This study was approved by the Animal Care and Use Committee of Qingdao University (Qingdao, China) and conducted in compliance with the guidelines of the National Institutes of Health for the care and the use of laboratory animals.

The strain A2 of human RSV was propagated in HeLa cells (both from the Virology Institute of Wuhan, Wuhan, China) in minimal essential medium supplemented with 2% heat-inactivated fetal bovine serum (both from Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). When 75% of the cells formed syncytia, the cells were harvested and disrupted by sonication using the same culture medium. The suspension was clarified by centrifugation at 10,000 × g for 15 min at 4°C and stored in aliquots at −70°C. Sham inoculum was prepared from uninfected HeLa cell lysate using the same procedure. The viral titer in the inoculums was determined by plaque assay (8) on the same day as the mice were inoculated.

Ten-week-old male C57BL/6 mice (n=80; weight, 20–25 g) were purchased from the Affiliated Hospital of Qingdao University (Qingdao, China). Mice were given free access to food and water and were maintained with a 12-h light/dark cycle. Mice in the RSV group were endotracheally injected 100 µl of PBS and 100 µl of RSV (2×10⁶ pfu/ml). Mice in the bleomycin group were endotracheally injected with 100 µl bleomycin (1 mg/ml) in sterile PBS and 100 µl Sham inoculum. Mice in the RSV+bleomycin group were endotracheally injected RSV (100 µl, 2×10⁶ pfu/ml) and bleomycin (100 µl, 1 mg/ml) (9). Combined sham inoculums in place of RSV, and PBS in place of bleomycin were used as a control. At 7, 14 or 21 days following injection, the animals were sacrificed, and analyzed. All experimental animals used in the current study were maintained under the protocol approved by the Institutional Animal Care and Use Committee of Qingdao University.

BALFs were centrifuged at 1,000 × g for 5 min at room temperature and the cell pellets were smeared onto glass slides. Cells were air dried and stained with Wright stain, which allows differential counting of various cells. In each sample, ≥300 cells per sample were counted under a light microscope.

Interleukin (IL)-1, -5 and -8 productions in BALF were measured using ELISA kits for mouse IL-1β (70-EK201B2/2), mouse IL-5 (70-EK2052) (both from Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China) and mouse IL-8 (kt21138; MoShaKe Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Briefly, a total of 100 µl BALF supernatants were added into a 96-well plate and incubated for 1 h, followed by 100 µl enzyme-linked antibodies for 30 min at 37°C. Following three washes with washing buffer, the chromogenic reagent was added and incubated for 30 min. 2 M H₂SO₄ was added to terminate reaction. Absorbance was determined at 450 nm using a microplate reader [Tecan Sunrise; Tecan (Shanghai) Trading Co., Ltd., Shanghai, China]. Each sample was repeated three times.

Mice were sacrificed with an intraperitoneal injection of sodium pentobarbitone (100 mg/kg) and the right lung lobes were obtained. Lobes were inflated (10% paraformaldehyde perfusion via trachea), harvested, fixed in 4% paraformaldehyde overnight, embedded in paraffin and cut into 5 µm sections. Endogenous peroxidase was inhibited by soaking tissue sections in 3% H₂O₂. Following rinsing in PBS, sections were incubated with goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) to block nonspecific binding of antibodies, and were then incubated overnight at 4°C with rabbit anti-human RSV polyclonal antibodies (orb101035; Biorbyt LLC, San Francisco, CA, USA; dilution, 1:250). Following washing in PBS, the sections were incubated with biotinylated goat anti rabbit IgG (Vector BA-1,000; Vector Laboratories, Inc., Burlingame, CA, USA) at 37°C for 1 h, and washed again. Following washing again in PBS, the signal was detected with 3,3′-diaminobenzidine. Omission of primary or secondary antisera was included as a method of providing a control for each biopsy.

Following soaking the sections in 3% H₂O₂ for 15 min and 10 µl of goat serum for 30 min, sections were incubated overnight at 4°C with rabbit anti collagen type I polyclonal antibodies (1:150; SAB4500362; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Following washing in PBS, the sections were incubated with fluorescein isothiocyanate-labeled goat anti rabbit IgG (1:1,000; A22120; Beyotime Institute of Biotechnology, Haimen, China) for 1 h, and washed again. The slides were finally mounted with mounting media (Thermo Fisher Scientific, Inc.) and examined by fluorescence microscopy.

