Identification of gene biomarkers for respiratory syncytial virus infection in a bronchial epithelial cell line
© US Government 2009
Received: 20 January 2009
Accepted: 24 April 2009
Published: 15 May 2009
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.
Respiratory syncytial virus (RSV), a negative-stranded RNA virus, is a common viral pathogen capable of causing respiratory tract infection and acute lung injury. In children, RSV bronchiolitis is estimated to cause 91,000 hospital admissions per year in the United States (Openshaw 2005). RSV is the pathogen in up to 50% of children hospitalized with bronchiolitis and 25% of children with pneumonia (Hall and McCarthy 1995). In adults, RSV may cause severe pneumonia with high mortality, especially in patients with underlying cardiopulmonary illnesses, the elderly and the immunocompromised (Falsey and Walsh 2000; La Montagne 1997). RSV infection also predisposes to the development of acute lung injury (Hammer et al. 1997; Hertz et al. 1989; Zaroukian et al. 1988).
The bronchial epithelial cell is the primary target of RSV. When RSV enters the respiratory tract, the surface proteins G (attachment) and F (fusion) of the virus interact with a heparan sulfate located on the bronchial epithelial cell surface (Feldman et al. 2000) (Levine et al. 1987). Once inside the cells, RSV replicates and assembles using the actin cytoskeleton (Ulloa et al. 1998) (Burke et al. 1998; Burke et al. 2000) (Werling et al. 1999) (Parton et al. 1994). Although the mechanisms by which RSV causes epithelial infection and injury are not entirely clear, there is increasing evidence that much of the pathology caused by RSV is due to the immune response to the infection (Openshaw 1995, 2005). Replication and proliferation of RSV inside the cells induce Th1/Th2 immune responses (Peebles and Graham 2005) (Welliver et al. 2002). The infected epithelial cells and immune cells release proinflammatory cytokines and chemokines that may result in acute lung injury (Krishnan et al. 2004). Inappropriate activation of the Th2 cellular immune response and deposition of antigen–antibody immune complexes may trigger complement activation and modulate the severity of RSV-induced cellular injury (Graham 1995; Openshaw 1995, 2005; Polack et al. 2002).
Our knowledge of the pathogenesis of RSV-induced lung infection is incomplete in part due to the complex virus-host cell interplay following the infection. Many previous studies have focused on specific aspects of the host response against RSV. Several microarray studies also have been reported (Kong et al. 2003; Martinez et al. 2007; Zhang et al. 2001). These studies provided a global view of gene expression during the complex virus-host cell interactions, but they were performed in transformed human alveolar epithelial cells using smaller arrays (A549 cells). It is unclear whether or not these events in distal lung cells could be extrapolated to the epithelial cells of proximal airways, which are early targets of RSV infection. In addition, clinical diagnosis of RSV infection still relies on the demonstration of the virus in the biological specimens. It is unclear how these gene biomarkers obtained in distal lung cells may help clinical diagnosis since these cells are less available. Thus in this study, we examined gene expression profile in human bronchial epithelial cells using microarray technology. The main goals were to explore signaling pathways and genes that may be used as biomarkers of infection in sputum specimens, which contain proximal airway cells.
For microarray experiments, human bronchial epithelial cell line (BEAS-2B, subclone S6) was obtained from the laboratory of Dr. Curtis C. Harris and maintained in serum-free growth medium (KGM, Clonetics, San Diego, CA) in T75 tissue culture flasks. The cells were used in experiments in their 62nd to 73rd passage. Cells were plated at 100 mm-diameter tissue culture plates (Costar, Cambridge, MA) in KGM and incubated at 37°C and 5% CO2 for 72 h before they were used for microarray experiments.
Primary human bronchial epithelial cells (HBEC) were also used for all non-microarray experiments (including PCR confirmation and siRNA).These primary cells were obtained from normal volunteers through bronchoscopic bronchial brushings (Ghio et al. 2000; Huang et al. 2003). Subjects were informed of the procedures and potential risks and each signed an informed consent. The protocol was approved by the University of North Carolina School of Medicine Committee on Protection of the Rights of Human Subjects and US Environmental Protection Agency. All brushings were processed by a single experienced technician following the established standard of procedures in our laboratory. The cells (passage 2 or 3) were maintained in bronchial epithelial growth medium (BEGM) (Clonetics, San Diego, CA), supplemented with bovine pituitary extract, insulin 5 μg/ml, hydrocortisone 0.5 μg/ml, gentamicin 50 μg/ml, retinoic acid 0.1 ng/ml, transferrin 10 μg/ml, triiodothyrodine 6.5 ng/ml, epinephrine 0.5 μg/ml, human epidermal growth factor 0.5 ng/ml.
Preparation of RSV
RSV (Long strain/lot 15D) was obtained from American Type Culture Collection (Bethesda, MD) and was propagated in mycoplasma-free HEp2 cells (ATCC 23-CCL, Bethesda, MD) as previously described (Becker et al. 1997). HEp2 supernatants containing infectious RSV were collected and the virus was precipitated using 10% polyethylene glycol (Sigma, St. Louis, MO). The precipitate was dissolved in NTE (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA), pH 7.4, and overlaid on a discontinuous 60, 45, and 30% sucrose gradient made up in NTE. After centrifugation for 90 min at 85,000×g in a Sorvall TH-641 rotor, the virus was collected from the 45–60% interface. This preparation contained ~1 × 107 plaque-forming units (PFU)/ml of RSV when tested for syncytia formation on HEp2 cells. The virus was snap frozen in liquid nitrogen and stored in small aliquots at −70°C until use.
