A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Intracellular TLR7 is activated in human oligodendrocytes in response to Borrelia burgdorferi exposure.

Lyme neuroborreliosis, caused by the gram-negative bacterium Borrelia burgdorferi, may affect the central and/or peripheral nervous systems. In previous studies, we showed that human oligodendrocytes exposed to the bacteria undergo apoptosis in an inflammatory environment, and that inflammatory pathways trigger cell-death pathways. We further demonstrated that several receptor tyrosine kinases were involved in triggering downstream effects, leading to inflammation and apoptosis. Toll-like receptors TLR2 and TLR5, which are commonly studied receptors in Lyme disease, only had a minimal role in inflammatory processes. To delineate the role of other TLRs, if any, real-time RT-PCR array experiments were carried out as an initial screen. Along with several inflammatory genes, TLR7 mRNA was upregulated in cells exposed to B. burgdorferi. Further analysis by immunohistochemistry showed that the TLR7 protein is present in readily detectable amounts, although no discernible differences could be seen between medium and B. burgdorferi-exposed cells by this technique. Nevertheless, use of specific inhibitors and siRNA showed that TLR7 is involved in inducing IL-6 and CCL2 in a dose dependent manner, and likely CXCL8. Triggering an intracellular receptor such as TLR7, which senses RNA, in typically non-phagocytic oligodendrocytes indicates either a niche for the bacterium inside the cell or novel uptake of nucleic acids to initiate inflammatory responses.

The Microbiome-Gut-Behavior Axis: Crosstalk Between the Gut Microbiome and Oligodendrocytes Modulates Behavioral Responses.

Environmental and dietary stimuli have always been implicated in brain development and behavioral responses. The gut, being the major portal of communication with the external environment, has recently been brought to the forefront of this interaction with the establishment of a gut-brain axis in health and disease. Moreover, recent breakthroughs in germ-free and antibiotic-treated mice have demonstrated the significant impact of the microbiome in modulating behavioral responses in mice and have established a more specific microbiome-gut-behavior axis. One of the mechanisms by which this axis affects social behavior is by regulating myelination at the prefrontal cortex, an important site for complex cognitive behavior planning and decision-making. The prefrontal cortex exhibits late myelination of its axonal projections that could extend into the third decade of life in humans, which make it susceptible to external influences, such as microbial metabolites. Changes in the gut microbiome were shown to alter the composition of the microbial metabolome affecting highly permeable bioactive compounds, such as p-cresol, which could impair oligodendrocyte differentiation. Dysregulated myelination in the prefrontal cortex is then able to affect behavioral responses in mice, shifting them towards social isolation. The reduced social interactions could then limit microbial exchange, which could otherwise pose a threat to the survival of the existing microbial community in the host and, thus, provide an evolutionary advantage to the specific microbial community. In this review, we will analyze the microbiome-gut-behavior axis, describe the interactions between the gut microbiome and oligodendrocytes and highlight their role in the modulation of social behavior.

Receptor tyrosine kinases play a significant role in human oligodendrocyte inflammation and cell death associated with the Lyme disease bacterium Borrelia burgdorferi.

