Cell culture and pre-activation
Murine B16-F10 melanoma cells (B16 cells) and human A549 lung adenocarcinoma epithelial cells (A549 cells) were provided by the Department of Molecular Biology, College of Basic Medical Sciences, Jilin University. The cells were maintained in RPMI-1640 medium containing 10% (v/v) Fetal Bovine Serum (FBS) and antibiotics (100 IU of penicillin/ml and 100 IU of streptomycin/ml). All cells were maintained routinely in 5% CO2 and 95% air at 37 ℃.
For the Cell Counting Kit-8 (CCK-8), crystal violet staining, and flow cytometry assays, cells were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate. For protein and RNA extraction, cells were seeded at a density of 1 × 105 cells/well in a 12-well plate.
Cells were pre-treated with CpG ODN (CpG5805)or CCT ODN for various time prior to influenza A virus (FM1 strain) infection. To evaluate the expression level of surface TLR9 (sTLR9) on the surface of B16 cells pretreated with CpG5805, CCT ODN, or their combination, the experiment was conducted as follows: Cells were seeded in 48-well plates at a density of 2 × 10⁴ cells per well and cultured overnight. Subsequently, quadruplicate wells were set up and treated with either CpG5805 (6 µg/mL), CCT ODN (6 µg/mL), their combination, or PBS control. After 24 h of incubation, single-cell suspensions were prepared using trypsin digestion method for subsequent TLR9 surface staining and flow cytometric detection and analysis.
Oligodeoxynucleotides (ODNs)
TLR9 agonist CpG5805 (5′-TCGACGAACGTTCGGTCGCCGCGG-3′)57 and TLR9 inhibitor CCT ODN (5′-CCTCCTCCTCCTCCTCCTCCTCCT-3′)58 were used in this study. All single-stranded oligodeoxynucleotides (ODNs) were synthesized, purified, and fully phosphorothioate-modified by Takara Biotech (Takara Biotech, Dalian, China). The fully phosphorothioate-modified ODNs were diluted in PBS.
Influenza virus
H1N1 influenza A virus (IAV), a mouse-adapted influenza virus strain A/Fort Monmouth/1/1947 virus (FM1), was propagated in the allantoic cavities of 9-day-old embryonated specific pathogen-free chicken eggs cultured at 37 ℃. After 48 h, the allantoic fluid was harvested from the infected eggs and stored at − 70 °C for subsequent experiments.
To determine the optimal viral infection concentration for B16 cells, a titration experiment was performed. Briefly, B16 cells seeded in a 96-well plate were inoculated with serial dilutions of IAV FM1 (ranging from 10⁻¹ to 10⁻⁶) for 1 h. The cells were then washed twice with cold 1× PBS and replaced with RPMI-1640 medium containing 2% fetal bovine serum. Microscopic observations were conducted every 24 h. Pronounced cytopathic effects and cell mortality were observed in the groups infected with the 10⁻¹ to 10⁻³ dilutions, with severity correlating with the viral concentration. In contrast, cells exposed to 10− 4 or lower virus concentrations exhibited no significant infection, resembling the blank control group. Based on these results, the 10⁻³ dilution, which represented the minimum concentration that produced a clear infectious outcome, was selected for subsequent infections of B16 cells in this study.
Cell viability assessment
Cell viability was assessed using three methods: microscopic observation, crystal violet staining, and CCK-8 assay.
Microscopic observation
Cellular morphology of B16 or A549 cells in 96-well plates was examined under an optical microscope at 24, 48, 72, and 96 h post-infection. Observations focused on identifying changes indicative of infection or cell death, such as reduced cell density, cell rounding, and fragmentation. Following the observation of these morphological changes, the cells were subsequently subjected to crystal violet staining and CCK-8 assay.
Crystal violet staining
B16 cells seeded in 96-well plates were treated with CpG5805 and infected with IAV FM1. After treatments, the culture supernatant was removed. The cells were then stained with 0.25% crystal violet (100 µL/well) for 10 min at room temperature (RT). After discarding the supernatant, the plate was rinsed several times with distilled water until the blank wells were clear. For destaining, 100 µL of 0.9% sodium citrate buffer was added per well and left at RT for 1 h. Finally, the absorbance was measured at 570 nm using a microplate reader (Synergy H1 Hybrid Reader, BioTek).
