1. Structural and Functional Characterisation of Interleukin 8 in Cattle
PhD abstract
Bojan Stojkovic
Accumulation of leukocytes in the tissue is one of the hallmarks of inflammation. In bovine mastitis and
uterine disease neutrophils are rapidly recruited to either the mammary gland or the uterus. This leads to an
increase in the number of somatic cells in milk as well as uterine tissue damage and infertility problems, both
of which constitute massive constraints to global agriculture. Interleukin 8 (IL8) is a chemokine specifically
involved in neutrophil recruitment and activation of anti-microbial function. It is secreted by a range of cell
types including monocytes, epithelial and endothelial cells, as well as neutrophils. IL8 plays a major role in the
immunopathology of mastitis and uterine disease and genetic polymorphisms within IL8 have been associated
with susceptibility to a number of infectious diseases in both humans and cattle.
Previously, analysis by our group, of a 2.1 kilobase spanning the upstream region of the IL8 gene transcription
start site, uncovered 29 novel polymorphisms in the promoter region of IL8, which segregate into two distinct
blocks, namely IL8 haplotype 1 and IL8 haplotype 2. The aim of this project was to characterize the population
genetic variation present at the IL8 gene, in a range of cattle breeds from both dairy and beef production
systems, as well as to test an association between the IL8 haplotype and both somatic cell count and calving
interval, traits of great agricultural importance for the Irish dairy sector. A functional analysis was then
performed in order to compare the ability of both IL8 promoter haplotypes to drive IL8 expression, both in
vitro, in bovine endometrial epithelial cells and monocyte-derived macrophages; and in vivo, in LPS challenged
Holstein-Friesian (HF) calves. Finally, the role of IL8 dose and IL8 haplotype on neutrophil recruitment and
activation was investigated.
Population genetic analysis of the IL8 haplotype distribution revealed significant differences in frequencies
among breeds. IL8 haplotype 1 was more prevalent in Shorthorn, whereas haplotype 2 was dominant in
Charolais. The Holstein-Friesian and Brahman population showed balanced frequencies of both IL8 haplotypes.
Highly significant associations between the IL8 haplotype and both somatic cell count and calving interval were
identified. The results suggest that artificial selection might have played a role in shaping the genetic structure
of the bovine IL8 gene.
Functional analysis of IL8 expression in endometrial epithelial cells showed significantly higher responsiveness
of the IL8 haplotype 2 promoter, to LPS and TNFα. Additionally, the critical regions in the bovine IL8 promoter
responsible for IL8 regulation, as well as the number of potential transcription factor binding sites in both IL8
haplotypes, were identified. Haplotype-specific IL8 expression was then confirmed in HF calves in vivo,
demonstrating higher IL8 gene and protein expression in whole blood and serum, in animals of IL8 haplotype
2. Interestingly, this haplotype was also associated with higher somatic cell count, suggesting that increased
IL8 expression could be responsible for attracting more neutrophils to the mammary gland. This hypothesis
was tested in vitro, using different doses of recombinant IL8 to drive neutrophil chemotaxis and a dose
dependent effect of recombinant IL8 on neutrophil migration was identified. However, neutrophil incubation
with serum collected from calves of the two IL8 haplotypes produced comparable levels of neutrophil
migration and killing of bacteria, possibly due to low systemic IL8 levels. This suggests that alternative
methods, using a local infection model should be employed in order to characterise neutrophil activation
profiles in the two IL8 haplotypes.

2. In conclusion, functional characterization of the genetic polymorphisms in the bovine IL8 gene has identified
differences in the proinflammatory responsiveness between the two IL8 haplotypes, both in vitro and in vivo.
Additionally, significant associations of the IL8 promoter variants with somatic cell count and calving interval
were also identified. These data strongly suggest that differential IL8 signalling profiles may confer differential
ability to clear mastitis or post-partum infection, which could be exploited via selection for enhanced immunity
in cattle.