Influenza A virus infects pulmonary microvascular endothelial cells leading to microvascular leakage and release of pro-inflammatory cytokines

Virus strains

Influenza A/Puerto Rico/8/34 (H1N1) virus was donated by the Guangzhou Institute of Respiratory Diseases. The virus was propagated in 9 days-old specific pathogen-free (SPF) embryonated hen eggs. Each chicken embryo was inoculated with 0.1ml of 100-fold diluted virus solution, and the hole was sealed with wax liquid. Following successful inoculation of the chicken embryos with the virus, they were incubated at 37 °C, for 48 h. The allantoic fluid was collected, centrifuged at 3000 rp for 5 min, to obtain the supernatant. The viral titer was detected by plaque assays (Furuta et al., 2002). The virus was packaged and stored at −80 °C.

Cell culture and viral infection of cells

Madin-Darby canine kidney (MDCK) cell was a gift from the Institute of Respiratory Diseases of Guangzhou Medical University and the State Key Laboratory of Respiratory Diseases (Guangzhou, China). MDCK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin at 37 °C under 5% CO₂ (Lai et al., 2021). The human lung microvascular endothelial cell (HULEC-5a) was obtained from The Global Bioresource Center (ATCC CRL-3244). The HULEC-5a cells were maintained in Endothelial Cell Medium (ECM) at 37 °C under 5% CO₂. The ECM contained basal medium, 5% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and 1% Endothelial cell growth supplement (ECGS). Unless otherwise stated, and in the priming experiment, influenza virus was added to the MDCK cells and HULEC-5a cells in serum-free media at a multiplicity of infection (MOI) of 0.00001, 0.0001, 0.001, and 0.01 for two hours. The virus was removed, and DMEM containing 2 µg/ml tosylphenylalanyl chloride methyl ketone (TPCK) treated trypsin and 1% bovine serum albumin was added. All infections occurred within 48 hours (Li et al., 2011).

Plaque formation assay

The experimental method of plaque reduction was based on a previous description (Furuta et al., 2002), as follows: MDCK cells (6 ×10⁵ cells/well) were seeded in 12-well plates and cultured at 37 °C under 5% CO₂ for 24 h (Lai et al., 2021). After infection with influenza virus, MOI 0.01, MDCK, and HULEC-5a cells were incubated for 48 h. The cells were then washed with phosphate buffer saline (PBS) and the supernatants collected from the virus-infected HULEC-5a and MDCK cells. The supernatants were diluted 10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold, and 10⁷-fold. The virus inoculums were removed, and the cells were washed three times with PBS. Next, the cell monolayers were overlaid with agar overlay medium (DMEM supplemented with 1% low melting point agarose and 2.5 µg/ml TPCK-treated trypsin) and incubated at 37 °C for 3 days. The cell monolayers were fixed with 4% paraformaldehyde for 2 h, the agarose overlays were then removed, and the cell monolayers were stained with 2%(w/v) crystal violet (Lai et al., 2021).

Real-Time quantitative PCR analysis

Total cellular RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using the reverse transcription kit (TaKaRa, Shiga, Japan). SYBR Green real-time PCR amplification and detection were performed using the ABI 7500 system (Applied Biosystems, Foster City, USA). The PCR system comprised of, 25 µl reaction buffer (RNA17 µl RNA template, 5 µl M-MLV 5 × reaction buffer, primer 50p molar buffer, 0.1mMd nucleotide, 25 units ribonuclease inhibitor and 200 units M-MLVRT polymerase). 18s rRNA was regarded as a house-keeping gene (Armstrong et al., 2012; Chunlian et al., 2014; Lai et al., 2020; Plotnikova, Klotchenko & Vasin, 2016; Yang et al., 2015).

