Abortively Infected Astrocytes Appear To Represent the Main Source of Interferon Beta in the Virus-Infected Brain.

Interferon beta (IFN-β) is a key component of cellular innate immunity in mammals, and it constitutes the first line of defense during viral infection. Studies with cultured cells previously showed that almost all nucleated cells are able to produce IFN-β to various extents, but information about the in vivo sources of IFN-β remains incomplete. By applying immunohistochemistry and employing conditional-reporter mice that express firefly luciferase under the control of the IFN-β promoter in either all or only distinct cell types, we found that astrocytes are the main producers of IFN-β after infection of the brain with diverse neurotropic viruses, including rabies virus, Theiler's murine encephalomyelitis virus, and vesicular stomatitis virus. Analysis of a panel of knockout mouse strains revealed that sensing of viral components via both RIG-I-like helicases and Toll-like receptors contributes to IFN induction in the infected brain. A genetic approach to permanently mark rabies virus-infected cells in the brain showed that a substantial number of astrocytes became labeled and, therefore, must have been infected by the virus at least transiently. Thus, our results strongly indicate that abortive viral infection of astrocytes can trigger pattern recognition receptor signaling events which result in secretion of IFN-β that confers antiviral protection

Characterization of the murine myeloid precursor cell line MuMac-E8

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies

Relative mRNA expression of several pluripotency and differentiation markers.

<p>(A) Relative expression of the marker of pluripotency Nanog and Nucleostemin (NST) determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. Nanog was hardly regulated. There were only small increases or decreases in expression. Nucleostemin (NST) showed on day 10 and 25 a 4-fold increase in expression. On all other days of culture variations were found around the control value. (B) Relative expression of the hematopoietic markers EPCR, Sca-1 and the mesenchymal marker CD105 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. EPCR showed in the first days after synchronization a significant decrease in expression and approached back to the expression level of control cells. In Sca-1 a slightly increased expression was observed, while it approached to the control value 30 days after treatment. CD105 expression rate decreased slightly within 30 days. (C) Relative expression of the hematopoietic markers CD11b and CD45 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. CD11b expression showed a clear increase up to day 2. Until day 30 the expression levels of CD11b approached to the control value. For CD45, only minor fluctuations were determined around the control value. (D) Relative expression of the neuronal markers Ezrin and Pax-6 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. Ezrin showed very moderate expression increases and reductions, which followed no clear pattern. Pax-6 expression was found to be decreased up to 5-fold of the control level between days 3 and 30.</p

Immunophenotyping of MuMac-E8 cells.

<p>MuMac-E8 cells were stained with fluorochrome-labeled monoclonal antibodies recognizing the surface marker proteins CD11b, F4/80 (A), CD14, and CD64 (B) in order to identify the functional phenotype of this cell line. It could be shown that MuMac-E8 cells in general express CD11b and that the majority of cultured MuMac-E8 cells express also F4/80 (91%). The number of CD11b⁺/F4/80⁺ cells increased following co-incubation with heat-killed <i>S</i>.E. (96%; A, right-hand panel). CD11b⁺/F4/80⁺ MuMac-E8 cells revealed high or moderate surface expression of CD14 or CD64, respectively. Moreover, the expression of both CD14 and CD64 on CD11b⁺/F4/80⁺ MuMac-E8 cells was additionally inducible by stimulation with <i>Salmonella</i> antigen (B).</p

Kaplan-Meier survival curve and number of leukocytes in peripheral blood after transplantation of MuMac-E8 cells into mice.

<p>(A) Survival of lethally irradiated mice after administration of different cell numbers of MuMac-E8. Animals that have received a transplantation dose of 2×10⁶ cells survived up to 17 days. Following the administration of 1×10⁶ the survival decreased down to 14 days. The control animals died within 12 days. (n = 4 per group) (B) Level of leukocytes in peripheral blood determined by analysis of blood, n = 4 per group, mean ± SD. Leukocytes decreased within 13 days post transplantation in all groups (no engraftment).</p

CFC assay after 12 days of culture.

<p>(A) MuMac-E8 cells were cultured for 12 days in methylcellulose-based semi-solid medium. The initial cell number was 2×10⁵ cells (magnification 100-fold). Cells revealed a distinct colony formation. The colonies consist of numerous cells of predominantly round shape. By comparison with example images from the manufacturer's instructions MuMac-E8 colonies were identified as CFU-M. (B) May-Grünwald-Giemsa stained MuMac-E8 cells harvested from bulk culture and allowed to adhere in chamber slides.</p

Optimum cell culture conditions and morphology of cultured MuMac-E8 cells.

<p>By means of real-time cell analysis using the xCELLigence system the optimum cell density (n = 12 per value of cell density, mean ± SD; A) and the optimum serum supplementation for cell synchronization through serum deprivation (B) were determined. Typical morphology of MuMac-E8 cells in culture was shown by inverse phase contrast microscopy (Axiovert, Zeiss). The majority of the cells were found to grow adherent until confluence (C). Visualisation of cell spreading after fluorescence staining of F-actin with Phalloidin-Alexa was performed by confocal laser-scanning microscopy (LSM 510 Meta, Zeiss) (D). Figure was reprint from Biochemica, 4, 14–16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113743#pone.0113743-Pfefferkorn1" target="_blank">[12]</a> with permission from the journal.</p

Experimental strategy for gene expression analyses.

<p>MuMac-E8 cells harvested from bulk culture were synchronized by serum deprivation for 48 h. The cell cycle was re-entered by supplementation of the culture medium with 10% FCS. At indicated time points total RNA was isolated for subsequent gene expression analysis by real-time RT-PCR.</p

Collagen and methylene blue staining of MuMac-E8 cells.

<p>Used cell number was 1×10⁴. (A) The cultivation was carried out in osteogenic differentiation medium and in (B) normal medium. Isolated yellow-colored cells are visible in both cases. (C) The cultivation was carried out in osteogenic differentiation medium and in (D) normal medium. Isolated blue-colored cells are visible in both cases. There was no colony formation.</p

Overview of the expression of investigated marker genes.

<p>Overview of the expression of investigated marker genes.</p