Integrin-mediated SPP1 signaling from macrophages orchestrates extracellular matrix remodeling and chondrogenesis in heterotopic ossification

Differentiation of mesenchymal progenitor cells (MPCs) is critical pre-requisition for the progression of heterotopic ossification (HO). However, the factors that initiate MPC differentiation remain elusive. This study aims to investigate the temporal crosstalk between macrophages and MPCs during HO, and identified the critical regulatory mechanism. Single-cell transcriptome data of mouse ossified hindlimb Achilles’ tendon was acquired from the Gene Expression Omnibus (GEO). MPC and macrophage clusters were visualized, followed by pseudo-time analysis, gene set variation analysis, transcription factor activity analysis, and cell communication analysis. Achilles’ tendon transection and dorsal burn injury was used to induce HO to validate the bioinformatic results. Macrophage-specific delete of Spp1 mouse model was established to observe the effects of macrophage-derived SPP1 on extracellular matrix remodeling and chondrogenesis of MPCs. Pharmacological blocking SPP1-Integrin receptors axis was performed both in vitro and in vivo to identify the essential role of SPP1. Single-cell transcriptome profiling analysis revealed Col12a1+ MPCs and SPP1+ macrophages were the critical clusters to initiate and maintain HO. Mechanistically, integrin receptors, especially Itgαv and Itgβ5, were the essential mediator to associate SPP1+ macrophage with MPCs to drive chondrogenesis. In vitro experiments revealed that Spp1 was involved in extracellular matrix (ECM) remodeling and chondrocyte differentiation of mesenchymal progenitor cells. Single cell RNA sequencing combined with in vivo experiments further confirmed that knocking out Spp1 in macrophages significantly alleviated cartilage formation within mouse Achilles’s tendon tissues and HO progression. Specific inhibiting SPP1-Itgαvβ5 axis or knocking out Spp1 gene in macrophages alleviated the progression of HO. Our findings revealed a novel crosstalk between MPC and macrophages via SPP1-integrin receptors axis to promote ECM remodeling and chondrogenic differentiation of MPCs during HO, which may be a potential target for a medical intervention to treat HO.