Pseudorabies virus induces Natural killer cell depletion by GSDMD-mediated inflammation and pyroptosis to promote infection and lung injury

To further explain why GSDMD deletion can significantly weaken the pathogenic ability of PRV and explore what role do GSDMD play and how do GSDMD act in the process of PRV infection and induction of tissue injury, we firstly sequenced the transcriptome of over 13,275 unselected cells of inflammatory cells isolated from the lung of PRV-infected WT mice and PRV-infected Gsdmd-/- mice using scRNA-seq . Overall design: WT mice and Gsdmd-/- mice (n = 5, per group) were inoculated intraperitoneally with PRV-RJ strain (2 ×103 TCID50) for 48 h, respectively. For the lung single-cell suspension, the lungs were resuspended with PBS containing 0.1 % Collagenase type I (SCR103, Sigma-Aldrich), 0.05 % DNase I (D5025, Sigma-Aldrich), and 5 % FBS (13011-8611, Tianhang Biotechnology). After incubation at 37 °C for 60 min, the samples were pestled and filtrated with a 200-mesh sieve, pelleted (300 g, 5 minutes), and resuspended in ACK red blood cell lysis buffer (Gibco A1049201) for 3 minutes, after which the buffer was inactivated by adding excess processing buffer. Cells were then filtered through a 70 µm strainer, pelleted again (300 g, 5 minutes), and resuspended in PBS buffer, counted, and immediately processed for single-cell RNA sequencing.