Reagents and antibodies
Commercial andrographolide (purity 99.67%) was purchased from APExBIO (N1855, USA), with high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) quality control. Other reagents included dimethyl sulfoxide (DMSO) (D8371, Solarbio, China), and HO-1 inducer hemin (HY-19424, MCE, China). The primary antibodies included HO-1 (R24541, Zen-Bio, China), RSV (AB1128, Millipore, Germany), IRF3 (ab68481, Abcam, UK), and GAPDH (60004-1-Ig, Proteintech Group, USA). The secondary antibodies included alkaline phosphatase-conjugated goat anti-mouse antibody (SA00002-1, Proteintech Group, USA), goat anti-rabbit antibody (SA00002-2, Proteintech Group, USA), and donkey anti-goat antibody (SA00002-3, Proteintech Group, USA).
Cell culture and virus preparation
The human lung carcinoma epithelial cell line A549 (CCL-185) and human laryngeal cancer epithelial cell line HEp-2 (CCL-23) were obtained from the American Type Culture Collection (ATCC, USA). The human bronchial epithelial cell line 16HBE was reserved in the laboratory. Cells were detected to exclude mycoplasma contamination using a Mycoplasma Detection Kit (Yise Med, China). The cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (AusgeneX, Australia) at 37 °C in a 5% CO2 humidified incubator. The RSV-A2 strain (VR-1540, ATCC, USA) was grown in HEp-2 cells with 2% FBS and purified by density gradient[18]. Working stocks of RSV were stored in liquid nitrogen.
Cell counting kit-8
To investigate whether the reagents (andrographolide, and HO-1 activator hemin) induced cytotoxicity, a cell counting kit-8 (CCK-8) assay was conducted. The A549 cells were seeded in 96-well plates at a density of 3 × 104 cells/mL overnight in 37℃, then serial concentrations of the tested reagents were added. Wells incubated with 10% FBS culture medium were employed as the control, and a blank group was also set. After incubation in 37℃ for 24 h, each well was incubated with 100 μL of medium plus 10 μL of CCK-8 reagent (Dojindo, Japan) for an additional 2 h. Absorbance was detected at 450 nm using a microplate reader and cell viability was calculated. Results are presented as the mean of six biological replicates in Supplementary Fig 1. Andrographolide concentration no more than 20μM and hemin concentration no more than 40μM showed no cytotoxicity to cells.
RSV infection and treatment
To establish an in vitro RSV infection model, the A549 and 16HBE cells were seeded in 12- or 6-well plates at a density of 3 × 105 cells/mL overnight in 37℃. When the monolayer cell confluency reached ~80%, RSV-A2 was added at a multiplicity of infection (MOI) of 1. After incubation in 37℃ for 2 h, the culture medium was replaced with DMEM containing 2% FBS, then andrographolide, hemin or PBS was added for another 24 h.
RNA-seq
Mock and RSV-infected A549 cells with or without andrographolide (10 μM) treatment (three samples in each group) were harvested 24 h after RSV infection. Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Japan) following the manufacturer’s instructions. In total, 1 μg of RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using a NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature. To select 250–300-bp cDNA fragments, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μL of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C. The polymerase chain reaction (PCR) products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on the Illumina NovaSeq platform (GeneChem, Shanghai, China) and 150-bp paired-end reads were generated.
Analysis of differentially expressed genes (DEGs)
DEGs analysis between two groups was performed using the DESeq2 R package (v1.16.1). DESeq2 was used to determine differential expression of digital gene expression data using a model based on negative binomial distribution. The resulting P values were adjusted using the Benjamini-Hochberg approach for controlling the false discovery rate (FDR). Genes with an adjusted P < 0.05 found by DESeq2 were assigned as differentially expressed.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
GO and KEGG enrichment analysis of DEGs was implemented using the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected P < 0.05 and KEGG pathways with P < 0.05 were considered significantly enriched.
RNA isolation and real-time quantitative real-time PCR (qRT-PCR)
Total RNA from the A549 cells was extracted using RNAiso Plus reagent (TaKaRa, Japan) following the manufacturer’s protocols. After quantitation, 1 µg of RNA was reverse transcribed for 15 min at 37 °C and 15 s at 85 °C using a TaKaRa kit. The RSV N gene was quantified using TaqMan qRT-PCR, as described earlier[19]. Known concentrations of the RSV-A2 plasmid were used to derive a standard curve. The PCR cycle conditions were: 50 ℃ for 2 min, 95 ℃ for 10 min, 40 cycles at 95 ℃ for 15 s and 60 ℃ for 1 min. The RSV N gene probe and primers are listed in Table 1. The relative expression levels of human genes IL-6, IL-8, CXCL10, IFNα, IFNβ, IFNL1-3, and HO-1 were detected by RT-PCR and calculated using the 2-∆∆Ct formula. GAPDH expression was quantified as a reference. The reaction conditions were: 95 °C for 5 min, then 40 cycles at 95 °C for 10 s, 59 °C for 15 s, and 72 °C for 1 min. Primers are listed in Supplementary Table 1.
Protein extraction and western blot analysis
Total protein of A549 cells was extracted using RIPA buffer containing Phenylmethanesulfonyl fluoride (CST, USA). Samples with equal quantities of protein were separated and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and probed with primary antibodies against RSV, HO-1, and IRF3 (1:1 000) at 4 °C overnight. The membranes were then incubated with alkaline phosphatase-conjugated secondary antibodies (1:5 000) at room temperature for 1 h. Protein bands were detected using an Enhanced Chemiluminescence (ECL) Kit (Millipore, USA). GAPDH was used as the reference protein for the whole-lysate proteins. Image collection was performed by Quantity One (v4.6.2, USA) and densitometry analysis was carried out using ImageJ (v2.0.0, USA).
Enzyme-linked immunosorbent assay (ELISA)
Cell supernatants were collected after centrifugation at 3 000 rpm for 5 min at 4 °C. Cytokine levels of IL-6, IL-8 and CXCL10 were measured using Human ELISA kits (NeoBioscience, China) in accordance with the manufacturer’s instructions.
Knockdown of HO-1 with lentiviral transfection
The siCON and siHO-1 lentiviruses used to knockdown HO-1 were constructed by GeneChem Co., Ltd. (Shanghai, China). The sequence of siHO-1 is TGCCAGTGCCACCAAGTTCAA. The A549 cells were seeded in 12- or 6-well plates at a density of 1 × 105 cells/mL overnight and transfected with lentivirus (MOI = 10). After 72 h of transfection, total RNA and protein of the A549 cells were extracted to verify knockdown efficiency (Supplementary Fig 2). Stable cell lines were obtained after puromycin (2 μg/mL) selection for the andrographolide intervention experiments.
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Differences between or among groups were compared with Student’s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (v7.0, GraphPad Software Inc., USA). Here, P < 0.05 was considered statistically significant. In each experiment, at least three replicates were used for statistical analysis.