Identification of gene biomarkers for respiratory syncytial virus infection in a bronchial epithelial cell line

Abstract

Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to Affymetrix HU133 plus 2.0 microarrays (n = 4). At 4 and 24 h post-infection, 277 and 900 genes (RSV/control ratio >/=2.0 or </=0.5), and 1 and 12 pathways respectively were significantly altered. Twenty-three and 92 genes at 4 and 24 h respectively matched respiratory disease biomarkers with ARG2 flagged at 24 h and SCNN1G, EPB41L4B, CSF1, PTEN, TUBB1 and ESR2 at both time points. Hierachical clustering showed a cluster containing ARG2 and IL8. In human bronchial epithelial cells, RSV upregulated arginase II protein. Knockdown of ARG2 increased RSV-induced IL-8, LDH and histone release. With microarray, we identified novel proximal airway epithelial cell genes that may be tested in the sputum samples as biomarkers of RSV infection.

Fig. 1

Confirmation of expression of 7 upregulated genes by Q-PCR. n = 4 for microarray; n = 4 experiments in bronchial epithelial cells from 4 different donors for Q-PCR

Fig. 2

Effects of RSV on the release of CSF2 (GMCSF) and interleukin-8 (IL-8). BEAS-2B cells were incubated with 0.1, 0.3 and 1.0 moi of RSV for 48 h. * p < 0.05 vs. no RSV. n = 4–6 each

Fig. 3

Venn diagrams comparing probe sets differentially expressed at 4 and 24 h post-infection. Numbers in each area represent the numbers of probe sets

Fig. 4

Expression of six genes that were significantly altered at both 4 and 24 h post-infection and were also biomarkers for respiratory diseases based on the biomarker filter of ingenuity pathway analysis

Fig. 5

Hierarchical clustering of the 912 differentially expressed genes at 24 h after RSV infection. a The heat map. The arrow indicates the location of the cluster that contains IL-8 and arginase II; b genes in this cluster

Fig. 6

Up-regulation of arginase II by RSV in primary human bronchial epithelial cells. Human bronchial epithelial cells were treated with RSV at multiplicity of infection (MOI) of 1.0 and 2.0. Arginase II protein expression was measured at 48 h post-infection. Upper panel shows a representive western blot. Lower panel is the densitometry result. n = 3–4 independent experiments. * p < 0.05 vs. time 0 or MOI = 0

Fig. 7

Effects of arginase II knockdown on RSV-induced injury in primary human bronchial epithelial cells. a Western blot analysis of arginase II in cells with or without arginase II siRNA pretreatment. Knockdown of arginase II gene expression with siRNA increased the release of IL-8; b LDH; c and histone; d induced by RSV. n = 4 independent experiments. * p < 0.05 vs. control; # p < 0.05 vs. RSV