Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV‐infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV‐mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.


| INTRODUC TI ON
Respiratory syncytial virus (RSV), a single-strand negative sense RNA virus, is the most important cause of lower respiratory tract infection in early childhood 1 ; especially in preterm neonates with chronic lung diseases like bronchopulmonary dysplasia (BPD), these infections can be clinically severe being associated with high morbidity and mortality. In addition, RSV increases the long-term risk for subsequent wheezing and elevated susceptibility to asthma.
Underlying molecular mechanisms are not yet fully known. 2 Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor (TGF)β, is a matricellular signaling modulator and a key molecule in tissue remodeling. 3 As upregulation of CTGF has been detected after mechanical ventilation and hyperoxia in the neonatal lung, 4 in proliferating type II lung epithelial cells, and activated fibroblasts of fibrotic lungs, 5 it has been suggested that CTGF may contribute to BPD. Moreover, a role of RSV in activation of TGFβ gene expression has been described, 6 suggesting a possible involvement of CTGF during RSV infection.
Administration of glucocorticoids to attenuate severe forms of BPD in preterm infants is controversially discussed, 7 and the potential impacts of glucocorticoids as well as RSV infection on remodeling processes of the lung are barley defined. Caffeine, a methylxanthine commonly used to minimize apnea of prematurity, has been associated with the ability to reduce the incidence of BPD. 8 We recently described increase in CTGF expression by various glucocorticoids in lung epithelial and fibroblast models. 9 In addition, we have shown that TGF-β1-induced upregulation of CTGF can be antagonized by caffeine in A549 lung epithelial cells. 10 As anti-inflammatory agents appear to offer no clinical benefit, 11 the interplay of inflammatory trigger, inflammation, and novel agents positively influencing remodeling processes of the lung is the subject of intense research.
We hypothesized that caffeine might counteract detrimental effects of glucocorticoids and RSV infection. Using the human lung epithelial cell line H441 and the fetal lung fibroblast strain IMR-90, the aim of this study was to define if RSV has an impact on the expression of CTGF mRNA in the simultaneous presence of dexamethasone and caffeine.

Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells
Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.

K E Y W O R D S
Caffeine, CCN2, dexamethasone, lung remodeling, poly(I:C)

| Cells and virus
NCI-H441 (H441) and IMR-90 cells were purchased from ATCC (LGC Standards, Teddington, UK) and cultured as described. 9 Recombinant green fluorescent protein-expressing RSV (rgRSV) was a kind gift of Peter L. Collins (National Institute of Allergy, Immunology, and Infectious Diseases, National Institutes of Health, Bethesda, MD).

| Infection and transfection
For infection, cells were seeded on six-well plates (Greiner, Frickenhausen, Germany) until 80% confluence was reached and infected with 5 × 10 5 pfu of rgRSV. After 2 hour, medium was removed and cells were incubated with substances in growth medium as indicated. Images of cells were captured using a DM IRE 220 microscope (Leica, Solms, Germany). For transfection, 5 × 10 5 cells were seeded on six-well plates (Greiner) and 16 hour later transfected with 10 μg poly(I:C) using 20 μg PEI in a total volume of 1 mL Opti-MEM I (Gibco, Thermo Fisher Scientific, Waltham, MA). After 4 hour, medium was removed and cells were treated for 24 hour with substances in growth medium as indicated.

| RNA extraction, RT-PCR, and quantitative real-time PCR (qPCR)
RNA extraction, RT-PCR, and qPCR were performed as described previously. 9 Levels of mRNAs were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences for qPCR are listed in Table 1. Mean fold changes in mRNA expression were calculated by the ΔΔC T method.

| Statistical analysis
Results are given as means ± SD. Unless otherwise stated, data were analyzed using one way ANOVA with Bonferroni's multiple comparison post hoc test. A P-value ≤.05 was considered significant. All statistical analyses were performed using Prism ® version 6 (GraphPad Software, San Diego, CA).

| D ISCUSS I ON
We report an upregulation of CTGF mRNA by RSV infection in H441 lung epithelial cells which was additive to the recently described induction by glucocorticoids. 9 Different to work describing a reduction of vanadium pentoxide-induced fibrosis by RSV via reduction of growth factors, 12 our data indicate that RSV infection might conceivably be able to induce fibrotic conditions by increasing CTGF mRNA in lung epithelial cells which might be further enhanced by glucocorticoids. 3,13 It has been shown that RSV-induced exacerbation of airway hyperresponsiveness in a mouse model was only partially suppressed by steroid treatment. 14 According to our observations in H441 cells, the simultaneous presence of glucocorticoids and RSV infection might provoke aggravated remodeling processes in the lower airways via increase in CTGF expression, which has been demonstrated in bleomycin-induced fibrosis. 13 Furthermore, in addition to the prevention of glucocorticoid-mediated increase, 9 our data suggest that caffeine treatment is able to block RSV-as well as dexamethasone/ RSV-caused upregulation of CTGF mRNA expression. Apart from its preventive effects on the development of BPD, 8  . IL-6 (C and D) and IL-8 (E and F) mRNA levels of H441 (C and E) and IMR-90 cells (D and F), respectively, were normalized to GAPDH mRNA and fold differences compared to corresponding untreated cells were calculated. Means + SD of n = 4 independent experiments are shown. *P < .05; **P < .01; ***P < .001 compared to untreated controls; # P < .05; ## P < .01; ### P < .001 compared to cells treated with dexamethasone; $$$ P < .001 compared to cells infected with rgRSV a maximum of infection could be reached was too early to detect CTGF protein expression in H441 cells.

| CON CLUS ION
We report a transcriptional attribute of increased CTGF mRNA expression in H441 cells following infection with RSV potentially prevalent for and restricted to lung epithelial cells. Caffeine might have the ability to support anti-inflammatory actions of glucocorticoids but antagonize initiation of CTGF expression, thereby pushing the overall outcome from long-term morbidity to healing.

ACK N OWLED G EM ENTS
We thank Peter L. Collins (National Institute of Allergy, Immunology, and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) for providing rgRSV and Theresa Kreuzahler for excellent technical support.