α7 nicotinic acetylcholine receptor signaling modulates the inflammatory phenotype of fetal brain microglia: first evidence of interference by iron homeostasis.

Neuroinflammation in utero may result in life-long neurological disabilities. Microglia play a pivotal role, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of microglia. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal microglia will augment their neuroprotective transcriptome profile, while the antagonistic stimulation of α7nAChR will achieve the opposite. Using an in vivo - in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep microglia cultures re-exposed to LPS in presence of a selective α7nAChR agonist or antagonist. Our RNAseq and protein level findings show that a pro-inflammatory microglial phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, antagonistic α7nAChR stimulation potentiates the pro-inflammatory microglial phenotype. Surprisingly, under conditions of LPS double-hit an interference of a postulated α7nAChR - ferroportin signaling pathway may impede this mechanism. These results suggest a therapeutic potential of α7nAChR agonists in early re-programming of microglia in neonates exposed to in utero inflammation via an endogenous cerebral cholinergic anti-inflammatory pathway. Future studies will assess the role of interactions between inflammation-triggered microglial iron sequestering and α7nAChR signaling in neurodevelopment.

RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain.

BACKGROUND: The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. NEW METHOD: Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. RESULTS: We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1ß) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. COMPARISON WITH EXISTING METHODS: This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. CONCLUSIONS: The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.

Strategy for anti-aquaporin-4 auto-antibody identification and quantification using a new cell-based assay.

NMO-IgG is a specific biomarker of neuromyelitis optica (NMO) that targets the aquaporin-4 (AQP4) water channel protein. The current gold standard for NMO-IgG identification is indirect immunofluorescence (IIF). Our aim in this study was to develop a new quantitative cell-based assay (CBA) and to propose a rational strategy for anti-AQP4 Ab identification and quantification. We observed an excellent correlation between the CBA and IIF for NMO-IgG/anti-AQP4 detection. The CBA appeared more sensitive than IIF but on the other hand, IIF allows the simultaneous detection of various auto-Abs, underlining the complementarity between both methods. In conclusion, we propose to use IIF for the screening of patients at diagnosis in order to identify auto-Abs targeting the central nervous system. A highly sensitive, AQP4 specific and quantitative assay such as our CBA could be used thereafter to specifically identify the target of the Ab and to monitor its serum concentration under treatment.

TLR-mediated B cell activation results in ectopic CLIP expression that promotes B cell-dependent inflammation.

Infectious pathogens produce compounds called Toll ligands that activate TLRs on lymphocytes. Acute activation triggered by certain TLRs appears to "jump start" the innate immune response, characterized by the release of inflammatory cytokines and cellular expansion. In some individuals, there is a failure to control acute inflammation, resulting in postinfectious, chronic inflammation. Susceptibility to chronic inflammation is strongly associated with an individual's MHC genes. Recent clinical trials for several autoimmune diseases characterized by chronic inflammation suggest that B lymphocyte depletion therapies dampen chronic immune activation. However, currently, there is no known mechanism that accounts for the correlation among TLR activation, MHC genetics, and a pathological role for B-lymphocytes. Our hypothesis is that TLR-activated B cells (B cells that have been polyclonally activated in the absence of antigen-specific signals) are not controlled properly by T cell-dependent B cell death, thereby causing B cell-dependent chronic inflammation. Here, we show that treatment with Toll ligands results in polyclonal B cell activation accompanied by ectopic expression of CLIP. Furthermore, by adoptively transferring purified CLIP+ B cells in syngeneic animals, we find that CLIP+ B cells induce production of TNF-α by host T cells. Finally, we demonstrate that CLIP-targeted peptide competition results in the death of polyclonally activated CLIP+ B cells.

Glatiramer acetate-reactive T lymphocytes regulate oligodendrocyte progenitor cell number in vitro: role of IGF-2.

Glatiramer acetate (GA) is an immunomodulator approved for therapy of relapsing-remitting multiple sclerosis (RRMS), but recent findings indicate that it may also have additional, neurotrophic effects. Here, we found that supernatants from human GA-reactive T lymphocytes potentiated oligodendrocyte numbers in rodent and human oligodendrocyte progenitor (OPC) cultures. Effects of Th2-polarized lines were stronger than Th1-polarized cells. Microarray and ELISA analyses revealed that neurotrophic factors induced in Th2- and Th1-polarized GA-reactive lines included IGF-2 and BMP-7 respectively, and functional studies confirmed IGF-2 as trophic for OPCs. Our results support the concept that GA therapy may result in supportive effects on oligodendrocytes in RRMS patients.

