Innate immune proteins regulating microparticle-mediated lung inflammation

原理：从细胞中释放的微粒可以发挥调节作用。肺天然免疫收集素(如表面活性蛋白D、SP-D)在调节肺内细胞死亡和凋亡微粒生成中的重要性尚未明确。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类(如磷脂酰肌醇)和死亡细胞；然而，死亡细胞上存在的靶点并不清楚。*初步数据：上一次NSERC拨款期间产生的数据表明，SP-D(I)调节caspase-8介导的细胞凋亡，(Ii)结合于凋亡细胞上的微粒位置，以及(Iii)促进这些死亡细胞释放微粒。*假设：SP-D与凋亡细胞上存在的特定碳水化合物或脂肪成分结合，促进MP的产生，并促进肺泡巨噬细胞摄取这些微粒来调节炎症。为了验证这一假设，我有三个具体的目标。*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，我们将确定暴露在濒死细胞表面的脂类和碳水化合物配体(例如，凋亡的T细胞和中性粒细胞)。特别是，我们将进行竞争和下拉分析来确定表面配体(交联法、免疫沉淀法、质谱仪)。为了验证候选基因，我们将使用基因敲除或基因敲除的方法。*目标2：确定SP-D对微粒子介导的巨噬细胞内信号的影响。在SP-D存在或不存在的情况下，我们将从中性粒细胞或T细胞(外在途径为FasL，内在途径为UV)中产生微粒，并允许这些成分与巨噬细胞相互作用(焦点是肺泡巨噬细胞)。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，独特的下垂途径是巨噬细胞中一个很好的活性靶点。因此，我们将通过免疫细胞化学的方法检测这些巨噬细胞中的1L-1b和溶血体相关标记。一旦确定，我们将使用通路特异性阻滞剂来验证我们的发现。*目标3：建立SP-D对体内微粒清除的影响。为了确定SP-D在肺中的存在是否是必需的，我们将从野生型和SP-D缺陷小鼠(可在我的实验室获得)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，我们将把微粒注入野生型和SP-D缺陷小鼠的肺中，并检测巨噬细胞死亡的类型和细胞因子谱的变化。*意义：这些研究应该建立调控SP-D介导的微粒产生的基本原理，以及SP-D在肺部炎症中的作用。

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.