The goal of this project is to elucidate the mechanisms by which interleukin-32 (IL-32) enhances immunity against Mycobacterium tuberculosis (MTB). A comprehensive understanding of IL-32 action will not only advance our knowledge of the host-protective immune response to tuberculosis (TB), but also drive future investigations designed to enrich vaccine therapy and cultivate innovative, immune-modifying treatments. IL-32 is a recently described cytokine. We have shown that IL-32 is induced by MTB infection and that it protects host cells against MTB. We discovered that IL-32 inhibits intracellular growth of MTB in THP-1 macrophages through caspase-3-dependent apoptosis and yet-to-be identified caspase-3-independent cell death pathways. While IL-32 plays a potentially critical role in controlling TB, important questions remain. We hypothesize that IL-32 reduces viability of intracellular MTB by inducing macrophages to undergo various types of programmed cell death (PCD), including caspase-3-dependent and caspase-independent apoptosis as well as pyroptosis, a unique form of inflammatory PCD mediated by caspase-1 and inflammasome complexes. Both apoptosis and pyroptosis are known killing mechanisms of intracellular pathogens including mycobacteria. While a mouse homolog of IL-32 has not been found, murine macrophages are activated by human IL-32. RAW 264.7 mouse macrophages incubated with IL-32 have a lower burden of intracellular MTB. To study the anti-TB effect of IL-32 in vivo, we generated transgenic mice that express IL-323 in the lungs under the control of the SPC promoter (SPC-IL-323Tg). Aerosol infection with the hypervirulent MTB W-Beijing HN878 showed that the SPC-IL-323Tg mice have reduced bacterial burden in the lungs and spleen, a more protective immune phenotype, and increased survival compared to wild type (WT) C57BL/6 mice. Recently, our collaborators developed a Tg mouse strain in which IL-323 is under the control of the 2-actin gene promoter. IL-32 is widely expressed in the tissues of these mice, including the lung, spleen, and white blood cells. To begin to address the clinical relevance of IL-32 in humans, we will test the efficacy of IL-32 against a virulent clinical strain of MTB in primary human alveolar macrophages (AM). We propose to more fully describe the mechanisms by which IL-32 kills intracellular MTB as well as the complex immunological pathways affected by IL-32 through the following three aims.
Aim 1. Elucidate how programmed cell death pathways contribute to IL-32 induced inhibition of intracellular MTB. We hypothesize that caspase-3-independent apoptosis and caspase-1-mediated pyroptosis are additional mechanisms by which IL-32 exerts its anti-TB effects.
Aim 2. Determine how IL-32 transgenic mice are protected against MTB infection. We hypothesize that SPC-IL-323Tg mice will have sustained resistance to MTB at longer times of infection, 2-actin-IL-323Tg mice will also resist MTB infection, and both knock-in models will survive longer and exhibit a more protective immune response with greater induction of PCD in the AM than infected WT mice.
Aim 3. Define the anti-mycobacterial activity of IL-32 in primary human cells and determine whether the anti-TB effects of IL-32 can be enhanced. We hypothesize that IL-32 will antagonize intracellular MTB in primary human AM due to its ability to induce caspase-3-dependent apoptosis and caspase-1-mediated pyroptosis. Since W-Beijing strains are less likely to induce host cell apoptosis, we hypothesize that IL-32 will be more efficient in killing MTB H37Rv than W-Beijing HN878. Additionally, enhancing the bioactivity of IL-32 should increase induction of PCD and MTB killing.

Public Health Relevance

Tuberculosis (TB) is one of the most deadly infectious diseases in the world, especially in health resource- poor nations where U.S. troops have been or are currently deployed. As a result, many veterans from World War II, Korean War, Vietnam War, and the Gulf Wars are at increased risk of developing active or latent disease. The evolution of highly drug-resistant TB has re-awakened interest in enhancing immunity against Mycobacterium tuberculosis. We have found that IL-32, a newly described protein, enhances the clearance of intracellular M. tuberculosis in human cells and mice. Proposed experiments will delineate the mechanisms behind this protective effect. Knowledge gained from these studies will facilitate future investigations on how to optimize the immune response to drug-resistant TB. Findings will promote veterans'health and support the VA mission by discovering new ways to augment immunological control of M. tuberculosis, thereby reducing treatment time and improving patient outcomes.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX001028-03
Application #
8598018
Study Section
Infectious Diseases B (INFB)
Project Start
2011-10-01
Project End
2015-09-30
Budget Start
2013-10-01
Budget End
2014-09-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
VA Eastern Colorado Health Care System
Department
Type
DUNS #
003252830
City
Denver
State
CO
Country
United States
Zip Code
80220