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Programmed Cell Removal (PrCR) by Macrophages: recognition and phagocytosis of target cells

巨噬细胞的程序性细胞去除（PrCR）：靶细胞的识别和吞噬作用

基本信息

批准号：
10576906
负责人：
IRVING L. WEISSMAN
金额：
$ 40.47万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10576906
关键词：
Adhesions Affect Aging Agonist American Society of Clinical Oncology Apoptotic Asialoglycoproteins Atherosclerosis BCL2 gene Binding Binding Sites Biochemical Biochemical Pathway Blocking Antibodies Blood Bone Marrow C-terminal CD47 gene Cell Communication Cell Membrane Proteins Cell secretion Cell surface Cells Cellular biology Clinical Clinical Trials Degenerative Disorder Development Disease Disease remission Eating Enzymes Event Excision Exposure to Extracellular Space Fibroblasts Generations Glycoproteins Hematopoietic stem cells Heterogeneity Homeostasis Human In Vitro Individual Inflammation Inflammatory Invaded Investigation KDEL Motif Knowledge Label Lesion Life Ligands Longevity Macrophage Macrophage Activation Malignant Neoplasms Mediating Membrane Molecular Chaperones Multipotent Stem Cells Mus Neoplasms Neuraminidase Non-Malignant Normal tissue morphology Pathogenicity Pathologic Pathway interactions Peritoneal Phagocytes Phagocytosis Phase Process Proteins Proteolytic Processing Proteomics Publishing Reporting Research Rest Role Sialic Acids Sialoglycoproteins Signal Transduction Smooth Muscle Smooth Muscle Myocytes Sterility Surface Systemic Scleroderma TLR3 gene Testing Therapeutic Tissues Toll-like receptors Translating calreticulin cancer cell cancer type disorder prevention in vivo leukemic stem cell lysyl-aspartyl-glutamyl-leucine neoplastic cell neutrophil new therapeutic target nonalcoholic steatohepatitis novel overexpression prevent receptor therapeutic development

项目摘要

Project Summary Macrophage-mediated programmed cell removal (PrCR) allows clearance of living cells. We have shown that this phagocytic process can eliminate cancer cells that present an ‘eat me’ signal and have their dominant 'don't eat me' molecules blocked. We then further extended this to the clearance of other pathogenic or ‘expired’ cells. The key novel findings of our recent studies are: 1. Activated macrophages produce and secrete calreticulin (CRT); 2. Secreted CRT binds to surface asialoglycans on target cells to create ‘eat me signals' for macrophages. 3. Cancer cells and neutrophiles modulate their surface to expose asialoglycan binding sites for CRT that acts as an ‘eat me’ signal for macrophages. We propose that by binding both to asialoglycans and to pro-phagocytic receptors on macrophages (CD91/LPR1), CRT can bridge target cells to macrophages for clearance via PrCR. CRT is normally a resident ER protein containing a C-terminal KDEL retention signal, but it’s been shown that in dying cells CRT can be translocated to the cell surface. We found that upon macrophage activation via toll-like receptors (TLR), CRT can both translocate to the cell surface and be secreted, leading to increased PrCR of either WT (dying) or Bcl-2+ (viable) peritoneal neutrophils and cancer cells on which the ‘don’t eat me’ signal CD47 is either absent or blocked. Based on these findings we proporse: (1) to elucidate the signals affecting the macrophage that result in CRT translocation to the cell surface and secretion of soluble forms of CRT; (2) to elucidate the mechanisms regulating the availability of asialoglycan-containing binding sites for CRT on target cells. Elucidating the individual mechanisms in macrophages and target cells required for PrCR and understanding the cross-talk within macrophage:target-cell interaction can have broad therapeutic implications. In Aim 1 we will investigate which signals stimulate macrophages to increase cell surface expression and secretion of CRT for PrCR and the heterogeneity of macrophages that can carry out PrCR in vitro and sterile inflammation in vivo. In Aim 2 we will employ proteomic analysis to define and characterize the different proteoforms of CRT originating in macrophages before or after stimulation: the ER form vs. cell-surface- bound, vs. soluble secreted CRT, vs. CRT that is bound to asialoglycans on target cells. We have preliminary evidence that the secreted form of CRT does not contain the KDEL motif and that potential proteolytic processing leads to the formation of the different CRT forms. Lastly in Aim 3 we will study the initiating signaling events and enzymes that affect the addition or removal of sialic acid, the activity of which determines the level of exposed asialoglycans and thus the binding of CRT to the surface of cells destined for elimination. These studies will shed light on a novel mechanism by which macrophages detect cells that are to be removed from the body. Our findings will have a broad relevance to cancer, degenerative disease, and inflammatory lesions and will likely reveal new therapeutic targets for life altering disease conditions.

