Cell line

HULEC-5a cells were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a specialized medium (Procell, CM-0565) designed for optimal growth of HULEC-5a cells at 37 °C with 5% CO2.

HAdV-7 strain

The HAdV-7 strain (CQ45_2019, MT113943) was obtained from nasopharyngeal aspirates of a pediatric patient with adenoviral pneumonia. The strain was cultured in A549 cells, purified [14], and quantified [15, 16] using standard procedures, resulting in a stock concentration of 3 × 1011 viral particles per milliliter.

Animal model

The hCD46 mice (C57BL/6 J) were obtained from Cyagen Biotechnology Co., LTD (Suzhou, China) and housed in a specific pathogen-free facility at the Experimental Animal Center of Chongqing Medical University. The mice were maintained under a 12-h light–dark cycle, with a humidity of 55 ± 5% and a temperature of 23 ± 1 °C, and provided with ad libitum access to food and water. Prior to infection with HAdV-7 (80 μl/mouse, nasal drip) [17], hCD46 homozygous mice (6–8 weeks old) were pretreated with intraperitoneal injections of rapamycin (RAPA, MedChemExpress, HY-17589A), 3-methyladenine (3MA, MedChemExpress, HY-19312), and chloroquine (CQ, MedChemExpress, HY-17589A) one hour. Mock-infected mice received PBS treatment for comparison. RAPA was dissolved in a mixture containing 2% DMSO (Sigma-Aldrich) and PEG300 and Tween at a concentration of 2.5 mg/mL and injected at a dose of 6 mg/kg [18]. 3MA was dissolved in PBS at a concentration of 4 mg/mL and administered at a dose of 30 mg/kg [19], while CQ was dissolved in a mixture containing 2% DMSO (Sigma-Aldrich), PEG300, and Tween at a concentration of 2.5 mg/mL and injected at a dose of 60 mg/kg [18]. Regarding the timing of sample collection, during the previous study on constructing an acute lung injury model in hCD46 mice infected with HAdV-7, we observed that inflammation, weight loss, and reduced activity were evident on the first day post-infection. By the third day, severe lung inflammation, decreased body weight, hypothermia, and mortality were observed. From the 5th to 7th day post-infection, a gradual resolution of lung inflammation along with recovery in body weight and mouse activity was noted. Based on these empirical findings, we opted to collect samples on day 3 post-infection [17, 20].

RNA extraction library construction and sequencing

Total RNA was extracted using Trizol reagent (Thermos Fisher, 15596018) following the manufacturer’s protocol. The samples were then flash-frozen in liquid nitrogen for 30 min and shipped on dry ice to LC-Bio Technology Co., Ltd. (Hangzhou, China) for transcriptome sequencing analysis. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. mRNA was purified from 5 μg of total RNA using Dynabeads Oligo (dT) (Thermos Fisher, CA, USA) with two rounds of purification. The purified mRNA was then fragmented into short pieces using divalent cations at 94 ℃ for 5–7 min with Magnesium RNA Fragmentation Module (NEB, MA, USA). These fragmented RNA pieces were reverse-transcribed into cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, CA, USA). The average insert size of the final cDNA libraries was 300 ± 50 bp. Finally, 2 × 150 bp paired-end sequencing (PE150) was carried out on an Illumina NovaSeq™ 6000 by LC-Bio Technology Co., Ltd. (Hangzhou, China) following the vendor’s recommended protocol.

Differential expression genes (DEGs) and GO enrichment analysis

DEGs analysis was performed by DESeq2 software (version 1.26.0) between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. GO analysis was conducted using Omic Studio tools (https://www.omicstudio.cn/tool), with terms considered significantly enriched at a cutoff of P < 0.05.

Gene set enrichment analysis

We performed gene set enrichment analysis using software GSEA (version 4.3.2) and MSigDB to identify whether a set of genes in specific GO terms shows significant differences in two groups. Briefly, we input gene expression matrix and rank genes by Signal2Noise normalization method. Enrichment scores and p value was calculated in default parameters. GO terms meeting this condition with |NES|> 1, NOM P-val < 0.05, FDRq-val < 0.25 were considered to be different in two groups.

