Strain-Dependent Apoptotic Gene Expression in Influenza-Infected Lung Epithelial Cells

Abstract

Background: Influenza viruses induce multiple programmed cell death pathways in respiratory epithelial cells. These pathways are associated with host antiviral defense but may also contribute to tissue damage through inflammation and increased disease severity, thereby functioning as a double-edged sword. Apoptosis is a major cell death pathway that regulates the fate of infected cells following viral infection. Objectives: This study evaluated the mRNA expression of BAX, BAK1, and Bcl-2, three key apoptosis-regulating genes, in A549 lung epithelial cells infected with influenza A/H1N1, A/H3N2, and influenza B viruses. Methods: A549 cells were cultured and inoculated with 100 TCID50 of influenza A/H1N1, A/H3N2, and influenza B viruses for 48 hours. Results were compared with those for uninfected control cells. At 48 hours post-infection, RNA extraction (RNJIA, ROJE) and cDNA synthesis were performed, and the mRNA expression levels of BAX, BAK1, and Bcl-2 were assessed using SYBR Green reverse transcription quantitative PCR. All analyses were based on log fold change (logFC). Differential gene expression versus control was evaluated using the Wilcoxon signed-rank test and a one-sample t test, whereas time-dependent changes within each virus group were assessed using the Kruskal-Wallis test and one-way analysis of variance, followed by pairwise post-hoc tests with Benjamini-Hochberg correction. Results: At 48 hours post-infection, BAX expression was significantly upregulated in A/H1N1-infected cells (log fold change = 2.85, P = 0.031). Although A/H3N2 also upregulated BAX expression (log fold change = 2.35, P = 0.061), this change was not significant. Minor downregulation of BAX was observed in influenza B-infected cells (log fold change = -0.26, P = 0.463). BAK1 expression was significantly upregulated in A/H3N2-infected cells (log fold change = 6.20, P = 0.019). A/H1N1-infected cells also showed BAK1 upregulation (log fold change = 2.10, P = 0.431), although this change was not significant, whereas influenza B-infected cells showed significant BAK1 upregulation (log fold change = 6.21, P = 0.025). Bcl-2 was significantly downregulated in all three infected groups (A/H1N1: log fold change = -6.05, P = 0.040; A/H3N2: log fold change = -4.02, P = 0.002; influenza B: log fold change = -0.70, P = 0.032). Conclusions: This experiment demonstrated strain-dependent regulation of apoptosis-related genes in A549 cells following infection with different influenza viruses. Significant upregulation of BAX and BAK1 after A/H1N1 infection, upregulation of BAK1 after A/H3N2 and influenza B infection, and downregulation of Bcl-2 in all infected groups suggest activation of pro-apoptotic signaling. These findings are preliminary transcriptional observations from an in vitro system and may provide insight into mechanisms involved in the regulation of pro-apoptotic genes in the apoptosis pathway.