Cell counting Kit-8 (CCK-8) assay
The effects of CpG5805 and H1N1 IAV on B16 or A549 cell viability were evaluated using the CCK-8 reagent (K1018, APExBIO). After treatments, 10 µL of CCK-8 reagent was directly added to each well containing 100 µL of the original culture medium.The plates were incubated at 37 °C for 1.5 h, and the absorbance was then measured at 450 nm using a microplate reader (Synergy H1 Hybrid Reader, BioTek).
Immunofluorescence assay
Infection of cells with the FM1 strain of IAV was detected by immunofluorescence. Cells were harvested and centrifuged onto glass slides. Subsequently, the cells were fixed and stained with FITC-labeled anti-IAV antibody from D3 Ultra DFA Respiratory Virus Screen & ID Kit (Diagnostic Hybrids, Athens, Ohio, USA), incubating at 37 ℃ for 20 min. After staining, the slides were washed, mounted, and visualized under a fluorescence microscope (BX53, Olympus, Tokyo, Japan).
Hemagglutination assay
Viral titers in culture supernatants were determined by hemagglutination assay. Briefly, 50 µL of supernatant from infected cells was serially diluted twofold with 1× PBS in a 96-well plate. Subsequently, 50 µL of a 0.5% chicken erythrocyte suspension was added to each well. The plate was gently agitated to mix the contents and incubated at RT for 30 min. The hemagglutination phenomenon in each well of the plate was then observed and recorded.
Western blotting and quantitative analysis of the immunoblot bands
B16 cells were lysed in ice-cold RIPA buffer (P0013C, Beyotime) containing 0.1 mmol/L PMSF (ST507, Beyotime) and a phosphatase inhibitor cocktail (CW2383S, CWBIO) used at a 1:100 dilution. The protein concentration of the samples was determined using a BCA protein assay kit (CW0014S, CW Biotech). Equal amounts of protein from each sample were separated by 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with Tris buffered saline containing 5% skim milk at RT for 1 h, the membranes were incubated with antibodies against STING (13647, CST), Phospho-STING (AF7416, Affinity), β-Actin (66009-1-Ig, Proteintech) at 4 ℃ overnight. After washing three times with TBST (0.05% Tween 20) for 5 min each, the membranes were incubated at RT for 1 h with either Goat anti-Rabbit (RGAR001, Proteintech) or Goat anti-Mouse (RGAM001, Proteintech) IgG HRP Antibody. To enable the combined analysis of data from different experimental batches, a protein lysate from untreated B16 cells was routinely loaded in one lane on each gel as a reference standard, designated as “Med” in the results.
For quantitative analysis of immunoblot bands, we utilized ImageJ software (version 1.54f) with the following protocol: Converted raw images to 8-bit grayscale mode to reduce computational complexity and improve analytical precision. Applied “Subtract Background” function with a rolling ball radius set at 50 pixels to eliminate nonspecific signal interference. Selected “Set Scale” in the Analyze module to define pixel as the unit length, ensuring subsequent measurements were recorded as absolute pixel values. Manually delineated target protein bands using rectangular selection tools to ensure complete coverage of regions of interest. Generated lane-specific grayscale distribution curves via Analyze > Gels > Select First Lane > Plot Lanes. Peaks were automatically identified using the Magic Wand tool, with system-recorded peak areas (Area). Repeated the above procedures for all target bands (p-STING, STING, and β-Actin). The data were exported to Excel, and the band intensity ratios (p-STING/β-Actin and STING/β-Actin) were calculated. For bands from different membranes, the ratio of the untreated “Med” band was set to 1, and all other lanes on the same membrane were normalized accordingly to enable the pooled analysis of results from independent experiments.
Quantitative reverse-transcription polymerase chain reaction (RT-qPCR)
The mRNA levels of Tnf-α, Il-6, Cxcl-2, Cxcl-10, Irf3, and Ifn-β were quantified by RT-qPCR. Total RNA was extracted from B16 cells using Trizol reagent (ET101-01, TransGen Biotech). The RNA was then reverse-transcribed into cDNA using a cDNA synthesis kit (AE301-03, TransGen Biotech). Quantitative PCR was performed using a SYBR Green kit on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with gene-specific primers (Table 1). The β-actin gene was used as an endogenous reference for normalization. Gene expression levels were calculated using the comparative Ct (2^(-ΔΔCt)) method.
