Hideya Kawasaki

Cytomegalovirus Infection of the Central Nervous System Stem Cells from Mouse Embryo: A Model for Developmental Brain Disorders Induced by Cytomegalovirus

Cytomegalovirus (CMV) is the most frequent infectious cause of developmental disorders of the central nervous system (CNS) in humans. Infection of the CNS stem cells seems to be primarily responsible for the generation of the brain abnormalities. In this study, we evaluated the infectivity of murine CMV (MCMV) in epidermal growth factor (EGF)-responsive CNS stem cells prepared from fetal mouse brains, and studied the effect of infection on growth and differentiation of the stem cells. The CNS stem cells were permissive for MCMV infection, although MCMV replication was slower than in mouse embryonic fibroblasts. MCMV infection inhibited the growth and DNA replication of the stem cells. A clonogenic assay revealed that MCMV infection suppressed generation of colonies from single stem cells. When uninfected stem cells were induced to differentiate, a decrease in expression of the primitive neuroepidermal marker nestin was observed by immunocytochemistry and flow cytometry, whereas expression of neurofilament and glial fibrillary acidic protein (GFAP) were induced. In virus-infected CNS stem cells, nestin expression was retained, whereas the expression of neurofilament was more severely inhibited than that of GFAP in these cells. Two-color flow cytometry showed that differentiated glial precursor cells were preferentially susceptible to MCMV infection. MCMV-infected and uninfected CNS stem cells were transplanted into the neonatal rat brains. The reduced number of infected stem cells were engulfed into the subventricular zone and expressed GFAP, but did not migrate further, in contrast to the uninfected stem cells. These results suggest that suppression of the growth of the CNS stem cells and inhibition of the neuronal differentiation by CMV infection may be primary causes of disorders of brain development in congenital CMV infection.

Innate Immune Responses to Cytomegalovirus Infection in the Developing Mouse Brain and Their Evasion by Virus-Infected Neurons

Cytomegalovirus (CMV) is the most frequent infectious cause of developmental brain disorders in humans. Here we show the role of innate immune responses caused by natural killer (NK) cells and nitric oxide (NO) derived from brain macrophages during murine CMV (MCMV) infection of the developing brain. Viral replication in the brain of newborn mice was significantly enhanced by administration of anti-asialo-GM1 antibody, specific for NK cells, or L-N6-(1-imminoethyl)-lysine, a specific inhibitor of NO synthase 2 (NOS2). These results suggest that NK cells and NO contribute to the viral clearance from the brain. At 3 days postinfection (dpi) MCMV early antigen (Ag)-positive cells were immunohistochemically detected in the periventricular area, where most of the positive cells were macrophages. At 7 dpi MCMV-Ag was found not only in cells of the periventricular area but also in neurons of the hippocampus and cortex. At 11 dpi MCMV-Ag disappeared from the periventricular area, but persisted in neurons. In the periventricular area, NK cells and NOS2-positive macrophages were associated with MCMV-Ag-positive cells. In contrast, there were very few NK cells and NOS2-positive macrophages around the MCMV-Ag-positive neurons. In situ hybridization for MCMV DNA demonstrated that positive signals were found mostly in the periventricular cells, and rarely in neurons. These results suggest that the innate immune responses are restricted to the virus-replicating cells, and do not affect MCMV-infected neurons. Therefore, evasion of the innate immune responses by MCMV-infected neurons may be an important factor in supporting the viral persistence in the developing brain.

