Macrophage-fibroblast crosstalk shapes wound repair signaling in vitro

Here, we investigated macrophage-fibroblast communication using complementary 2D and 3D in vitro wound healing models combined with live-cell calcium imaging and single-cell RNA sequencing (scRNA-seq). We show that bone marrow-derived macrophages (BMDMs) promote fibroblast scratch closure in a contact-dependent manner independent of connexins, whereas fibroblasts reciprocally regulate macrophage cytokine secretion through distinct mechanisms.

Direct cell-cell contact with fibroblasts enhanced macrophage IL10 production via connexin 43 (Cx43)-dependent signaling, whereas TNFα secretion was suppressed through paracrine interactions. We further demonstrate that fibroblast-macrophage contact induces connexin-dependent intermittent calcium signals selectively in macrophages. ScRNA-seq revealed that wounding reshapes macrophage and fibroblast populations, uncovering dynamic regulation of cell adhesion molecules (CAMs) and intercellular signaling pathways.