Depression is a major disease burden worldwide and, despite its prevalence and socioeconomic costs, around 30% of patients do not respond to currently available treatments. Inflammation is increasingly associated with, not only depressive illness but also resistance to existing therapies. This highlights the need for investigation of the mechanisms of neuro-inflammation, particularly in the context of peripheral inflammatory stimuli. Specifically, the chemokine molecular family is increasingly associated with human depressive illness, and neuro-inflammation and behavioural change in rodent models, making this an attractive molecular family for study. This thesis describes research aimed at investigating the association of these molecules with human depression and analysis of their role in an animal model of peripherally stimulated neuro-inflammation, the Aldara model of psoriasis-like inflammation. Systematic review and meta-analysis of the human biomarker literature using a random effects, inverse variance model revealed that a number of chemokines (CCL2, CCL3, CCL4, CCL11, CXCL4, CXCL7, CXCL8) are significantly associated with depressive illness in a human population. However this work revealed that there are a number of limitations of the human literature primarily associated with the methodological challenges of studies in human populations and confounding factors. Alongside this work, the Aldara model, which utilises the toll-like receptor 7 (TLR7) ligand imiquimod (IMQ), was investigated as a tool for studying neuroinflammation. Initial time-course investigation revealed that significant chemokine and cytokine transcriptional alterations occur within four hours at the local site of cutaneous treatment, the peripheral tissues and the brain. In addition, protein quantification in the brain confirmed that many of these transcriptional responses are translated to protein. Interestingly, it was shown that the brain response was temporally distinct from that of the peripheral tissues, and that in general brain responses were induced slightly more slowly and persisted for a longer period of time than those in the periphery. Investigation of Iba1+ (microglia/monocytes), GFAP+ (astrocytes) and CD3+ (T-cells) cells within the brain revealed significant changes in the microglial and T-cell populations, which were consistent with microgliosis and T-cell recruitment to the brain parenchyma. Changes in astrocyte populations were more equivocal although there was evidence of astrogliosis. Mechanistic investigations into responses to the Aldara model in inflammatory chemokine receptor (iCCR) KO mice did not reveal significant alterations in chemokine and cytokine transcription or in microglial responses to cutaneous Aldara treatment in the absence of the iCCRs (CCR1, CCR2, CCR3 and CCR5), but there did appear to be evidence of reduced CD3+ T-cell recruitment. In contrast, investigations in type I interferon receptor (IFNAR) KO mice identified a clear role for type I IFN signalling through IFNAR in the induction of chemokine and cytokine gene expression in the brain, and associated changes in Iba1+ microglial and CD3+ T-cell populations in response to cutaneous Aldara treatment. Mass spectrometric analysis of IMQ, the main active ingredient of Aldara, revealed that within four hours it enters both the circulation and the brain. The finding of IMQ within the brain parenchyma suggests that, while it is not an appropriate tool for studying peripheral-central immune crosstalk, it is a useful non-invasive model of TLR7 mediated neuroinflammation. These data provide compelling evidence of a role for chemokines in human depression and in neuro-inflammation, although the precise actions of this family of molecules remain unclear. In addition, building on previous work, the Aldara model appears to be a suitable tool for the study of neuro-inflammation, particularly interferon-driven immune responses, but is less appropriate for studying peripherally driven CNS immune reactions. Further work into the specific role of chemokines and associated cellular populations will hopefully provide additional insight into how CNS immune reactions are co-ordinated.

Nematode infection is one of the major causes of disease in young sheep. Selective breeding of genetically nematode resistant sheep is an alternative method for controlling the nematode infection. This process could be simplified if loci that account for nematode resistance can be identified. MHC is one of the candidates and several studies have confirmed the association between MHC alleles and nematode resistance. The aim of this study is to establish the role of MHC class II genes in nematode resistance in Texel sheep. Thus, it can help endorse the usefulness of the MHC class II genes as a genetic marker of nematode resistance and extend the knowledge of the mechanism of resistance against nematodes. This study has been focused on three main areas; 1) description of MHC class II gene diversity, 2) description of haplotype and linkage disequilibrium pattern at MHC class II genes and 3) the association of MHC class II genes and nematode resistance. Sequence-based typing was applied to characterise MHC class II allelic diversity in 235 Texel lambs. The haplotype and linkage disequilibrium patterns were deduced from pedigree information. Finally, the association between MHC class II haplotypes and nematode resistance (FEC and IgE activity against L3) were investigated using a MIXED model approach. MHC class IIa genes were diverse in Texel, consistent with previous studies reported in sheep. The most polymorphic locus among MHC class IIa genes was DRB1. A total of 21 distinct DR-DQ haplotypes were obtained and strong linkage disequilibrium exhibited between DR-DQ genes. There were also statistically significant associations of specific haplotypes and nematode resistance in this population. The work in this thesis confirms the likely importance of MHC genes in regulating resistance against gastrointestinal nematodes, thus supporting the use of MHC as a genetic marker of nematode resistance in selective breeding. Sequence-based typing system for MHC class IIa has been established in this study.