For microarray experiments, confluent monolayer cultures of BEAS-2B were infected with RSV at a multiplicity of infection (MOI) of 1.0 PFU/cell. Virus was added to the cells for 2 h and then removed by a gentle wash with culture medium, followed by addition of 1 ml/well fresh KGM for 4 or 24 h (Soukup and Becker 2003). The control cells were incubated with vehicle.
Purification and hybridization of RNA
Total cellular RNA was extracted from the cells with Trizol reagent (GIBCO BRL Life Technologies, Gaithersburg, MD) and further purified with phenol/chloroform. The RNA integrity was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Inc, Pao Alto, CA). The 260 nm/280 nm ratios for all RNAs were >1.9. The RNAs were hybridized to Hu133 plus 2.0 gene chips (Affymetrix, Inc, Santa Clara CA). The chip contained probes for 38,500 human genes. The hybridization was performed by the Expression Analysis, Inc. (Durham, NC) according to the “Affymetrix Technical Manual”. Briefly, total RNA (10 μg) was converted into cDNA using Reverse Transcriptase (Invitrogen Corp, Carlsbad, CA) and a modified oligo(dT)24 primer that contains T7 promoter sequences (GenSet Corp, San Diego, CA). After first strand synthesis, residual RNA was degraded by the addition of RNaseH and a double-stranded cDNA molecule was generated using DNA polymerase I and DNA ligase. The cDNA was then purified and concentrated using a phenol: chloroform extraction followed by ethanol precipitation. The cDNA products were incubated with T7 RNA Polymerase and biotinylated ribonucleotide using an In Vitro Transcription kit (Enzo Diagnostics, Inc, New York, NY). One-half of the cRNA products were purified using an RNeasy column (Qiagen Inc, Valencia, CA) and quantified with a spectrophotometer. The cRNA target (20 μg) was incubated at 94°C for 35 min in fragmentation buffer (Tris, magnesium acetate, potassium acetate). The fragmented cRNA was diluted in hybridization buffer (2-morpholinoethanesulfonic acid, NaCl, EDTA, Tween 20, herring sperm DNA, acetylated bovine serum albumin) containing biotin-labeled OligoB2 and Eukaryotic Hybridization Controls (Affymetrix). The hybridization cocktail was denatured at 99°C for 5 min, incubated at 45°C for 5 min and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42°C for at least 16 h in a rotating oven at 60 rpm. GeneChips were washed with a series of non-stringent (25°C) and stringent (50°C) solutions containing variable amounts of 2-morpholinoethanesulfonic acid, Tween20 and SSPE (3 M NaCl, 0.2 M, NaH2PO4, 0.02 M EDTA). The microarrays were then stained with Streptavidin Phycoerythrin and the fluorescent signal was amplified using a biotinylated antibody solution. Fluorescent images were detected in a GeneChip® Scanner 3000 and expression data was extracted using the default settings in the MicroArray Suite 5.0 software (Affymetrix). All GeneChips were scaled to a median intensity setting of 500. A total of 9 microarrays were performed, including control and RSV (1.0 MOI) at 4 and 24 h (n = 3 biological replicates). The vehicle-treated cells were harvested at 24 h and used as the control for all RSV experiments.
Microarray data analysis
The microarray data were deposited in the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo/) (accession number GSE3397). Gene expression values were background-corrected and normalized globally using the default setting of the Affymetrix Microarray Suite version 5.0 and log2-transformed according to the “Affymetrix Statistical Algorithm Reference Guide”. The log2 signal ratios of RSV over control for all probe sets were compared to zero using the one class t-test with a p of <0.05 using the Multi experiment Viewer (MeV version 3.0, The Institute of Genomic Research, Rockville, MD). Genes that showed statistically significant expression over control and a cut-off fold change of >2.0 or <0.5 were retained. If more than one probe set for the same gene were flagged, their ratios were averaged. Only data from three microarray experiments were analyzed because one of the control data sets was found to be very different from the other three controls during the clustering analysis.
Pathway and biomarkers analysis
The differentially expressed genes at 4 and 24 h post-infection were categorized based on the KEGG_Pathway using the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 2006) (http://niaid.abcc.ncifcrf.gov/). Pathways with p ≤ 0.1 were considered significant. We also used Venn diagram and Ingenuity Pathway Analysis (http://www.ingenuity.com) (Ingenuity System, Inc, Redwood City, CA) to identify potential biomarkers for respiratory diseases.
Quantitative polymerase chain reaction (Q-PCR)
Q-PCR was performed for selected genes. cDNAs were synthesized from 0.4 μg of total RNA in 100 μl of buffer containing 5 μM random hexaoligonucleotide primers (Pharmacia, Piscataway, NJ), 10 U/μl Moloney murine leukemia virus reverse transcriptase (GIBCO BRL Life Technologies), 1 U/μl RNase inhibitor (RNasin®, Promega, Madison, WI), 0.5 mM dNTP (Pharmacia), 50 mM KCl, 3 mM MgCl2 and 10 mM Tris-HCl (pH 9.3) for 1 h at 39°C. Reverse transcriptase was heat-inactivated at 94°C for 4 min.
