Abstract
Objectives
The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs).
Methods
Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18 h before stimulation with lipopolysaccharide (LPS μg/ml), lipoteichoic acid (LTA μg/ml) or polyinosinic:polycytidylic acid (poly I:C-20 μg/ml) PAMPs for 6 h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR).
Results
RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, β-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.
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Data availability
The datasets analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors would like to acknowledge Moyvalley Meats, Kildare for their kind assistance with sample collection.
Funding
This work was funded by a Research Grant to KM from the Irish Department of Agriculture (DAFM grant 13/S/472).
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Conceived the study: KM. Developed primary culture model: MB, MOB and CB. Performed experiments and interpreted data: MB, MOB and CB. Wrote the manuscript: MB, MOB and KM. All authors read and approved the final manuscript.
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All procedures described were conducted post-mortem, and for which ethical approval and licences are not required.
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Boylan, M., O’Brien, M.B., Beynon, C. et al. 1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts. Vet Res Commun 44, 83–88 (2020). https://doi.org/10.1007/s11259-020-09775-y
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DOI: https://doi.org/10.1007/s11259-020-09775-y