M2 macrophage co-culture overrides viscoelastic hydrogel mechanics to promote IL-6-dependent fibroblast activation

Fibroblast activation drives fibrotic disease; however, the complex interplay of how tissue mechanics and macrophage signaling combine to influence fibroblast activation remains unclear. Using hyaluronic acid hydrogels to mimic lung stiffness and viscoelasticity, we investigated macrophage influence on fibroblast activation. Fibroblasts cultured on stiff (50 kPa) hydrogels mimicking fibrotic tissue exhibit increased activation, as measured by cell spreading and type I collagen and cadherin-11 expression, compared to fibroblasts cultured on soft (1 kPa) viscoelastic hydrogels mimicking normal lung. Macrophage-conditioned media did not alter these trends, however co-culture with M2 macrophages increased fibroblast activation independent of direct macrophage contact, even on soft viscoelastic hydrogels. Blocking interleukin 6 (IL6) signaling mitigated this pro-fibrotic effect but did not affect fibroblast-only cultures. These findings demonstrate that M2 macrophages override hydrogel viscoelasticity to promote fibroblast activation independent of direct contact in an IL6-dependent manner and highlight the utility of hydrogels in deconstructing complex tissue microenvironments.