Q-PCR of specimen and standard cDNA was completed using TaqMan pre-developed assay reagents. Quantitative fluorogenic amplification of cDNA was performed using the ABI Prism 7500 Sequence Detection System, primers and probes of interest and TaqMan Universal PCR Master Mix (Applied Biosystems). The relative abundance of mRNA levels was determined from standard curves generated from a serially diluted standard pool of cDNA prepared from control HPAEC cultures. The relative abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Unigene accession #544577) mRNA was used to normalize levels of the mRNAs of interest. For Q-PCR verification, RNA from six additional experiments was collected. RNA samples for microarray and Q-PCR were collected from different experiments and do not represent the same sample.
IL-8 and CSF2 (GMCSF) in the medium were measured using ELISA kits purchased from R&D Systems (Minneapolis, MN) according to the manufacturer’s directions. The detection limit for the kits was 10 pg/ml.
Western blot analysis
After the exposure, the cells were washed once with ice cold phosphate-buffered saline (PBS) and then lysed with radioimmunoprecipitation (RIPA) buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 0.1 mM vandal sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml E-64, 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). The cell lysates were then centrifuged at 3,000g for 10 min at 4°C. Protein concentration of supernatant was measured with Bio-Rad protein assay reagent. Cellular proteins were separated by 10% SDS–PAGE and transferred to a polyvinylidene difluoride membrane. The blot was blocked with 5% milk in PBS with 0.05% Tween-20 for 1 h at room temperature, washed briefly, and then probed with a rabbit antibody against human arginase II (Catalog# sc-20151, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. This was followed by incubation with HRP-conjugated secondary antibodies. Bands were detected by using ECL and films.
Transfection of HBEC with arginase II siRNA
Cells were grown to ~60–70% confluency in 6-well plates. They were then transfected with Gene Silencer® transfecting agent plus (Gene Therapy System, Inc, San Diego, CA) with arginase II siRNA (100 nM) (Catalog# sc-29729, Santa Cruz Biotechnology, Santa Cruz, CA) in serum-free EGM-2 medium for 3 h according to the manufacturer’s recommendation. Control cells were incubated with Gene Silencer® negative control #1 siRNA (Ambion, Inc, TX). Fresh EGM-2 medium with 2% fetal bovine serum was then added and cells were cultured for an additional 24 h. Cells were then treated with RSV or vehicle as described above. Cell lysates were collected at 24 and 48 h post-infection and arginase II protein expression was measured by western blotting.
All non-microarray data are expressed as mean ± standard errors (SE). Data from multiple groups were analyzed by one way analysis of variance (ANOVA) followed by the Tukey’s subtest for between-group comparisons. The statistical analysis was performed using StatView (version 5.0.1, SAS, Inc, Cary NC). A p value of <0.05 was considered statistically significant.
Gene expression profile induced by RSV
Genes in the neuroactive ligand-receptor interaction pathway that were differentially expressed at 4 h (4 H) after RSV infection. Fold changes at 24 h (24 H) are also shown
Hypocretin (orexin) receptor 1
Purinergic receptor p2x, ligand-gated ion channel, 2
Glutamate receptor, ionotropic, delta 1
Tachykinin receptor 1
Angiotensin II receptor, type 2
Neuropeptide FF receptor 1
Gastric inhibitory polypeptide receptor
Glutamate receptor, metabotropic 5
Gamma-aminobutyric acid (gaba) A receptor, gamma 2
Vasoactive intestinal peptide receptor 1
Arginine vasopressin receptor 1a
Pathways associated with RSV infection at 24 h
p53 signaling pathway
Ubiquitin mediated proteolysis
Jak-STAT signaling pathway
Cytokine–cytokine receptor interaction
Small cell lung cancer
Hematopoietic cell lineage
Genes in P53 signaling pathway that were differentially expressed at 24 h (24 H) after RSV infection. Fold changes at 4 h (4 H) are also shown
Damage-specific DNA binding protein 2, 48 kDa
MDM2, transformed 3T3 cell double minute 2, p53 binding protein (mouse)
BCL2 binding component 3
Growth arrest and DNA-damage-inducible, beta
Phorbol-12-myristate-13-acetate-induced protein 1
Phosphatase and tensin homolog (mutated in multiple advanced cancers 1)
Cyclin-dependent kinase inhibitor 1A (p21, CIP1)
Seven in absentia homolog 1 (Drosophila)
Caspase 8, apoptosis-related cysteine peptidase
CHK1 checkpoint homolog (s. Pombe)
Genes in the apoptosis pathway that were differentially expressed at 24 h (24 H) after RSV infection. Fold changes at 4 h (4 H) are also shown
Baculoviral IAP repeat-containing 3
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
Phosphoinositide-3-kinase, regulatory subunit 5, p101
X-linked inhibitor of apoptosis
Interleukin-1 receptor-associated kinase 2
Interleukin 1 receptor, type I
Tumor necrosis factor receptor superfamily, member 10D, decoy with truncated death domain
Fas (TNFRSF6)-associated via death domain
Caspase 8, apoptosis-related cysteine peptidase
