This proposal is in response to the program announcement "Research on molecular immunology of STDs (ROMIS)". Interferon (IFN)-gamma 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 interleukin-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 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 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 and otherwise effective immunologic response, and to understand regulation of this response in order to overcome Chlamydia's evasive mechanisms.