Functional consequences of S1P receptor modulation in rat oligodendroglial lineage cells.

Fingolimod (FTY720) and its phosphorylated form FTY720P are modulators of sphingosine-1-phosphate (S1P) receptors, which are G-protein coupled receptors linked to cell migration and vascular maturation. The efficacy of FTY720 in autoimmune diseases such as multiple sclerosis and its animal models has been attributed to its inhibition of lymphocyte trafficking to target organs. In this study, we examined the role of S1P receptors in cultured rat oligodendrocytes (OLGs) and OLG progenitor cells (OPCs) using the active phosphorylated form of FTY720. We found that (1) FTY720P improves the survival of neonatal rat OLGs during serum withdrawal, which is associated with the phosphorylation of extracellular signal regulated kinases (ERK1/2) and Akt; (2) FTY720P regulates OPC differentiation into OLGs in a concentration-dependent manner; and (3) S1P receptors are differentially modulated by platelet-derived growth factor (PDGF) resulting in downregulation of S1P5 and upregulation of S1P1 in OPCs. In addition, siRNA studies revealed that S1P1 participates in PDGF-induced OPC mitogenesis. We conclude that S1P1 and S1P5 serve different functions during oligodendroglial development, and possibly during remyelination.

Inhibition of medulloblastoma cell invasion by Slit.

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.

Upregulation of TRAIL expression on human T lymphocytes by interferon beta and glatiramer acetate.

We measured the in vivo and in vitro effects of interferon (IFN)beta and glatiramer acetate (GA) on the expression of the regulatory molecule, tumor necrosis factor related apoptosis inducing ligand (TRAIL), in patients with multiple sclerosis (MS). We confirmed the prior observation that TRAIL is enhanced on anti-CD3 activated T cells by the in vitro addition of IFNbeta. T cells from IFNbeta-treated patients stimulated with anti-CD3 only, had higher levels of TRAIL than untreated patients, suggesting that in vivo IFNbeta exposure has an effect on TRAIL expression in association with T cell activation. In vitro IFNbeta-induced TRAIL upregulation on anti-CD3 or phytohemagglutinin-activated T cells was comparable for IFNbeta-treated and non-treated MS patients and controls, indicating that IFN receptors were neither saturated nor down-regulated by current IFNbeta therapy. Although GA in vivo or in vitro did not induce TRAIL, the IFNbeta +GA combination in vitro enhanced TRAIL expression to higher levels than IFNbeta alone on CD4+ T cells obtained from MS patients, regardless of GA treatment status, and healthy donors, and on GA reactive T cell lines derived from GA-treated patients or controls. Whether any observed therapeutic effects of GA/IFNbeta combination therapy will correlate with TRAIL expression and function remains to be determined.

MRI lesion volume heterogeneity in primary progressive MS in relation with axonal damage and brain atrophy.

OBJECTIVES: To investigate whether axonal damage in primary progressive (PP) multiple sclerosis (MS), as measured by proton magnetic resonance spectroscopy (HMRS) imaging and brain atrophy, is a function of T2 weighted brain lesion volume. METHODS: 34 PP MS patients were divided into two categories: low (<3 cm(3), n = 18) or high (>or=3 cm(3), n = 16) T2 lesion load (LL). An Index of Brain Atrophy (IBA) was calculated and HMRS metabolite ratios were derived from a central brain area centred at the corpus callosum. RESULTS: Patient groups did not differ with regard to clinical characteristics and showed lower mean IBA and mean N-acetylaspartate:creatinine (NAA:Cr) ratios compared to healthy controls. CONCLUSION: PP patients with low and high brain T2LL have detectable brain atrophy and NAA:Cr reduction compared to healthy controls. In PP MS, T2 lesions alone are insufficient to explain the presence of brain atrophy and decrease in NAA:Cr.

Persistence of immune responses to altered and native myelin antigens in patients with multiple sclerosis treated with altered peptide ligand.

