(607c) Fibroblasts Promote Macrophage Migration in 3D Collagen Matrices through Tunnel Formation and Fiber Alignment
To investigate how normal macrophages and fibroblasts migrate within a 3D environment as a function of co-culture as well as the connectivity of collagen fibers, we assembled collagen hydrogels with dense (DCN) or with loosely connected networks (LCN). These networks were found to have elastic moduli values of 0.23 kPa (LCN) and 2.16 kPa (DCN), closely matching the properties of neural, lung and breast tissues. RAW 264.7 macrophages and BALB/3T3 fibroblasts were encapsulated within these networks and their migratory behavior, ECM remodeling capabilities and inflammatory states were assessed. In DCNs, macrophages in monocultures were virtually stationary. However, macrophages co-cultured with fibroblasts exhibited approximately 3-fold higher displacements than those in monocultures. Confocal reflectance microscopy revealed tunnels roughly 15 Âµm in diameter and up to 110 Âµm in length created by fibroblasts within the DCNs, providing conduits for macrophage migration. A similar increase in macrophage migration was observed in LCN co-cultures where fibroblasts aligned fibers up to a distance of 100 Âµm providing tracks for macrophages. Matrix reorganization was accompanied by intra-cellular and extra-cellular fluorescent fragments of degraded collagen detected inside both cell types as well as around their cell peripheries. Macrophages in co-culture expressed significantly higher levels of urokinase-type plasminogen activator receptor associated protein (uPARAP)/mannose receptor 1 (CD206), as well as a2Î²1 indicating that collagen internalization in these cells occurred via integrin-independent and integrin-dependent mechanisms. Higher uPARAP in co-cultured macarophages led to increased clearance of degraded collagen. Furthermore, upregulation of CD206 was accompanied by downregulation of inducible nitric oxide synthase (iNOS) in macrophages co-cultured with fibroblasts, suggesting a shift towards an anti-inflammatory state. This work unveils new roles for normal fibroblasts in forming tunnels and aligning fibers in networked ECM to modulate macrophage migration and alter macrophage phenotype. Investigations into the critical contributions of fibroblasts in initiating and enhancing macrophage migration promise to have a significant impact in improving our understanding of wound healing, the foreign body response and in tumor progression.