Measles virus hemagglutinin triggers intracellular signaling in CD150-expressing dendritic cells and inhibits immune response

International audienceMeasles virus (MV) is highly contagious pathogen, which causes a profound immunosuppression, resulting in high infant mortality. This virus infects dendritic cells (DCs) following the binding of MV hemagglutinin (MV-H) to CD150 receptor and alters DC functions by a mechanism that is not completely understood. We have analyzed the effect of MV-H interaction with CD150-expressing DCs on the DC signaling pathways and consequent phenotypic and functional changes in the absence of infectious context. We demonstrated that contact between CD150 on human DCs and MV-H expressed on membrane of transfected CHO cells was sufficient to modulate the activity of two major regulatory pathways of DC differentiation and function: to stimulate Akt and inhibit p38 MAPK phosphorylation, without concomitant ERK1/2 activation. Furthermore, interaction with MV-H decreased the expression level of DC activation markers CD80, CD83, CD86, and HLA-DR and strongly downregulated IL-12 production but did not modulate IL-10 secretion. Moreover, contact with MV-H suppressed DC-mediated T-cell alloproliferation, demonstrating profound alteration of DC maturation and functions. Finally, engagement of CD150 by MV-H in mice transgenic for human CD150 decreased inflammatory responses, showing the immunosuppressive effect of CD150-MV-H interaction in vivo. Altogether, these results uncover novel mechanism of MV-induced immunosuppression, implicating modulation of cell signaling pathways following MV-H interaction with CD150-expressing DCs and reveal anti-inflammatory effects of CD150 stimulation

Expression pattern of MRPS18 family genes in medulloblastoma: a case report

Medulloblastoma is one of the most prevalent brain tumors in children. Due to alterations in the gene expression patterns, medulloblastomas display diversity in the transcriptional, genetic, and clinical markers. However, these markers are few. Hence, there is an urgent need for other molecular, preferentially, non-invasive markers to propose the personalized treatment. One of the putative markers can be the mitochondrial ribosomal protein MRPS18-2. Purpose: In the present work we aimed to study expression of the MRPS18 family genes at mRNA and protein levels, in serum and tissue of the medulloblastoma.  Materials and methods. To do so, a real-time quantitative polymerase chain reaction (qPCR) was used to assess the relative expression of RB and MRPS18 family genes at mRNA levels in patient sera and tissue. Protein signals were detected by immunohistochemistry. The relative expression of MRPS18 genes was lower, when assessed in serum of the tumor patient compared with the control. Results. Thus, MRPS18-1 expression level, detected in serum, is up to 8.5-folds lower than in the control sample, while in tissue it is quite similar in both samples. The MRPS18-2 gene was detected at up to 26-fold lower levels in the serum of the tumor patient. Importantly, MRPS18-2 and MRPS18-3 are elevated by 13- and 7.2-fold, respectively, in tumor tissue, compared to the control. Moreover, the MRPS18-2 protein signal is dramatically elevated in medulloblastoma cells, compared with the conditionally healthy brain tissue. Concluding, the members of the MRPS18 protein family, especially MRPS18-2, are the putative candidates for molecular prognostic markers. More experiments should be done on a study on this family, and on a large cohort

Measles virus (MV) does not infect human glioma cell lines.

<p>(A) Measles virus cytopathic effect in glioma and control cell lines. U87 glioma cell line and Vero-SLAM cells (positive control) were infected with wild type MV strain G954 and laboratory MV strain Edmonston, at MOI of 1. Images show syncytia formation (white arrows) at 24 h (for Vero-SLAM cells) or 96 h (for U87 cells) post-infection. Microscopic magnification of 400× was used for all images. One of five experiments. (B-C) Wild type (B) and laboratory strain (C) MV production in glioma and control cell lines, determined daily by plaque assay on Vero-SLAM cells, 24 h (1), 48 h (2), 72 h (3) and 96 h (4) post-infection. Vero-SLAM and Vero cells were used as positive and negative control respectively. One of three representative experiments. All studied glioma cell lines were not sensitive to the infection with wild type measles virus (MV), which uses CD150 receptor for its entry. At the same time all cell lines were infected and produced high titers of laboratory adapted Edmonston MV strain that also uses ubiquitously expressed CD46 as entry receptor.</p

The expression of a novel CD150 splice isoform (nCD150).

