Asthma and allergies are complex diseases with a strong familial component suggestive of a genetic predisposition. Genetic variants in the human TLR2 locus, environmental factors and their interactions are strongly associated with susceptibility to asthma in children exposed to microbial products. However, genetic heterogeneity within outbred populations prevents association studies from revealing which variants affect disease susceptibility. Our goal is to characterize the mechanism(s) underlying the impact of natural variants on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model gene is human TLR2, a key effector in innate immunity and modulator of asthma and allergy. We showed that: (1) human TLR2 variant rs4696480 is strongly associated with protection against asthma and allergy;(2) the human TLR2 locus includes other polymorphisms linked to rs4696480. We are convinced that, ultimately, human SNPs need to be studied in vivo within a physiologic genomic context. Here we wish to test the hypothesis that the interplay between human TLR2 genetic variation and environmental exposures that results in asthma susceptibility can be effectively modeled and dissected in BAC transgenic (TG) mice carrying asthma/allergy-associated human TLR2 haplotypes. More specifically, we propose:
Aim 1 : Generation, validation and analysis of murine Tlr2-deficient BAC TG lines carrying human TLR2 locus haplotypes involved in gene-environment interactions that affect asthma susceptibility. This work will capitalize on our analysis of variation in TLR2 and our skills in BAC recombineering, and will lead the generation of TG lines exhibiting faithful tissue-specific and copy number-dependent expression of human TLR2, but lacking murine Tlr2.
Aim 2 : Identification of the mechanisms underlying gene-environment interactions between hTLR2 variants and exposure to microbial products, and assessment of their impact on asthma/allergy susceptibility. This work will rely on in vitro and ex vivo models in order to determine whether variation acts on expression and functions of hTLR2 at transcriptional and/or post-transcriptional level. The murine tlr2-deficient background will be ideal for the characterization of human TLR2-dependent phenotypes. By providing a controlled genetic background, the TG model we propose will define the haplotypes involved in TLR2-dependent phenotypes and their modifying effects on TLR2 expression and/or function, paving the way for strategies aimed at neutralizing the effects of genetic TLR2/environment impact on asthma and allergies.
The overall goal of our work is to characterize the mechanisms underlying the impact of natural genetic variation and environmental factors on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model is TLR2, a receptor involved in innate immunity whose genetic variation interacting with microbial products affects the susceptibility to asthma and allergy. We have found that a TLR2 polymorphism, rs4696480, is strongly associated with susceptibility to asthma and allergy in children exposed to microbial molecules. We have performed an extensive analysis of TLR2 variation and identified polymorphisms in linkage with rs4696480. To elucidate the mechanisms involved in the interaction between human TLR2 genetic variation and exposure to microbial products that result in asthma/allergy susceptibility powerful and physiologic approaches are required. Ultimately, polymorphisms need to be studied in vivo within the physiologic genomic context. In this proposal we wish to explore the hypothesis that (1) human TLR2 polymorphisms associated with asthma susceptibility phenotypes are sufficient to induce appreciable alterations of TLR2 expression and/or function in defined and controlled environmental conditions, and (2) the interactions between natural genetic TLR2 variation and environment that affect human TLR2 regulation/function can be effectively modeled and dissected in mouse models carrying defined human TLR2 haplotypes on a murine Tlr2-deficient background. The fact that haplotype/environment patterns affecting TLR2 expression/function are assessed against controlled genetic background, ensures that this in vivo model will allow us to determine which polymorphisms are necessary and sufficient to induce altered TLR2 expression and/or function activity and therefore leading to the molecular mechanisms responsible for TLR2.
