The role of mechanosensation in monocyte differentiation

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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.