5B)

5B). switch from apoptosis to pyroptosis by inhibiting apoptosis possibly through the induced expression of the anti-apoptotic gene. Further, the inhibition of JAK-STAT signaling repressed pyroptosis but enhanced apoptosis in infected PL16T cells. Collectively, we propose that type I IFN signaling pathway triggers pyroptosis but not apoptosis in the respiratory BC-1215 epithelial cells in a mutually exclusive manner to initiate proinflammatory responses against influenza virus infection. IMPORTANCE Respiratory epithelium functions as a sensor of infectious agents to initiate inflammatory responses along with cell death. However, the exact cell death mechanism responsible for inflammatory responses by influenza virus infection is still unclear. We showed that influenza virus infection induced apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced at early phases of infection, but the cell death pathway was shifted to pyroptosis at late phases of infection under the regulation of type I IFN signaling to promote proinflammatory cytokine production. Taken together, our results indicate that the type I IFN signaling pathway plays an important role to induce pyroptosis but represses apoptosis in the respiratory epithelial cells to initiate proinflammatory responses against influenza virus infection. BC-1215 anti-apoptotic gene. Further, the inhibition of the JAK-STAT pathway, which is downstream of type I IFN, repressed pyroptotic cell death but enhanced apoptotic cell death in PL16T cells. Collectively, we propose that type I IFN signaling pathway triggers pyroptosis but not apoptosis in the respiratory epithelial cells in a mutually exclusive manner to initiate proinflammatory responses against IAV infection. RESULTS AND DISCUSSION Precancerous respiratory epithelial cells induce pyroptotic cell death in response to infection. To determine whether respiratory epithelial cell lines are susceptible to the cell death induced by IAV infection, we carried out trypan blue dye exclusion assays at 24 h postinfection with different types of human malignant tumor respiratory epithelial cells (A549, PC9, H1975, H1650, and HCC827), human atypical adenomatous hyperplasia (AAH) respiratory epithelial cells (PL16T), human nonneoplastic respiratory epithelial cells (PL16B), and primary normal human bronchial epithelial cells (NHBE). The cell death in all malignant tumor cell lines was rarely induced by IAV infection, whereas the number of dead cells in PL16T, PL16B, and NHBE lines was 30 to 40% of total cells at 24 h postinfection (Fig. 1A). PL16T is an immortalized cell line that was established from a precancerous region of a lung adenocarcinoma patient (24). It has been reported that PL16T cells do not have any tumorigenic activity and there are no mutations or abnormal expressions of oncogenesis-related genes, such as (25). To determine what kinds of cell death pathways are activated by IAV infection, we treated infected PL16T, NHBE, and BC-1215 A549 cells with each type of cell death inhibitor: Z-DEVD-FMK (caspase-3 inhibitor) (Fig. 1B, ?,D,D, ?,F,F, and ?andH),H), VX-765 (caspase-1 inhibitor) (Fig. 1C, ?,E,E, ?,G,G, and ?andI),I), and GSK-872 (RIP3 inhibitor) with Z-VAD-FMK (pancaspase inhibitor) (Fig. 1J, ?,K,K, and ?andL).L). In infected PL16T cells, the number of dead cells either stained with trypan blue dye (Fig. 1B) or having fragmented DNA (Fig. 1D) was reduced by the addition of the caspase-3 inhibitor at 12 and 24 h postinfection, but not after 36 h postinfection. In contrast, the caspase-1 inhibitor repressed cell death even at 36 h postinfection in infected PL16T cells (Fig. 1C and ?andE).E). These results suggest that apoptosis is induced in infected PL16T cells at early phases of infection but the cell death pathway is shifted to pyroptosis at late phases of infection. Similar results were obtained with infected NHBE cells (Fig. 1F and ?andG).G). Furthermore, the number of dead cells in infected A549 cells was decreased by the caspase-3 inhibitor in both early and late phases of infection, but not by the caspase-1 inhibitor (Fig. 1H and ?andI).I). Thus, it is likely that IAV infection triggers both apoptotic and pyroptotic cell deaths in precancerous or normal BC-1215 human respiratory epithelial cells but only apoptotic cell death in malignant tumor cells. GSK-872 did not inhibit cell death by IAV infection in PL16T cells, NHBE cells, and A549 cells (Fig. 1J, ?,K,K, and ?andL).L). These results indicate that necroptosis merely occurs in response to IAV infection in the cultured cells that we used. However, it has been reported that necroptosis is triggered by IAV infection in a mouse model (26) and immortalized murine cells, including lung epithelial cells and Tgfbr2 embryonic fibroblast cells (27). Thus, it is possible that necroptosis is stimulated by IAV infection in a species- and/or cell type-dependent manner as previously reported in other virus infections such as herpes simplex virus (28), human cytomegalovirus (29), and human immunodeficiency virus (30) infections. Note that the expression level of viral protein NP in PL16T cells was similar to that.