Porcine Reproductive and Respiratory Syndrome Virus Infection Upregulates Negative Immune Regulators and T-Cell Exhaustion Markers

Abstract

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for PRRSV, but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystanders PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP) and GFP + (PRRSV infected) and GFP – (bystander) cells were sorted for RNA-sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP + and 0.06% reads from GFP – PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP + as compared to GFP – PAMs. Importantly, negative immune regulators including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3), and T-cell exhaustion markers (PD-L1, PD-L2, IL10, IDO1, and TGFB2) were highly upregulated in GFP + cells as compared to GFP – cells. By using in situ hybridization assay, RNA transcripts of TNF and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. Importance PRRSV is widespread in many swine producing countries, causing substantial economic loses to the swine industry. PAM is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAM from an acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV-GFP strain to infect pigs and sorted infected- and bystander- PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of and NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

The transcriptional characteristics of NADC34-like PRRSV in porcine alveolar macrophages

“…IFN-I activated by several transcription factors such as NF-κB and IRFs triggers the production of numerous ISGs through this pathway, yet PRRSV can counteract it through viral proteins ( Schoggins and Rice, 2011 ; Wang et al, 2021 ). In the present study, both IFN-ALPHAOMEGA and IFNB1 were extremely significantly up-regulated (LFCs >5) at three timepoints after YC-2020 infection, which has been detected in some previous RNA-Seqs toward PRRSV-2 infection ( Lim et al, 2020 ; Chaudhari et al, 2021 ). Intriguingly, most of the downstream ISGs’ up-regulation levels were much lower in the comparison between YC-2020 and NC, with some even exhibiting a reversal at 38 hpi.…”

“…Meanwhile, anti-inflammatory factors such as TNFAIP3, NFKBIA, NFKBIZ, SOCS1, SOCS3, and IL-10 were elevated, with the reverse trends for the inflammation-inducing genes PPBP and MARCO at 38 hpi, suggesting that the pro-and anti-inflammatory responses might coexist in PAMs during YC-2020 infection. These phenomena were also captured in a recent study (Chaudhari et al, 2021), where the author thought that the production of pro-inflammatory factors originates from the body's immune defenses, while anti-inflammatory factors are attributed to PRRSV-induced negative regulation of immunity. The majority of inflammatory cytokines presented lower expression levels except for TNF, IL1α, and some chemokines (CCL4, CCL20) in the YC-2020 vs. JXA1 groups, but expression of the anti-inflammatory gene markers (NFKBIA, NFKBIZ, and TNFAIP3) was higher, indicating that YC-2020 PRRSV may cause a weaker inflammatory response than HP-PRRSV through stronger inhibition of the NF-κB signaling pathway (Oeckinghaus and Ghosh, 2009;Das et al, 2018).…”

“…We also discovered that the YC-2020-derived viral reads accounted for a significantly higher percentage than JXA1, once reaching more than 60% of the belonging sample’s total data volume. The proportion of viral-derived reads in existing PRRSV-associated transcriptome studies typically ranges from 1 to 20% ( Liang et al, 2017 ; Wu et al, 2020 ; Chaudhari et al, 2021 ); however, a recent analysis of SARS-CoV-2 transcriptome exhibited that the percentage of viral data approached 70% ( Kim et al, 2020 ). Therefore, without exogenous contamination, the dramatically transcriptional differences between the two PRRSV strains may be attributed to the discrepancy in the viral transcriptional capacity.…”

PRRSV-1 induced lung lesion is associated with an imbalance between costimulatory and coinhibitory immune checkpoints

“…Moreover, a marked up-regulation of PDL1 and CTLA4 genes has been also observed in the thymus of piglets infected with the virulent PRRSV-1 Lena strain ( Ruedas-Torres et al, 2021b ). In this context, transcriptional genome analysis performed by our research group and others have also described an overexpression of some of these costimulatory and coinhibitory molecules in target organs from PRRSV-1 and PRRSV-2 infected pigs ( Chaudhari et al, 2020 , 2021 ; Fleming et al, 2020 ; Sánchez-Carvajal et al, 2021a ). In this sense, Chaudhari et al (2020) already described an up-regulation of coinhibitory molecules such as TIGIT, PD1, TIM3, and IDO1 in the inguinal lymph node from pigs infected with a live-attenuated PRRSV-2 strain.…”

Porcine Reproductive and Respiratory Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1

“…PRRSV has a restricted tropism for monocyte/macrophage lineage in lungs and other tissues and preferentially targets porcine alveolar macrophages (PAMs) ( 27 ). Therefore, primarily isolated and cultured PAMs act as an appropriate infection model, which has the comparable soundness of a pig model with minimal animal usage, for pathogenesis research on PRRSV ( 28 , 29 ) (including this study) and some other major porcine viruses, such as African swine fever virus (ASFV) ( 30 ) and porcine circovirus (PCV) ( 31 ). PAMs are close to pig tissues in vivo and provide a clear platform to reveal the interaction or regulation between host and PRRSV without interference from many other factors in pig infection, such as other pathogen infection and factors from environment ( 27 , 32 ).…”