This was a collaborative grant between four academic partners and funded jointly by BBSRC, Scottish Executive ERAD and Pfizer Animal Health, under a Genesis Faraday (now KTN Biosciencs) earmarked project. The partners were: Prof David Haig, now at University of Nottingham (UoN), previously Moredun Research Institute (MRI) (with Dr George Russsell, as Co-PI at MRI), Drs Tracey Coffey and Jayne Hope, at the Institute for Animal Health (IAH), Prof Dirk Werling at Royal Veterinary College ((RVC) and Prof Liz Glass, at The Roslin Institute & R(D)SVS, University of Edinburgh (RI). Thus this grant is joint with BBD5240591.The Consortium placed particular emphasis on the pathogen-recognition receptors (PRR), Toll-like receptors (TLR), in the ruminant livestock species, cattle and sheep. These receptors are expressed on specialised immune cells, dendritic cells (DC), that are poised to detect pathogen invasions, and are key to initiating and maintaining immune responses against pathogens. Once pathogens are detected by TLR, signals are delivered to the DC which then set off a cascade of inflammatory and immune pathways.
We focused on the genetic polymorphisms and the evolutionary selection that livestock specific pathogens place on ruminant TLR ligand binding domains. The research sought to understand the influence that these sequence variants could have on disease resistance traits in livestock. Differing expression profiles of TLRs on cattle and sheep DC derived ex vivo as well as bovine monocytes, and monocyte-derived DCs and macrophages were observed for the various subsets within DCs obtained from afferent lymph, suggesting important functional consequences. The results gained in this project have important consequences for vaccine design as well as breeding for disease resistance.
By mapping the positions of genes for TLRs and their down-stream signalling molecules, we identified a number of functional and positional candidates for disease resistance traits for several species. Our detailed analysis of TLR sequence variation both within ruminant breeds and between species suggested single nucleotide polymorphisms (SNPs)) may reflect co-evolution of ruminants with pathogens encountered in distinct geographic and micro-organism environments. Non-synonymous (ns) SNPs (those that changed the amino acid) which were in highly positively selected sites mayhave the greatest effect on the function of the molecule. We tested whether these SNPs were associated with disease resistance and susceptibility and found that such SNPs in several TLRs are significantly associated with immunity.
Activation of TLR5 may be important in relation to pathogens invading mucosal sites, such as Salmonella species, which remain a major issue for food security in relation to livestock species. Sequencing revealed that four ns SNPs cause premature stop codons withinTLR5, and a further six ns SNPs are within positively selected sites, three of which are within the intracellular signalling domain. These SNPs are surprisingly frequent, and we are investigating their functional significance in in vitro and in vivo assays in cattle and sheep. This information may have functional implications and may reveal new insights into host-pathogen interactions, as despite close similarity in the sequences for cattle and sheep TLRs these species respond very differently to many pathogens.
In summary (1) cattle TLR2 and TLR5 (at least) have multiple breed-specific variants, suggesting ruminant PRR have co-evolved with pathogens to which they have been exposed, so that the design of optimum ruminant TLR-agonist combinations should account for both species and breed variants; (2) soluble, secreted forms of TLRs may act as decoys to block pathogen engagement with membrane-bound receptors; (3) allelic variants of TLRs and their down-stream signalling molecules may genetically influence disease resistance against multiple pathogens in a range of host species.
In cattle and sheep all 1-10 TLRs together with genes the important TLR signalling molecules for have been mapped, isolated, and all 10 sheep and cattle TLRs cloned and sequenced, and relevant assays – qRT-PCR - developed for subsequent use.
Detailed phylogenetic and SNP polymorphism studies revealed that there has been evolutionary pressure (potentially from pathogens) on putative ligand binding domains. The implication is that appropriate adjuvant ligands need to be carefully tailored to host species’ variation.
A5-species comparative genomics study has identified “hot” candidate genes for disease resistance, and the resulting open access publication has been highly accessed.
In vitro HEK cell assays have explored ligand-receptor interactions. A number of bovine TLRs have been expressed in HEK cells including TLR2, TLR4 and TLR5. In addition as many TLRs are heterodimers constructs were developed that would allow combined expression of TLR2 together with TLR6, dectin-1 and TLR10. Using a variety of down-stream functional assays such as NFkB signalling and cytokine production, clear species differences were observed.
TLR expression has been determined on bovine and ovine Dendritic cells (DC) obtained ex vivo from afferent lymph. In general afferent DC from sheep and cattle expressed TLR1-10 to apparently similar degrees, although some differences were observed in distinct subsets, with potentially functional consequences.
Anti-bovine TLR2 antibodies have been identified, validated and shown to cross-react with sheep.