[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To compare the in vivo and in vitro effects of DMF and MMF on human monocytes. BACKGROUND: DMF therapy reduces relapse rates in multiple sclerosis (MS). In experimental models, DMF inhibits production of pro-inflammatory molecules, and induces anti-oxidant responses. In vivo, DMF is rapidly metabolized to MMF, thought to be the biologically active agent. Expression of the pro-inflammatory cytokine regulator microRNA-155 (miR-155) is up-regulated in monocytes in untreated MS patients compared to healthy controls. DESIGN/METHODS: Monocytes obtained from healthy controls, DMF-treated or untreated MS patients were isolated using CD14-coated immunomagnetic beads. Cells were used for RNA extraction or cultured up to 24 hours under basal or LPS activated conditions ± DMF or MMF (1-50μM). Tumor necrosis factor (TNF) production was measured by ELISA. Expression of miR-155 and mRNAs encoding anti-oxidant genes OSGIN1, HO-1, and NQO1 were measured by qPCR. Cell viability was determined by trypan blue or flow cytometry. RESULTS: MiR-155 levels were significantly decreased in monocytes of DMF-treated patients, compared to untreated MS patients. MMF (50μM) in vitro did not modulate miR-155 expression and TNF secretion in monocytes obtained from healthy controls. DMF induced significant reduction of miR-155 expression and TNF secretion, but only at concentrations (10-50 uM) also associated with significant cytotoxic effects. No such effects were seen using monocyte-derived macrophages. Absolute monocyte numbers, viability up to 24 hours after isolation, and anti-oxidant gene expression were comparable between DMF treated and untreated MS patients, as was LPS-induced TNF secretion. CONCLUSIONS: Our in vitro results indicate that the in vivo effects of DMF on miR-155 cannot be ascribed to direct effects of MMF on monocytes. We cannot exclude a non-cytotoxic effect of residual DMF on monocyte properties in vivo, or an indirect effect of fumarate therapy on the overall inflammatory response, in vivo.
American Academy of Neurology, Washington, DC; 04/2015
[Show abstract][Hide abstract] ABSTRACT: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.Results:In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.Discussion: We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.
[Show abstract][Hide abstract] ABSTRACT: Dimethyl fumarate (DMF, Tecfidera™) has been demonstrated to reduce lesion formation and lesion load in relapsing–remitting multiple sclerosis. DMF is metabolized to monomethyl fumarate (MMF) in vivo and is thought to have anti-inflammatory and anti-oxidative effects. The former have been demonstrated to involve the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), an inflammation master-regulator responsible for the transcription of inflammation-associated molecules such as tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 10 (IL-10). Microglia are CNS-resident myeloid lineage cells that exhibit a range of activated phenotypes depending on signals present in their local environment. Microglia can cause oligodendroglial and neuronal damage via secretion of soluble factors, but have also been shown to improve remyelination in some toxin-induced demyelination models. In this study, we investigated the anti-inflammatory effects of DMF and MMF in primary human microglia isolated from 14 to 18 week-old fetal CNS tissue. These results were compared with responses in primary cultures of monocyte-derived macrophages. Following lipopolysaccharide (LPS) stimulation, enzyme-linked immunosorbance assays (ELISA) were used to detect TNF, IL-6, and IL-10 in the culture supernatants. DMF dose-dependently suppressed secretion of TNF, IL-6, and IL-10 from microglia, but MMF did not. Macrophages exhibited robust cytokine responses to LPS that were not attenuated by DMF or MMF treatment. DMF inhibited expression of miR-155, a known inhibitor of suppressor of cytokine secretion 1 (SOCS1) in microglia, as assessed by quantitative polymerase chain reaction (qPCR). Our studies indicate that DMF inhibition of inflammatory properties may be myeloid sub-type specific and involves regulation of miR-155 expression.
International Congress of Neuroimmunology (ISNI), Mainz, Germany; 10/2014
[Show abstract][Hide abstract] ABSTRACT: Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.
[Show abstract][Hide abstract] ABSTRACT: Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.
[Show abstract][Hide abstract] ABSTRACT: Background
FTY720 (fingolimod, Gilenya™) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1β.
Astrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1β-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA.
We observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1β. Recurrent FTY720 treatments did not inhibit serum- or IL-1β-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1β were unaffected by FTY720 treatment(s).
Our results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.
Journal of Neuroinflammation 03/2013; 10(1):41. DOI:10.1186/1742-2094-10-41 · 4.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca(2+)) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca(2+)-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca(2+) concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca(2+) concentrations. By contrast, the rise in intracellular Ca(2+) concentrations produced by N-methyl-D-aspartate receptor stimulation and voltage gated Ca(2+) channel activation were markedly attenuated by XIAP over-expression. The release of Ca(2+) from intracellular stores induced by the sarco/endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca(2+) concentrations elicited by L-glutamate suggesting that XIAP influences Ca(2+) signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca(2+) by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.
Neurochemical Research 02/2013; 38(4). DOI:10.1007/s11064-013-0989-0 · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory disease characterized by central nervous system (CNS) demyelination and axonal degeneration. Although the cause of MS is still unknown, it is widely accepted that novel drug targets need to focus on both decreasing inflammation and promoting CNS repair. In MS and experimental autoimmune encephalomyelitis, non-coding small microRNAs (miRNAs) are dysregulated in the immune system and CNS. Since individual miRNAs are able to down-regulate multiple targeted mRNA transcripts, even minor changes in miRNA expression may lead to significant alterations in gene expression. Herein, we review miRNA signatures reported in CNS tissue and immune cells of MS patients and consider how altered miRNA expression may influence MS pathology.
Frontiers in Genetics 01/2013; 3. DOI:10.3389/fgene.2012.00311
[Show abstract][Hide abstract] ABSTRACT: Microglia are an important component of the innate immune system within the central nervous system (CNS). Isolation and in vitro culturing of microglia can provide insight towards the basic biology of these cells as well as their interactions with neurons, astrocytes, and oligodendrocytes. While studies of rodent microglia and microglial cell lines have provided a basis for our understanding of these cells, human adult microglia exhibit distinct properties when compared to rodent cells. Furthermore, the study of human fetal microglia provides a window into the developing CNS. This chapter describes the protocols used to isolate, purify, and culture both human adult and fetal microglia. Under basal culture conditions, human microglia survive for extended periods in the absence of growth factors, thus allowing their properties to be investigated under resting conditions. In addition, both human adult and fetal microglia can be used to study how they respond to different polarization conditions. As is the case with macrophages, it is also possible to polarize microglia towards the pro-inflammatory "M1" and the anti-inflammatory "M2" phenotypes, as described in this chapter.
[Show abstract][Hide abstract] ABSTRACT: The pannexin-1 (Panx1) channel (often referred to as the Panx1 hemichannel) is a large-conductance channel in the plasma membrane of many mammalian cells. While opening of the channel is potentially detrimental to the cell, little is known about how it is regulated under physiological conditions. Here we show that stomatin inhibited Panx1 channel activity. In transfected HEK-293 cells, stomatin reduced Panx1-mediated whole-cell currents without altering either the total or membrane surface Panx1 protein expression. Stomatin coimmunoprecipitated with full-length Panx1 as well as a Panx1 fragment containing the fourth membrane-spanning domain and the cytosolic carboxyl terminal. The inhibitory effect of stomatin on Panx1-mediated whole-cell currents was abolished by truncating Panx1 at a site in the cytosolic carboxyl terminal. In primary culture of mouse astrocytes, inhibition of endogenous stomatin expression by small interfering RNA enhanced Panx1-mediated outward whole-cell currents. These observations suggest that stomatin may play important roles in astrocytes and other cells by interacting with Panx1 carboxyl terminal to limit channel opening.