IL-1β (forward primer) 5′-AGCTGATGGCCCTAAACAGA-3′

IL-1β (reverse primer) 5′-TGGTGGTCGGAGATTCGTAG-3′

TNF-α (forward primer) 5′-TGTAGCCCATGTTGTAGCAAA-3′

TNF-α (reverse primer) 5′-CAAAGTAGACCTGCCCAGACT-3′

NP (forward primer) 5′-GCACCAAACGGTCTTACGAA-3 ′

NP(reverse primer) 5′-TTTGGATCAACCGTCCCTCA-3 ′

18s rRNA(forward primer) 5′-GATGGAAAATACAGCCAGGTCCTA-3′

18s rRNA(forward primer) 5′-TTCTTCAGTCGCTCCAGGTCTT-3′

IL-4(forward primer) 5′-AGCTGATCCGATTCCTGAAAC-3 ′

IL-4 (reverse primer) 5′-AACGTACTCTGGTTGGCTTC-3 ′

IL-6(forward primer) 5′-CCACTCACCTCTTCAGAACG-3 ′

IL-6(forward primer) 5′-CATCTTTGGAAGGTTCAGGTTG-3 ′

Immunofluorescence staining

HULEC-5a (1.5 ×10⁵ cells/well) and MDCK cells (1 ×10⁵ cells/well) were cultured on coverslips for 24 h. After infection with the influenza virus (MOI 0.01), they were incubated for 48 h. The cells were washed three times with phosphate buffer saline (PBS), fixed with 4% (v/v) paraformaldehyde in PBS for 15 min, and permeabilized with 0.5% Triton X-100 (prepared in PBS) permeate at room temperature for 20 minutes (Kim et al., 2015). For immunocytochemistry analysis of the H1N1 virus, the cells were incubated with mouse anti-NP protein antibody, followed by Alexa Fluor 488-conjugated anti-mouse IgG. The nucleus was fluorescently labeled with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO) (Kim et al., 2015). Coverslips were mounted on the slide, with a mounting solution containing an anti-fluorescence quencher. Fluorescent images were obtained using a fluorescence microscope.

Western blot

Lysates were prepared using the Radio Immunoprecipitation Assay (RIPA) lysis buffer and separated by 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked for 1 h using 5% BSA, and probed overnight with a primary antibody at 4 °C. After washing, the blots were incubated with HRP-conjugated secondary antibodies for 1 h, washed, and visualized by advanced chemiluminescence (Amersham). Band intensity was quantified using Image J (NIH) and normalized to the loading control after background correction (Sugiyama et al., 2016). The NP antibody used to detect the influenza virus and the antibody to detect GAPDH were all purchased from GENE Tex (Southern California, Unites States). The goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Jackson Immuno Research Laboratories Inc. (Pennsylvania, Unites States).

Permeability measurement

HULEC-5a cells (3 ×10⁵ cells/well) were seeded on a 24-hole polyester transfer hole (Costar) with a 0.4 mm pore size for 24 h. The cells were then infected with the influenza virus, MOI 0.01. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) at 0, 8, 16, 24, and 48 h after stimulation using the EVOM2 Epithelial Voltohmmeter (Bio-Rad, Hercules, CA, USA). Untreated HULEC-5a cells were used as a negative control, and medium alone was used as a blank control. Relative TEER was expressed as follows: [(resistance in the experimental group) - (resistance in medium alone)]/[(resistance in untreated HUVECs)-(resistance in medium alone)]; resistance was expressed in ohms.

Target gene for pulmonary microvascular leakage caused by H1N1

We performed text mining in the GeneCards database (version 5.0) (https://www.genecards.org/Search/Keyword?queryString=Pulmonary%20microvascular%20lea kage%20caused%20by%20H1N1). The GeneCards is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes. The knowledgebase automatically integrates gene-centric data from 150 web sources, including genomic, transcriptomic, proteomic, genetic, clinical, and functional information. The keyword was “Pulmonary microvascular leakage caused by H1N1”. All targets were restricted to human origin.

Functional and signal pathway enrichment analysis

To systematically understand the biological processes of the H1N1 virus causing pulmonary microvascular leakage, we conducted enrichment analysis of potential targets using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). DAVID Bioinformatics Resources (version 6.8) provides a comprehensive set of functional annotation tools for investigators to understand the biological meaning behind a large list of genes. DAVID (https://david.ncifcrf.gov/tools.jsp) was used to perform GO analysis, including biological process (BP), cellular component (CC), molecular function (MF), and KEGG Pathway analysis. The terms with a p-value of less than 0.05 were selected for functional annotation and signaling pathway clustering (Huang, Sherman & Lempicki, 2009a; Huang, Sherman & Lempicki, 2009b; Lai et al., 2021).

