Modulating plasticity of primary human macrophages by extracellular matrix signals of biomimetic 3D matrices

摘要

Cues of the extracellular matrix - including topology, mechanics and composition - are considered to tightly control the polarization of macrophages along inflammatory versus regulatory pathways during wound healing. While in vivo studies do not allow for high-resolution in-depth analysis, in vitro systems frequently lack important physiological cues of the microenvironment like 3D matrices and controlled stiffness. We are interested whether mimicking important biophysical and biomolecular properties of the extracellular matrix (ECM) in in vitro 3D matrices can influence macrophage polarization and function in order to implement those features in the design of biomaterials scaffolds to support wound healing. In our in vitro approach, we mimic in vivo ECM by reconstruction of 3D fibrillar networks based on collagen Ⅰ (Coll Ⅰ) with precisely controlled topology, elasticity as well as glycosaminoglycan (GAG) or fibronectin (FN) content. Primary monocytes from human peripheral blood (CD14 depletion) were differentiated into inflammatory macrophages using GM-CSF either prior to or during cultivation inside the defined matrices. Polarization and function of macrophages depending on matrix characteristics were analyzed based on gene and protein (secreted and intracellular) expression of relevant cytokines (IL10, IL12, TNFα), as well as gene expression of matrix remodeling marker (MMP2/9/14, FN, Coll Ⅰ) after activation by lipopolysaccharide. Collagen based networks were prepared with defined pore and fibril diameter (7 urn, 0.63 μm) and modulated stiffness increase by a factor of 4 using carbodiimide crosslinking. Leaving the mechanical and topological networks properties unaffected, the matrices were optionally functionalized with stable modifications of GAG (synthetically sulfated or non-sulfated hyaluronan (HA)) or FN. Cytokines IL10 and IL12 were found with a higher IL10/IL12 ratio in stiff networks and reduced IL10/IL12 ratio in case of GAG presence for macrophage differentiation inside networks. For pre-differentiated macrophages we additionally found an enhanced IL10/IL12 ratio on FN functionalized networks. Furthermore, enhanced gene expression of the matrix remodeling genes FN, Coll Ⅰ and MMP14 were found in dependence on presence of sulfated HA functionalization. In sum these findings demonstrate the importance of analyzing macrophage plasticity in 3D microenvironments and suggest a support of a regulatory phenotype of macrophages (so-called M2) in stiffer 3D environments and a regulatory function of GAG functionalization on macrophage polarization.