BACKGROUND: In previous studies, human oligodendrocytes were demonstrated to undergo apoptosis in the presence of Borrelia burgdorferi under an inflammatory milieu. Subsequently, we determined that the MEK/ERK pathway played a significant role in triggering downstream inflammation as well as apoptosis. However, the identity of receptors triggered by exposure to B. burgdorferi and initiating signaling events was unknown. METHODS: In this study, we explored the role of several TLR and EGFR/FGFR/PDGFR tyrosine kinase pathways in inducing inflammation in the presence of B. burgdorferi, using siRNA and/or inhibitors, in MO3.13 human oligodendrocytes. Cell death and apoptosis assays were also carried out in the presence or absence of specific receptor inhibitors along with the bacteria to determine the role of these receptors in apoptosis induction. The expression pattern of specific receptors with or without B. burgdorferi was also determined. RESULTS: TLRs 2 and 5 had a minimal role in inducing inflammation, particularly IL-6 production. Rather, their effect was mostly inhibitory, with TLR2 downregulation significantly upregulating CXCL8, and CXCL (1,2,3) levels, and TLR5 likely having a similar role in CXCL8, CXCL(1,2,3), and CCL5 levels. TLR4 contributed mostly towards CCL5 production. On the other hand, inhibition of all three EGF/FGF/PDGF receptors significantly downregulated all five of the inflammatory mediators tested even in the presence of B. burgdorferi. Their inhibition also downregulated overall cell death and apoptosis levels. The expression pattern of these receptors, as assessed by immunohistochemistry indicated that the PDGFRß receptor was the most predominantly expressed receptor, followed by FGFR, although no significant differences were discernible between presence and absence of bacteria. Interestingly, inhibition of individual EGFR, FGFR, or PDGFR receptors did not indicate an individual role for any of these receptors in the overall downregulation of pathogenesis. Contrarily, suppression of FGFR signaling alone in the presence of bacteria significantly upregulated inflammatory mediator levels indicating that it might control an inhibitory pathway when triggered individually. CONCLUSIONS: Unlike TLRs, EGF/FGF/PDGF receptors collectively play a significant role in the inflammation and apoptosis of human oligodendrocytes as mediated by B. burgdorferi. It is likely that these three receptors need to be triggered simultaneously to achieve this effect.

Non-viable Borrelia burgdorferi induce inflammatory mediators and apoptosis in human oligodendrocytes.

In previous studies, exposure to live Borrelia burgdorferi was shown to induce inflammation and apoptosis of human oligodendrocytes. In this study we assessed the ability of non-viable bacteria (heat killed or sonicated) to induce inflammatory mediators and cell death. Both heat-killed and sonicated bacteria induced release of CCL2, IL-6, and CXCL8 from oligodendrocytes in a dose dependent manner. In addition, non-viable B. burgdorferi also induced cell death as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and another cell viability assay. These results suggest that spirochetal residues left after bacterial demise, due to treatment or otherwise, may continue to be pathogenic to the central nervous system.

Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis.

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

A possible role for inflammation in mediating apoptosis of oligodendrocytes as induced by the Lyme disease spirochete Borrelia burgdorferi.

BACKGROUND: Inflammation caused by the Lyme disease spirochete B. burgdorferi is an important factor in the pathogenesis of Lyme neuroborreliosis. Our central hypothesis is that B. burgdorferi can cause disease via the induction of inflammatory mediators such as cytokines and chemokines in glial and neuronal cells. Earlier we demonstrated that interaction of B. burgdorferi with brain parenchyma induces inflammatory mediators in glial cells as well as glial (oligodendrocyte) and neuronal apoptosis using ex vivo and in vivo models of experimentation. METHODS: In this study we evaluated the ability of live B. burgdorferi to elicit inflammation in vitro in differentiated human MO3.13 oligodendrocytes and in differentiated primary human oligodendrocytes, by measuring the concentration of immune mediators in culture supernatants using Multiplex ELISA assays. Concomitant apoptosis was quantified in these cultures by the in situ terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) assay and by quantifying active caspase-3 by flow cytometry. The above phenomena were also evaluated after 48 h of stimulation with B. burgdorferi in the presence and absence of various concentrations of the anti-inflammatory drug dexamethasone. RESULTS: B. burgdorferi induced enhanced levels of the cytokine IL-6 and the chemokines IL-8 and CCL2 in MO3.13 cells as compared to basal levels, and IL-8 and CCL2 in primary human oligodendrocytes, in a dose-dependent manner. These cultures also showed significantly elevated levels of apoptosis when compared with medium controls. Dexamethasone reduced both the levels of immune mediators and apoptosis, also in a manner that was dose dependent. CONCLUSIONS: This finding supports our hypothesis that the inflammatory response elicited by the Lyme disease spirochete in glial cells contributes to neural cell damage. As oligodendrocytes are vital for the functioning and survival of neurons, the inflammation and subsequent apoptosis of oligodendrocytes induced by B. burgdorferi could contribute to the pathogenesis of Lyme neuroborreliosis.