Optical microscope and transmission electron microscopy assays

Morphological characteristics of cells were observed using an optical microscope. Images were captured with a Nikon microscope (Nikon, Japan) and processed for analysis using Image Viewer software (version 4.5). For the observation of mitochondrial ultrastructure and viral particles, the treated HULEC-5a cells were fixed with 3% glutaraldehyde at 4 °C overnight, followed by fixation with 1% osmium tetroxide at 4 °C for 1 h. Dehydration steps using acetone and embedding in Epon-812 were then performed. Ultra-thin sections were prepared using a Leica ultramicrotome (LEICA, Buffalo Grove, IL, USA) and stained with uranyl acetate and lead citrate. TEM images were acquired with a JEM-1400FLASH transmission electron microscope (JEOL Ltd., Tokyo, Japan).

ELISA

Endothelial injury markers in the cell supernatant and bronchoalveolar lavage fluid (BALF)/blood were quantified using ELISA. BALF was obtained by rinsing the right lung three times with 0.5 mL of PBS, resulting in a 90% recovery rate. Levels of VEGF (NeoBioscience, EHC108.96, EMC103.96.2), sICAM-1 (4A Biotech, CHE0052), sVCAM-1 (finetest, EM1382), E-selectin (finetest, EM0229), ESM1 (finetest, EH0125, EM0075), MCP1 (NeoBioscience, EHC113.96, EMC105.96), and IL-1β (NeoBioscience, EMC113.96) were determined using ELISA kits according to the manufacturer’s instructions. Absorbance was read at 450 nm with a microplate reader (Biotek Epoch, VT, USA), and protein concentrations were calculated using ELISA calc software (version 0.1).

Cell viability assays

HULEC-5a cells were pre-treated with RAPA (20 nM), 3MA (5 mM), CQ (20 μM), and Z-VAD-FMK (40 μM, Selleck, S7023) for one hour before infection with HAdV-7(MOI = 105). Subsequently, the cells were incubated with 10% Cell Counting Kit-8 (CCK8) reagent (MedChemExpress, HY-K0301) for 0.5 to 2 h. Absorbance was read at 450 nm with a microplate reader (Biotek Epoch, VT, USA).

Immunofluorescence staining

For the Immunofluorescence assay, HULEC-5a cells were cultured on confocal dishes, while Paraffin-embedded tissue sections underwent deparaffinization and antigen retrieval treatment. The cells and lung tissues were then fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Solarbio, 9002-93-1). Subsequently, after blocking with 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, V900933), the cells and lung tissues were incubated overnight at 4 °C with primary antibodies LC3B (Cell Signaling Technology, 83506S, 1:500), SQSTM1 (Cell Signaling Technology, 16177S, 1:500), Anti-Adenovirus antibody (Abcam, ab7428, 1:1000) and CD31 (Abcam, ab124432, 1:500). Following this, secondary antibodies Cy3-labeled goat anti-mouse/rabbit IgG (Beyotime, A0521/ A0516, 1:500), FITC-labeled goat anti-rabbit IgG (Beyotime, A0562, 1:500), and Alexa Fluor488 labeled goat anti-mouse IgG (Beyotime, A0428, 1:500) were applied for 1 h at room temperature, followed by DAPI staining (Beyotime, C1005). Fluorescent images were captured using a Nikon A1R + /A1 confocal microscope (Nikon, Tokyo, Japan), and fluorescence intensity analysis was performed using NIS ELEMENTS Viewer software (version 4.5).

Flow cytometry

The lungs were digested in Roswell Park Memorial Institute 1640 culture medium (Gibco) at 37 °C for 30 min with collagenase IV (Sigma, C0130) at a concentration of 2 mg/mL and DNase I (Roche, 10104159001) at a concentration of 30 mg/mL. The samples were then filtered through a 40-μm cell strainer to obtain single-cell suspension. Subsequently, the cells were blocked with rat serum for 30 min and stained in the dark for one hour with fixable viability dye eFluor 660 (Invitrogen, 65-0864-14), PE-Cy7-conjugated anti-CD45 antibody (eBioscience, 17-0451-82), and FITC-conjugated anti-CD31 antibody (eBioscience, 11-0311-82). The samples were analyzed using BD FACSCanto plus equipped with 50-mW 405-nm, 50-mW 488-nm, and 20-mW 633-nm lasers and an ND1.0 filter in front of the forward scatter photodiode.FlowJo software (version 10, TreeStar, USA) was used for further data analysis.

