Tuberculosis remains a major cause of death, in part because of lack of an effective vaccine. Dendritic cells (DC) are critical in priming T cell responses through production of IL-12 and IL-23, which favor expansion of IFN-g-producing Th1 cells and IL-17-producing Th17 cells, respectively. The early secreted antigenic target of 6-kD (ESAT-6) protein of Mycobacterium tuberculosis (M. tb) is a candidate vaccine antigen, but our preliminary data show that ESAT-6 inhibits IL-12 and enhances IL-23 secretion by stimulated DC, by reducing p35 and enhancing p19 transcription. ESAT-6-treated DC favors T-cell production of IL-17 over IFN-g, in response to M. tb. Based on these data, we hypothesize that ESAT-6 affects binding of critical transcription factors to the p35 and p19 promoters, and differentially regulates production of IL-12 and IL-23 by DC. To identify the molecular mechanisms underlying these important effects, we propose to: 1. Identify ESAT-6 response elements (EREs) in the promoters of p35 and p19. We will first determine if treatment of immature DC with ESAT-6 during maturation reduces p35 and increases p19 promoter-controlled luciferase expression, using p35 and p19 promoter-controlled reporter construct expression systems in human primary DC (aim 1.1). Next, we will identify EREs in the promoters of p35 and p19 by serial deletion and point mutations of potential transcription factor binding sites that respond differentially to ESAT-6 (aim 1.2). Finally, we will determine if the EREs in the p35 and p19 promoters of primary human DC respond differentially to M. tb wildtype and an ESAT-6 deletion mutant strain (aim 1.3). 2. Identify proteins by which ESAT-6 differentially regulate p35 and p19. Based on the EREs delineated in aim 1, we will identify proteins that bind to ERE by EMSA and ERE-pulldown assays (aim 2.1) and verify ESAT-6-mediated binding of these proteins to the EREs in live DC by chromatin immunoprecipitation (aim 2.2). We will determine how ESAT-6 affects expression and activation of ERE-binding proteins in DC, using Western blot (aim 2.3). Finally, we will determine if knockdown of the ERE-binding proteins with RNAi make DC resistant to ESAT-6-mediated differential expression of p35 and p19 mRNA (aim 2.4). Understanding the mechanisms by which ESAT-6 inhibits IL-12 and stimulates IL-23 production by DC will provide insight into novel mechanisms for regulation of Th1 and Th17 responses, which contribute to immune protection and immunopathology. In addition, these studies will facilitate design of more effective antituberculosis vaccines and identification of novel therapeutic targets for immunotherapy.

Public Health Relevance

Tuberculosis remains an urgent public health problem world wide due to lack of effective vaccine. Developing such vaccine requires detailed understanding of the interaction between host immune cells and Mycobacterium tuberculosis, the causative agent of tuberculosis. Our preliminary data demonstrate that ESAT-6, a secreted protein of Mycobacterium tuberculosis, primes human dendritic cells to stimulate inflammatory Th17 cells at the expense of protective Th1 cells, which can lead to extensive tissue damage that is characteristic of tuberculosis. Because ESAT-6 is a candidate vaccine antigen, it is important to understand how it may cause tissue damage and reduce immune protection. The findings from this proposal will uncover how Mycobacterium tuberculosis reduces protective immune responses and increases tissue-damaging effects through ESAT-6. This information will lead to improved and safer ESAT-6-based vaccines against tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI099345-02
Application #
8448065
Study Section
Immunity and Host Defense Study Section (IHD)
Program Officer
Parker, Tina M
Project Start
2012-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
2
Fiscal Year
2013
Total Cost
$176,250
Indirect Cost
$51,250
Name
University of Texas Health Center at Tyler
Department
None
Type
Organized Research Units
DUNS #
800772337
City
Tyler
State
TX
Country
United States
Zip Code
75708