Innate immune proteins regulating microparticle-mediated lung inflammation

先天免疫蛋白调节微粒介导的肺部炎症

• 批准号: RGPIN-2018-06575
• 负责人: Palaniyar, Nades
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686672
• 关键词: Innate; immune; proteins; regulating; microparticle

Rationale: Microparticles released from cells can exert regulatory effects. Importance of pulmonary innate immune collectins (e.g., surfactant protein D, SP-D) in regulating cell death and generation of apoptotic microparticles in the lungs have not been clearly established. SP-D has collagen and lectin (or carbohydrate recognition) domains. It recognizes carbohydrate headgroup-containing proteins and lipids (e.g., Phosphatidylinositol) and dying cells; however, the targets present on the dying cells are not clearly established.******Preliminary data: Data generated in the last NSERC grant period have identified that SP-D (i) regulates caspase-8-mediated apoptosis, (ii) binds at the loci of microparticles on apoptotic cells, and (iii) promotes microparticle release from these dying cells.******Hypothesis: SP-D binds to specific carbohydrate or lipid components present on apoptotic cells to promote MP generation, and facilitates the uptake of these particles by alveolar macrophages to regulate inflammation. To test this hypothesis, I have 3 specific aims.******Aim 1: To identify the molecules that SP-D recognizes on apoptotic cells and microparticles. To achieve this aim, we will identify the lipids and carbohydrate ligands exposed on the surface of dying cells (e.g., apoptotic T-cells and neutrophils). Particularly, we will conduct competition and pull-down assays to identify the surface ligands (cross-linking, immunoprecipitation, mass spec). To validate the candidates, we will use knockout or knockdown approaches.******Aim 2: To determine the effect of SP-D on microparticle-mediated intracellular signaling of macrophages. We will generate microparticles from neutrophils or T cells (FasL for extrinsic and UV for intrinsic pathways) in the presence or absence of SP-D, and allow these components to interact with macrophages (focus is on alveolar macrophages). Then we will study the changes in macrophages by morphological and cytokine profile changes. Our initial data indicates that the unique pyroptosis pathway is a good target to be active in macrophages. Therefore, we will examine 1L-1b, and pyroptosome-related markers in these macrophages by immunocytochemistry. Once identified, we would conduct pathway specific blockers to validate our findings.******Aim 3: To establish the effect of SP-D on microparticle clearance in vivo. To determine whether the presence of SP-D is essential in the lungs, we will isolate alveolar macrophages from the wild type and SP-D-deficient mice (available in my lab). We will conduct a comparative analysis on these cells. As the last step, we will instill microparticles into the lungs of wildtype and SP-D-deficient mice lungs, and examine the type of macrophage death and changes in cytokine profiles.******Significance: These studies should establish the fundamental principles regulating SP-D-mediated microparticle generation, and the role of SP-D in lung inflammation.

理由：细胞释放的微粒可以发挥调节作用。肺部先天免疫集合素（例如表面活性蛋白 D、SP-D）在调节肺部细胞死亡和凋亡微粒生成方面的重要性尚未明确。 SP-D 具有胶原蛋白和凝集素（或碳水化合物识别）结构域。它识别含有碳水化合物头基的蛋白质和脂质（例如磷脂酰肌醇）和垂死细胞；然而，死亡细胞上存在的目标尚未明确确定。******初步数据：上一次 NSERC 资助期间生成的数据已确定 SP-D (i) 调节 caspase-8 介导的细胞凋亡，(ii) 结合在凋亡细胞上的微粒位点，以及 (iii) 促进这些死亡细胞释放微粒。******假设：SP-D 结合特定的 凋亡细胞上存在的碳水化合物或脂质成分可促进 MP 生成，并促进肺泡巨噬细胞摄取这些颗粒以调节炎症。为了检验这个假设，我有 3 个具体目标。*****目标 1：识别 SP-D 在凋亡细胞和微粒上识别的分子。为了实现这一目标，我们将识别垂死细胞（例如凋亡 T 细胞和中性粒细胞）表面暴露的脂质和碳水化合物配体。特别是，我们将进行竞争和下拉分析来鉴定表面配体（交联、免疫沉淀、质谱）。为了验证候选者，我们将使用基因敲除或敲低方法。 *****目标 2：确定 SP-D 对微粒介导的巨噬细胞细胞内信号传导的影响。我们将在存在或不存在 SP-D 的情况下从中性粒细胞或 T 细胞（FasL 代表外在途径，UV 代表内在途径）生成微粒，并允许这些成分与巨噬细胞相互作用（重点是肺泡巨噬细胞）。然后我们将通过形态和细胞因子谱的变化来研究巨噬细胞的变化。我们的初步数据表明，独特的细胞焦亡途径是巨噬细胞中活跃的良好靶点。因此，我们将通过免疫细胞化学检查这些巨噬细胞中的 1L-1b 和焦亡体相关标记。一旦确定，我们将使用途径特异性阻断剂来验证我们的发现。******目标 3：确定 SP-D 对体内微粒清除的影响。为了确定 SP-D 的存在是否在肺部是必需的，我们将从野生型和 SP-D 缺陷型小鼠（我的实验室中有）中分离出肺泡巨噬细胞。我们将对这些细胞进行比较分析。最后一步，我们将微粒注入野生型和 SP-D 缺陷小鼠的肺部，并检查巨噬细胞死亡的类型和细胞因子谱的变化。 *****意义：这些研究应建立调节 SP-D 介导的微粒生成的基本原理，以及 SP-D 在肺部炎症中的作用。