Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation.

The formation of traumatic heterotopic ossification (HO) is an abnormal repair process after soft tissue injury. Recent studies establish the involvement of immune cells and cellular metabolism in the tissue healing process; however, their role in HO remains unknown. Here, by using murine burn/tenotomy model in vivo and tendon stem/progenitor cells (TSPCs) osteogenic differentiation model in vitro, together with techniques including transgenic knockout, gene knockdown, transcriptome and proteome sequencings, mass spectrometry, co-immunoprecipitation, seahorse, etc., we reveal a novel p21-activated kinase 4 (Pak4) mediated crosstalk where the necroptotic macrophages arouse TSPCs with reduced fatty acid β-oxidation (FAO), to promote aberrant osteogenic differentiation during HO formation. Necroptosis blockade with Mlkl knockout (C57BL/6JGpt-Mlkl(em1Cd1679)/Gpt) significantly reduces HO than WT mice. Extracellular vesicle (EVs) secreted from necroptotic bone marrow-derived macrophages (BMDMs, NecroMφ-EVs) are determined to motivate FAO reduction in TSPCs and result in higher osteogenic activity. Pak4 conditional knockout (C57BL/6JGpt-Pak4(em1Cflox)/Gpt) in macrophage significantly increases FAO and reduces HO than Flox mice, as well as local injection of PAK4(-/-)-EVs (NecroMφ-EVs with Pak4 knockout) than NecroMφ-EVs, and the protective effects are reversed after transfection of Fabp3(S122D), a phosphomimetic mutant of S122 on fatty acid binding protein 3 (Fabp3) phosphorylation site. Mechanically, after soft tissue injury, macrophages infiltrate, and necroptosis occurs, accompanied by paracrine EVs-derived Pak4, which binds directly to Fabp3 and phosphorylates it at the S122 site in TSPCs, results in reduced FAO, finally osteogenic behavior, and HO formation. This study adds perceptiveness into abnormal regeneration-based theory for traumatic HO and raises treatment strategy development.