The role of mechanosensation in monocyte differentiation

背景和研究问题：巨噬细胞是许多组织内稳态的关键免疫细胞。许多组织巨噬细胞亚群不断被骨髓来源的单核细胞取代。这些细胞通过血液和淋巴系统在体内循环，并迁移到不同的组织，在那里M-CSF和其他信号诱导它们分化为巨噬细胞。在从血液向实质迁移的过程中，单核细胞会遇到各种不同硬度的化学底物。然而，目前尚不清楚这些张力变化如何促进细胞发育和功能的变化。我们的初步数据表明，组织僵硬是单核细胞向巨噬细胞分化的重要线索。这项拟议的研究的目标是关注巨噬细胞的分化和功能如何受到机械力的影响，并确定调节其发育的关键机械传感途径。特定目的平台I：在静态体外培养平台中研究机械转导对巨噬细胞发育的影响II：采用多层体外培养方法研究内皮细胞对巨噬细胞表型、功能和发育的影响。纤维连接蛋白是一种基质蛋白，被用来包裹这些水凝胶，以模拟组织环境。C57BL/6小鼠的骨髓来源的巨噬细胞(BMMF)将在水凝胶上培养7天后分化，并收集用于下游应用。内皮细胞激活和细胞黏附的作用将通过与辐射内皮瘤细胞的多层培养来评估。最后，将使用振荡闭合细胞培养系统来评估剪切力的作用。为了探讨机械僵硬在巨噬细胞发育中的作用，采集的骨髓间充质干细胞将被量化，并通过流式细胞仪和定量聚合酶链式反应检测单核/巨噬细胞分化标志。机械传感通路，包括整合素/FAK/Pyk2，Piezo1和TRPV4，将通过Western印迹、抑制剂、激动剂和KO小鼠进行评估。影响：在动态平衡条件下，决定单核细胞分化为组织驻留巨噬细胞的线索知之甚少。在这里，我们建议研究从液体介质(血液或淋巴管)到固体(僵硬)组织在单核细胞向巨噬细胞分化过程中的作用，这是巨噬细胞生理学中知之甚少的一个方面。

Background and Research Problems: Macrophages are key immune cells responsible for the homeostasis of many tissues. Many of the tissue macrophage subsets are continuously replaced by bone marrow-derived monocytes. These circulate the body via the blood and lymphatic system and migrate to different tissues where M-CSF and other signals induce their differentiation to macrophages. During migration from the blood to the parenchyma, monocytes encounter a variety of chemical substrates of different stiffness. However, it is unclear how these changes in tension can promote changes in cell development and function. Our preliminary data demonstrates that tissue stiffness is an essential cue for monocyte-to-macrophage differentiation. The goal of the proposed research is to focus on how macrophage differentiation and function is influenced by mechanical forces, and to identify key mechanosensing pathways that modulate their development. Specific Aims Platform I: Investigate the impact of mechanotransduction on macrophage development in a static in vitro culture Platform II: Investigate the impact of the endothelium on macrophage phenotype, function and development using multilayered in vitro culture. Methodology: Our lab has optimized a cell culturing system that uses polymerized polydimethylsiloxane (PDMS) hydrogels on plastic tissue culture plates that mimic softer, compliant tissues at 2 kPa, as well as those that resemble stiffer organs at 50 kPa. Fibronectin, a matrix protein, is used to coat these hydrogels to resemble tissue environments. Bone marrow-derived macrophages (BMMFs) from C57BL/6 mice will be differentiated in a 7 day culture on hydrogels and harvested for downstream applications. The role of endothelial cell activation and cell adhesion will be assess using a multilayer culture with irradiated endothelioma cells. Finally, the role of shear stress will be assessed using an oscillating closed cell culture system. To address the role of mechanical stiffness on the development of macrophages, the harvested BMMFs will be quantified and stained for monocyte/macrophage differentiation markers by flow cytometry and qPCR. Mechanosensing pathways, including Integrin/FAK/Pyk2, Piezo1 and TRPV4, will be assessed by Western blot, inhibitors, agonists and KO mice. Impact: The cues that determine the differentiation of monocytes into tissue resident macrophages under homeostatic conditions are poorly understood. Here, we propose to study the role of the transition from a fluid media (blood or lymphatics) into a solid (stiff) tissue in monocyte differentiation to macrophage, which is an aspect of macrophage physiology poorly understood. In addition, these observations will change our interpretation of much research done on the biology of macrophages as the stiffness of plastic substrates (thousands of times higher that any tissue) has rarely been taken into account when describing phenotypic observation of in vitro cultured BMMFs.