Genes in JAK-STAT signaling pathway that were differentially expressed at 24 h (24 H) after RSV infection. Fold changes at 4 h (4 H) are also shown
Colony stimulating factor 2 (granulocyte–macrophage)
Phosphoinositide-3-kinase, regulatory subunit 5, p101
Oncostatin M receptor
Thymic stromal lymphopoietin
Cas-Br-M (murine) ecotropic retroviral transforming sequence b
Sprouty homolog 2 (Drosophila)
Interferon gamma receptor 1
Suppressor of cytokine signaling 4
Interferon (alpha, beta and omega) receptor 1
Interleukin 12 receptor, beta 1
Genes in cytokine–cytokine receptor interaction pathway that were differentially expressed at 24 h (24 H) after RSV infection. Fold changes at 4 h (4 H) are also shown
Colony stimulating factor 2 (granulocyte–macrophage)
Nerve growth factor receptor (TNFR superfamily, member 16)
Oncostatin M receptor
Lymphotoxin beta (TNF superfamily, member 3)
Thymic stromal lympho poietin
Colony stimulating factor 1 (macrophage)
Vascular endothelial growth factor
Inhibin, beta A (activin A, activin AB alpha polypeptide)
Chemokine (c-c motif) ligand 23
Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
Interferon gamma receptor 1
Chemokine (c-X-c motif) ligand 2
Interleukin 1 receptor, type I
Tumor necrosis factor receptor superfamily, member 10D, decoy with truncated death domain
Interferon (alpha, beta and omega) receptor 1
Interleukin 12 receptor, beta 1
Pathways and biomarkers analysis
Top ten genes that were up- and down-regulated at both 4 and 24 h after RSV infection
ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d isoform 2
Sodium channel, non-voltage-gated 1, gamma
Solute carrier family 9 (sodium/hydrogen exchanger), isoform 1 (antiporter, Na+/H+, amiloride sensitive)
Cytochrome P450, family 4, subfamily F, polypeptide 8
Erythrocyte membrane protein band 4.1 like 4B
Signal transducer and activator of transcription 3 interacting protein 1
Protein kinase C, epsilon
HMG-box transcription factor 1
Dual specificity phosphatase 2
Tubulin, beta 1
tRNA nucleotidyl transferase, CCA-adding, 1
ELOVL family member 5, elongation of long chain fatty acids
Protein tyrosine phosphatase, receptor type, G
Mitogen-activated protein kinase 7
Testis specific, 10
Keratin 12 (Meesmann corneal dystrophy)
Signal-regulatory protein beta 1
Role of arginase II in RSV-induced epithelial injury
At 4 and 24 h after RSV infection of human bronchial epithelial cells, approximately 5 and 12.5% of the probe sets were altered respectively. There were 277 unique genes at 4 h and 912 genes at 24 h with a RSV/control ratio of ≥2.0 or ≤0.5. Three previous microarray studies with a smaller number of probe sets and genes have reported gene expression profile associated with RSV infection in distal airway epithelial cells (A549 cells) (Kong et al. 2003; Martinez et al. 2007; Zhang et al. 2001). Our study characterized gene expression pattern in proximal airway cells, an early target of RSV. Compared to these previous studies, our results showed some similarity and differences. All studies showed significant induction of inflammation-related genes, including many cytokines and chemokines, and induction of genes involved in cell growth/proliferation, cellular protein metabolism and cytoskeleton organization. Some of the unique results from our study were early effects on genes involved in neuroactive ligand-receptor interactions and later effects on a repair enzyme, arginase II.
The neuroactive ligand-receptor interaction pathway contains 11 highly significant genes with a RSV/control ratio of ≥2.0 or ≤0.5 (Table 1). These genes included six that encode G protein-related receptors (HCRTR1, TACR1, AGTR2, NGFFR1, GRM5 and AVPR1A), three that encode neurotransmission-related receptors (P2RX2, GRID1 and GABRG2) and two that encode receptors with endocrine and paracrine functions (GIPR and VIPR1). Two genes (TACR1, VIPR1) are known to be related to RSV infection. TACR1 belongs to a family of genes that function as receptors for tachykinin substance P (neurokinin 1). RSV infection increases the mRNA expression of TACR1 (Auais et al. 2003; King et al. 2001; Piedimonte 2003). Upregulation TACR1 would further enhance the substance P-mediated signaling during RSV-induced airway inflammation and dysfunction (Amadesi et al. 2001; Dakhama et al. 2005; Joos et al. 1994; Tan et al. 2008; Tripp et al. 2000). Persistent RSV infection in mice decreases VIP signaling (Tan et al. 2008). Downregulation of VIPR1 in our study is consistent with this observation. None of the other neuroactive ligand-receptor genes have been reported previously to be associated with RSV infection. These results showed a much more extensive network of neuroactive ligand-receptor interaction that can be activated early after RSV infection. These characteristic patterns underscore the unique localization of dendritic cells within bronchial epithelium and their critical role in respiratory viral infections (Grayson and Holtzman 2007). Early changes in these receptor genes are consistent with alterations in neuroimmune mechanisms that have been linked to airway inflammation and airway hyper responsiveness following RSV infection (Krishnan et al. 2004; Piedimonte 2003).