项目概要巨噬细胞介导的程序性细胞去除 (PrCR) 可以清除活细胞。我们已经证明这种吞噬过程可以消除发出“吃我”信号并具有显性“别”信号的癌细胞吃掉我’分子被封锁。然后，我们进一步将其扩展到清除其他致病或“过期”细胞。我们最近研究的主要新发现是： 1. 活化的巨噬细胞产生并分泌钙网蛋白（显像管）； 2. 分泌的 CRT 与靶细胞表面的脱唾液酸聚糖结合，产生“吃我信号” 巨噬细胞。 3. 癌细胞和中性粒细胞调节其表面以暴露脱唾液酸聚糖结合位点 CRT 充当巨噬细胞“吃掉我”的信号。我们建议通过结合去唾液酸聚糖和巨噬细胞上的促吞噬细胞受体 (CD91/LPR1)，CRT 可以将靶细胞与巨噬细胞桥接起来，通过 PrCR 清除。 CRT 通常是一种驻留 ER 蛋白，含有 C 端 KDEL 保留信号，但是研究表明，在垂死细胞中，CRT 可以转移到细胞表面。我们发现巨噬细胞通过 Toll 样受体 (TLR) 激活，CRT 既可以转移到细胞表面并被分泌，从而导致 WT（死亡）或 Bcl-2+（存活）腹膜中性粒细胞和癌细胞的 PrCR 增加 “吃我”信号 CD47 要么不存在，要么被阻断。基于这些发现，我们建议：（1）阐明影响巨噬细胞的信号导致 CRT 易位到细胞表面并分泌可溶性 CRT 形式； (2) 阐明调节含脱唾液酸聚糖的结合位点可用性的机制用于靶细胞上的 CRT。阐明 PrCR 所需的巨噬细胞和靶细胞的个体机制并了解巨噬细胞内的串扰：靶细胞相互作用可以具有广泛的治疗作用影响。在目标 1 中，我们将研究哪些信号刺激巨噬细胞增加细胞表面积 PrCR的CRT表达和分泌以及可进行PrCR的巨噬细胞的异质性体外和体内无菌炎症。在目标 2 中，我们将采用蛋白质组学分析来定义和表征刺激之前或之后源自巨噬细胞的 CRT 的不同蛋白质形式：ER 形式与细胞表面形式结合，与可溶性分泌型 CRT 相比，与与靶细胞上的脱唾液酸聚糖结合的 CRT 相比。我们有初步的有证据表明 CRT 的分泌形式不包含 KDEL 基序，并且潜在的蛋白水解加工导致了不同CRT形式的形成。最后，在目标 3 中，我们将研究启动信号事件和影响唾液酸添加或去除的酶，其活性决定了暴露水平脱唾液酸聚糖，从而使 CRT 与注定要消除的细胞表面结合。这些研究将揭示揭示了巨噬细胞检测要从体内移除的细胞的新机制。我们的研究结果将与癌症、退行性疾病和炎症病变具有广泛的相关性，并且可能揭示改变生活的疾病的新治疗靶点。