Innate immune proteins regulating microparticle-mediated lung inflammation

点击翻译按钮获取中文摘要

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.