(607c) Fibroblasts Promote Macrophage Migration in 3D Collagen Matrices through Tunnel Formation and Fiber Alignment

The extracellular matrix (ECM) provides cells in vivo with a myriad of chemical and mechanical cues, which influence cell function and phenotype. ECM protein composition and organization lead to differences in network porosity, fiber architecture, and elasticity of the ECM changing the landscape through which cells migrate and adhere. As cells navigate through the ECM, they can alter its structure through contraction, alignment, and cross-linking of fibers or by degradation of proteins through protease dependent and independent mechanisms. During migration, cell-cell contacts as well as soluble signaling molecules can influence speed, direction and modes of migration. Macrophages and fibroblasts are two migratory cell types found throughout the body, which play important roles in wound healing as well as in fibrosis, inflammation and the progression of cancer. Since these cells are frequently located within the same tissue, their biochemical and physical interactions likely influence each other’s ability to navigate through the complex structure of the ECM. Despite their relevance to tissue homeostasis and disease progression, few studies have examined the interactions between macrophages and fibroblasts in 3D ECMs and many of these studies have been conducted in cancerous systems.

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 µm in diameter and up to 110 µm 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 µm 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 a₂β₁ 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.