Reactivation of Latent Cytomegalovirus Infection in Mouse Brain Cells Detected after Transfer to Brain Slice Cultures

ABSTRACT Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans involving the developing brain. It is hypothesized that the brain disorders occur after recurrent reactivation of the latent infection in some kinds of cells in the brains. In order to test this hypothesis, we examined the reactivation of latent murine CMV (MCMV) infection in the mouse brain by transfer to brain slice culture. We infected neonatal and young adult mice intracerebrally with recombinant MCMV in which the lacZ gene was inserted into a late gene. The brains were removed 6 months after infection and used to prepare brain slices that were then cultured for up to 4 weeks. Reactivation of latent infection in the brains was detected by β-galactosidase (β-Gal) staining to assess β-galactosidase expression. Viral replication was also confirmed by the plaque assay. Reactivation was observed in about 75% of the mice infected during the neonatal period 6 months after infection. Unexpectedly, reactivation was also observed in 75% of mice infected as young adults, although the infection ratio in the brain slices was significantly lower than that in neonatally infected mice. β-Gal-positive cells were observed in marginal regions of the brains or immature neural cells in the ventricular walls. Immunohistochemical staining showed that the β-Gal-positive reactivated cells were neural stem or progenitor cells. These results suggest that brain disorders may occur long after infection by reactivation of latent infection in the immature neural cells in the brain.

Cyclosporine Inhibits Mouse Cytomegalovirus Infection via a Cyclophilin-Dependent Pathway Specifically in Neural Stem/Progenitor Cells

ABSTRACT The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.

Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences

BackgroundMyofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions.ResultsWe demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts.ConclusionsThis is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

Roles of neural stem progenitor cells in cytomegalovirus infection of the brain in mouse models

Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans. Although the brain is the principal target organ for CMV infection in infants with congenital infection and in immunocompromised patients, little has been known about cellular events in pathogenesis of the brain disorders. Mouse models have been developed by the authors for studying the cell tropism, infectious dynamics of CMV infection and the effects of CMV infection on proliferation, regeneration and differentiation of neural cells. It has been shown, using brain slice cultures and neurospheres, that neural stem progenitor (NSP) cells are the most susceptible to CMV infection in developing brains. The NSP cells are also susceptible to CMV infection in adult and aged brains. The susceptibility can be enhanced by stimulation of neurogenesis. It was shown that latent murine CMV infection occurs in NSP cells by demonstrating the reactivation in brain slice culture or neurospheres. It is hypothesized that CMV brain disorder such as microcephaly is caused by disturbance of cellular events in the ventricular regions, including proliferation and differentiation of the neural stem cells, whereas neurons are also targets in persistent CMV infection, presumably resulting in functional disorders such as mental retardation.

Activation of murine cytomegalovirus immediate‐early promoter in cerebral ventricular zone and glial progenitor cells in transgenic mice

Cytomegalovirus (CMV) is the most common infectious cause of congenital anomalies of the CNS in humans. We recently reported that the murine cytomegalovirus (MCMV) immediate‐early (IE) gene promoter directs astrocyte‐specific expression in adult transgenic mice. In the present study, we analyzed the activation of the MCMV IE promoter in developing transgenic mouse brains and compared the activation with that of the Musashi 1 (Msi1) gene, which is expressed in neural progenitor cells, including neural stem cells. During the early phase of neurogenesis, the transgene was expressed predominantly in endothelial cells of the vessels, but not in neuroepithelial cells in which Msi1 was expressed. During later stages of gestation, expression of the transgene was largely restricted to the ventricular zone (VZ) in the CNS, similar to the expression of Msi1. In neurosphere cultures from transgenic embryos in the late phase of neurogenesis, the transgene was expressed in some cells of neurospheres expressing Msi1 and nestin. In neural precursor cells induced to differentiate from stem cells, expression of the transgene was detected in glial progenitor cells, expressing GFAP, nestin, and Msi1, but not in cells expressing MAP2 or MAG. In postnatal development, persistent expression of the transgene was observed in astrocyte lineage cells as was Msi1. These spatiotemporal changes of the MCMV IE promoter activity during development of transgenic mice correlated with susceptible sites in congenital HCMV infection. Moreover, this transgenic mouse model may provide useful model for analysis of the regulation of the switching of neuronal and astrocyte differentiation, and the maintenance of the astrocyte lineage. GLIA 35:41–52, 2001.