Altered peptide ligands (APLs) can modulate responses of T cells to native peptide antigens implicated in the pathogenesis of autoimmune diseases. An APL of the putative target antigen myelin basic protein (MBP) peptide 83-99 has been used in abbreviated clinical trials in patients with multiple sclerosis (MS). Our objective was to assess the long-term persistence, and characteristics, of the APL-induced immune response in such patients. We measured the ex vivo proliferative frequency to the APL and native MBP, the cross-reactivity, and the cytokine production by these lines. We found that a 4- to 16-week course of APL therapy could induce a persistent (2-4.5 year) increase in the frequency of T cells responsive to both the APL and the native MBP in a select number of patients. These T cells produced high levels of IL-5, contrasting with the pretreatment observation that the responses to either antigen were IFNgamma (Th1) dominant. Our results indicate that APL therapy can induce persistent Th2-directed immune deviation. Understanding the impact of such APL-induced immune responses on MS disease activity will require additional clinical trials that incorporate careful monitoring of both clinical and immunological parameters.

Malignant glioma cells use MHC class II transactivator (CIITA) promoters III and IV to direct IFN-gamma-inducible CIITA expression and can function as nonprofessional antigen presenting cells in endocytic processing and CD4(+) T-cell activation.

Malignant gliomas (MGs), lethal human central nervous system (CNS) neoplasms, contain tumor infiltrating lymphocytes (TIL). Although MHC class II molecules are frequently detected on MG cells, suggesting that they may be capable of antigen (Ag) presentation to CD4(+) T cells, deficiencies in CD4(+) T-cell activation are associated with these nonimmunogenic tumors. We evaluated regulation of the MHC class II transactivator (CIITA), the key intermediate that controls class II expression, in MG cells and tested whether MG cells could process native Ag. After interferon-gamma (IFN-gamma) stimulation, MG cells upregulated CIITA and class II molecules. IFN-gamma-inducible CIITA expression in MG cells, as well as primary human astrocytes, was directed by two CIITA promoters, pIV, the promoter for IFN-gamma-inducible CIITA expression in nonprofessional antigen-presenting cells (APC), and pIII, the promoter that directs constitutive CIITA expression in B cells. Both pIII and pIV directed CIITA transcription in vivo in MGs and ex vivo in IFN-gamma-activated primary MG cultures. We also demonstrate for the first time that MG cells can process native Ag for presentation to CD4(+) MHC class II-restricted Th1 cells, indicating that MG cells can serve as nonprofessional APC. CIITA may be a key target to modulate MHC class II expression, which could augment immunogenicity, Ag presentation, and CD4(+) T-cell activation in MG therapy.

Glial cell influence on the human blood-brain barrier.

The blood-brain barrier (BBB) is a specialized structure of the central nervous system (CNS) that restricts immune cell migration and soluble molecule diffusion from the systemic compartment into the CNS. Astrocytes and microglia are resident cells of the CNS that contribute to the formation of the BBB. In this article, we consider the influence of these glial cells on the immune regulatory functions of the microvascular endothelium, with special emphasis on the human BBB. A series of in vitro studies demonstrate that soluble factors produced by glial cells, under basal culture conditions, help restrict development of inflammation within the CNS. These soluble factor effects include upregulating expression of molecules including HT7, UEA-1 lectin-binding sites, and angiotensin receptors that help define the phenotype of endothelial cells. These factors also induce tight junction formation between brain endothelial cells, contributing to the restricted permeability of the BBB. In contrast, these factors have little effect on expression of molecules by ECs that either promote lymphocyte migration, such as chemokines and adhesion molecules or molecules that are required for competent antigen presentation, such as MHC and co-stimulatory molecules. Glial cells that become activated in response to signals derived from the immune system or generated within the CNS, produce an array of inflammatory molecules that increase permeability and promote lymphocyte trafficking and persistence. These observations emphasize the bidirectional nature of neural-immune interactions; this dynamic system should be amenable to therapeutic interventions.

Unusual imaging findings in progressive myoclonus epilepsy.

We describe a patient with progressive myoclonus epilepsy (PME), white matter hyperintensities in the corpus callosum, cerebral hemispheres, and left cerebral peduncle on magnetic resonance imaging (MRI), and positive oligoclonal bands. A phosphorus magnetic resonance spectrum was compatible with mitochondrial dysfunction. Abnormal white matter signals are not a feature of the known PME syndromes, although they occur in Leber's hereditary optic neuropathy (LHON). These abnormalities oriented the diagnosis toward mitochondrial disease.

