Abstract
Resistance and susceptibility to infectious diseases are well-documented in a variety of host species. While environmental modifiers and pathogen virulence influence disease severity following infection, a major contributor is the genotype of the host. Johne’s disease is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP) and manifests as a chronic inflammatory bowel disease in ruminants such as cattle, deer, goats and sheep. The Disease Research Laboratory (DRL) has previously identified breed lines of deer that express either a resistant or susceptible phenotype for Johne’s disease.
The aim of this PhD was to investigate immunological differences between red deer that have polarised genotypes for resistance (R) or susceptibility (S) to Johne’s disease. The macrophage was selected as the representative leukocyte to assess the immune response of these animals, due to its central role in formulating an immune response to MAP. Monocyte-derived macrophages were obtained from blood samples of animals, infected with MAP in vitro and analysed for the expression of candidate genes (iNOS, IL-1α, TNF-α, IL-23p19, IL-10 and IL-12p35) by quantitative PCR. Genetically susceptible animals, with no reactivity to MAP at the time of sampling, exhibited an inflammatory transcriptional profile in response to MAP infection, characterised by significantly higher upregulation of the inflammatory markers (iNOS, IL-1α, TNF-α and IL-23p19), compared to genetically resistant animals.
Analysis of the whole transcriptome of macrophages from two naïve R and two naïve S animals by the Illumina HiSeq 2000 next-generation sequencing system was also undertaken. This confirmed the trends seen from the candidate gene expression study where macrophages from S animals upregulated more genes than macrophages from R animals in response to MAP. Gene expression profiles in S animals were biased towards an M1-inflammatory transcriptional profile in response to MAP while macrophages from R animals showed a more balanced M1-inflammatory/M2-regulatory macrophage transcriptional signature. This was characterised by a greater increase in molecules associated with type 1 interferon signalling and neutrophil recruitment in S animals compared with R animals. The amplified inflammatory transcriptional profile of macrophages from susceptible animals may reflect less efficient clearance of MAP bacilli and ultimately cause an exacerbation of Johne’s disease symptoms. By contrast, the more controlled upregulation of inflammatory pathways in macrophages from resistant animals may result in effective containment of infection by the innate immune system and the establishment of protective adaptive immunity.
These data affirm the value of both transcriptomic technologies and candidate gene approaches to enable the identification of robust, reliable transcriptional markers of resistance or susceptibility to Johne’s disease. Work of this kind has never been performed before and has produced initial data that may advance the understanding of the biology of Johne’s disease and of mycobacterial disease in general, which will ultimately inform the design of more effective vaccines, improved diagnostics and treatments for these conditions. Further, this study has highlighted that heritability of resistance or susceptibility to Johne’s disease is polygenic in nature, involving a complex interplay of innate and adaptive immune systems rather than single genes.