Innate immune potential of bovine respiratory epithelial cells: role in viral infection

Respiratory epithelial cells have important defence activities against respiratory viruses. While their physical barrier function is well described, the molecular mechanisms responsible for how epithelial cells initiate, mediate and regulate local immune activity to viral infection are poorly defined, particularly in livestock species. This lack of knowledge contributes to the challenge in controlling respiratory infections, including bovine herpesvirus 1 (BoHV-1) in cattle. Our study hypothesises that bovine respiratory epithelial cells can respond directly to viral infections and have potent innate molecular antiviral activity. The overall aim was to characterise innate antiviral immune potential of primary bovine respiratory epithelial cells in responding to purified live BoHV-1 and viral ligands. A cell culture model of primary bovine tracheal epithelial cells was established in order to provide physiologically relevant insight into immune mechanisms. These cultures retain the same genetic and physiological characteristics of their in-vivo host. While demonstrating significant inter-animal variation reflective of the outbred animal population, our primary culture model is reproducible and easily available, with low costs, from abattoir by-products. Using this model, we demonstrated that BoHV-1 is able to infect primary tracheal epithelial cells and went on to examine the transcriptomic changes induced early in infection. Bulk next generation RNA sequencing analysis of epithelial cells infected with purified live BoHV-1 (MOI 7) for 6 hours revealed after correction or multiple testing, differential expression of nine genes. The heat shock protein family A (Hsp70) member 6 gene HSPA6 demonstrated a fold change of 16 (adj. p-value = 2.59E-45) and was the most upregulated gene. We went on to investigate other heat shock proteins (GRP78/HSPA5 and GRP94/HSP90B1) that are involved in stress responses occurring during viral infections as part of the unfolded protein response (UPR). The UPR is a fundamental cellular defence mechanism activated by disrupted ER homeostasis caused by the accumulation of mis- and unfolded proteins during viral replication. Previously considered reflective of non-specific stress in the host promoting cell survival or initiating cell death, it has become clear that viruses can disarm the UPR in order to assist their replication. We found that infection with purified live BoHV-1 virus downregulated genes involved in the UPR in primary bovine tracheal epithelial cells (ATF6, DDIT3, sXBP1 and HERPUD1). Thapsigargin (Tg) is an ER stressor which inhibits the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump. Pre-treatment of primary cells with Tg partly counteracted the virally-induced downregulation of GRP78, sXBP1, HERPUD1 and GRP94. Pre-treatment with Tg also significantly decreased viral genome expression. Further investigations of the UPR manipulation by viruses and UPR targeting agents may identify new strategies for preventing infection by respiratory viruses. Innate immune mechanisms active in bovine tracheal epithelial cells were further investigated by qPCR analysis and results showed that these cells are equipped with membrane associated and cytosolic pathogen recognition receptors (PRRs). While infection with BoHV-1 failed to upregulate expression of IL6, TNF, IFNB1 and IFNB3 genes in primary bovine tracheal epithelial cells, the gene encoding interferon ?3 (IFNL3), was upregulated by > 30 fold after 24 hours. Interferon stimulated genes RSAD2, ISG15 and USP18 were also significantly upregulated as was IDO1, an important enzyme in several metabolic pathways and a key immunosuppressor. In recent years, modulation of cellular metabolic pathways, including the synthesis of cholesterol, polyamines and tryptophan by ISGs has been discovered to have exciting potential as a novel antiviral strategy and our results support this approach. Our results also emphasise an important role for IFN-? at epithelial surfaces during BoHV-1 infection. However, it is unclear what signalling pathways lead to IFN-? upregulation and to which extent it might contribute to antiviral innate immunity in epithelial cells. We found that stimulation with Poly(dA:dT), a synthetic dsDNA sequence capable of stimulating multiple cytosolic DNA sensors, and 3p-hpRNA, a 5?triphosphate hairpin RNA as a RIG-I agonist, induced significant expression of IFNL3 in tracheal epithelial cells, while type I interferons were not significantly induced. Stimulation with Poly(I:C), a synthetic nucleic acid molecule with similar structure to viral dsRNA and TLR3 agonist, upregulated the viral sensing genes DDX58, IFIH1, IFI16 and MB21D1 and induced the interferon-stimulated genes ISG15, RSAD2, USP18 and IDO1. Regulation of IFNB1 or IFNB3 was not detected. In contrast, IFNL3 expression was significantly increased by over 9,000-fold in response to Poly(I:C) at 6 hours. A 6-hour pre-treatment with Poly(I:C) also significantly decreased viral genome abundance. This inhibition indicates that activation of the TLR3/RIG-like receptor signalling pathways induces an antiviral state effective against BoHV-1 replication in tracheal epithelial cells. Our study highlights the importance of epithelial cells for local innate antiviral activity in the respiratory tract due to their ability to detect and respond to viral infection. Our studies using the infection model of primary bovine tracheal epithelial cells demonstrate the significant innate immune potential of bovine tracheal epithelial cells against viral pathogens characterised by significant IFNL3 upregulation. We demonstrate protective antiviral potential against viral infection in these cells through activation of the TLR3/RIG-like receptor signalling pathway. Further characterisation of antiviral innate immune potential of respiratory epithelial cells including definition of the signalling pathways underpinning inter-animal variation will ultimately contribute to breeding and immunotherapeutic strategies capable of preventing and better treatment of BoHV-1 infection in livestock.

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