Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosis.

BACKGROUND: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a 'surface-in' gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell-myeloid cell interactions to propagate progressive MS is of considerable interest. METHODS: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied. FINDINGS: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation. INTERPRETATION: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells. FUNDING: Stated in Acknowledgments section of manuscript.

MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes.

BACKGROUND: Astrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest. METHODS: Laser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse. RESULTS: Here, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b). CONCLUSIONS: We conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

Multiple Sclerosis as a Syndrome-Implications for Future Management.

We propose that multiple sclerosis (MS) is best characterized as a syndrome rather than a single disease because different pathogenetic mechanisms can result in the constellation of symptoms and signs by which MS is clinically characterized. We describe several cellular mechanisms that could generate inflammatory demyelination through disruption of homeostatic interactions between immune and neural cells. We illustrate that genomics is important in identifying phenocopies, in particular for primary progressive MS. We posit that molecular profiling, rather than traditional clinical phenotyping, will facilitate meaningful patient stratification, as illustrated by interactions between HLA and a regulator of homeostatic phagocytosis, MERTK. We envisage a personalized approach to MS management where genetic, molecular, and cellular information guides management.

Species differences in immune-mediated CNS tissue injury and repair: A (neuro)inflammatory topic.

Cells of the adaptive and innate immune systems in the brain parenchyma and in the meningeal spaces contribute to physiologic functions and disease states in the central nervous system (CNS). Animal studies have demonstrated the involvement of immune constituents, along with major histocompatibility complex (MHC) molecules, in neural development and rare genetic disorders (e.g., colony stimulating factor 1 receptor [CSF1R] deficiency). Genome wide association studies suggest a comparable role of the immune system in humans. Although the CNS can be the target of primary autoimmune disorders, no current experimental model captures all of the features of the most common human disorder placed in this category, multiple sclerosis (MS). Such features include spontaneous onset, environmental contributions, and a recurrent/progressive disease course in a genetically predisposed host. Numerous therapeutic interventions related to antigen and cytokine specific therapies have demonstrated effectiveness in experimental autoimmune encephalomyelitis (EAE), the animal model used to define principles underlying immune-mediated mechanisms in MS. Despite the similarities in the two diseases, most treatments used to ameliorate EAE have failed to translate to the human disease. As directly demonstrated in animal models and implicated by correlative studies in humans, adaptive and innate immune constituents within the systemic compartment and resident in the CNS contribute to the disease course of neurodegenerative and neurobehavioral disorders. The expanding knowledge of the molecular properties of glial cells provides increasing insights into species related variables. These variables affect glial bidirectional interactions with the immune system as well as their own production of "immune molecules" that mediate tissue injury and repair.

Astrocytes in the Pathogenesis of Multiple Sclerosis: An In Situ MicroRNA Study.

Astrocytes are increasingly recognized as active contributors to the disease process in multiple sclerosis (MS), rather than being merely reactive. We investigated the expression of a selected microRNA (miRNA) panel that could contribute both to the injury and to the recovery phases of the disease. Individual astrocytes were laser microdissected from brain sections. We then compared the miRNAs' expressions in MS and control brain samples at different lesional stages in white versus grey matter regions. In active MS lesions, we found upregulation of ischemia-related miRNAs in white but not grey matter, often with reversion to the normal state in inactive lesions. In contrast to our previous findings on MS macrophages, expression of 2 classical inflammatory-related miRNAs, miRNA-155 and miRNA-146a, was reduced in astrocytes from active and chronic active MS lesions in white and grey matter, suggesting a lesser direct pathogenetic role for these miRNAs in astrocytes. miRNAs within the categories regulating aquaporin4 (-100, -145, -320) and glutamate transport/apoptosis/neuroprotection (-124a, -181a, and -29a) showed some contrasting responses. The regional and lesion-stage differences of expression of these miRNAs indicate the remarkable ability of astrocytes to show a wide range of selective responses in the face of differing insults and phases of resolution.

Immunology of oligodendrocyte precursor cells in vivo and in vitro.

Remyelination following myelin/oligodendrocyte injury in the central nervous system (CNS) is dependent on oligodendrocyte progenitor cells (OPCs) migrating into lesion sites, differentiating into myelinating oligodendrocytes (OLs), and ensheathing axons. Experimental models indicate that robust OPC-dependent remyelination can occur in the CNS; in contrast, histologic and imaging studies of lesions in the human disease multiple sclerosis (MS) indicate the variable extent of this response, which is particularly limited in more chronic MS lesions. Immune-mediated mechanisms can contribute either positively or negatively to the presence and functional responses of OPCs. This review addresses i) the molecular signature and functional properties of OPCs in the adult human brain; ii) the status (presence and function) of OPCs in MS lesions; iii) experimental models and in vitro data highlighting the contribution of adaptive and innate immune constituents to OPC injury and remyelination; and iv) effects of MS-directed immunotherapies on OPCs, either directly or indirectly via effects on specific immune constituents.

