Toll-like receptors play a critical role in the initiation of the innate and adaptive immune responses. Members of the TLR family recognize conserved microbial structures and activate signaling pathways that result in immune responses against microbial infections. All TLRs activate common pathways to induce a core set of stereotyped responses, such as inflammation. However, individual TLRs can also induce immune responses that are tailored to a given microbial infection. The mechanisms and components of these varied responses are poorly understood. Given the importance of TLRs in host defense, dissection of these pathways is key to the rational design of immunomodulators and adjuvants. To address the complexity of the TLR signaling network, it is imperative to put in place technologies enabling systematic examination of the signal transduction. ATTAGENE Inc. has developed a reversible genetic approach that affords screening expression libraries of tens of thousands of cDNAs to identify signaling intermediates. We used this methodology to identify a number of novel components of the pathways that mediate interleukin-1-inducible activation of the transcription factor NF-kB. Our studies indicate that the reversible genetic approach offers a highly versatile tool for a systematic, genome-wide identification of signal transduction. This comprehensive approach does not rely on preconceived notions and it has built-in procedures that eliminate false-positive background. In this study, we will adapt the reversible genetic approach to systematic identification of components of the TLR signaling network. Our objectives are (1) to identify the components of signal transduction that link individual members of the TLR family with activation of the transcription factor NF-kB; (2) to annotate those components as positive/negative and differential/common intermediates; (3) to examine biological functions of the identified mediators in innate immune responses in vitro; and (4) to create knock-out animal models in order to assess the identified mediators as potential targets to modulate the innate and adaptive immune responses to different pathogens, including Listeria monocytogenes and Salmonella typhimurium. Successful implementation of the proposed plan should provide comprehensive knowledge of molecular mechanisms controlling immune responses to pathogens, thus greatly facilitating the development of highly specific immunomodulators and adjuvants. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI061360-02
Application #
6921353
Study Section
Special Emphasis Panel (ZAI1-LR-M (M1))
Program Officer
Winter, David B
Project Start
2004-07-15
Project End
2009-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
2
Fiscal Year
2005
Total Cost
$2,242,952
Indirect Cost
Name
Attagene, Inc.
Department
Type
DUNS #
147164193
City
Research Triangle Park
State
NC
Country
United States
Zip Code
27516
Martin, Matthew T; Dix, David J; Judson, Richard S et al. (2010) Impact of environmental chemicals on key transcription regulators and correlation to toxicity end points within EPA's ToxCast program. Chem Res Toxicol 23:578-90
Hargreaves, Diana C; Horng, Tiffany; Medzhitov, Ruslan (2009) Control of inducible gene expression by signal-dependent transcriptional elongation. Cell 138:129-45
Kagan, Jonathan C; Su, Tian; Horng, Tiffany et al. (2008) TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat Immunol 9:361-8
Romanov, Sergei; Medvedev, Alexander; Gambarian, Maria et al. (2008) Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nat Methods 5:253-60
Diatchenko, Luda; Romanov, Sergei; Malinina, Inga et al. (2005) Identification of novel mediators of NF-kappaB through genome-wide survey of monocyte adherence-induced genes. J Leukoc Biol 78:1366-77