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: $ 5.25万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757972
• 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.

原理：从细胞释放的微粒可以发挥调节作用。肺先天免疫聚集素的重要性（例如，表面活性剂蛋白D，SP-D）在调节肺中细胞死亡和凋亡微粒产生中的作用尚未明确确立。SP-D具有胶原和凝集素（或碳水化合物识别）结构域。它识别含碳水化合物头基的蛋白质和脂质（例如，初步数据：在最后一个NSERC资助期产生的数据已经确定SP-D（i）调节caspase-8介导的凋亡，（ii）结合在凋亡细胞上的微粒位点，（iii）促进这些垂死细胞的微粒释放。假设：SP-D与凋亡细胞上存在的特定碳水化合物或脂质成分结合以促进MP生成，并促进肺泡巨噬细胞对这些颗粒的摄取以调节炎症。为了验证这一假设，我有三个具体的目标：目标1：鉴定SP-D在凋亡细胞和微粒上识别的分子。为了实现这一目标，我们将鉴定暴露在垂死细胞表面的脂质和碳水化合物配体（例如，凋亡T细胞和嗜中性粒细胞）。特别是，我们将进行竞争和下拉分析，以确定表面配体（交联，免疫沉淀，质谱）。为了验证候选者，我们将使用knockout或knockdown的方法。目的2：确定SP-D对微粒介导的巨噬细胞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用于外源性途径，UV用于内源性途径）中产生微粒，并允许这些组分与巨噬细胞相互作用（重点是肺泡巨噬细胞）。然后通过形态学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pyroptosis途径是一个很好的目标，是活跃在巨噬细胞。因此，我们将通过免疫细胞化学检查这些巨噬细胞中的1 L-1b和焦浆体相关标志物。一旦确定，我们将进行途径特异性阻断剂，以验证我们的发现。目的3：建立SP-D微粒清除在体内的影响。为了确定SP-D的存在是否在肺中是必需的，我们将从野生型和SP-D缺陷小鼠（在我的实验室中可获得）中分离肺泡巨噬细胞。我们将对这些细胞进行比较分析。作为最后一步，我们将微粒灌输到野生型和SP-D缺陷小鼠的肺，并检查巨噬细胞死亡的类型和细胞因子profiles.Significance的变化：这些研究应该建立调节SP-D介导的微粒生成的基本原则，以及SP-D在肺部炎症中的作用。