Using Genetics to Understand Toll Receptors and Innate Immunity

Bacterial pathogens and inflammatory cytokines play an important role in the innate immune response. Our focus of research is using forward genetics approaches to elucidate the mechanisms of detecting pathogens by mammalian hosts, with particular emphasis on lipopolysacharide (LPS), the most abundant and toxic activator of innate immunity.

In order to protect mammalian hosts from invading microorganisms, the innate system has to respond fast. In this response, it relies on specific receptors that are capable of recognizing conserved patterns unique to microbes and therefore referred to as “pattern recognition receptors” (PRR). One of the PRR classes is the family of Toll-like receptors (TLR), which are evolutionary conserved between insects and mammals. To date, 10 human and 9 mouse TLRs have been reported. Each TLR carries a number of extracellular leucine-rich repeats (LRRs) and intracellular Toll/IL-1 receptor (TIR) domains, which determine the TLR specificity toward different pathogens. The name Toll appeared in the literature in the early 1980-s and was given to one of the mutants in Drosophila genetic screen performed by C.Nusslein-Volhard and E.Wieschaus. The phenotype of Toll (from the German “Toll!” meaning crazy, extravagant, wild) was first described as an essential molecule involved in fly development. Later, Drosophila Toll was shown to control innate immunity in flies by activating a signaling cascade, which protects the flies from fungi. The human homologue of Drosophila Toll (TLR4) was the first characterized mammalian Toll and was shown to induce activation of the NF kB signaling pathway. The function of TLR4 as an endotoxin (LPS, lipopolysacharide) transducer was discovered by positional cloning of mouse Tlr4.

Five years after the cloning of Tlr4, it remains the most studied receptor of all family of Tlrs. However, many details of transducing signal from LPS through Tlr4 are still unclear and therefore are in focus of our research. Despite the ability of TLR4 to recognize LPS directly, the TLR4 LPS interaction is much more complex (See figure) than the sensing of other pathogen ligands as several accessory proteins are required. LPS is first bound to an LBP (LPS binding protein), which functions by transferring LPS monomers to CD14. CD14 was originally cloned as a high-affinity LPS receptor that is a GPI-linked protein, does not have a cytoplasmic domain, and cannot transduce signal from LPS by itself. On the other hand, LBP is not essential for LPS detection in vivo, suggesting that parallel mechanisms for presentation of the monomeric LPS to cell surface must also exist. A small protein MD2 associates with Tlr4 and is required for LPS signaling but its precise role in the signal transduction has yet to be revealed. Several other molecules (CD11b/CD18, heat shock proteins and others) are being recruited to “LPS-cluster” on the cell surface. However, it remains unclear as to how all these molecules interact with each other and, particularly, how Tlr4 interacts with CD14-LPS to transduce the signal. Upon priming with LPS, Tlr4, via its cytoplasmic TIR domain, recruits at least 3 different adapters (MyD88, TIRAP, and Trif) but it is not clear what determines the specificity of such recruitment.

We are using a combination of forward and reverse genetic approaches in order to find new components of LPS signaling pathway as well as to elucidate functional importance of already known components. We are planning to use genetically engineered mutants carrying reporter molecules in fusion with the signaling molecules in order to monitor their interaction with other proteins. In our attempt to elucidate interaction of the Tlr4 with other intracellular components, we are planning to generate transgenic lines with amino acid substitutions in TIR domain. Phenotypic analysis of such mutants will help to assess the roles of the TIR domain containing adapter molecules. The broad variation of LPS response observed in inbred strains of mice encouraged us to extend these studies to the wild-derived mouse strains, which are thought to possess greater genetic diversity than inbred laboratory mice. We are conducting phenotypic analysis of the innate immune response in MSM/ms, SPRET/Ei, MOLF/Ei, CALB/RK, CAST/Ei, CZHECHII/Ei and other strains. As a result of this screening, we found low response to TLR agonists in MSM/Ms and MOLF/Ei mice and currently are mapping new genetic loci. Finding these genes will reveal new components of innate immune system and advance our understanding of how TLR signaling works.