Innate immune proteins regulating microparticle-mediated lung inflammation

原理：细胞释放的微粒具有调节作用。肺先天免疫集合素（如表面活性剂蛋白D， SP-D）在调节肺细胞死亡和凋亡微粒产生中的重要性尚未明确确立。SP-D具有胶原蛋白和凝集素（或碳水化合物识别）结构域。它识别含碳水化合物头基团的蛋白质和脂质（如磷脂酰肌醇）和垂死细胞；然而，存在于死亡细胞上的靶标尚未明确确定。******初步数据：在上一次NSERC拨款期间产生的数据已经确定SP-D (i)调节caspase-8介导的凋亡，（ii）结合凋亡细胞上的微粒位点，以及（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，促进MP的产生，并促进这些颗粒被肺泡巨噬细胞摄取，调节炎症。为了验证这个假设，我有三个具体的目标。******目的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，我们将鉴定暴露在死亡细胞表面的脂质和碳水化合物配体（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试验来鉴定表面配体（交联，免疫沉淀，质谱）。为了验证候选药物，我们将使用敲除或击倒方法。******目的2：研究SP-D对巨噬细胞微颗粒介导的胞内信号传导的影响。我们将在SP-D存在或不存在的情况下，从中性粒细胞或T细胞（FasL为外源性途径，UV为内在途径）中产生微粒，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，我们将从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，独特的焦亡途径是巨噬细胞活跃的良好靶点。因此，我们将通过免疫细胞化学检测这些巨噬细胞中的1L-1b和焦体相关标志物。一旦确定，我们将进行通路特异性阻断剂来验证我们的发现。******目的3：建立SP-D对体内微粒清除的影响。为了确定SP-D的存在是否在肺中必不可少，我们将从野生型和SP-D缺乏小鼠（在我的实验室中可用）中分离肺泡巨噬细胞。我们将对这些细胞进行对比分析。作为最后一步，我们将在野生型和sp -d缺陷小鼠的肺中注入微颗粒，并检测巨噬细胞死亡的类型和细胞因子谱的变化。******意义：本研究应确立SP-D介导的微粒生成的基本调控原理，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.