Inflammation can be beneficial for a normal immune response to microbial pathogens. However, prolonged inflammation can promote tissue damage during infection and has been closely linked to a diverse range of diseases, including cancer, atherosclerosis, and several inflammatory autoimmune diseases. Although a number of anti-inflammatory drugs are available, none of them are considered to be ideal for a variety of reasons, including insufficient target specificity. Therefore, new strategies are needed for the development of selective inhibitors of pro-inflammatory genes and proteins. One major limitation in pursuing pharmaceuticals that inhibit the transcription of specific pro-inflammatory genes is that our understanding of the molecular mechanisms responsible for selective gene regulation is surprising limited. Signaling pathways such as the NF-?B and AP-1 pathways are known to contribute to the activation of many pro-inflammatory genes. However, because of their broad functions, these pathways are not appropriate targets for the selective modulation of individual genes. Because it has proved to be difficult to uncover the mechanisms of selective regulation through the use of conventional experimental strategies, we have begun to attack the selectivity question using a new strategy that should lead to a much broader appreciation of this issue, with the possibility of identifying therapeutic lead compounds. Specifically, we are generating macrophage cell lines from mice in which fluorescent protein reporter genes are regulated by cytokine gene control regions in their native chromatin environment. High-throughput screens will then be performed to identify small molecules that differentially alter the expression of key cytokine genes, including the genes encoding IL-12 p40, IL-12 p35, IL-23 p19, and IL-10. The rationale for inserting fluorescent protein reporter genes into a native chromatin environment is that our past studies have revealed that conventional promoter-reporter plasmids, often used for high-throughput screens, fail to assemble into physiologically relevant chromatin structures upon stable transfection. Furthermore, the fluorescent protein reporter assay is preferable to an ELISA assay that monitors endogenous cytokine secretion because the ELISA is susceptible to misleading effects on cytokine translation, processing, and secretion. By testing small-molecule libraries in which the molecular targets are known, we hope to gain unprecedented insight into the signaling pathways that contribute to selective gene regulation. The signaling pathways identified will then be examined in greater depth to elucidate selectivity mechanisms. Larger libraries of compounds whose targets are unknown will also be screened to gain further insight into the potential for selective regulation, with the possibility of identifying therapeutic lead compounds.

Public Health Relevance

The objective of the proposed research is to explore the feasibility of a novel high-throughput screening strategy that may lead to the discovery of small molecules capable of modulating the expression of proteins involved in inflammation. The small molecules identified will facilitate our ongoing studies of the signaling pathways that regulate the selective expression of inflammatory genes. Furthermore, the proposed screens may lead to the discovery of therapeutic lead compounds for the treatment of diseases associated with aberrant inflammation, including atherosclerosis, cancer, and a number of inflammatory autoimmune disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI076921-02
Application #
7842635
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Leitner, Wolfgang W
Project Start
2009-05-15
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
2
Fiscal Year
2010
Total Cost
$182,430
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Smale, Stephen T (2012) Transcriptional regulation in the innate immune system. Curr Opin Immunol 24:51-7
Smale, Stephen T (2011) Hierarchies of NF-ýýB target-gene regulation. Nat Immunol 12:689-94
Kobayashi, Taku; Matsuoka, Katsuyoshi; Sheikh, Shehzad Z et al. (2011) NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity. J Immunol 186:4649-55
Smale, Stephen T (2010) Selective transcription in response to an inflammatory stimulus. Cell 140:833-44
Sen, Ranjan; Smale, Stephen T (2010) Selectivity of the NF-{kappa}B response. Cold Spring Harb Perspect Biol 2:a000257