Lung tissues were washed by ice-cold PBS twice and extracted in radioimmunoprecipitation assay buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride, 5 mM β-glycerophosphate and 1% standard protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min. Protein concentrations were measured by Bradford assay (Thermo Fisher Scientific, Inc.) and adjusted to a final concentration of 10 mg/ml. Lung homogenates (50 µg) were subjected to separation by 12% SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) and blocked with 5% skimmed milk. After heat-induced epitope retrieval (Microwave on high for 15 min), the slides were incubated with rabbit anti-mouse p-PERK (1:150; sc-13073), anti-GRP78 (1:250; sc-13968), anti-CHOP (1:200; sc-575), anti-ATF6α (1:100; sc-22799), anti-XBP-1 (1:150; sc-7160) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit anti-collagen type I polyclonal antibodies (1:200; SAB4500362; Sigma-Aldrich; Merck KGaA) overnight at 4°C. Blots were developed using corresponding horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:2,000; A0208; Beyotime Institute of Biotechnology) at 37°C for 1 h, and detected using an enhanced chemiluminescence system (Amersham ECL Plus; GE Healthcare Life Sciences, Chalfont, UK). Band intensities were quantified by Image Gauge (version 3.4) software with the LAS-1,000 plus system (Fujifilm Holdings Corporation, Tokyo, Japan). β-actin served as a loading control.

The statistical significance of the data was analyzed using the SPSS software (version 14.0; SPSS Inc., Chicago, IL, USA). Data were expressed as means ± standard deviation. Statistical analyses were performed using an unpaired Student's t-test for the comparison of data from different treatment groups. P<0.05 was considered to indicate a statistically significant difference.

The results of immnohistochemistry indicated that RSV (dark brown staining) was evident in the airway epithelium and peaked at day 7 and decreased with infection time in the RSV alone group. In RSV+bleomycin group, the RSV infection was localized predominantly to alveoli and was persistent in the lungs from day 7 to day 21 (Fig. 1). No viruses were observed in uninfected animals (data not shown).

The lungs of mice infected with RSV presented significant increases in the numbers of eosinophils at day 7 (P<0.01), when compared with the control group, before returning to normal levels. In addition, the bleomycin group demonstrated obvious increases in macrophages and neutrophils at day 7 (both P<0.01), only macrophages at day 14 (P<0.01), when compared with the control group before returning to normal levels of all red blood cell types at day 21. However, the RSV+bleomycin group exhibited obvious increases in macrophages, neutrophils and lymphocytes at day 7, 14 and 21 (P<0.01) when compared with bleomycin group (Fig. 2A). In pursuit of the effects of RSV on the process of bleomycin-induced pulmonary fibrosis, the authors investigated the production of the pro-inflammatory cytokines IL-1, -5 and -8 in BALF. As demonstrated in Fig. 2B, RSV promoted the production of IL-1 and -5 at day 7 (both P<0.01), when compared with the control group, before returning to normal levels. Bleomycin induced release of IL-1 and -8 at day 7 and day 14 (all P<0.01), when compared with the control group, before returning to normal levels at day 21. However, RSV+bleomycin group presented obvious increases in IL-1 and -8 at days 7, 14 and 21 (all P<0.01), when compared with control or the bleomycin group.

Excessive production and deposition of extracellular matrix components is crucial for the development of pulmonary fibrosis. As demonstrated in Fig. 3, immunofluorescence (Fig. 3A) and western blot analyses (Fig. 3B and C) identified that RSV or bleomycin alone promoted the accumulation of collagen type I in the lungs at day 7, when compared with the control group (Fig. 3C; P<0.01); these effects decreased with time. However, RSV+bleomycin promoted more collagen type I over the time course compared with bleomycin alone.

Using western blot analysis, GRP78, p-PERK, CHOP, XBP-1 and ATF6α were detectable at low levels in control lung tissues at days 7, 14 and 21 (Fig. 4). Mice treated with RSV demonstrated significantly elevated levels of p-PERK, ATF6α and GRP78 at day 7 compared with those of controls and returned to normal levels at days 14 and 21 (Fig. 4; all P<0.01). Mice treated with bleomycin demonstrated significantly elevated levels of CHOP, XBP-1, GRP78 and ATF6α at day 7, CHOP, XBP-1 and GRP-78 at day 14, when compared with those of controls, before returning to normal levels at day 21 (Fig. 4; all P<0.01). Mice treated with combined RSV+bleomycin demonstrated significantly elevated levels of p-PERK, XBP-1, ATF6α and GRP78 from day 7 to day 21, when compared to the control group (Fig. 4; all P<0.01).

In order to further identify the effects of ER stress-related proteins induced by RSV in the lung fibrosis, the correlations of p-PERK, ATF6α and GRP78 with accumulation of collagen type-1 were examined. The results indicated that p-PERK, (P=0.0093), ATF6α (P=0.0097) and GRP78 (P=0.0089) were closely related with accumulation of collagen type-1 (Fig. 5).