By 24 h after RSV infection, many more genes and signaling pathways were altered (Table 2). Alterations in some of these pathways were qualitatively similar to those shown in previous microarray studies. The p53 signaling pathway regulates cell growth and apoptosis induced by genotoxic and non-genotoxic stresses. Twelve genes were in the p53 pathway (Table 3), including upregulation of MDM2, a critical negative regulator of p53, that may contribute to the inhibition of p53 signaling and the anti-apoptotic effects in lung epithelial cells (Bitko et al. 2007; Groskreutz et al. 2007; Monick et al. 2005; Takeuchi et al. 1998; Thomas et al. 2002). Downregulation of pro-apoptotic genes (e.g., FADD, CASP 8) and upregulation of antiapoptotic genes (e.g., BIRC3, XIAP) may further inhibit apoptosis during RSV infection (Table 4). The JAK-STAT signaling pathway is another important pathway known to be activated by RSV in response to type I interferon’s (Kong et al. 2003) (Ramaswamy et al. 2004). There were 17 genes in this pathway (Table 5). Another DNA microarray study in A549 cells also identified genes containing STAT-binding elements (Kong et al. 2003). Activation of the JAK-STAT pathway also has been shown in previous non-gene profiling studies (Nguyen et al. 1997; Ramaswamy et al. 2004; Tarnawski and Jones 1998). The cytokine-cytokine receptor interaction pathway contained genes that are well known to be induced by RSV in lung epithelial cells (Table 6) (Arnold et al. 1994) (Becker et al. 1991) (Casola et al. 2001) (Fiedler et al. 1995) (Pazdrak et al. 2002) (Saito et al. 1997) (Soukup and Becker 2003) (Thomas et al. 1998) (Thomas et al. 2000) (Zhang et al. 2001). Genes in the cytokine–cytokine receptor interaction pathways included three TNF/TNF receptor family genes (LTB, NGFR, TNFRSF10D), two IFN-related genes (IFNAR1, IFNGR1), six interleukin family genes (IL12RB1, IL1R1, IL2, IL20, IL24, IL8) and 4 cytokine/chemokine genes (CCL23, CSF1, CSF2, CXCL2). We also noted upregulation of VEGF and FLT1, a VEGF receptor gene. Upregulation of these two genes may explain the activation of VEGF signaling responsible for increased permeability of bronchial epithelial cells during RSV infection (Kilani et al. 2004; Lee et al. 2000).
We also matched the altered genes with respiratory disease biomarkers in the Ingenuity Pathway Analysis. Twenty-three and 92 genes were identified at 4 and 24 h post-infection. Six genes were present at both time points. Four genes (SCNN1G, EPB41L4B, CSF1, PTEN) were upregulated, one gene (TUBB1) was down-regulated at 4 and 24 h and one gene (ESR2) was upregulated at 4 h, but downregulated at 24 h post-infection. These six genes may not only be pathogenetically important in RSV infection (e.g., CSF2 and TUBB1) (Arnold et al. 1994; Huang et al. 2005; Noah and Becker 1993), but may also serve as biomarkers of RSV infection.
The 92 respiratory disease biomarker genes also contain arginase II gene (ARG2). Arginase II is the mitochondrial isoform of arginase that catalyzes the conversion of L-arginine to L-ornithine and urea (Morris 2006). Arginase II is expressed constitutively in a number of extra-hepatic tissues, including bronchial epithelial cells (Que et al. 1998). Hierarchical clustering linked ARG2 closely to IL8, a major RSV-induced chemokine gene. The same cluster also contains other cytokine genes that are important in inflammation, including IL-24 (4.52-fold) and CXCL2 (2.18-fold). Upregulation of ARG2 during RSV infection is important in limiting cell injury since knockdown of arginase II gene expression by siRNA increased the release of IL-8, histone and LDH induced by RSV. This is consistent with the putative role of arginase II in tissue repair (Morris 2005). The potential implication of upregulation of arginase II during RSV infection may also extend to the pathogenesis of human asthma. Increased arginase expression and activity has been linked to the pathogenesis of asthma (King et al. 2004) (Vercelli 2003) (Zimmermann and Rothenberg 2006). Using gene expression profiling, Zimmerman et al. found genes related to metabolism of arginine, including cationic amino acid transporter 2, arginase I and arginase II, prominently expressed in an experimental model of asthma (Zimmermann et al. 2003). Since RSV is an important exacerbating factor and trigger for asthma (Kalina and Gershwin 2004; Openshaw 2003; Stein et al. 1999), arginase II may be one of the important mechanistic links between RSV infection and asthma.
In conclusion, using DNA microarray, we provided the gene expression profile in human bronchial epithelial cells during RSV infection. The gene expression profile in proximal airway cells share some similarity with those shown in distal lung cells, primarily in inflammation and cell growth and cell death. Our results also reveal novel genes and pathways that may be important in the pathogenesis of RSV infection and the associated complications. More significantly, we identified a set of genes that may be used as biomarkers of RSV infection. Since these genes are expressed in proximal airway cells, the clinical utility of these biomarker genes can be readily tested in the sputum samples in the future.
The authors would like to thank Lisa Dailey for assistance in cell culture and Dr. ZhengZheng Wei of Microarray Facility of the Duke Institute of Genomic Sciences and Policy for assistance in genomic statistical and pathway analyses.
