Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality.

Marie-Christine Bartens,Graham J Etherington,Angela Holder,Sam Willcocks,Dirk Werling,Amanda J Gibson,Federica Di Palma

doi:10.3389/fimmu.2021.764390

Abstract

Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses containing H326Q were found to be partially recovered compared to human TLR2. The creation of human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific response to mycobacterial antigens, but furthermore, that there are clear differences between the response seen in different cattle breeds, which may contribute to their enhanced or reduced susceptibility to mycobacterial infection.

Highlights

Recognition of pathogens and activation of the innate immune response is critical in determining the outcome of infection
We report on the species- and breed- specific phenotypic consequence of this mutation in response to canonical TLR2 ligands and in the context of challenge with M. tuberculosis and M. bovis
For M. bovis culture only, growth media was supplemented with sodium pyruvate; for M. tuberculosis culture only, media was supplemented with glycerol (Sigma-Aldrich, UK)

Summary

Introduction

Recognition of pathogens and activation of the innate immune response is critical in determining the outcome of infection. One of the major groups of PRRs are the toll-like receptors (TLR), ten of which have been discovered common to both bovine and human immune systems [2,3,4,5], and are expressed on myeloid antigen presenting cells such as macrophages (MØ). Each of these TLR has evolved to recognise specific pathogen associated molecular patterns (PAMP) – and docking of these ligands to the extracellular domain (ECD) of the TLR triggers an intracellular signalling cascade [6]. While the amino acid sequence of the ECD varies considerably between species [9], the TIR domain is highly conserved [7, 9]

Methods

Results

Conclusion