CRISPR/Cas9-mediated PD-L1 knockout reshapes temporal transcriptomic responses to PRRSV infection in porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses innate antiviral immunity in porcine alveolar macrophages, and the PD-1 / PD-L1 axis is thought to contribute to this immune dysregulation. This study investigated how PD-L1 knockout (KO) alters the host transcriptional response to PRRSV infection compared to wild-type (WT) immortalized porcine alveolar macrophages (iPAMs). PD-L1 KO iPAMs were generated by CRISPR/Cas9 targeting exon 2 of porcine CD274 locus, and knockout was confirmed in clone #35. After PRRSV-2 infection, KO#35 cells exhibited approximately 5-fold reduced viral RNA accumulation at 48 h post-infection compared with WT cells. Time-course RNA sequencing at 0, 6, and 48 h post-infection was then performed to characterize the transcriptional consequences of PD-L1 KO during acute PRRSV-2 infection. PD-L1 KO iPAMs exhibited downregulation of immune checkpoint and exhaustion-related genes ( LAG3 , PDCD1 , TIGIT , TOX family) and NF-κB inhibitors ( SOCS4 , NFKBIA ), alongside upregulation of antigen presentation (MHC I/II genes like SLA-2 , SLA-5 , B2M , SLA-DMA , SLA-DMB , HLA-DRA ) and co-stimulatory molecules ( CD80 , CD86 , CD40 ). Cytokine profiling indicated a reprogrammed response favoring antiviral signaling, including sustained elevation of CXCL10/11 , ISGs ( MX1/2 , ISG15 , IFIT1–3 ), and select interleukins ( IL15 , IL6 ), while suppressing pro-inflammatory TNF and CXCL8 across time points. Pathway enrichment analysis revealed significant enrichment of the cytokine–receptor interaction pathway. Weighted gene co-expression network analysis (WGCNA) further identified a key co-expression module containing 1,763 DEGs that was associated with innate immune activation. Moreover, protein-protein interaction (PPI) network analysis detected PXDN as a key hub gene connected to the PI3K-Akt signaling and natural killer cytotoxicity pathways. These findings provide transcriptomic insight into the association between PD-L1 deficiency and altered macrophage transcriptional responses to PRRSV infection in an in-vitro iPAM model and provide a basis for future mechanistic investigation of PD-L1 in PRRSV pathogenesis.