Significant upregulation of GSDMD and TfR-1 genes following A/H1N1, A/H3N2, and Flu-B infections

Influenza viruses, as important respiratory viruses for seasonal flu, are responsible for yearly mortality among human populations. Among different and complex interactions within the cell, pyroptosis and ferroptosis have emerged as two main cell death pathways that are correlated to inflammation following influenza infection. In this study, we have investigated GSDMD (pyroptosis marker) and TfR-1 (ferroptosis marker) mRNA expression, 48 h post-infection (hpi) in A549 infected cells with different types of influenza viruses. A549 cells were infected with A/H1N1, A/H3N2, and Flu-B with 100 TCID₅₀. RNA was extracted by RNJIA RNA extraction kit (RNJIA, ROJE, Iran) 48 hpi, and mRNA expression of GSDMD and TfR-1 was assessed by SYBR Green qRT-PCR. Gene expression data were analyzed using both non-parametric and parametric statistical tests, followed by pairwise post-hoc comparisons. Significant upregulation of GSDMD expression was observed in all infected groups compared to the controls (A/H1N1: mean fold change = 5.55, P = 0.018; A/H3N2: mean fold change = 4.69, P < 0.001; Flu-B: mean fold change = 5.28, P = 0.016). In addition, TfR-1 expression was also increased in all three infected groups compared to the controls (A/H1N1: mean difference = 11.98, P = 0.006; A/H3N2: mean difference = 13.17, P = 0.003; Flu-B: mean difference = 12.69, P < 0.001). The consistent upregulation of GSDMD and TfR-1 across A/H1N1, A/H3N2, and Flu-B compared to the control groups suggests the involvement of these pro-pyroptotic and pro-ferroptotic factors associated to pyrotposis and ferroptosis cell death pathways following inflammation, 48 hpi. While GSDMD had sharpest upregulation in A/H1N1 compared to A/H3N2 and Flu-B, TfR-1 upregulation showed different pattern with highest upregulation in A/H3N2. In total, our study supports the potential involvement of GSDMD and TfR-1 which are associated with pyroptosis and ferroptosis that are active following different types of influenza viruses.