Protein-Protein interaction (PPI) analysis and gene module analysis

The STRING search tool was used to provide a key assessment of the protein-protein functional associations, with the species limited to “Homo sapiens.” The protein-protein interaction network (PPI) among the potential targets was constructed using the STRING search tool (version 11.0) (https://www.string-db.org/cgi/input?sessionId=bvSr4bpeU0nK&input_page_active_form=multiple_sequences), and the dates were saved as “CSV”. Cytoscape app was used for subsequent analysis. The molecular complex detection (MCODE) algorithm, was used to discover the closely related areas in the PPI network, by calculating the node information of each node in the network. The dates were run on the MCODE and STRING modules in Cytoscape, and the top one protein interaction module was saved for the next analysis (Halary et al., 2010).

Drug-gene interaction analysis

DGIdb database (version 3.0) (https://dgidb.org/search_interactions) was used to search for potential targets with existing associations with existing drugs or small organic compounds. We selected the genes of the top one protein interaction module and click “Find Drug-Gene Interactions” to identify the drugs. Here, we set the preset filter to all approved drugs (Cotto et al., 2018).

H1NI virus infects human lung microvascular endothelium

MDCK and HULEC-5a cells were infected with different dilutions of the A/PR/8/34 (H1N1) influenza virus for 48 h. Pathological changes of endothelial cells under the microscope were not obvious (Fig. 1A). Immunofluorescence staining results showed that A/PR/8/34 (H1N1) influenza virus-infected both MDCK and HULEC-5a cells (Fig. 1B). The supernatants collected from MDCK and HULEC-5a cells were used to detect the virus titer. The results showed that the virus titer for the MDCK cells was 1.9*10⁵ PFU/ml, while that for the endothelial cell was 0 PFU/ml (Fig. 2A). We determined the replication of A/PR/8/34 (H1N1) influenza virus nucleoprotein (NP) mRNA in endothelial cells by real-time quantitative PCR. The results showed that endothelial cells infected with the virus (MOI 0.00001, 0.0001, 0.0001, 0.001, 0.01) had increased NP mRNA (Fig. 2B). Finally, western blotting was used to detect the expression of viral nucleoprotein. At different virus titers, endothelial cells did not produce NP protein within 48 h (Fig. 2C, Supplement File S1, File S2). A/PR/8/34 (H1N1) influenza virus was found to infect HULEC-5a cells and initiate viral gene transcription but did not express a viral protein or produce infectious offspring.

Viral infection of microvascular endothelial cells leads to increased permeability

The fused HULEC-5a cells formed a compact monolayer on the semipermeable membrane, and the initial resistance value was measured before the cells were infected with the virus. Afterward, the old medium in the upper and lower chambers was removed, and influenza virus was added to the upper chamber at a multiplicity of infection (MOI) of 0.01, and maintenance solution was added to the lower chamber. Two hours after infection with the virus, the liquid in the upper and lower chambers was withdrawn, rinsed with PBS, and maintenance liquid added to both the upper and lower chambers. Resistance was determined at 0, 8, 16, 24, and 48 h after virus removal. A decrease in TEER was associated with increased permeability, while increased TEER suggested enhanced integrity of HULEC-5a cells (Fig. 3A). We observed that the TEER level significantly dropped at 16 h after influenza virus infection, and was at its lowest at 48 h. This indicated that viral infection increased the permeability of lung endothelial cells.

H1N1 virus induces the production of inflammatory cytokines

Cytokines are considered to be useful indicators of the severity of influenza infection and lung pathological characteristics, and endothelial cells are at the center of cytokine expansion during influenza virus infection (Teijaro et al., 2011). Real-time quantitative PCR was used to detect the mRNA relative expression of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-4, and IL-6 in the supernatant of endothelial cells 48 h after infection with influenza virus. The results showed that the relative mRNA expression levels of TNF-α, IL-1β, IL-4, and IL-6 were significantly higher than that of the control cells (Fig. 3B). The levels of inflammatory cytokines TNF-α, IL-1β, IL-4, and IL-6 were significantly higher than those in the control cells (Fig. 3B). This indicated that endothelial cells could induce the production of inflammatory cytokines after infection with the influenza virus.

