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

描述(由申请人提供)： 本项目的目的是阐明白介素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疗法和培育创新的免疫调节疗法的研究。IL-32是最近发现的一种细胞因子。我们已经证明IL-32是由结核分枝杆菌感染诱导的，它保护宿主细胞免受结核分枝杆菌的侵袭。我们发现IL-32通过caspase-3依赖的细胞凋亡和caspase-3非依赖的细胞死亡途径抑制THP-1巨噬细胞中MTB的细胞内生长。尽管IL-32在控制结核病方面发挥着潜在的关键作用，但重要的问题仍然存在。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的活性，包括caspase-3依赖和caspase非依赖的细胞凋亡，以及由caspase-1和炎症体复合体介导的一种独特的炎症性PCD-下垂。细胞凋亡和下垂都是已知的杀死包括分枝杆菌在内的细胞内病原体的机制。虽然还没有发现IL-32的小鼠同源物，但小鼠巨噬细胞可以被人的IL-32激活。与IL-32共同孵育的RAW 264.7小鼠巨噬细胞胞内结核分枝杆菌负荷较低。为了研究IL-32在体内的抗结核作用，我们建立了在SPC启动子(SPC-IL-323Tg)控制下在肺部表达IL-323的转基因小鼠。用超强毒力结核分枝杆菌W-Beijing HN878气溶胶感染SPC-IL-323Tg小鼠表明，与野生型(WT)C57BL/6小鼠相比，SPC-IL-323Tg小鼠减少了肺和脾中的细菌负荷，具有更强的保护性免疫表型，并提高了存活率。最近，我们的合作者开发了一种受2-肌动蛋白基因启动子控制的IL-323的TG小鼠品系。IL-32在这些小鼠的组织中广泛表达，包括肺、脾和白细胞。为了解决IL-32在人类中的临床相关性，我们将在原代人肺泡巨噬细胞(AM)中测试IL-32对一种毒力临床株MTB的疗效。我们建议通过以下三个目标更全面地描述IL-32杀死细胞内结核分枝杆菌的机制以及IL-32影响的复杂免疫途径。目的1.阐明程序性细胞死亡途径在IL-32诱导的细胞内MTB抑制中的作用。我们推测，caspase-3非依赖的细胞凋亡和caspase-1介导的下垂是IL-32发挥抗结核作用的另一种机制。目的2.研究IL-32转基因小鼠对结核分枝杆菌感染的保护作用。我们假设SPC-IL-323Tg小鼠将在更长的感染时间内对MTB具有持续的抵抗力，2-Actin-IL-323Tg小鼠也将抵抗MTB感染，两种敲入模型都将比感染WT小鼠存活更长时间，并表现出更具保护性的免疫反应，在AM中诱导更多的PCD。目的3.确定IL-32在原代人体细胞中的抗分枝杆菌活性，并确定IL-32的抗结核作用是否可以增强。我们推测，IL-32能够诱导caspase-3依赖的细胞凋亡和caspase-1介导的下垂，从而拮抗原代人AM细胞内的MTB。由于W-Beijing株诱导宿主细胞凋亡的可能性较小，我们推测IL-32在杀死结核分枝杆菌H37Rv方面将比W-Beijing HN878更有效。此外，增强IL-32的生物活性应该会增加对PCD和MTB的诱导杀伤。

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.