Divergent <i>cis-</i> regulatory haplotypes at <i>Tlr2</i> are associated with immune responsiveness

Positive and balancing selection on pattern recognition receptors (PRRs) is widely thought to target ligand-binding domains and affect the specificity of recognition of different pathogens. Alternatively, positive/balancing selection on PRRs could affect general responsiveness by targeting for example signaling domains or cis-regulatory variation. Studies of a wild rodent (the bank vole, Clethrionomys glareolus) have shown that Tlr2-a lipoprotein-binding PRR-is highly polymorphic with divergent haplotypes and signatures of balancing selection, and that Tlr2 genotype is associated with susceptibility to Borrelia afzelii infection in the wild. To investigate what aspect of Tlr2 function has been under selection, we here perform integrated population genetic and functional analyses. Ex vivo infection experiments show that the protective Tlr2 haplotype produces a stronger proinflammatory response to B. afzelii compared to the haplotype associated with susceptibility. Tlr2 genotype has a similar, albeit not statistically significant, effect on responsiveness to the phylogenetically distant pathogen Streptococcus pyogenes. We find that the strongest signature of balancing selection is 4.6 kb upstream of the Tlr2 coding sequence, near a putative enhancer, and that Tlr2 exhibits allele-specific expression such that the protective haplotype is more expressed. Collectively these results indicate that balancing selection has primarily acted on cis-regulatory variation affecting the general responsiveness via Tlr2-signaling rather than on polymorphisms affecting Tlr2 ligand-binding specificity.