Infectious agents and multiple sclerosis--are Chlamydia pneumoniae and human herpes virus 6 involved?

A good deal of evidence suggests an infectious component in the development of multiple sclerosis (MS) and, to date, some 20 bacteria and viruses have been associated with the disease. Recent independent sets of studies have implicated the respiratory bacterium Chlamydia pneumoniae and human herpes virus 6 (HHV-6) in the pathogenesis of MS. However, as is the case for essentially all earlier microbial associations, experimental evidence linking either this bacterium or this virus to MS is equivocal. We review the published reports concerning involvement of C. pneumoniae and HHV-6 in MS, and data relating to possession of the APOE epsilon 4 allele, which some studies indicate might influence how these or other pathogens affect disease genesis. Based on the large set of inconsistent observations available and given important new information regarding the neuropathology of MS, we contend that no conclusion is possible at this point regarding the potential role of either C. pneumoniae or HHV-6 in MS. We therefore propose future studies that should clarify whether, and if so how, these and other organisms function in the pathogenesis of this disease.

[RT-PCR: a tool for the generation of clones for research on canine distemper]. / RT-PCR: Ein Hilfsmittel zur Herstellung von Klonen für die Staupeforschung.

In the present work an adapted method of the reverse transcriptase polymerase chain reaction (RT-PCR) is described. This procedure was an important tool for cloning a variety of gene sequences derived from oligodendrocytes and canine distemper virus (CDV). Among other applications, the obtained clones were used to produce probes for in situ hybridization and to sequence the inserted DNAs. Due to these molecularbiological techniques new insights were gained into the pathogenesis of CDV-induced demyelination and CDV-persistence in the central nervous system.

Infection of murine fetal brain cell cultures with Listeria monocytogenes.

The uptake of Listeria monocytogenes by different brain cells was studied in primary dissociated brain cell cultures derived from murine fetuses. In respect to the supposed intraaxonal migration of Listeria monocytogenes in the pathogenesis of listeric focal brain stem encephalitis, it was examined whether the bacterium was internalized by neurons. Infection rates of distinct cell types were determined by double immunofluorescence with antibodies against cell type-specific markers and the bacterial pathogen. Because of the changing composition of the cultures and time-dependent expression of the oligodendrocyte marker galactocerebroside (GC), infections were carried out on day 4, 6, 8, and 15 in vitro. Listeria monocytogenes was detected predominantly within macrophages. Astrocytes, oligodendrocytes, and fibronectin-expressing cells were infected to a lesser extent. The lowest rates of infection were observed in neurons. A tropism of Listeria monocytogenes for neurons was not detected in vitro.

Identification of the infected target cell type in spongiform myeloencephalopathy induced by the neurotropic Cas-Br-E murine leukemia virus.

The Cas-Br-E murine leukemia virus (MuLV) induces a progressive hindlimb paralysis accompanied by a spongiform myeloencephalopathy in susceptible mice. In order to better understand the pathological process leading to these neurodegenerative lesions, we have investigated the nature of the cell type(s) infected by the virus during the course of the disease in CFW/D and SWR/J mice. For this purpose, we used in situ hybridization with virus-specific probes in combination with cell-type-specific histochemical (lectin) and immunological markers as well as morphological assessment. In the early stage of infection, endothelial cells represented the main cell type expressing viral RNA in the central nervous system (CNS). With disease progression and the appearance of lesions, microglial cells became the major cell type infected, accounting for up to 65% of the total infected cell population in diseased areas. Morphologically, these cells appeared activated and were frequently found in clusters. Infection and activation of microglial cells were almost exclusively restricted to diseased regions of the CNS. Neurons in diseased regions were not discernibly infected with virus at either early or late times of disease progression. Similarly, the proportion of infected astrocytes was typically < 1%. Although some endothelial cells and oligodendrocytes were infected by the virus, their infection was not limited to diseased CNS regions. These results are consistent with a model of indirect motor neuron degeneration, subsequent to the infection of nonneuronal CNS cells and especially of microglial cells. Infected microglial cells may play a role in the disease process by releasing not only virions or viral env-gene-encoded gp70 proteins but also other factors which may be directly or indirectly toxic to neurons. Parallels between microglial cell infection by MuLV and by lentiviruses, and specifically by human immunodeficiency virus, are discussed.