NK cell-mediated lysis of autologous human oligodendrocytes.

Although considered an autoimmune disease, the mechanisms underlying oligodendrocyte (OL)/myelin injury in multiple sclerosis (MS) remain to be established. We utilized in vitro assays to demonstrate that human OLs, as well as other glial elements (astrocytes, microglia), were susceptible to injury mediated by peripheral blood-derived mononuclear cell preparations (MNCs) enriched for natural killer (NK cells) by depleting CD3(+) +/- CD19(+) cells through use of either magnetic beads or cell sorting. Cytotoxic effects of the NK cell-enriched effectors were dependent on pre-exposure of these cells to IL-2. Furthermore, we found that autologous OLs were as susceptible to injury mediated by IL-2 activated NK cells as were heterologous OLs. In context of the tissue injury that occurs in MS, our results suggest that the inflammatory milieu in MS lesions could provide conditions required for NK cell activation and that such effector cells can bypass the putative protective effects of self-MHC class I molecules that may be expressed on OLs.

T lymphocytes conditioned with Interferon beta induce membrane and soluble VCAM on human brain endothelial cells.

Vascular cell adhesion molecule (VCAM)-1 plays a critical role in mediating inflammatory cell adhesion and migration. Factors regulating the expression of membrane (m)VCAM and its cleaved counterpart soluble (s)VCAM are poorly understood. We previously demonstrated that serum sVCAM levels are increased in multiple sclerosis (MS) patients treated with interferon beta 1b (IFNbeta1b), which correlated with a reduction in gadolinium enhancing lesions on magnetic resonance imaging. However, subsequent studies have shown that IFNbeta does not directly induce VCAM expression on endothelial cells. We demonstrate here that co-culture with IFNbeta-conditioned T cells induces mVCAM on human brain endothelial cells (HBEC). Further, rapid shedding of sVCAM occurs, which mirrors the response after in vivo IFNbeta treatment. The VCAM induction is mediated partially through tumor necrosis factor (TNF)alpha and can be abrogated by sTNF receptor. VCAM could also be induced on astroglioma lines using IFNbeta-conditioned T cells, which suggests the effect is not specific for HBEC. Kinetic studies demonstrated an increase in the sVCAM to mVCAM ratio over time, which may contribute to the ultimate therapeutic effect of IFNbeta in patients. These data have important implications for understanding the events occurring at the blood brain barrier in vivo, and for determining the mechanism of action of IFNbeta in MS.

Caspase 8 expression and signaling in Fas injury-resistant human fetal astrocytes.

Fas (APO-1/CD95) is a cell surface receptor initially identified in lymphoid cells, but more recently detected in the central nervous system under pathological, usually inflammatory, conditions. In most Fas expressing cells, triggering of Fas by its ligand or by antagonistic antibodies leads to apoptosis. Human fetal astrocytes (HFA) constitutively express Fas yet are resistant to cell death following Fas ligation. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we attempted to identify a basis for HFA resistance to Fas-mediated injury. We compared the components of the Fas signaling pathway of HFA to those of two human cell lines susceptible to Fas-mediated injury, U251 glioma and Jurkat T-cells. We found that HFA did not express caspase 8 (FLICE), the caspase primarily activated on Fas signaling. Although we could induce caspase 8 in HFA with the inflammatory cytokines IFNgamma and TNFalpha, HFA remained resistant to Fas-mediated injury. Addition of inflammatory cytokines to the extracellular milieu also increased FLIP mRNA (FLICE inhibitory protein). Furthermore, upon triggering of cytokine-treated cells with FasL, we observed upregulation of the cleavage product of FLIP (p43-FLIP) previously shown to associate with the DISC and to block caspase 8 recruitment, thereby inhibiting Fas-mediated death. Our findings indicate that caspase 8 and its regulators play a central role in determining the response to Fas ligation of HFA and support a role for Fas signaling in the developing central nervous system other than related to cytotoxicity.

Evidence of axonal damage in the early stages of multiple sclerosis and its relevance to disability.

