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

Abstract

Objective: To investigate the replication of influenza A virus A/Puerto Rico/8/34 (H1N1) in pulmonary microvascular endothelial cells and its effect on endothelial barrier function.

Methods: Human pulmonary microvascular endothelial cells were infected with influenza A/Puerto Rico/8/34 (H1N1) virus. Plaque reduction assay, real-time quantitative PCR, immunofluorescence staining, and western blot were used to elucidate the replication process of virus-infected endothelial cells. In addition, real-time quantitative PCR was used to detect the relative expression levels of mRNA of some inflammatory factors. The endothelial resistance assay was used to determine the permeability of the endothelial monolayer. Excavation and analysis of data from open databases, such as the GeneCards database, DAVID Bioinformatics Resources, STRING search tool, and DGIdb database determined the genes, proteins, and signal pathways related to microvascular leakage caused by the H1N1 virus, and predicted the drugs that could be effective for treatment.

Results: In vitro experiments showed that the influenza virus can infect endothelial cells, leading to a significant increase in the permeability of pulmonary microvascular endothelial cells and the release of pro-inflammatory cytokines, but does not efficiently replicate in endothelial cells. A total of 107 disease-related target genes were obtained from the Gene-cards database. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes mainly affected the pathways related to "Inflammatory bowel disease" (IBD), "Chagas disease" (American trypanosomiasis), "Influenza A", and also played a key role in anti-inflammation and regulation of immunity. After enrichment analysis, 46 hub genes were screened. A total of 42 FDA-approved drugs corresponding to the hub genes were screened from the DGIdb database, and these could be formulated for topical application. In addition, these drugs can be used to treat other diseases, including cancer, inflammatory diseases, immune system disorders, and cardiovascular diseases.

Conclusion: H1N1 influenza virus affects the barrier function of endothelial cells indirectly. Combined with bioinformatics tools, we can better understand the possible mechanism of action of influenza A (H1N1) virus causing pulmonary microvascular leakage and provide new clues for the treatment of pulmonary microvascular leakage.

Keywords: Endothelial cells; Influenza A; Pulmonary microvascular; Vascular leakage.

Figure 1. Influenza virus can infect endothelial cells, but cannot cause obvious cytopathic changes.

(A) 48 h after infection of MDCK cells and HULEC-5a with influenza virus of different dilutions, pathological changes were observed under the microscope. (B) 48 h after infection of MDCK cells and HULEC-5a with influenza virus of MOI 0.01. The cells were fixed and immunostained with nucleoprotein (green). Counterstaining was performed using DAPI (blue).

Figure 2. Influenza virus can initiate RNA replication after infecting endothelial cells, but cannot initiate protein replication, and cannot produce progeny viruses.

(A) 48 h after infection, the virus titer in MDCK cells infected with influenza virus and HULEC-5a supernatant was measured by the plaque formation method. The dilution factor of the virus group in the figure is 10⁵-fold and the results represent three experiments. (B) After infection with influenza virus of different dilutions, real-time quantitative PCR analysis using nucleoprotein specific primers confirmed that the virus can replicate on endothelial cells and MDCK, *p < 0.05. The results are representative of three experiments. (C) After influenza virus infection, HULEC-5a cells and MDCK cells were incubated for 48 h, and the level of NP protein was determined by WB analysis, as described in the “Materials and methods” section. The gray level of each band was measured by Image J software to detect the difference of NP protein expression. Data are mean ± SD of three experiments. Compared with the control group, *p < 0.05. The picture shows the representative data of three independent experiments.

Figure 3. After influenza virus infects endothelial cells, it can cause vascular leakage and induce endothelial cells to produce pro-inflammatory factors.

(A) After being infected with influenza virus (MOI 0.01), HULEC-5a cells were incubated for 0, 8, 16, 24 and 48 h. Then the trans-endothelial resistance is detected. Results (mean, SE) are from 3 experiments and are normalized to control, *p < 0.05. B: After infected with influenza virus (MOI 0.01), HULEC-5a cells were incubated for 48 h, and the mRNA level of TNF-α, IL-1β, IL-4, IL-6 was detected by real-time quantitative PCR. Data are mean ± SD of three experiments. *p < 0.05, ^##p < 0.01 versus control group.

Figure 4. The top five of GO enrichment and KEGG pathway enrichment and PPI network.

The top three of GO enrichment and KEGG pathway enrichment of hub genes. (A) The top five of GO enrichment and KEGG pathway enrichment. (B) PPI network. the 107 common genes and network of hub genes. (C) The top three of GO enrichment and KEGG pathway enrichment of hub genes.