- Amadesi S, Moreau J, Tognetto M et al (2001) NK1 receptor stimulation causes contraction and inositol phosphate increase in medium-size human isolated bronchi. Am J Respir Crit Care Med 163(5):1206–1211PubMedView ArticleGoogle Scholar
- Arnold R, Humbert B, Werchau H et al (1994) Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology 82(1):126–133PubMedPubMed CentralGoogle Scholar
- Auais A, Adkins B, Napchan G et al (2003) Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats. Am J Physiol Lung Cell Mol Physiol 285(1):L105–L113PubMedView ArticleGoogle Scholar
- Becker S, Quay J, Soukup J (1991) Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages. J Immunol 147(12):4307–4312PubMedGoogle Scholar
- Becker S, Reed W, Henderson FW et al (1997) RSV infection of human airway epithelial cells causes production of the beta-chemokine RANTES. Am J Physiol(3 Pt 1) 272:L512–L520Google Scholar
- Bitko V, Shulyayeva O, Mazumder B et al (2007) Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth. J Virol 81(4):1786–1795. doi:10.1128/JVI.01420-06PubMedPubMed CentralView ArticleGoogle Scholar
- Burke E, Dupuy L, Wall C et al (1998) Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus. Virology 252(1):137–148. doi:10.1006/viro.1998.9471PubMedView ArticleGoogle Scholar
- Burke E, Mahoney NM, Almo SC et al (2000) Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA. J Virol 74(2):669–675. doi:10.1128/JVI.74.2.669-675.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Casola A, Burger N, Liu T (2001) Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus Role in viral-induced interferon regulatory factor activation. J Biol Chem 276(23):19715–19722. doi:10.1074/jbc.M101526200PubMedView ArticleGoogle Scholar
- Curran JN, Winter DC, Bouchier-Hayes D (2006) Biological fate and clinical implications of arginine metabolism in tissue healing. Wound Repair Regen 14(4):376–386. doi:10.1111/j.1743-6109.2006.00151.xPubMedView ArticleGoogle Scholar
- Dakhama A, Park JW, Taube C et al (2005) Alteration of airway neuropeptide expression and development of airway hyperresponsiveness following respiratory syncytial virus infection. Am J Physiol Lung Cell Mol Physiol 288(4):L761–L770. doi:10.1152/ajplung.00143.2004PubMedView ArticleGoogle Scholar
- Falsey AR, Walsh EE (2000) Respiratory syncytial virus infection in adults. Clin Microbiol Rev 13(3):371–384. doi:10.1128/CMR.13.3.371-384.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Feldman SA, Audet S, Beeler JA (2000) The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J Virol 74(14):6442–6447. doi:10.1128/JVI.74.14.6442-6447.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Fiedler MA, Wernke-Dollries K, Stark JM (1995) Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells. Am J Physiol(6 Pt 1) 269:L865–L872Google Scholar
- Ghio AJ, Kim C, Devlin RB (2000) Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. Am J Respir Crit Care Med(3 Pt 1) 162:981–988View ArticleGoogle Scholar
- Graham BS (1995) Pathogenesis of respiratory syncytial virus vaccine-augmented pathology. Am J Respir Crit Care Med 152:S63–S66PubMedView ArticleGoogle Scholar
- Grayson MH, Holtzman MJ (2007) Emerging role of dendritic cells in respiratory viral infection. J Mol Med 85(10):1057–1068. doi:10.1007/s00109-007-0212-3PubMedView ArticleGoogle Scholar
- Groskreutz DJ, Monick MM, Yarovinsky TO et al (2007) Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells. J Immunol 179(5):2741–2747PubMedView ArticleGoogle Scholar
- Hall CB, McCarthy CA (1995) In: Mandel GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases. Churchill Livingston, New YorkGoogle Scholar
- Hammer J, Numa A, Newth CJ (1997) Acute respiratory distress syndrome caused by respiratory syncytial virus. Pediatr Pulmonol 23(3):176–183. doi:10.1002/(SICI)1099-0496(199703)23:3%3C176::AID-PPUL2%3E3.0.CO;2-MPubMedView ArticleGoogle Scholar
- Hertz MI, Englund JA, Snover D et al (1989) Respiratory syncytial virus-induced acute lung injury in adult patients with bone marrow transplants: a clinical approach and review of the literature. Medicine (Baltimore) 68(5):269–281. doi:10.1097/00005792-198909000-00002View ArticleGoogle Scholar
- Huang YC, Ghio AJ, Stonehuerner J et al (2003) The role of soluble components in ambient fine particles-induced changes in human lungs and blood. Inhal Toxicol 15(4):327–342. doi:10.1080/08958370304460PubMedView ArticleGoogle Scholar
- Huang YC, Li Z, Brighton LE et al (2005) 3-nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line. Am J Physiol Lung Cell Mol Physiol 288(5):L988–L996. doi:10.1152/ajplung.00378.2004PubMedView ArticleGoogle Scholar
- Joos GF, Germonpre PR, Kips JC et al (1994) Sensory neuropeptides and the human lower airways: present state and future directions. Eur Respir J 7(6):1161–1171PubMedGoogle Scholar