OBJECTIVE: To assess axonal damage and its contribution to disability at different stages of multiple sclerosis (MS). BACKGROUND: Recent in vivo imaging and in situ pathologic studies have demonstrated that substantial axonal damage accompanies the inflammatory lesions of MS. However, the relation of axonal damage to the duration of MS and its contribution to disability at different stages of the disease remain poorly defined. DESIGN: We performed proton magnetic resonance spectroscopic imaging in 88 patients with a wide range of clinical disability and disease duration to measure N-acetylaspartate (NAA, an index of axonal integrity) relative to creatine (Cr) in a large central brain volume that included mostly normal-appearing white matter on magnetic resonance imaging. RESULTS: We observed that the NAA/Cr values were abnormally low in the early stages of MS, even before significant disability (measured using the Expanded Disability Status Scale [EDSS]) was evident clinically, and declined more rapidly with respect to EDSS at lower than at higher EDSS scores (P<.001). The correlation of NAA/Cr values with EDSS score was significantly (P<.03) stronger in patients with mild disability (EDSS score <5, Spearman rank order correlation = -0.54, P<.001) than in patients with more severe disability (EDSS score >/=5, Spearman rank order correlation = -0.1, P<.9). When similar analyses were performed in patients with MS grouped for duration of disease, the subgroup with early disease duration (<5 years) also showed central brain NAA/Cr resonance intensity ratios significantly lower than healthy controls (P<.001). CONCLUSION: Cerebral axonal damage begins and contributes to disability from the earliest stages of the disease.

Axonal metabolic recovery in multiple sclerosis patients treated with interferon beta-1b.

Patients with multiple sclerosis (MS) can benefit from treatment with interferon beta-1b. However, the mechanisms of action of this drug are incompletely understood and effects of interferon beta-lb on axonal injury are not known. A measure of axonal injury can be obtained in vivo using magnetic resonance spectroscopy to quantify the resonance intensity of the neuronal marker, N-acetylaspartate (NAA). In a small pilot study, we performed combined magnetic resonance imaging and magnetic resonance spectroscopic imaging on 10 patients with relapsing-remitting MS before and 1 year after starting treatment with subcutaneous interferon beta-lb. Resonance intensities of NAA relative to creatine (Cr) were measured in a large, central brain volume. These measurements were compared with those made in a group of 6 untreated patients selected to have a similar range of scores on the Expanded Disability Status Scale and mean NAA/Cr at baseline. NAA/Cr in the treated group [2.74 (0.16), mean (SD)] showed an increase of 5.5% 12 months after the start of therapy [2.89 (0.24),p = 0.05], while NAA/Cr in the untreated group decreased, but not significantly [2.76 (0.1) at baseline, 2.65 (0.14) at 12 months,p > 0.1]. NAA/Cr had become significantly higher in the treated group at 12 months than in the untreated group (p = 0.03). Our data suggest that, in addition to losing axons, patients with chronic multiple sclerosis suffer from chronic, sublethal axonal injury that is at least partially reversible with interferon beta-lb therapy.

Regulation of Th1 and Th2 lymphocyte migration by human adult brain endothelial cells.

Endothelial cells of the blood-brain barrier (BBB) have the ability to regulate and restrict the passage of cells and molecules from the periphery to the CNS. We have used an in vitro assay of lymphocyte migration across monolayers of human adult brain endothelial cells (HBEC) as a model of lymphocyte migration across the BBB. We found that human allogeneic or MBP-reactive Th2-polarized lymphocytes migrate more avidly than Th1-polarized lymphocytes. Migration of Th2 but not Th1 cells across brain endothelium was inhibited by antibodies directed at MCP-1, a chemokine produced by HBECs. We could detect CCR2, a chemokine receptor that recognizes MCP-1 on Th2 but not Th1 lymphocytes. ICAM-1 and VCAM-1 molecules were expressed on the surface of HBECs under basal conditions and were upregulated by Th1 but not Th2 cell-derived supernatants. Migration of both lymphocyte subsets was dependent on LFA-1/ICAM-1 interactions. Blocking VLA-4/VCAM-1 binding did not influence actual trans-endothelial migration. These results suggest that HBECs composing the BBB favor the migration of Th2 cells. We postulate that this selectivity may help prevent activated Th1 lymphocytes, the putative CNS autoimmune disease initiating cells, from reaching the CNS parenchyma and favor entry of Th2 cells, a putative means to induce bystander suppression in the CNS.