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

Restricted canine distemper virus infection of oligodendrocytes.

BACKGROUND: Canine distemper virus, a morbillivirus induces multifocal demyelination in the central nervous system. The acute demyelination correlates with virus replication in brain cells, especially astrocytes. Observations in vivo and in vitro demonstrated degeneration of oligodendrocytes, the myelin producing cells. However, the mechanism of oligodendroglial degeneration in distemper remained unexplained. Infection of the myelin producing cells, the most obvious explanation for the phenomenon of demyelination, could not be supported by extensive searches for viral particles or antigens in these cells neither in vivo nor in vitro. EXPERIMENTAL DESIGN: In the present study, we combined in situ hybridization to visualize viral nucleic acid sequences with immunofluorescence for oligodendroglial antigens. RESULTS: The nonradioactive in situ hybridization technique in combination with contrast enhanced video microscopy allowed us to unequivocally demonstrate the presence of canine distemper virus nucleic acid sequences in cultured oligodendrocytes. Many oligodendrocytes close to infected foci in the brain cell cultures were found to contain viral nucleic acid sequences. Only 1% of the viral nucleic acid sequences containing oligodendrocytes also contained viral antigen. Canine distemper virus replication in these cells is clearly restricted. CONCLUSIONS: Different possibilities why oligodendrocytes do not support a productive virus infection and mechanisms by which such a restricted infection leads to oligodendroglial degeneration and ensuing demyelination are discussed. While our results have advanced our understanding of the pathogenesis of acute demyelination in distemper, they may also offer a possible explanation for the chronic progressive or even relapsing course of the disease. A restricted infection of the oligodendrocytes may be the mechanism by which canine distemper virus persists in the central nervous system. Virus persistence is probably a key event in many chronic viral induced inflammatory demyelinating diseases.

Neurotropism of human coronavirus 229E.

The 299E prototype strain of human coronavirus (HCV-229E) has so far been mainly associated with infections of the respiratory tract. In the present study, we show evidence for infection of the central nervous system (CNS) by HCV-229E, both in vitro and in vivo. Various human cell lines of CNS origin were tested for their susceptibility to infection by HCV-229E. Production of viral antigens was monitored by indirect immunofluorescence with monoclonal antibodies and infectious progeny virions by plaque assay on the L132 human embryonic lung cell line. The SK-N-SH neuroblastoma and H4 neuroglioma cell lines were highly susceptible to infection. The U-87 MG and U-373 MG astrocytoma cell lines were also infectable by HCV-229E. We could also demonstrate infection of the MO3.13 cell line, which was established by fusion of human oligodendrocytes with a thioguanine-resistant mutant of the TE671 (RD) human rhabdomyosarcoma cell line. An apparently more extensive infection of the MO3.13 cells, when compared to the parental cells, supports the notion that human oligodendrocytes are differentially susceptible to infection by this virus. We also tested for HCV-229E gene expression in pathological brain specimens. For that purpose, we developed a reverse transcription-polymerase chain reaction (RT-PCR) assay to amplify a portion of the mRNA encoding the viral nucleocapsid protein. Using stringent laboratory conditions, viral RNA was detectable in brain tissue of 4 of 11 multiple sclerosis patients and none of 6 neurological and 5 normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The restricted nature of HIV-1 tropism for cultured neural cells.