- Kalina WV, Gershwin LJ (2004) Progress in defining the role of RSV in allergy and asthma: from clinical observations to animal models. Clin Dev Immunol 11(2):113–119. doi:10.1080/10446670410001722131PubMedPubMed CentralView ArticleGoogle Scholar
- Kilani MM, Mohammed KA, Nasreen N et al (2004) Respiratory syncytial virus causes increased bronchial epithelial permeability. Chest 126(1):186–191. doi:10.1378/chest.126.1.186PubMedView ArticleGoogle Scholar
- King KA, Hu C, Rodriguez MM et al (2001) Exaggerated neurogenic inflammation and substance P receptor upregulation in RSV-infected weanling rats. Am J Respir Cell Mol Biol 24(2):101–107PubMedView ArticleGoogle Scholar
- King NE, Rothenberg ME, Zimmermann N (2004) Arginine in asthma and lung inflammation. J Nutr 134(10):2830S–2836S discussion 2853SPubMedGoogle Scholar
- Kong X, San Juan H, Kumar M et al (2003) Respiratory syncytial virus infection activates STAT signaling in human epithelial cells. Biochem Biophys Res Commun 306(2):616–622. doi:10.1016/S0006-291X(03)01008-8PubMedView ArticleGoogle Scholar
- Krishnan S, Halonen M, Welliver RC (2004) Innate immune responses in respiratory syncytial virus infections. Viral Immunol 17(2):220–233. doi:10.1089/0882824041310612PubMedView ArticleGoogle Scholar
- La Montagne JR (1997) RSV pneumonia, a community-acquired infection in adults. Lancet 349(9046):149–150. doi:10.1016/S0140-6736(05)60974-9PubMedView ArticleGoogle Scholar
- Lee CG, Yoon HJ, Zhu Z et al (2000) Respiratory syncytial virus stimulation of vascular endothelial cell growth Factor/Vascular permeability factor. Am J Respir Cell Mol Biol 23(5):662–669PubMedView ArticleGoogle Scholar
- Levine S, Klaiber-Franco R, Paradiso PR (1987) Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 68(Pt 9):2521–2524. doi:10.1099/0022-1317-68-9-2521PubMedView ArticleGoogle Scholar
- Martinez I, Lombardia L, Garcia-Barreno B et al (2007) Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells. J Gen Virol 88(Pt 2):570–581. doi:10.1099/vir.0.82187-0PubMedView ArticleGoogle Scholar
- Monick MM, Cameron K, Staber J et al (2005) Activation of the epidermal growth factor receptor by respiratory syncytial virus results in increased inflammation and delayed apoptosis. J Biol Chem 280(3):2147–2158. doi:10.1074/jbc.M408745200PubMedView ArticleGoogle Scholar
- Morris SM Jr (2005) Arginine metabolism in vascular biology and disease. Vasc Med 10(Suppl 1):S83–S87. doi:10.1177/1358836X0501000112PubMedView ArticleGoogle Scholar
- Morris SM Jr (2006) Arginine: beyond protein. Am J Clin Nutr 83(2):508S–512SPubMedGoogle Scholar
- Nguyen H, Lin R, Hiscott J (1997) Activation of multiple growth regulatory genes following inducible expression of IRF-1 or IRF/RelA fusion proteins. Oncogene 15(12):1425–1435. doi:10.1038/sj.onc.1201318PubMedView ArticleGoogle Scholar
- Noah TL, Becker S (1993) Respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line. Am J Physiol 265:L472–L478PubMedGoogle Scholar
- Openshaw PJ (1995) Immunopathological mechanisms in respiratory syncytial virus disease. Springer Semin Immunopathol 17(2–3):187–201. doi:10.1007/BF00196165PubMedGoogle Scholar
- Openshaw PJ (2003) RSV bronchiolitis, gammadelta T cells and asthma: are they linked? Clin Exp Immunol 131(2):197–198. doi:10.1046/j.1365-2249.2003.02086.xPubMedPubMed CentralView ArticleGoogle Scholar
- Openshaw PJ (2005) Antiviral immune responses and lung inflammation after respiratory syncytial virus infection. Proc Am Thorac Soc 2(2):121–125. doi:10.1513/pats.200504-032AWPubMedView ArticleGoogle Scholar
- Parton RG, Joggerst B, Simons K (1994) Regulated internalization of caveolae. J Cell Biol 127(5):1199–1215. doi:10.1083/jcb.127.5.1199PubMedView ArticleGoogle Scholar
- Pazdrak K, Olszewska-Pazdrak B, Liu T et al (2002) MAPK activation is involved in posttranscriptional regulation of RSV- induced RANTES gene expression. Am J Physiol Lung Cell Mol Physiol 283(2):L364–L372PubMedView ArticleGoogle Scholar
- Peebles RS Jr, Graham BS (2005) Pathogenesis of respiratory syncytial virus infection in the murine model. Proc Am Thorac Soc 2(2):110–115. doi:10.1513/pats.200501-002AWPubMedPubMed CentralView ArticleGoogle Scholar
- Piedimonte G (2003) Contribution of neuroimmune mechanisms to airway inflammation and remodeling during and after respiratory syncytial virus infection. Pediatr Infect Dis J 22(2):S66–S74. doi:10.1097/00006454-200302001-00010 discussion S74-65PubMedView ArticleGoogle Scholar
- Polack FP, Teng MN, Collins PL et al (2002) A role for immune complexes in enhanced respiratory syncytial virus disease. J Exp Med 196(6):859–865. doi:10.1084/jem.20020781PubMedPubMed CentralView ArticleGoogle Scholar
- Que LG, Kantrow SP, Jenkinson CP et al (1998) Induction of arginase isoforms in the lung during hyperoxia. Am J Physiol(1 Pt 1) 275:L96–L102Google Scholar