Gene mouse identification

The genotyping procedure is as follows: 98 μl lysis buffer and 2 μl proteinase K (20 mg/mL) are added to the toe of each tested mouse and incubated at 56 ℃ overnight. Then, they are placed in a metal bath at 98 ℃ for 15 min, centrifuged at 12,000 rpm at 4 ℃ for 15 min, and the supernatant contains DNA. Next, PCR amplification detection is performed using Premix Taq enzyme (TAKALA, RR901Q). The PCR cycle consists of heating at 94 ℃ for 3 min, denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 35 s, and extension at 72 ℃ for 35 s with a total of 35 cycles. CD46 primer sequences used are Forward1: 5′-GCTAAGTCTGCAGCCATTACTAAAC-3′, Reverse1: 5′-GAAATCAGGCTGCAAATCTCAGC-3′; Forward2: 5′-GGTTGGCTATAAAGAGGTCATCAG-3′, Reverse2: 5′-GAAATCAGGCTGCAAATCTCAGC-3′. Gel electrophoresis is then conducted by preparing a 1.5% agarose gel supplemented with GoldView (3–5 μl), which is cooled and solidified before adding the test DNA and DNA ladder(both in amounts of 5 μl). Electrophoresis takes place under 120v voltage for 25 min, followed by visualization under ultraviolet light to determine genotype based on band appearance.

Quantification of HAdV-7 fiber gene copy number

The total viral nucleic acid was extracted using a QIAamp mini-viral DNA extraction kit (Qiagen, 51306) following the manufacturer's instructions. Polymerase Chain Reaction (PCR) amplification assays were performed with TaqMan Universal Master MixII (Applied Biosystems, 4440040) on a Real-Time PCR Detection System (Bio-Rad, CFX96 Touch). The HAdV-7 fiber gene copies were amplified using the forward primer 5′-CATAAGTGCCACCACACCAC-3′, reverse primer 5′-GTGCGCTTAACTCCTGTCCA-3′, and probe sequence 5′-FAM-5′-TGTCGCTAGGACCCGGATTAGAAACAA-3-BHQ1-3′. PCR cycles included 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Absolute quantification of HAdV-7 gene copies was done using a plasmid DNA standard curve.

Western blotting assays

Total protein extraction from HULEC-5a cells was conducted using the Total Protein Extraction Kit (Key GEN, KGP2100). Protein concentrations in the cell lysates were quantified with the NanoDrop One instrument (Thermo Fisher, 840-317400). Following separation by SDS-PAGE, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010). The membranes were then blocked with Quick Blocking Buffer (Beyotime, P0252) and probed with specific primary antibodies, including LC3B (Cell Signaling Technology, 83506S, 1:1000), SQSTM1 (Cell Signaling Technology,16177S, 1:1000), GAPDH (Zenbio, 380626, 1:10,000), and β-actin (Zenbio, 700063, 1:10,000). The membranes were then incubated with the secondary antibody (Zenbio, 511103, 511203, 1:10,000) at room temperature for 1 h. Signal detection employed a super sensitive ECL luminescence reagent (MeilunBio, MA0186). Densitometry analysis was performed using Image Lab software (version 5.0).

Histopathology

The lung of hCD46 mice was fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned (4um thickness), and stained with hematoxylin and eosin (H&E). The staining method and pulmonary pathology evaluation followed a previously established protocol [17]. A Slide Scan System (SQS-40P, Shengqiang) and image Viewer G software (version G1.1.7.3107) were utilized for scanning and analysis, respectively.

Detection of pulmonary vascular permeability

hCD46 mice were intravenously injected with 0.5% Evans blue (EB, MedChemExpress, HY-B1102) at a dose of 3 mL/kg 1 h prior to euthanasia. Lung tissue weighing approximately 100 mg was then homogenized and mixed with dimethylformamide (1 mL/100 mg). The mixture was incubated at 60 °C for 24 h. The absorbance at a wavelength of 620 nm was measured using a microplate reader (Biotek Epoch, VT, USA), and the concentration of EB content was calculated using Curve Expert software (version 1.4.0.0). Total protein concentration in bronchoalveolar lavage fluid was determined following the instructions provided by the Bicinchoninic acid (BCA) assay kit (Beyotime, P0010).

Statistical analysis

The quantitative data were reported as mean ± standard error of the mean. Unpaired Student’s t-test was used for comparing two groups, while One-way analysis of variance, two-way analysis of variance, or Kruskal–Wallis test were employed for multiple group comparisons. Data analysis was conducted using GraphPad Prism (version 9.4.1). Statistical significance was defined as a P-value less than 0.05.