Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection

Abstract

Objective To investigate the inductive effect of human adenovirus type 7 (HAdV-7) infection on ferroptosis in alveolar epithelial cells. Methods Human alveolar epithelial carcinoma cells A549 and human pulmonary type Ⅱ alveolar epithelial cells HPAEpiC were infected with HAdV-7, respectively, and in 24, 48 and 72 h after infection, cell viability was measured using CCK-8 assay, mitochondrial morphology was observed by transmission electron microscopy (TEM), productions of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured with ROS probe and Lipid Peroxidation, respectively, and intracellular Fe2+ level were observed by Fe2+ fluorescence probe. RT-qPCR and Western blotting were applied to determine the expression changes of key genes in ferroptosis. After CD46 humanized mice were infected with HAdV-7 by nose dropping, Perls staining and Western blotting were applied to detect iron deposition and the expression of ferroptosis-related proteins in the lung tissue. After administration of ferroptosis inhibitor, deferoxamine mesylate (DFO), in the mice with HAdV-7 infection, CCK-8 assay and HE staining were performed to observe the alveolar epithelial cell death and lung pathology. Results After HAdV-7 infection, cell viability were reduced in the A549 and HPAEpiC cells, and smaller mitochondria and decreased even disappeared mitochondrial crista were observed in the ferroptotic cells. ROS level (A549 and HPAEpiC cells: 48 h and 72 h, P < 0.01) and MDA level (both A549 and HPAEpiC cells: P < 0.05) were significantly higher in the HAdV-7 group than the control group. The intracellular iron concentration was obviously higher in the infected HPAEpiC cells than the control cells (P < 0.01). RT-qPCR results showed that the mRNA levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11, also known as xCT) (P < 0.01), glutathione peroxidase 4 (GPX4) (P < 0.05), ferroportin (SLC40A1) (P < 0.05) and ferritin heavy chain (FTH1) (A549 cells: 72 h, P < 0.01; HPAEpiC cells: 48, 72 h, P < 0.01) were down-regulated after HAdV-7 infection, whereas that of HAMP was up-regulated (A549 cells: 72 h, P < 0.01; HPAEpiC cells: P < 0.05). The protein levels of xCT and GPX4 were also decreased in the infected HPAEpiC cells than the control group. HAdV-7 infection in the CD46 humanized mice resulted in deepened iron stains in the lung tissue, and decreased protein levels of xCT (P < 0.05) and Gpx4 (P < 0.01). Ferroptosis inhibitor DFO could partially prevent HAdV-7-induced cell death (HPAEpiC: P < 0.01), and obviously enhance Fer-1 inhibited cell death (A549 cells: P < 0.05; HPAEpiC cells: P < 0.01). In addition, Fer-1 treatment could effective ameliorate pathological damages in the CD46 humanized mice after HAdV-7 infection. Conclusion HAdV-7 infection increases iron accumulation and lipid peroxides, and thus induces ferroptosis in alveolar epithelial cells, which may be involved in lung injury caused by HAdV-7 infection.

Keywords

• human adenovirus type 7
• alveolar epithelial cells
• ferroptosis