The role of mechanosensation in monocyte differentiation

背景和研究问题：巨噬细胞是负责许多组织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。它们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。在从血液迁移到实质的过程中，单核细胞遇到各种不同硬度的化学底物。然而，目前还不清楚这些张力的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，组织硬度是单核细胞向巨噬细胞分化的重要线索。拟议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，并确定调节其发展的关键机械传感途径。具体目标平台一：研究机械转导对静态体外培养中巨噬细胞发育的影响平台II：使用多层体外培养研究内皮对巨噬细胞表型、功能和发育的影响。方法学：我们的实验室已经优化了一种细胞培养系统，该系统在塑料组织培养板上使用聚合的聚二甲基硅氧烷（PDMS）水凝胶，模拟2 kPa下较软的顺应性组织，以及50 kPa下类似较硬器官的组织。纤连蛋白是一种基质蛋白，用于包覆这些水凝胶以模拟组织环境。来自C57 BL/6小鼠的骨髓源性巨噬细胞（BMMF）将在水凝胶上的7天培养物中分化，并收获用于下游应用。内皮细胞活化和细胞粘附的作用将使用多层培养与辐射内皮瘤细胞进行评估。最后，将使用振荡封闭细胞培养系统评估剪切应力的作用。为了解决机械刚度对巨噬细胞发育的作用，将通过流式细胞术和qPCR对收获的BMMF进行定量和单核细胞/巨噬细胞分化标志物染色。将通过蛋白质印迹、抑制剂、激动剂和KO小鼠评估机械感测途径，包括整联蛋白/FAK/Pyk 2、Piezo 1和TRPV 4。影响：在稳态条件下，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，我们建议研究的作用，从流体介质（血液或抗凝剂）转化为一个坚实的（硬）组织单核细胞分化为巨噬细胞，这是一个方面的巨噬细胞生理学知之甚少。此外，这些观察结果将改变我们对巨噬细胞生物学研究的解释，因为在描述体外培养BMMF的表型观察时，很少考虑塑料基质的硬度（比任何组织高数千倍）。

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.