Functional Relevance of Interleukin 8 Haplotype for the Innate Immune System in Holstein-Friesian Cattle

Abstract

Calves rely heavily on their innate immune response in the post-birth period before their adaptive immune system develops. Interleukin 8 (IL-8) is a major chemoattractant predominantly associated with neutrophils response to infection. The genetic variation at this gene promoter is likely to have functional consequences for both inflammation and infectious disease susceptibility. Previous work by our group identified two distinct haplotypes (IL8-h1 and IL8-h2) in the IL-8 gene promoter region carried at equal frequencies (~ 50%) in the Holstein-Friesian breed. Calves carrying IL8-h2 produce significantly higher levels of IL-8 after in vivo systemic stimulation with lipopolysaccharide (LPS). However, little is understood about IL8 haplotype effect on the IL-8 response and the wider innate immune system within the first six months of life. It also remains unknown if levels of IL-8 secreted by local cell types differ between haplotypes. Additionally, vitamin D has been described to modulate the IL-8 response but its role in calf immunity and the specific effects of IL8 haplotype has not been previously investigated. The aims of this project were I. To investigate immune cell populations and the systemic innate immune response in calves with different IL8 haplotypes to bacterial, viral and fungal PAMPs over the first year of life; II. To establish the relationship between IL8 and circulating vitamin D levels in peripheral blood and expression of vitamin D pathway genes; and III. To establish a local primary cell model to examine the effect of IL8 haplotype on innate immune gene expression and the response to vitamin D.

Haematological profiling of genotyped Holstein-Friesian calves showed neutrophils, monocytes, eosinophils and basophils were increased at specific time points in IL8-h2 calves across the first ten months of life in comparison to IL8-h1 calves. Using whole blood culture systems, peripheral blood from these calves was exposed to a bacterial (LPS), viral (poly (I:C)) and fungal (zymosan) PAMPs. No significant differences in IL-8 were identified in response to any PAMP which may be due to the absence of a cell type in peripheral blood which produces IL-8. Examination of basal IL-8 levels in serum along with vitamin D showed that IL8-h2 calves produced significantly higher levels of IL-8, concurrently with lower circulating levels of vitamin D. The opposite relationship was apparent for IL8-h1 calves which produce relatively lower levels of IL-8 but higher levels of vitamin D. Further analysis in vitro showed vitamin D to significantly reduce IL-8 from monocytes at a basal level. Finally, primary dermal fibroblasts isolated from genotyped cattle were stimulated with LPS and Pam3CSK4. IL8-h1 cells showed significantly increased levels of IL-8 in response to Pam3CSK4 when compared with IL8-h2 fibroblasts. This indicates a cell specific and PAMP specific IL-8 response which is dependent on IL8 haplotype. To conclude, the results here support a significant effect of IL8 haplotype on both systemic and local innate immune response in Holstein-Friesian cattle and further demonstrates functional interaction between IL8 haplotype and vitamin D levels in peripheral blood. Therefore, IL8 haplotype is likely to have important relevance for immune competence and in vivo susceptibility of cattle to multiple diseases.