Infection of the central nervous system by HIV-1, the agent of AIDS, is characterized by the presence of infected and giant microglial cells as well as astrocytosis, demyelination, and neuronal loss. To determine whether cells of neuroectoderm origin can be infected by HIV-1, we have inoculated primary cultures derived from adult human brain with a lymphotropic virus (LAV) or a neurotropic virus (Jr-FL) isolated from a patient with AIDS dementia. While Jr-FL invariably causes productive infection of cultured brain microglia, neither astrocytes nor oligodendrocytes became productively infected by these viral strains. Moreover, the cultured oligodendrocytes develop a normal network of processes and express differentiation antigens in the presence of an ongoing lytic infection of microglial cells. No HIV-1 proviral DNA was detected in primary astrocyte cultures devoid of microglial after inoculation of either HIV-1 strain. Similarly, the neuronal cell line HCN-1 in its differentiated state did not allow the virus to go through cycles of reverse transcription and replication. LAV, however, was able to replicate in undifferentiated HCN-1 cells. Thus, tropism of HIV-1 appears tightly restricted to only one type of differentiated cell in the CNS, the microglia.

Canine distemper virus-immune complexes induce bystander degeneration of oligodendrocytes.

Demyelination in chronic canine distemper encephalitis may be the result of a bystander effect in which the antiviral immune response is involved. In the present report we demonstrate that canine distemper virus-antiviral antibody immune complexes induce oligodendroglial degeneration in mixed brain cell cultures, particularly at the level of the cell processes. The involvement of macrophages as effector cells in this process was confirmed by depletion of these cells from the cultures which prevented the immune complex-mediated oligodendroglial degeneration. Canine distemper virus-immune complex-induced oligodendroglial pathology is thought to be mediated by toxic factors released from stimulated macrophages, this bystander effect demonstrated here in vitro may be relevant to the mechanisms of demyelination in vivo, in which virus persistence plays an important role.

In situ analysis of proteolipid protein gene transcripts during persistent Theiler's virus infection.

SJL/J mice inoculated intracranially with the DA strain of Theiler's virus exhibit a persistent demyelinating disease of the central nervous system. To investigate the effect of persistent infection of oligodendrocytes on the expression of myelin genes, we analyzed the level of PLP mRNA in infected as well as uninfected oligodendrocytes. This study was performed at the single-cell level using the simultaneous detection of viral antigens by immunocytochemistry and PLP mRNAs by in situ hybridization with 35S-labeled oligonucleotide probes. Our data indicate that viral infection of oligodendrocytes reduces the level of PLP mRNA by about 80%.

Infection by coronavirus JHM of rat neurons and oligodendrocyte-type-2 astrocyte lineage cells during distinct developmental stages.

Primary telencephalic cultures derived from neonatal Wistar Furth rats were able to support the growth of coronavirus JHM if a viable neuronal population was maintained. This occurred under serum-free defined, but not serum-supplemented, growth conditions. The importance of neurons in establishing infections in mixed cultures was confirmed by immunocytochemical and electron microscopic studies. Glia, although more abundant than neurons in these cultures, were less frequently infected during the initial 48 h postinoculation. The two glial lineages present in mixed telencephalic cultures were separated into type-1 astrocytes and oligodendrocyte-type-2 astrocyte (O-2A) lineage cells and individually assessed for their ability to support virus growth. Infection could not be established in type-1 astrocytes regardless of the culture conditions employed, consistent with our previous study (S. Beushausen and S. Dales, Virology 141:89-101, 1985). In contrast, infections could be initiated in selected O-2A lineage cells grown in serum-free medium. Virus multiplication was however significantly reduced by preconditioning the medium with mixed telencephalic or enriched type-1 astrocyte cultures, suggesting that intercellular interactions mediated by soluble factor(s) can influence the infectious process in O-2A lineage cells. This presumption was supported by eliciting similar effects with basic fibroblast growth factor and platelet-derived growth factor, two central nervous system cytokines known to control O-2A differentiation. The presence of these cytokines, which synergistically block O-2A cells from differentiating into oligodendrocytes was correlated with specific and reversible resistance to JHM virus (JHMV) infection. These data, combined with our finding that accelerated terminal differentiation of the oligodendrocyte phenotype confers resistance to JHMV (Beushausen and Dales, Virology, 1985), suggest that the permissiveness of O-2A cells for JHMV is restricted to a discrete developmental stage.