ESTABLISHING THE ROLE OF BONE MARROW STROMAL ANTIGEN 2 IN HEMATOPOIETIC STEM CELL NICHE INTERACTIONS AND ACTIVATION

摘要 干扰素γ是一种促炎细胞因子，在慢性感染和铅中毒时上调。 导致造血干细胞(HSCs)的激活、分化和最终丧失。我们之前已经证明了 这些影响可能导致慢性感染期间骨髓(BM)不足和细胞减少。 它的临床意义，干扰素γ激活和耗尽造血干细胞的机制尚不清楚。因为HSCs 受到它们与BM微环境相互作用的严重影响2，我们先前研究了 干扰素γ对造血干细胞-生态位相互作用的影响。我们的初步数据显示，由干扰素γ激活的造血干细胞在骨髓中重新定位 远离静止的促进CXCL12丰富的网状(CAR)细胞。我们使用转录分析来 骨髓基质抗原2是干扰素γ刺激后唯一上调的表面蛋白 在HSC迁移的早期阶段。到目前为止，我们的工作表明BST2基因缺陷的小鼠有更多的HSC 在骨髓中，它们的HSC比野生型(WT)的HSCs增殖更少，BST2 KO HSCs不再 在干扰素γ诱导过程中定位于CAR细胞，提示BST2可能具有调节激活的作用。 以前的报道表明，BST2可以作为一种黏附蛋白，促进其他 细胞类型，我们怀疑它可能在造血干细胞中执行类似的功能。这项提案的首要目标是 目的是通过确定BST2与蛋白质的相互作用及其对HSC活化的影响来阐明BST2在HSC激活中的作用 HSC在生态位内的相互作用。我们假设BST2的表达增加促进了HSC 通过促进与其他类型的小生境细胞的结合，在炎症应激期间激活。要测试 假设，在目标1中，我们将利用交联剂辅助建立HSCs中BST2-表面蛋白的相互作用 亲和纯化质谱学。我们将进一步确定阻止这些互动是否会影响 通过测量HSC的数量、增殖和细胞周期状态来激活HSC。在目标2中，我们将确定BM 通过使用各种骨髓进行活体成像来促进BST2介导的HSC激活的利基细胞 报告小鼠来确定HSCs相对于其他壁龛细胞的空间位置。之间的直接交互作用 BST2阳性的造血干细胞和壁龛细胞也将接受测试。总而言之，这里提出的实验旨在 扩大我们对BST2改变HSC-生态位相互作用机制的理解。阐明如何 在干扰素γ刺激下，肝星状细胞被激活，这将有助于深入了解炎症的机制 导致HSC衰竭，并可能导致骨髓衰竭综合征的新治疗策略 与过度的炎症有关。

Abstract Interferon gamma (IFNγ) is a pro-inflammatory cytokine that is upregulated during chronic infections and leads to activation, differentiation, and eventual loss of hematopoietic stem cells (HSCs). We previously showed that these effects may contribute to bone marrow (BM) insufficiency and cytopenias during chronic infection.1 Despite its clinical significance, the mechanism by which IFNγ activates and depletes HSCs is unknown. Since HSCs are heavily influenced by their interaction with the BM microenvironment2, we previously studied the impact of IFNγ on HSC-niche interactions. Our preliminary data show that HSCs activated by IFNγ re-localize in the BM away from quiescent-promoting CXCL12-abundant reticular (CAR) cells. We used transcriptomic analysis to identify Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated upon IFNγ stimulation during the early stages of HSC migration. Our work to date shows that BST2-deficient mice have more HSCs present in the BM, their HSCs are less proliferative than wild type (WT) HSCs, and BST2 KO HSCs do not re- localize from CAR cells during IFNγ induction suggesting that BST2 could play a role in regulating activation. Previous reports have suggested that BST2 could serve as an adhesion protein to facilitate migration in other cell types, and we suspect that it may perform a similar function in HSCs. The overarching goal of this proposal is to elucidate the role of BST2 in HSC activation by determining BST2-protein interactions and its impact on HSC interactions within the niche. We hypothesize that increased expression of BST2 promotes HSC activation during inflammatory stress by facilitating binding to alternative niche cell types. To test the hypothesis, in Aim 1, we will establish BST2-surface protein interactions in HSCs using cross-linking aided affinity purification mass spectrometry. We will further determine whether blocking these interactions will affect HSC activation by measuring HSC numbers, proliferation and cell cycle status. In Aim 2, we will identify the BM niche cells that facilitate BST2-mediated HSC activation by performing intravital imaging using a variety of BM reporter mice to identify the spatial location of HSCs relative to the other niche cells. Direct interaction between BST2-positive HSCs and niche cells will also be tested. Collectively, the experiments proposed here aim to expand our understanding of the mechanism by which BST2 alters HSC-niche interactions. Elucidating how HSCs become activated during IFNγ stimulation will provide insight into the mechanisms by which inflammation drives HSC exhaustion and will potentially lead to novel therapeutic strategies for bone marrow failure syndromes associated with excessive inflammation.