PLoS ONE 06/2012; 7(6):e39489. DOI:10.1371/journal.pone.0039489 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both microglia, the resident myeloid cells of the CNS parenchyma, and infiltrating blood-derived macrophages participate in inflammatory responses in the CNS. Macrophages can be polarized into M1 and M2 phenotypes, which have been linked to functional properties including production of inflammation association molecules and phagocytic activity. We compare phenotypic and functional properties of microglia derived from the adult human CNS with macrophages derived from peripheral blood monocytes in response to M1 and M2 polarizing conditions. Under M1 conditions, microglia and macrophages upregulate expression of CCR7 and CD80. M2 treatment of microglia-induced expression of CD209 but not additional markers CD23, CD163, and CD206 expressed by M2 macrophages. M1-polarizing conditions induced production of IL-12p40 by both microglia and macrophages; microglia produced higher levels of IL-10 under M1 conditions than did macrophages. Under M2 conditions, microglia ± LPS produced comparable levels of IL-10 under M1 conditions whereas IL-10 was induced by LPS in M2 macrophages. Myelin phagocytosis was greater in microglia than macrophages under all conditions; for both cell types, activity was higher for M2 cells. Our findings delineate distinctive properties of microglia compared with exogenous myeloid cells in response to signals derived from an inflammatory environment in the CNS.
[Show abstract][Hide abstract] ABSTRACT: Tissue inhibitors of metalloproteinases (TIMPs) are pleiotropic extracellular proteins. TIMPs are recognized as endogenous regulators of matrix metalloproteinases (MMPs), a large family of extracellular enzymes with proteolytic activities that participate in cellular homeostasis, adaptation, and tissue remodeling. In addition to their roles as endogenous potent MMP inhibitors, accumulating evidence indicates important physiological roles for TIMPs that are independent of their ability to block MMP activities. For instance, MMP-independent actions of TIMP-1 in the central nervous system have been implicated in synaptic plasticity, neuroprotection, oncogenesis, and oligodendrocyte differentiation. Expression of TIMP-1 is dramatically increased in response to a variety of injurious and inflammatory insults. In the context of disease pathogenesis, MMP and TIMP expression are interpreted with respect to the proteolytic consequences of increased MMP/TIMP ratios. Here, we provide an alternative perspective on the homeostatic balance of TIMP and MMP proteins, whereby consideration is given to the possible role of MMPs as cognate inhibitors of the signaling functions of TIMPs. Thus, MMPs may regulate the receptor-mediated actions of TIMPs, inasmuch as TIMPs are themselves inhibitors of MMP-mediated proteolytic activities. This broader view reflects our emerging understanding that TIMP signaling and MMP inhibition represent two important functions of TIMPs that have the potential to affect tissue pathology.
American Journal Of Pathology 01/2012; 180(1):12-6. DOI:10.1016/j.ajpath.2011.09.008 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1 knock-out (KO) mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1 KO mice, a situation that coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1 KO mice revealed a specific deficit of NG2(+) oligodendrocyte progenitor cells. Application of recombinant murine TIMP-1 (rmTIMP-1) to TIMP-1 KO neurosphere cultures evoked a dose-dependent increase in NG2(+) cell numbers, while treatment with GM6001, a potent broad-spectrum MMP inhibitor did not. Similarly, administration of rmTIMP-1 to A2B5(+) immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1(+) oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5(+) oligodendrocyte progenitors grown in conditioned media derived from TIMP-1 KO primary glial cultures resulted in reduced differentiation of mature O1(+) oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2011; 31(16):6247-54. DOI:10.1523/JNEUROSCI.5474-10.2011 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over a century ago, hypertrophy of astrocytes was noted as a pathology of multiple sclerosis (MS) and was hypothesized to play an important role in this disease, yet the contribution of astrocytes has been largely underemphasized in the pathophysiology of CNS demyelination. Astrocytes perform many homeostatic functions within the developing and adult CNS, including enhancing formation and maintenance of the blood-brain barrier, moderating neuronal connections through the tripartite synapse, and perhaps even offering intercellular communication independently of neurons. Although there is a significant body of literature characterizing different types of MS lesions, the inflammatory demyelination in an active MS lesion is accompanied by the presence of macrophages, lymphocytes, and large reactive astrocytes. The astrocyte has long been viewed as a cell that promotes inflammation and demyelination, while also forming the glial scar, thus hindering remyelination and axon growth. Renewed interest in the astrocyte has been brought about by recent studies demonstrating that astrocytes can also function as cellular mediators of CNS myelination by promoting oligodendrocyte progenitor migration, proliferation, and differentiation. Thus, refining our knowledge of astrocytic functions in the regulation of CNS myelination may help us to better understand why remyelination fails in MS.
Journal of Neuroscience Research 01/2011; 89(1):13-21. DOI:10.1002/jnr.22482 · 2.73 Impact Factor