Neuron-Specific Activation of Murine Cytomegalovirus Early Gene e1 Promoter in Transgenic Mice

The brain is the main target in congenital cytomegalovirus (CMV) infection and immunocompromised patients. No definite evidence that a CMV has special affinity for the central nervous system (CNS) has been published. Here, we generated transgenic mice with an e1 promoter/enhancer region connected to the reporter gene lacZ. Surprisingly, expression of the transgene was completely restricted to the CNS in all lines of transgenic mice. The transgene was expressed in subpopulation of neurons in the cerebral cortex, hippocampus, diencephalon, brainstem, cerebellum, and spinal cord in all of the lines. Non-neuronal cells in the CNS were negative for transgene expression. Activation of the transgene was first observed in neurons of mesencephalon in late gestation, and then the number of positive neurons increased in various parts of the brain as development proceeded. Upon infection of the transgenic mouse brains with MCMV, the location of the activated neurons became more extensive, and the number of such neurons increased. These results suggest that there are host factor(s) that directly activate the MCMV early gene promoter in neurons. This neuron-specific activation may be associated with persistent infection in the brain and may be responsible for the neuronal dysfunction and neuronal cell loss caused by CMV infection.

Induction of cytomegalovirus-infected labyrinthitis in newborn mice by lipopolysaccharide: a model for hearing loss in congenital CMV infection

Congenital cytomegalovirus (CMV) infection is the most common infectious cause of sensorineural hearing loss in children. Here, we established an experimental model of hearing loss after systemic infection with murine CMV (MCMV) in newborn mice. Although almost no viral infection was observed in the inner ears and brains by intraperitoneal (i.p.) infection with MCMV in newborn mice, infection in these regions was induced in combination with intracerebral (i.c.) injection of bacterial lipopolysaccharide (LPS). The susceptibility of the inner ears was higher than that of the brains in terms of viral titer per unit weight. In the labyrinths, the viral infection was associated with the mesenchymal vessels and accompanied by inflammatory cells induced by LPS, causing hematogenous targets of infection in the labyrinths. Viral infection also spread in the perilymph regions such as the scala tympani and scala vestibuli, probably from infected brains via meningogenic and cochlear nerve routes. Viral infection was not observed in the scala media in the endolymph, including the Corti organ. However, viral infection was observed in the spiral limbus, including the stria vascularis. These results suggest that hearing loss caused by labyrinthitis after congenital CMV infection may be enhanced by inflammation caused by systemic bacterial infection in the neonatal period.

Cytomegalovirus infection inhibits the expression of N-methyl-D-aspartate receptors in the developing mouse hippocampus and primary neuronal cultures.

Cytomegalovirus (CMV) is the most significant infectious cause of developmental brain disorders in humans. The infection occasionally persists and causes neurological disorders. The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors is essential for the development and plasticity of synapses, but also is involved in neuronal excitotoxicity during viral infection. Here we investigated the effects of murine CMV (MCMV) infection on the expression of NMDA receptors in the hippocampal neurons of neonatal mice and primary neuronal cultures. Viral antigen was mostly found in hippocampal pyramidal neurons from the CA1 to CA3. Image analysis of immunohistochemistry demonstrated that the expression of NMDA receptor subunit 1 (NMDA-R1) protein in CA1 neurons of MCMV-infected brain was reduced to 40% of that in uninfected brain. The signal of in situ hybridization for NMDA-R1 mRNA was also decreased in CA1 neurons of MCMV-infected brain. In primary neuronal cultures, reduction of NMDA-R1 expression in MCMV-infected neurons was also detected by immunocytochemistry and Western blotting. These results suggest that reduction of NMDA receptor expression by MCMV infection may cause a decrease in the susceptibility of the neurons to excitotoxic cell death, and may be related to the establishment of viral persistence and functional disturbances in MCMV-infected neurons.