Toll-like Receptors (TLRs) and NOD-like receptors (NLRs) are critical components of the innate immune system, where they serve as sentinel receptors for the detection of microbes and endogenous danger signals. To date, 10 human and 13 mouse TLRs have been characterized and shown to recognize microbial products such as lipopeptides, flagellin, lipoteichoic acid, GPI anchors, various nucleic acids, and lipopolysaccharides. In many cases, the ectodomains of the TLRs, which consists of a series of Leucine-rich repeats (LRRs), directly bind microbe-derived compounds. The NLRs, which are triggered by various microbial compounds as well as numerous endogenous danger signals, use LRRs as putative ligand binding domains. In addition to mammalian TLRs/NLRs, the purple sea urchin (Strongylocentrotus purpuratus) has a greatly expanded repertoire of pattern recognition receptors, in lieu of an adaptive immune system, with 222 TLRs and 203 NLRs. These animals live in microbe-rich coastal waters, and this expanded receptor repertoire creates an even more diverse set of microbial ligands that they can detect. We propose to use high throughput cloning technology and our proven experience in producing recombinant TLR ectodomains to generate a robust library of microbial product binding proteins, with recombinant TLR and NLR-derived ectodomains (Aim 1). We will harness these proteins to produce MAbs to the mammalian TLR/NLRs (Aim 2). Moreover, the microbial recognition capabilities of novel TLR ectodomains will be characterized with PAMP arrays (Aim 3). Finally, these TLR ectodomains will be developed for use in diagnostics, for detection of microbes, microbial products, and endogenous danger signals in biological fluids (Aim 4) and potentially as antimicrobial therapies. This project will deliver to the scientific community recombinant TLR and NLR ectodomains, tools for rapidly expressing TLR/NLR ectodomains in a variety of systems, MAbs for mammalian TLRs and NLRs, arrayed libraries of microbial products and microbes, and will further develop these families of pattern recognition receptors as tools for clinical applications. Our plan for distributing these reagents to colleagues (Aim 5) is based upon our documented extensive history of sharing reagents within the TLR field.

Public Health Relevance

The innate immune response plays a critical role in the pathogenesis of many human diseases.
We aim to develop microbe-sensing innate immune receptors as probes to be used for further studies of innate immunity in the laboratory, for development of novel methods for detecting microbes in clinical samples, and as possible therapies for diseases caused by hyper-stimulation of innate immune response.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Resource-Related Research Projects--Cooperative Agreements (U24)
Project #
5U24AI082663-05
Application #
8448020
Study Section
Special Emphasis Panel (ZAI1-KE-I (J2))
Program Officer
Prabhudas, Mercy R
Project Start
2009-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$528,030
Indirect Cost
$207,039
Name
University of Massachusetts Medical School Worcester
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Gupta, Rahul; Ghosh, Shubhendu; Monks, Brian et al. (2014) RNA and β-hemolysin of group B Streptococcus induce interleukin-1β (IL-1β) by activating NLRP3 inflammasomes in mouse macrophages. J Biol Chem 289:13701-5
Meinander, Annika; Runchel, Christopher; Tenev, Tencho et al. (2012) Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling. EMBO J 31:2770-83