The Role of Bovine Cathelicidins in Host Defence

<p>Cattle are constantly exposed to environmental pathogens and are susceptible to a number of diseases which cause significant economic loss or animal welfare concerns. The mucosal surface is a key barrier to infection and a greater understanding of bovine mucosal immunology may lead to improved disease management strategies. The cathelicidins are a family of host defence proteins which may play an important role in this defensive barrier.  The cathelicidins are composed of a globular cathelin-like domain (CLD) and a C-terminal antimicrobial peptide (AMP) domain. The CLD is highly conserved across all species in which cathelicidins are found, yet the role of the CLD during infection has not been firmly established. The first aim of this thesis was to produce functional recombinant bovine CLD as a precursor to further experiments. However, the recombinant protein bound less lipopolysaccharide, was unable to agglutinate microbes, and was unable to permeabilise neutrophil membranes when compared to the activity of a native CLD preparation. Although further studies were not carried out with recombinant CLD, these results demonstrated that cell death induced by the native CLD and the agglutination of microbes potentially contribute to a broad anti-inflammatory role for the CLD during infection.  In contrast to humans and mice where only one cathelicidin isoform is expressed, bovine express seven cathelicidins, with variable AMP domains. Therefore the second aim of this thesis was to profile the effect of bovine cathelicidin AMPs on neutrophil function. The bovine AMPs were able to modify a number of activities. Migration and reactive oxygen species (ROS) production were enhanced by several peptides while ROS production was inhibited by others. When investigated in further detail, linear Bac1 (bactenecin), Bac5 and BMAP-34 (bovine myeloid antimicrobial peptide) were able to dose-dependently induce or inhibit several key neutrophil functions including migration, degranulation, respiratory burst and phagocytosis, indicating significant roles in differential modulation of immune responses. In particular, Bac5 was able to differentially modify neutrophil respiratory burst without significant disruption to cellular homeostasis, which suggested Bac5 was acting via an intracellular mechanism.  The third aim of this thesis was to investigate the mechanism by which Bac5 modulated neutrophil function. The results demonstrated the ability of Bac5 to be internalised by neutrophils and that Bac5 inhibition of p47phox binding to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a possible explanation for the differential induction and inhibition of extracellular ROS production. β-actin was identified as a major intracellular target for Bac5 and is consistent with the ability of Bac5 to modulate multiple neutrophil functions.  In summary, this thesis demonstrates that the bovine cathelicidins have multiple roles in host defence. The conserved CLD appears to have an anti-inflammatory role through an as yet undefined mechanism. The variable AMPs together have multifaceted roles which may act in concert to promote elimination of pathogens and regulate excessive detrimental neutrophil activity. The combined effect of these roles will facilitate clearance of pathogens during infection and aid in the resolution of the innate inflammatory response at mucosal surfaces.</p>