Target gene for pulmonary microvascular leakage caused by H1N1

The GeneCards database was used to perform text mining, and search for genes related to “pulmonary microvascular leakage caused by H1N1”, Through text mining, a total of 107 genes related to pulmonary microvascular leakage caused by H1N1 (Table 1, Supplement File S3) were identified. This gene set was saved for the next analysis.

Functional and signal pathway enrichment analysis

The top five BP, CC, MF, and KEGG pathways were determined using DAVID. For CC, the genes were mainly enriched in the extracellular space, extracellular region part, cell surface, and vesicle lumen (Supplement File S4). For BP the genes were closely related to “defense response”, “response to external stimulus”, “immune response”, “positive regulation of immune system process” and “regulation of immune system process” (Supplement File S5). The results of MF enrichment showed that they were primarily associated with “receptor binding”, “cytokine activity”, “cytokine receptor binding”, “growth factor receptor binding” and “identical protein binding” (Supplement File S6). The top five highly enriched Signal Pathways were “Tuberculosis”, “Chagas disease (American trypanosomiasis)”, “Toll-like receptor signaling pathway”, “Influenza A” and “Inflammatory bowel disease (IBD)” (Supplement File S7, Supplement File S8, Fig. 4A). These results showed that BP was mainly related to immune response, CC was mainly related to the extracellular region and vesicle lumen, MF was mainly related to cytokines, and KEGG Pathway was mainly related to inflammation signaling pathway and immune signaling pathway.

PPI and gene module analysis

To understand the relationship between the targets more intuitively, 107 targets were imported into the SRING database to construct a gene functional association network, and the minimum score of protein interaction was set to 0.400. The network contained 105 nodes, 1808 edges, and a network graph with an average node degree of 34.4. The nodes represented the target protein, and the edges represented the protein-protein interaction relationship. STRING was used to analyze the protein-protein interaction of the genes using a protein interaction network diagram. We saved the file in “TSV” format and used Cytoscape to analyze the file (Supplement 9). The levels of TNF, IL6, TLR4, VEGFR, ALB, IL-10, IL-1β, and CXCL8 were significantly higher than other targets that play an important role in the PPI network. The highest target in this network was TNF, with a degree value of 82, which may be the most important potential target for H1N1 in causing pulmonary microvascular leakage. We used the MCODE plug-in in Cytoscape to perform module analysis on the file and obtained 39.111 points, ranking first, cluster-1 with 46 core genes (Supplement File S10, Fig. 4B). Further analysis of cluster-1 showed that the first five genes TNF, IL6, TLR4, IL-1β, and IL-10 had the highest degree values, and play a very important role in the cluster-1 network.

Functional and signal pathway enrichment analysis of module

Cluster-1 was further analyzed by GO and KEGG and based on the p-value, the first three BP, CC, MF, and KEGG were identified. CC enrichment was mainly enriched in the extracellular space and the extracellular area. For BP the enrichment was closely related to “defense response”, “inflammatory response” and “immune response”. For MF, the enrichment was mainly related to “receptor binding”, “cytokine activity” and “cell Factor receptor binding”. The top 3 most enriched signal pathways were “Chagas disease (American trypanosomiasis), ”Influenza A”’ and “Inflammatory bowel disease (IBD)” (Fig. 4C). In summary, BP was mainly related to the immune response, CC to the extracellular area, MF to cytokines, and KEGG pathway analysis was mainly related to inflammatory signaling pathway and immune signaling pathway. H1N1 was found to mainly cause pulmonary microvascular leakage in the extracellular area, activate and produce cytokines, and cause inflammation and immune responses. The signal pathway was mainly related to the inflammatory signaling pathway and the immune signaling pathway.

Drug-gene interaction analysis

The DGIdb tool was used to search and screen 46 genes in cluster-1 for drug-gene interaction analysis (Supplement File S11). As shown in Table 2, a list of approved drugs that meet the requirements of treatment standards has been compiled. There were 42 potential genetic targets in this list, and the corresponding drugs were ranked first in the Interaction Score & Query Score. Common uses of these drugs included cancer treatment, anti-inflammation, vascular disease treatment, immune function regulation, and hormone regulation.