TY - JOUR A1 - Riedel, Alice A1 - Mofolo, Boitumelo A1 - Avota, Elita A1 - Schneider-Schaulies, Sibylle A1 - Meintjes, Ayton A1 - Mulder, Nicola A1 - Kneitz, Susanne T1 - Accumulation of Splice Variants and Transcripts in Response to PI3K Inhibition in T Cells JF - PLoS ONE N2 - Background Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression. KW - T cells KW - gene regulation KW - alternative splicing KW - measles virus KW - T cell receptors KW - reverse transcriptase-polymerase chain reaction KW - cell cycle and cell division KW - TCR signaling cascade Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130335 VL - 8 IS - 2 ER - TY - JOUR A1 - Morton, Charles Oliver A1 - Fliesser, Mirjam A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Bauer, Ruth A1 - Kneitz, Susanne A1 - Hope, William A1 - Rogers, Thomas Richard A1 - Einsele, Hermann A1 - Löffler, Jürgen T1 - Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface N2 - The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA. KW - aspergillus fumigatus KW - gene expression KW - immune receptors KW - immune response KW - denritic cells KW - B cell receptors KW - gene regulation KW - RNA extraction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112893 ER -