Mechanistic study of the host defense functions of IL-32 in tuberculosis

描述（由申请人提供）： 本课题旨在阐明白细胞介素-32（IL-32）增强抗结核分枝杆菌（MTB）免疫的机制。对IL-32作用的全面了解不仅将促进我们对结核病（TB）宿主保护性免疫反应的了解，还将推动未来旨在丰富疫苗治疗和培养创新免疫修饰治疗的研究。 IL-32是最近描述的细胞因子。我们已经表明IL-32由MTB感染诱导，并且它保护宿主细胞免受MTB的侵害。我们发现IL-32通过caspase-3依赖性细胞凋亡和尚未鉴定的caspase-3非依赖性细胞死亡途径抑制THP-1巨噬细胞中MTB的细胞内生长。虽然IL-32在控制结核病方面发挥着潜在的关键作用，但仍存在重要问题。我们假设IL-32通过诱导巨噬细胞经历各种类型的程序性细胞死亡（PCD）来降低细胞内MTB的活力，所述程序性细胞死亡包括半胱天冬酶-3依赖性和半胱天冬酶非依赖性细胞凋亡以及焦亡（由半胱天冬酶-1和炎性体复合物介导的独特形式的炎性PCD）。细胞凋亡和焦亡都是已知的细胞内病原体（包括分枝杆菌）的杀伤机制。 虽然尚未发现IL-32的小鼠同源物，但鼠巨噬细胞被人IL-32激活。用IL-32孵育的RAW 264.7小鼠巨噬细胞具有较低的细胞内MTB负荷。为了研究IL-32在体内的抗TB作用，我们产生了在SPC启动子控制下在肺中表达IL-323的转基因小鼠（SPC-IL-323Tg）。用高毒力MTB W-Beijing HN878进行的气溶胶感染显示，与野生型（WT）C57BL/6小鼠相比，SPC-IL-323Tg小鼠在肺和脾中具有降低的细菌负荷、更具保护性的免疫表型和增加的存活率。最近，我们的合作者开发了一种Tg小鼠品系，其中IL-323在2-肌动蛋白基因启动子的控制下。IL-32在这些小鼠的组织中广泛表达，包括肺、脾和白色血细胞。 为了开始解决IL-32在人中的临床相关性，我们将在原代人肺泡巨噬细胞（AM）中测试IL-32针对MTB的毒性临床菌株的功效。我们建议通过以下三个目标更全面地描述IL-32杀死细胞内MTB的机制以及受IL-32影响的复杂免疫途径。目标1.阐明程序性细胞死亡途径如何促进IL-32诱导的细胞内MTB抑制。我们推测，caspase-3非依赖性细胞凋亡和caspase-1介导的细胞凋亡是IL-32发挥其抗TB作用的额外机制。目标2.确定IL-32转基因小鼠如何保护免受MTB感染。我们假设SPC-IL-323Tg小鼠在较长时间的感染下将具有对MTB的持续抗性，2-肌动蛋白-IL-323Tg小鼠也将抵抗MTB感染，并且两种敲入模型将存活更长时间，并且表现出更具保护性的免疫应答，在AM中比感染的WT小鼠具有更大的PCD诱导。目标3：确定IL-32在原代人类细胞中的抗分枝杆菌活性，并确定IL-32的抗TB作用是否可以增强。我们假设IL-32将拮抗原代人AM中的细胞内MTB，这是由于其诱导半胱天冬酶-3依赖性凋亡和半胱天冬酶-1介导的焦亡的能力。由于W-北京菌株不太可能诱导宿主细胞凋亡，我们假设IL-32在杀死MTB H37Rv方面比W-北京HN 878更有效。此外，增强IL-32的生物活性应增加PCD和MTB杀伤的诱导。

DESCRIPTION (provided by applicant): 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.