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

项目摘要 巨噬细胞介导的程序性细胞清除（PrCR）允许清除活细胞。我们已经证明 这种吞噬过程可以消除呈现“吃我”信号的癌细胞， 吃我的分子被阻断了。然后，我们将其进一步扩展到其他致病性或“过期”细胞的清除。 我们最近的研究的关键新发现是：1。活化的巨噬细胞产生并分泌钙网蛋白 (CRT)2.分泌的CRT与靶细胞上的表面脱唾液酸聚糖结合，以产生“吃我信号”， 巨噬细胞3.癌细胞和嗜酸性粒细胞调节其表面以暴露脱唾液酸聚糖结合位点， CRT作为巨噬细胞的“吃我”信号。我们提出，通过结合脱唾液酸聚糖和脱唾液酸聚糖， 巨噬细胞上的促吞噬细胞受体（CD 91/LPR 1），CRT可以将靶细胞连接到巨噬细胞， 通过PrCR进行清除。CRT通常是含有C-末端KDEL保留信号的常驻ER蛋白，但 已经证明在垂死的细胞中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既可以易位到细胞表面，也可以分泌，导致细胞凋亡。 WT（死亡）或Bcl-2+（存活）腹膜中性粒细胞和癌细胞的PrCR增加， “吃了我”信号CD 47要么缺失要么被阻断基于这些发现，我们进行了以下研究：（1）阐明 信号影响巨噬细胞，导致CRT易位到细胞表面和分泌可溶性 CRT形式;（2）阐明调节含脱唾液酸聚糖结合位点可用性的机制 用于靶细胞的CRT。阐明PrCR所需的巨噬细胞和靶细胞中的个体机制 了解巨噬细胞内的相互作用：靶细胞相互作用可以有广泛的治疗作用， 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 PrCR的CRT表达和分泌以及能够进行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 刺激前后巨噬细胞中CRT的不同蛋白质形式：ER形式与细胞表面- 结合的CRT，相对于可溶性分泌的CRT，相对于与靶细胞上的脱唾液酸聚糖结合的CRT。我们有初步的 有证据表明，CRT的分泌形式不包含KDEL基序，潜在的蛋白水解加工 导致不同CRT形式的形成。最后，在目标3中，我们将研究启动信号事件， 影响唾液酸的添加或去除的酶，其活性决定暴露水平。 脱唾液酸聚糖，因此CRT与预定消除的细胞表面的结合。这些研究将使 揭示了一种新的机制，通过这种机制，巨噬细胞可以检测到要从体内清除的细胞。我们 研究结果将与癌症、退行性疾病和炎性病变有广泛的相关性， 揭示了改变生命的疾病的新的治疗靶点。

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.