- Ramaswamy M, Shi L, Monick MM et al (2004) Specific inhibition of type I interferon signal transduction by respiratory syncytial virus. Am J Respir Cell Mol Biol 30(6):893–900. doi:10.1165/rcmb.2003-0410OCPubMedView ArticleGoogle Scholar
- Saito T, Deskin RW, Casola A et al (1997) Respiratory syncytial virus induces selective production of the chemokine RANTES by upper airway epithelial cells. J Infect Dis 175(3):497–504PubMedView ArticleGoogle Scholar
- Satriano J (2003) Agmatine: at the crossroads of the arginine pathways. Ann N Y Acad Sci 1009:34–43. doi:10.1196/annals.1304.004PubMedView ArticleGoogle Scholar
- Soukup JM, Becker S (2003) Role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro. Clin Immunol 107(3):178–185. doi:10.1016/S1521-6616(03)00038-XPubMedView ArticleGoogle Scholar
- Stein RT, Sherrill D, Morgan WJ et al (1999) Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 354(9178):541–545. doi:10.1016/S0140-6736(98)10321-5PubMedView ArticleGoogle Scholar
- Takeuchi R, Tsutsumi H, Osaki M et al (1998) Respiratory syncytial virus infection of human alveolar epithelial cells enhances interferon regulatory factor 1 and interleukin-1beta-converting enzyme gene expression but does not cause apoptosis. J Virol 72(5):4498–4502PubMedPubMed CentralGoogle Scholar
- Tan YR, Yang T, Liu SP et al (2008) Pulmonary peptidergic innervation remodeling and development of airway hyperresponsiveness induced by RSV persistent infection. Peptides 29(1):47–56. doi:10.1016/j.peptides.2007.10.020PubMedView ArticleGoogle Scholar
- Tarnawski AS, Jones MK (1998) The role of epidermal growth factor (EGF) and its receptor in mucosal protection, adaptation to injury, and ulcer healing: involvement of EGF-R signal transduction pathways. J Clin Gastroenterol 27(Suppl 1):S12–S20. doi:10.1097/00004836-199800001-00004PubMedView ArticleGoogle Scholar
- Thomas LH, Friedland JS, Sharland M et al (1998) Respiratory syncytial virus-induced RANTES production from human bronchial epithelial cells is dependent on nuclear factor-kappa B nuclear binding and is inhibited by adenovirus-mediated expression of inhibitor of kappa B alpha. J Immunol 161(2):1007–1016PubMedGoogle Scholar
- Thomas LH, Wickremasinghe MI, Sharland M et al (2000) Synergistic upregulation of interleukin-8 secretion from pulmonary epithelial cells by direct and monocyte-dependent effects of respiratory syncytial virus infection. J Virol 74(18):8425–8433. doi:10.1128/JVI.74.18.8425-8433.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Thomas KW, Monick MM, Staber JM et al (2002) Respiratory syncytial virus inhibits apoptosis and induces NF-kappa B activity through a phosphatidylinositol 3-kinase-dependent pathway. J Biol Chem 277(1):492–501. doi:10.1074/jbc.M108107200PubMedView ArticleGoogle Scholar
- Tripp RA, Moore D, Winter J et al (2000) Respiratory syncytial virus infection and G and/or SH protein expression contribute to substance P, which mediates inflammation and enhanced pulmonary disease in BALB/c mice. J Virol 74(4):1614–1622. doi:10.1128/JVI.74.4.1614-1622.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Ulloa L, Serra R, Asenjo A et al (1998) Interactions between cellular actin and human respiratory syncytial virus (HRSV). Virus Res 53(1):13–25. doi:10.1016/S0168-1702(97)00121-4PubMedView ArticleGoogle Scholar
- Vercelli D (2003) Arginase: marker, effector, or candidate gene for asthma? J Clin Invest 111(12):1815–1817PubMedPubMed CentralView ArticleGoogle Scholar
- Welliver RC, Garofalo RP, Ogra PL (2002) Beta-chemokines, but neither T helper type 1 nor T helper type 2 cytokines, correlate with severity of illness during respiratory syncytial virus infection. Pediatr Infect Dis J 21(5):457–461. doi:10.1097/00006454-200205000-00033PubMedView ArticleGoogle Scholar
- Werling D, Hope JC, Chaplin P et al (1999) Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells. J Leukoc Biol 66(1):50–58PubMedGoogle Scholar
- Witte MB, Barbul A (2003) Arginine physiology and its implication for wound healing. Wound Repair Regen 11(6):419–423. doi:10.1046/j.1524-475X.2003.11605.xPubMedView ArticleGoogle Scholar
- Zaroukian MH, Kashyap GH, Wentworth BB (1988) Respiratory syncytial virus infection: a cause of respiratory distress syndrome and pneumonia in adults. Am J Med Sci 295(3):218–222. doi:10.1097/00000441-198803000-00011PubMedView ArticleGoogle Scholar
- Zhang Y, Luxon BA, Casola A et al (2001) Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays. J Virol 75(19):9044–9058. doi:10.1128/JVI.75.19.9044-9058.2001PubMedPubMed CentralView ArticleGoogle Scholar
- Zimmermann N, Rothenberg ME (2006) The arginine-arginase balance in asthma and lung inflammation. Eur J Pharmacol 533(1–3):253–262. doi:10.1016/j.ejphar.2005.12.047PubMedView ArticleGoogle Scholar
- Zimmermann N, King NE, Laporte J et al (2003) Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest 111(12):1863–1874PubMedPubMed CentralView ArticleGoogle Scholar