CRISPR/Cas9-mediated PD-L1 knockout reshapes temporal transcriptomic responses to PRRSV infection in porcine alveolar macrophages
- 1Animal Biotechnology and Genomics Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea.
- 2College of Veterinary Medicine, Jeonbuk National University, Iksan, Republic of Korea.
- 3Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea.
Abstract
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.
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BibTeX
@article{sheet2026crisprcas9mediated,
title = {{CRISPR/Cas9-mediated PD-L1 knockout reshapes temporal transcriptomic responses to PRRSV infection in porcine alveolar macrophages}},
author = {Sunirmal Sheet and Hwan-Ju Kim and Won-Il Kim and Seokho Kim and Poongyeon Lee and Ara Cho and Dahye Kim},
journal = {PLOS One},
year = {2026},
month = nov,
doi = {10.1371/journal.pone.0355142},
url = {https://doi.org/10.1371/journal.pone.0355142},
note = {CC BY},
}
RIS
TY - JOUR
TI - CRISPR/Cas9-mediated PD-L1 knockout reshapes temporal transcriptomic responses to PRRSV infection in porcine alveolar macrophages
AU - Sunirmal Sheet
AU - Hwan-Ju Kim
AU - Won-Il Kim
AU - Seokho Kim
AU - Poongyeon Lee
AU - Ara Cho
AU - Dahye Kim
PY - 2026
DA - 2026/11/10
JO - PLOS One
DO - 10.1371/journal.pone.0355142
UR - https://doi.org/10.1371/journal.pone.0355142
AB - 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.
SN - 1932-6203
ER -