This proposal is in response to the program announcement 'Research on molecular immunology of STDs (ROMIS).' Interferon (IFN)-y induces an effective antichlamydial mechanism in vitro by inducing indoleamine 2,3-dioxygenase (IDO) which depletes tryptophan that is essential for chlamydial growth. Although proinflammatory cytokines produced during infection enhance the amount of IDO induced by IFN, the presence of chronic disease suggests that Chlamydia is evading this response. The goals of this research project are to identify and characterize mechanisms by which Chlamydia evades the effect of IFN. Chlamydia may be affecting IDO regulation directly by interfering with transcriptional activation of the IDO gene by IFNS, or by blocking the effect of proinflammatory cytokines. Chlamydia also may be regulating IDO indirectly by stimulating production of interieukin-10 (IL-10) leading to inhibition of IDO transcription.
Specific aim 1 : Molecular mechanisms of IDO potentiation. IDO regulatory mechanisms will be evaluated using HeLa cells transfected with a green fluorescent protein reporter vector containing the IDO promoter. Identification of IDO promoter regions and DNA-binding proteins will be by DNase I footprinting, EMSA, and super-shift assays. Site-directed mutagenesis will be used to confirm promoter site function.
Specific aim 2 : Direct mechanisms of evasion. The effect of Chlamydia on IDO promoter activity and cytokine receptor expression will be assessed using two-color flow cytometric analysis of infected HeLa cells.
Specific aim 3 : Indirect mechanisms of evasion. The role of IL-10 in inhibition of IDO will be assessed by quantifying IL-10 production by Chlamydia-exposed cells using ELISA, assessing the effect of IL-10 on proinflammatory cytokine production by Chlamydia-exposed cells and by measuring the effect of IL-10 on IDO regulation using the fluorescent IDO promoter reporter. Thus, the aims are to dissect the process of IDO potentiation at the transcriptional level, and to assess the means by which Chlamydia interferes with this process. Accomplishment of the aims will help resolve the long- term objectives of this research project: to determine how Chlamydia evades an otherwise effective immunological response, and to understand regulation of this response in order to overcome Chlamydia's evasive mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI045836-04
Application #
6738186
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Deal, Carolyn D
Project Start
2001-06-01
Project End
2007-04-30
Budget Start
2004-05-01
Budget End
2007-04-30
Support Year
4
Fiscal Year
2004
Total Cost
$140,000
Indirect Cost
Name
Miami University Oxford
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
041065129
City
Oxford
State
OH
Country
United States
Zip Code
45056
Shirey, Kari Ann; Jung, Joo-Yong; Maeder, Gregory S et al. (2006) Upregulation of IFN-gamma receptor expression by proinflammatory cytokines influences IDO activation in epithelial cells. J Interferon Cytokine Res 26:53-62
Shirey, Kari Ann; Carlin, Joseph M (2006) Chlamydiae modulate gamma interferon, interleukin-1 beta, and tumor necrosis factor alpha receptor expression in HeLa cells. Infect Immun 74:2482-6
Robinson, Cory M; Hale, Phillip T; Carlin, Joseph M (2005) The role of IFN-gamma and TNF-alpha-responsive regulatory elements in the synergistic induction of indoleamine dioxygenase. J Interferon Cytokine Res 25:20-30
Robinson, Cory M; Shirey, Kari Ann; Carlin, Joseph M (2003) Synergistic transcriptional activation of indoleamine dioxygenase by IFN-gamma and tumor necrosis factor-alpha. J Interferon Cytokine Res 23:413-21