Sublethal oligodendrocyte injury: A reversible condition in multiple sclerosis?

OBJECTIVE: Degeneration of oligodendroglial distal processes has been identified as an early event in multiple sclerosis (MS) lesion development. Our objective was to further define the development of the "dying-back" oligodendrocyte lesion in situ and to model the development and potential reversibility of such responses using dissociated cultures of adult human brain-derived oligodendrocytes. METHODS: In situ analyses were performed on glutaraldehyde-fixed thin sections of clinically acute and pathologically active cases of MS. In vitro studies were conducted using adult human brain-derived oligodendrocytes challenged by metabolic stress conditions (low nutrient/glucose). RESULTS: In situ analyses indicated a spectrum of myelin changes in the presence of morphologically intact oligodendrocytes; these included degeneration of the inner cytoplasmic tongue with increasing sizes of intramyelinic bleb formation that could result in radial fractures of the myelin sheath. Macrophages with ingested myelin fragments were identified only once the fragmentation was established. In vitro studies indicated that oligodendrocyte process retraction, which was linked to reduced glycolytic respiratory activity, is reversible until a critical time point. Subsequent cell death was not linked to caspase-3-dependent programs. Gene expression studies conducted at the latest reversible time point revealed reduced expression of pathways associated with cell process outgrowth and myelination, as well as with metabolic activity. INTERPRETATION: Our findings reveal the potential to protect and possibly restore myelin elaborated by existent oligodendrocytes in early and evolving MS lesions, and suggest the necessity of ongoing studies of the mechanisms underlying subsequent adult human oligodendrocyte cell death. Ann Neurol 2017;81:811-824.

An updated histological classification system for multiple sclerosis lesions.

Multiple sclerosis is a complex and heterogeneous, most likely autoimmune, demyelinating disease of the central nervous system (CNS). Although a number of histological classification systems for CNS lesions have been used by different groups in recent years, no uniform classification exists. In this paper, we propose a simple and unifying classification of MS lesions incorporating many elements of earlier histological systems that aims to provide guidelines for neuropathologists and researchers studying MS lesions to allow for better comparison of different studies performed with MS tissue, and to aid in understanding the pathogenesis of the disease. Based on the presence/absence and distribution of macrophages/microglia (inflammatory activity) and the presence/absence of ongoing demyelination (demyelinating activity), we suggest differentiating between active, mixed active/inactive, and inactive lesions with or without ongoing demyelination. Active lesions are characterized by macrophages/microglia throughout the lesion area, whereas mixed active/inactive lesions have a hypocellular lesion center with macrophages/microglia limited to the lesion border. Inactive lesions are almost completely lacking macrophages/microglia. Active and mixed active/inactive lesions can be further subdivided into lesions with ongoing myelin destruction (demyelinating lesions) and lesions in which the destruction of myelin has ceased, but macrophages are still present (post-demyelinating lesions). This distinction is based on the presence or absence of myelin degradation products within the cytoplasm of macrophages/microglia. For this classification of MS lesions, identification of myelin with histological stains [such as luxol fast blue-PAS] or by immunohistochemistry using antibodies against myelin basic-protein (MBP) or proteolipid-protein (PLP), as well as, detection of macrophages/microglia by, e.g., anti-CD68 is sufficient. Active and demyelinating lesions may be further subdivided into the early and late demyelinating lesions. The former is defined by the presence in macrophages of major and small molecular weight myelin proteins, such as cyclic nucleotide diphosphoesterase (CNP), myelin oligodendrocyte glycoprotein (MOG), or myelin-associated protein (MAG), whereas macrophages in the latter demonstrate merely the presence of the major myelin proteins MBP or PLP. We discuss the histological features and staining techniques required to classify MS lesions, and, in addition, describe the histological hallmarks of cortical pathology and diffuse white matter changes, as well as of remyelination.

Astrocytes in multiple sclerosis.