Teladorsagia circumcincta is a common abomasal parasite of sheep in temperate regions and is one of the major causes of parasitic gastroenteritis (PGE) in growing lambs. Control of infection is achieved using anthelmintic drugs; however, this practice is rapidly becoming unsustainable due to widespread anthelmintic resistance within the T. circumcincta population. Sheep can acquire protective immunity against this parasite; immunity involves local and systemic antibodies and immune cells which can impair worm growth and fecundity and lead to expulsion of the parasites from the abomasum. Vaccination against this parasite is therefore a feasible option of control. A recent study showed that a recombinant vaccine cocktail containing 8 T. circumcincta antigens significantly reduced the faecal egg count and worm burdens of immunised sheep, compared to an adjuvant-only control group. However, the recombinant antigens induced a suboptimal antibody response to the recombinant antigens. This suggests that differencies between the native antigens and their recombinant versions may exist, possibly due to variations in structure and/or post-translational modifications (PTMs). The main aim of this work was to use a novel expression system, the free living nematode Caenorhabditis elegans, to generate alternative recombinant versions of two of the T. circumcincta antigens used in the 8-antigen vaccine, cathepsin F (Tci-CF-1) and monocyte Migration Inhibitory Factor (Tci-MIF-1). This was achieved by micro-injection of C. elegans worms with plasmids containing the cDNA sequences of Tci-cf-1 and Tci-mif-1 followed by purification of recombinant Tci-CF-1 and Tci-MIF-1 from the transformed worms. Immune recognition, enzyme activity and biological effects on sheep cells of the recombinant antigens were characterised. The results show that immunisation-induced antibodies bind to native Tci-CF-1 purified from T. circumcincta L4 ES, whereas infection-induced antibodies were unable to bind the recombinant Tci-CF-1 versions. Further characterisation of recombinant Tci-CF-1 versions expressed in C. elegans or Pichia pastoris showed that in order to be enzymically active, these proteins require cleavage of the pro-peptide by an exogenous enzyme and that some differences were present in the glycosylation of the recombinant versions and native Tci-CF-1. Characterisation of both recombinant Tci-MIF-1 versions showed that although both are enzymically active, neither showed a significant inhibitory effect on the migration of sheep monocytes or on the activation of sheep macrophages in vitro compared to unstimulated controls. It is speculated that Tci-MIF-1 may be involved in T. circumcincta larval development rather than host immunosuppression.

Regulatory T cells (Treg) are enriched in tumour tissue relative to other compartments. Anti-tumour immunity is promoted through their depletion. It is hypothesised that Treg are recruited to human colorectal cancer (CRC) via a specific combination of chemokine receptors and integrins, blockade of which reduces tumour Treg recruitment, ameliorating the anti-tumour immune response. A systematic examination was conducted of receptors expressed by CRC-isolated Treg and the cognate ligands expressed by CRC. The effects of receptor inhibition were tested in murine models of colorectal cancer. Human CRC-infiltrating Treg exhibit a specific chemokine receptor signature, expressing significantly higher levels of CCR5 than conventional T cells. CRC expresses the ligands for CCR5 at significantly higher levels than distal tissue. Isolated Treg migrated towards CCR5 ligands in vitro and suppressed allogeneic T cell proliferation. CCR5 inhibition in murine models of CRC led to delayed tumour growth but had no effect on tumour Treg infiltration compared with vehicle control. CCR5 inhibition is unlikely to provide any significant reduction in the infiltration of Treg into human CRC. Given the effects CCR5 inhibition had on tumour growth, CCR5 antagonists command further investigation into their potential role as novel therapeutic agents in the treatment armoury against human CRC.