS lesions, led to increased release of IL-1RA from primary human microglia and macrophages. In the CNS, IL-1RA+ macrophages/microglia were present at the rim of mixed active/inactive MS lesions. CONCLUSIONS: Results presented in this study demonstrate that IL-1RA is a novel exploratory biomarker in relapsing-remitting MS, which correlates with disability and provides mechanistic insights into the regulatory inflammatory responses within the demyelinated CNS.

Inhibitory milieu at the multiple sclerosis lesion site and the challenges for remyelination.

Regeneration of myelin, following injury, can occur within the central nervous system to reinstate proper axonal conductance and provide trophic support. Failure to do so renders the axons vulnerable, leading to eventual degeneration, and neuronal loss. Thus, it is essential to understand the mechanisms by which remyelination or failure to remyelinate occur, particularly in the context of demyelinating and neurodegenerative disorders. In multiple sclerosis, oligodendrocyte progenitor cells (OPCs) migrate to lesion sites to repair myelin. However, during disease progression, the ability of OPCs to participate in remyelination diminishes coincident with worsening of the symptoms. Remyelination is affected by a broad range of cues from intrinsic programming of OPCs and extrinsic local factors to the immune system and other systemic elements including diet and exercise. Here we review the literature on these diverse inhibitory factors and the challenges they pose to remyelination. Results spanning several disciplines from fundamental preclinical studies to knowledge gained in the clinic will be discussed.

MicroRNA-223 protects neurons from degeneration in experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.

Corrigendum: Phagocytosis in the Brain: Homeostasis and Disease.

[This corrects the article DOI: 10.3389/fimmu.2019.00790.].

Phagocytosis in the Brain: Homeostasis and Disease.

Microglia are resident macrophages of the central nervous system and significantly contribute to overall brain function by participating in phagocytosis during development, homeostasis, and diseased states. Phagocytosis is a highly complex process that is specialized for the uptake and removal of opsonized and non-opsonized targets, such as pathogens, apoptotic cells, and cellular debris. While the role of phagocytosis in mediating classical innate and adaptive immune responses has been known for decades, it is now appreciated that phagocytosis is also critical throughout early neural development, homeostasis, and initiating repair mechanisms. As such, modulating phagocytic processes has provided unexplored avenues with the intent of developing novel therapeutics that promote repair and regeneration in the CNS. Here, we review the functional consequences that phagocytosis plays in both the healthy and diseased CNS, and summarize how phagocytosis contributes to overall pathophysiological mechanisms involved in brain injury and repair.

miR-223 promotes regenerative myeloid cell phenotype and function in the demyelinated central nervous system.

In the injured central nervous system, myeloid cells, including macrophages and microglia, are key contributors to both myelin injury and repair. This immense plasticity emphasizes the need to further understand the precise molecular mechanisms that contribute to the dynamic regulation of myeloid cell polarization and function. Herein, we demonstrate that miR-223 is upregulated in multiple sclerosis (MS) patient monocytes and the alternatively-activated and tissue-regenerating M2-polarized human macrophages and microglia. Using miR-223 knock-out mice, we observed that miR-223 is dispensable for maximal pro-inflammatory responses, but is required for efficient M2-associated phenotype and function, including phagocytosis. Using the lysolecithin animal model, we further demonstrate that miR-223 is required to efficiently clear myelin debris and promote remyelination. These results suggest miR-223 constrains neuroinflammation while also promoting repair, a finding of important pathophysiological relevance to MS as well as other neurodegenerative diseases.

The roles of extracellular vesicle microRNAs in the central nervous system.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules that post-transcriptionally regulate protein expression and most biological processes. Mature miRNAs are recruited to the RNA-induced silencing complex (RISC) and target mRNAs via complementary base-pairing, thus resulting in translational inhibition and/or transcript degradation. Here, we present evidence implicating miRNAs within extracellular vesicles (EVs), including microvesicles and exosomes, as mediators of central nervous system (CNS) development, homeostasis, and injury. EVs are extracellular vesicles that are secreted by all cells and represent a novel method of intercellular communication. In glial cells, the transfer of miRNAs via EVs can alter the function of recipient cells and significantly impacts cellular mechanisms involved in both injury and repair. This review discusses the value of information to be gained by studying miRNAs within EVs in the context of CNS diseases and their potential use in the development of novel disease biomarkers and therapeutic strategies.

Effects of fumarates on inflammatory human astrocyte responses and oligodendrocyte differentiation.

OBJECTIVE: Dimethyl fumarate (DMF) is a fumaric acid ester approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). In both the brain and periphery, DMF and its metabolite monomethyl fumarate (MMF) exert anti-inflammatory and antioxidant effects. Our aim was to compare the effects of DMF and MMF on inflammatory and antioxidant pathways within astrocytes, a critical supporting glial cell in the central nervous system (CNS). Direct effects of fumarates on neural progenitor cell (NPC) differentiation toward the oligodendrocyte lineage were also assessed. METHODS: Primary astrocyte cultures were derived from both murine and human brains. Following pretreatment with MMF, DMF, or vehicle, astrocytes were stimulated with IL-1ß for 24 h; gene and microRNA expression were measured by qPCR. Cytokine production and reactive oxygen species (ROS) generation were also measured. NPCs were differentiated into the oligodendrocyte lineage in the presence of fumarates and immunostained using early oligodendrocyte markers. RESULTS: In both murine and human astrocytes, DMF, but not MMF, significantly reduced secretion of IL-6, CXCL10, and CCL2; neither fumarate promoted a robust increase in antioxidant gene expression, although both MMF and DMF prevented intracellular ROS production. Pretreatment with fumarates reduced microRNAs -146a and -155 upon stimulation. In NPC cultures, DMF increased the number of O4+ and NG2+ cells. INTERPRETATION: These results suggest that DMF, and to a lesser extent MMF, mediates the anti-inflammatory effects within astrocytes. This is supported by recent observations that in the inflamed CNS, DMF may be the active compound mediating the anti-inflammatory effects independent from altered antioxidant gene expression.

miRNAs As Emerging Regulators of Oligodendrocyte Development and Differentiation.

Chronic demyelination is a hallmark of neurological disorders such as multiple sclerosis (MS) and several leukodystrophies. In the central nervous system (CNS), remyelination is a regenerative process that is often inadequate during these pathological states. In the MS context, in situ evidence suggests that remyelination is mediated by populations of oligodendrocyte progenitor cells (OPCs) that proliferate, migrate, and differentiate into mature, myelin-producing oligodendrocytes at sites of demyelinated lesions. The molecular programming of OPCs into mature oligodendrocytes is governed by a myriad of complex intracellular signaling pathways that modulate this process. Recent research has demonstrated the importance of specific and short non-coding RNAs, known as microRNAs (miRNAs), in regulating OPC differentiation and remyelination. Fortunately, it may be possible to take advantage of numerous developmental studies (both human and rodent) that have previously characterized miRNA expression profiles from the early neural progenitor cell to the late myelin-producing oligodendrocyte. Here we review much of the work to date and discuss the impact of miRNAs on OPC and oligodendrocyte biology. Additionally, we consider the potential for miRNA-mediated therapy in the context of remyelination and brain repair.