Recent experimental and clinical studies on astrocytes are unraveling the capabilities of these multi-functional cells in normal homeostasis, and in central nervous system (CNS) disease. This review focuses on understanding their behavior in all aspects of the initiation, evolution, and resolution of the multiple sclerosis (MS) lesion. Astrocytes display remarkable flexibility and variability of their physical structure and biochemical output, each aspect finely tuned to the specific stage and location of the disease, participating in both pathogenic and beneficial changes seen in acute and progressive forms. As examples, chemo-attractive or repulsive molecules may facilitate the entry of destructive immune cells but may also aid in the recruitment of oligodendrocyte precursors, essential for repair. Pro-inflammatory cytokines may attack pathogenic cells and also destroy normal oligodendrocytes, myelin, and axons. Protective trophic factors may also open the blood-brain barrier and modulate the extracellular matrix to favor recruitment and persistence of CNS-specific immune cells. A chronic glial scar may confer structural support following tissue loss and inhibit ingress of further noxious insults and also inhibit migration of reparative cells and molecules into the damaged tissue. Continual study into these processes offers the therapeutic opportunities to enhance the beneficial capabilities of these cells while limiting their destructive effects.

Oligodendrogliopathy in Multiple Sclerosis: Low Glycolytic Metabolic Rate Promotes Oligodendrocyte Survival.

UNLABELLED: Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS. SIGNIFICANCE STATEMENT: The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.

MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells.

Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to TGF-ß compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional analysis indicated that TGF-ß-treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-ß-treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair.

MicroRNA Expression Patterns in Human Astrocytes in Relation to Anatomical Location and Age.

Anatomic distribution and age are variables linked to functions of astrocytes under physiologic and pathologic conditions. We measured the relative expression of a panel of microRNAs (miRNAs) in astrocytes captured by laser micro-dissection from normal human adult white and grey matter, human fetal white matter and germinal matrix samples. Although expression of most miRNAs was comparable between adult and fetal samples, regional differences were observed. In the adult cerebral cortex, expression of miRNAs in morphologically distinct inter-laminar astrocytes underlying the glial limitans differed from those in deeper cortical layers, suggesting functional specialization possibly related to structural stability and defense from potentially harmful factors in the cerebrospinal fluid. Differences between adult white and grey matter miRNA expression included higher expression of pro-inflammatory miRNAs in the former, potentially contributing to differences in inflammation between grey and white matter plaques in multiple sclerosis. Lower expression of miRNAs in fetal versus adult white matter astrocytes likely reflects the immaturity of these migrating cells. Highly expressed miRNAs in the fetal germinal matrix are probably relevant in development and also recapitulate some responses to injury. Future studies can address regional alterations of miRNA expression in pathological conditions.

Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation.

Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αß (LTαß) on Th17 cells and LTßR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTßR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.

P2Y12 expression and function in alternatively activated human microglia.

OBJECTIVE: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation. METHODS: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain. RESULTS: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection. CONCLUSION: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

miR-155 as a multiple sclerosis-relevant regulator of myeloid cell polarization.

OBJECTIVE: To define the functional significance of increased miR-155 expression in myeloid cells in multiple sclerosis (MS). METHODS: miR-155 expression levels were measured in CD14+ monocytes from untreated relapsing-remitting MS patients and compared to healthy controls. Similar microRNA (miRNA) analyses were performed in laser-captured CD68+ cells from perivascular (blood-derived macrophages) and parenchymal (microglia) brain regions in both active MS lesions and noninflammatory cases. Using human adult blood-derived macrophages and brain-derived microglia, in vitro experiments were performed to demonstrate how miR-155 influences the polarization state, phenotype, and functional properties of myeloid cells, in addition to their ability to subsequently impact adaptive T-cell responses. RESULTS: In MS, miR-155 expression was significantly increased in both peripheral circulating CD14+ monocytes and active lesions (CD68+ cells) compared to control donor monocytes and parenchymal microglia, respectively. In vitro, miR-155 was significantly increased in both M1-polarized primary human macrophages and microglia. Transfection of an miR-155 mimic increased proinflammatory cytokine secretion and costimulatory surface marker expression in both cell types; an miR-155 inhibitor decreased proinflammatory cytokine expression. Coculture experiments demonstrated that allogeneic T-cell responses were significantly enhanced in the presence of miR-155-transfected myeloid cells compared to controls. INTERPRETATION: Our results demonstrate that miR-155 regulates proinflammatory responses in both blood-derived and central nervous system (CNS)-resident myeloid cells, in addition to impacting subsequent adaptive immune responses. Differential miRNA expression may therefore provide insight into mechanisms responsible for distinct phenotypic and functional properties of myeloid cells, thus impacting their ability to influence CNS injury and repair.

Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E(+) oligodendrocytes and NKG2C(+) CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.