Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract.
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This study was supported by the Agriculture and Food Research Initiative competitive grant number 2013-68004-20360 from the USDA National Institute of Food and Agriculture. This work was also supported by the USDA National Institute of Food and Agriculture hatch project number KY014055 (College of Agriculture, Food and Environment, University of Kentucky), and partially supported by Louisiana State University, School of Veterinary Medicine start-up fund (PG 002165) to Dr. Udeni B. R. Balasuriya.
The RNA sequencing data from this study were deposited in the Gene Expression Omnibus (GEO, NCBI, NIH) database under study GSE114982 (accession numbers GSM3161940- GSM3161951).
Carossino, Mariano; Dini, Pouya; Kalbfleisch, Theodore S.; Loynachan, Alan T.; Canisso, Igor F.; Cook, R. Frank; Timoney, Peter J.; and Balasuriya, Udeni B. R., "Equine Arteritis Virus Long-Term Persistence Is Orchestrated by CD8+ T Lymphocyte Transcription Factors, Inhibitory Receptors, and the CXCL16/CXCR6 Axis" (2019). Veterinary Science Faculty Publications. 49.
S1 Fig. Gene ontology analysis of DEGs between long-term carrier (n = 3) and short-term carrier stallions (n = 6). (A) Biological process. (B) Molecular function. https://doi.org/10.1371/journal.ppat.1007950.s001
ppat.1007950.s002.tif (6421 kB)
S2 Fig. Inflammatory response in the ampullae during long-term EAV persistence. https://doi.org/10.1371/journal.ppat.1007950.s002
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S3 Fig. Gene ontology analysis (biological process) of DEGs between stallions homozygous and heterozygous for CXCL16S (n = 5) and stallions homozygous for CXCL16R (n = 4). https://doi.org/10.1371/journal.ppat.1007950.s003
ppat.1007950.s004.tif (307 kB)
S4 Fig. Hierarchical clustering of module eigengenes (MEs) for the modules identified. https://doi.org/10.1371/journal.ppat.1007950.s004
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S5 Fig. Immunohistochemical staining for EOMES, NFATC2, TBX21 (T-bet), BLIMP-1, pAkt, CTLA-4 and granzyme B on equine palatine tonsil (tissue control). The negative immunostaining control is labeled as isotype control. DAB. 100X. Bar = 80 μm. https://doi.org/10.1371/journal.ppat.1007950.s005
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S1 Table. Top 25 canonical pathways associated with common upregulated genes. Pathway analysis was performed using IPA. https://doi.org/10.1371/journal.ppat.1007950.s006
ppat.1007950.s007.xlsx (11 kB)
S2 Table. Transcription factors (TFs) identified by means of upstream regulator analysis performed on Ingenuity Pathway Analysis (IPA) in long-term carrier stallions. Target molecules in dataset are depicted. https://doi.org/10.1371/journal.ppat.1007950.s007
ppat.1007950.s008.xlsx (35 kB)
S3 Table. Target and correlated genes obtained from the Ingenuity Knowledgebase (IPA), the Immuno-Navigator database and literature search associated with (A) EOMES, (B) NFATC2, (C) TBX21 and (D) PRDM1. https://doi.org/10.1371/journal.ppat.1007950.s008
ppat.1007950.s009.xlsx (13 kB)
S4 Table. Genes positively correlated to CXCL16 and CXCR6 (R2 greater or equal to 0.5) and differentially expressed in long-term carrier stallions. https://doi.org/10.1371/journal.ppat.1007950.s009
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S5 Table. Gene members of modules correlated with the CD3+ T lymphocyte susceptibility phenotype/CXCL16 genotype (blue and lightyellow) following weighted gene co-expression network analysis of 12,303 genes. https://doi.org/10.1371/journal.ppat.1007950.s010
ppat.1007950.s011.xlsx (36 kB)
S6 Table. Network analysis parameters for the "hub" genes in the blue (n = 1135 total genes) and lightyellow (n = 130 total genes) modules (MM≥0.90, p-value<0.05, GS≥0.5). Network analysis was performed using Cytoscape. https://doi.org/10.1371/journal.ppat.1007950.s011
ppat.1007950.s012.xlsx (14 kB)
S7 Table. Gene members in each module (n = 5 modules) following weighted gene co-expression analysis of 494 transcription factor genes in CXCL16S stallions. https://doi.org/10.1371/journal.ppat.1007950.s012
ppat.1007950.s013.xlsx (12 kB)
S8 Table. RNA sequencing mapping statistics. https://doi.org/10.1371/journal.ppat.1007950.s013
ppat.1007950.s014.docx (19 kB)
S9 Table. Primers designed for gene expression analysis by RT-qPCR. https://doi.org/10.1371/journal.ppat.1007950.s014