<p>(A) Schematic representation of the CD150/SLAM gene structure and alternative splicing of mRNA. The exon designated Cyt-new is flanked with canonical splice sites (AG/GT marked in grey in the above genomic sequence). Exons are shown by filled rectangles, noncoding sequences—by solid line. Abbreviations: LS—leader sequence, V and C2—extracellular domains, TM—transmembrane domain, Cyt—cytoplasmic tail. (B) Expression of a nCD150 splice isoform was found in TE671, A172, U343, NCH92, NCH89 and U87 glioma cell lines, human tonsillar B cells (TBC) and cell lines of B cell origin, human acute monocytic leukemia cell line THP-1 and dendritic cells (DC), T cells (CD3⁺), monocytes (Mon) and macrophages (Mac), but it was not detected in human primary monocytes (Mon). The quality and quantity of cDNA was monitored by GAPDH expression. One of three representative experiments.</p

Real-time-PCR analysis of CD150 splice isoforms expression in glioma cell lines and primary tumors.

<p>We used 6 glioblastoma cell lines, four glioma tumor samples (GB1, GB2—glioblastoma, AODG—anaplastic oligodendroglioma, DA—diffuse astrocytoma), human tonsillar CD38⁺ B cells and lymphoblastoid cell line T5–1 for the analysis. Expression level of mRNA coding for each CD150 isoform was calculated using (ddCt) method, normalized to TBP and then expressed relative to respective isoform in CD38⁺ B cells, the value for which was set at 1. The results, presented as mean of triplicates (±SEM), are from one of three independent experiments. In glioma cell lines and glioma primary tumors the novel CD150 isoform is expressed at the high level, while the isoforms with the conventional cytoplasmic tail are absent or detected at the low level.</p

Cell lines transfected with mCD150 and nCD150 are susceptible to wild type measles virus infection.

<p>(A) Surface expression of mCD150 and nCD150 isoforms in HEK293T and U87 cell lines after transfection with respective plasmids measured by flow cytometry, after staining with anti-CD150 mAb IPO3. (B) Wild type MV (G954 strain) production in HEK293T cells transfected with mCD150 and nCD150, determined daily by plaque assay on Vero-SLAM cells, 24 h (1), 48 h (2), 72 h (3), 96h (4) and 120 h (5) post-infection. Vero-SLAM and non-transfected HEK293T cells were used as positive and negative control respectively. One of three independent experiments. The expression of both mCD150 and nCD150 isoforms on the surface of transfected cells allows the entry of wt measles virus to the cell.</p

Expression of CD150 in human glioma cell lines.

<p>(A) Flow cytometry study of CD150 surface expression on U87, U343 and A172 cells. One of four experiments. (B) Immunofluorescent analysis of CD150 expression in the cytoplasm of U87, U343 and A172 glioma cell lines. The cells were fixed, stained with anti-CD150 antibodies (IPO-3) followed by secondary antibody labelled with Alexa 488 (upper panel). Nuclei were visualized by staining with DAPI (middle panel). Results are representative of more than five experiments. Magnification: 630×. (C) Western blot analysis of CD150 expression in glioma cell lines using rabbit monoclonal anti-CD150 antibodies (Sino Biologicals Inc., China). Pre-B cell line REH and B-LCL MP-1 (in two different dilutions) were used as negative and positive controls respectively. One of five experiments is presented. The flow cytometry analysis of live cells does not show any CD150 expression in all used glioma cell lines, however fluorescent studies of fixed permeabilized cells and western blot detected CD150 expression in U87, U343, U251, TE671, NCH84, and NCH92 glioma cell lines.</p

Colocalization of CD150 with the markers of endoplasmic reticulum and Golgi apparatus.

<p>U87 or MP-1 cells were stained for CD150 (green) and the markers of endoplasmic reticulum (A) or Golgi apparatus (B) (red). Markers for endoplasmic reticulum—kinectin-1 and GRP78, for Golgi—furin. Nuclei were visualized by staining with DAPI (4’,6-diamidino-2-phenylindole). Colocalization coefficients were determined using the Manders algorithm (which ranges from 0 to 1, where 0 is defined as no colocalization and 1 as perfect colocalization) and are indicated within the panels. Confocal microscopy shows the similar high colocalisation of CD150 and ER markers in both types of cells, but significantly lower colocalisation of CD150 and Golgi marker in glioma cell line U87 comparing to B cell line MP-1. Microscopic magnification of 630× was used for all pictures. Digital magnification of 3150× was made for the insertions. The data are presented as mean ± SD (n = 7).</p

CD150 expression in CNS tumors.

<p>* Tumor grade according to WHO classification</p><p>CD150 expression in CNS tumors.</p

CD150 expression in serial sections of tumor and reactive brain tissues.

<p>(A) Diffuse astrocytoma with extensive microcyst formation. (B) Zone of infiltration with tumor cells of perifocal brain tissue. Scattered tumor cells are shown with arrows. Activated microglia and reactive astrocytes are CD150 negative. (C) Infiltration of subventricular zone with tumor cells (arrows), negative reaction in ependymal cells (Ep). DAB staining shows specific reaction in brown. Cell nuclei are counterstained with haematoxylin. Microscopic magnification 400×.</p