Using Wild Mice to Uncover the Genes Involved in Innate Immunity
The laboratory is using genetic analysis to better understand signaling pathways that lead to innate immune responses. To facilitate this approach we have elected to focus on the differences between wild-derived and classical laboratory mice. This approach takes advantage of the fact that the wild-derived strain we study, MOLF/Ei which shares only 11% of its genome with laboratory mice as compared to 90% of shared genome between classical inbred mice (Figure 1). Genetic analysis of innate responses in MOLF allowed us not only to reveal new components of the signaling pathways but also to identify interactions between different genes (i.e. epistatic interactions) that contribute to unique signature of immune responses in wild-derived mice. Several examples of gene interactions (also known as epistasis) revealed in human immune dysfunctions, such as autoimmunity and AIDS, illustrate the fundamental importance of this phenomenon in human disease and support a compelling necessity for uncovering gene-gene interactions in addition to the traditional approach of assigning biological functions to individual genes. Therefore, finding adequate genetic models of epistasis is instrumental in understanding these complex disorders.
Figure 1. This illustration shows the strategy we are using to identify novel genes.
Novel Phenotypes Identified in MOLF/Ei Mice
As homologous of human genetics, classical inbred strains of mice have been indispensable in analysis of simple, monogenic traits conferred by genes making significant “top-down” contributions to the network. However, the majority of the classical inbred strains were separated from a common ancestor around 120 years ago and therefore do not reach the level of diversity observed in humans. In contrast, wild-derived mice diverged from the common ancestor of classical strains more than one million years ago and therefore accumulated a lot of phenotypic differences. Most importantly, wild-derived mice offer the opportunity to observe novel phenotypes that have arisen in the evolutionarily driven process of sub-speciation. Finally, comparison of the genomes of wild-derived and laboratory mice might reveal differences in even relatively simple networks of genes. The figure below provides an example of recently identified differences in innate immune responses between MOLF and C57BL/6J mice and shows that whereas LPS-activated MOLF macrophages have significantly higher levels of IL-6 and IL-10, their response to CpG is very weak as compared to B6 macrophages.
Figure 2. One strain – two phenotypes: MOLF marophages are hyper-responsive to LPS but hypo-responsive to CpG: accumulation of mRNA (Northern blot) for TNF, IL-6, and IL-10 in activated C57BL/6 (B6) and MOLF/Ei macrophages. Note the high levels of RNA in the LPS-treated samples.
Both traits have been recently mapped to several genomic loci. By using levels of IL-6 protein as a phenotypic trait, we mapped the hyper-inflammatory responses to two main loci, termed Wild Derived Hyper-Response 1 and 2 (Why1 and Why2), and ultimately provided evidence that Irak1bp1 (a.k.a. Simpl) is the gene encoded by Why2 (where the MOLF allele is associated with a decrease in IL-6 production) and reported a critical role of SIMPL in preventing dangerous overproduction of pro-inflammatory cytokines in wild-derived but not laboratory inbred mice. Furthermore, SIMPL appears to be specifically up-regulated in macrophages of wild-derived but not classical inbred mice. Our latest data suggest that Why1 encodes IRAK2 that appears to play significantly more important pro-inflammatory role in MOLF than in B6 mice. Finally, we were able to identify several epistatic interactions between the loci that contain genes involved in innate immune signaling. We are currently trying to identify these genes and understand the mechanism by which they influence the immune response.
Figure 3. The figure at the left illustrates a simplified TLR4-mediated pathway with highlighted genes (yellow) contained within the loci, for which strong epistatic interaction have been identified.