Mouse experiments
Female ICR mice aged 6 to 8 weeks were purchased from Yisi Laboratory Animal Technology Co., Ltd., in Changchun, China. The mice were raised in an Animal Biosafety Level 2 Laboratory at the Laboratory Animal Center of Jilin University and provided with free access to food and water. At the experimental endpoint, mice were humanely euthanized via CO₂ inhalation. The mouse experiments were reviewed and approved by the Animal Ethics Committee of Jilin University College of Basic Medical Sciences (Approval ID: 2024 − 607). The study was conducted in strict accordance with ARRIVE guidelines 2.0 and the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB/T 35892 − 2018). All procedures were performed in compliance with institutional biosafety and animal welfare policies.
To establish the influenza infection model, mice were randomly divided into four main groups based on the pre-treatment time point (1, 6, 24, or 48 h before viral infection). Each main group was then further divided into two subgroups (n = 9): one receiving an intranasal instillation of CpG5805 (10 µg, 50 µL/mouse), and the other receiving an equal volume of PBS as the vehicle control. All mice were subsequently infected intranasally with the FM1 strain of IAV (10⁻², 50 µL/mouse). At 3 days post-infection, three mice from each subgroup were euthanized for sample collection. Bronchoalveolar lavage fluid (BALF), lung tissue, and liver tissue were harvested for subsequent analysis. The remaining mice in each subgroup were monitored daily for 12 days.
Pathological analysis
To prepare tissue paraffin sections, the lungs of mice were fixed in 10% neutral formalin for 48 h, embedded in paraffin and sliced. The sections were stained with haematoxylin & eosin, and observed under the microscope.
These criteria are used to evaluate pathological changes in the lung, based on observations at different magnifications during microscopic examination. To enable accurate assessment of pulmonary pathology in mice, each lung was sectioned into four equal quadrants. Pathological changes within each quadrant were then scored individually. The specific scoring criteria employed are detailed below. (1) 4× macroscopic view: 3 points: Abnormalities affecting > 75% of the lung. 2 points: Abnormalities affecting > 50% but ≤ 75% of the lung. 1 point: Abnormalities affecting > 25% but ≤ 50% of the lung. 0 points: Essentially no abnormalities in the lung. (2) High-magnification microscopic view: 1 point: Inflammatory cell infiltration. 1 point: Abnormal alveolar structure (e.g., structural disorder, interstitial thickening, etc.). 1 point: Presence of significant hemorrhage. The total lung pathology score is the sum of the scores obtained from the 4×macroscopic view and the high-magnification microscopic view of the 4 quadrants.
Flow cytometry
For analyzing the percentage of IAV FM1 positive (FM1+) cells, B16 cells were stained with FITC-labeled anti-IAV antibody from D3 Ultra DFA Respiratory Virus Screen & ID Kit (Diagnostic Hybrids, Athens, Ohio, USA) for flow cytometry analysis. For analyzing the immune cell composition in mouse BALF, fluorescence-conjugated monoclonal antibodies of Ly6G-PE (Cat: 551461), CD14-FITC (Cat: 553739), CD3e-APC (Cat: 558257), CD45-Percp (Cat: 550994) from BD Biosciences (NJ, USA) were used for flow cytometry. For analyzing the sTLR9 expression on B16 cells, mouse TLR9 Alexa Fluor 647-conjugated antibody (FAB7960R, R&D Systems) were used. Staining was performed for 30 min on ice in the dark, followed by two washes with PBS. All the stained cells were detected and analyzed using BD Accuri C6 flow cytometer (BD Biosciences, NJ, USA).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 for Windows (GraphPad Software, San Diego, California, USA), and experimental data were displayed as mean ± standard deviation. An unpaired t-test was used to compare groups. Survival curves of mice were estimated and compared using the Mantel-Cox test and Gehan-Breslow-Wilcoxon test. Differences were considered statistically significant for p values < 0.05.