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

## TL;DR

This study shows how necroptotic macrophages interact with tendon stem cells through Pak4 to cause abnormal bone formation after injury.

## Contribution

A novel Pak4-mediated crosstalk mechanism linking necroptotic macrophages and tendon stem cells in traumatic heterotopic ossification is identified.

## Key findings

- Necroptotic macrophages release EVs that reduce fatty acid oxidation in tendon stem cells, promoting osteogenic differentiation.
- Pak4 knockout in macrophages increases fatty acid oxidation and reduces heterotopic ossification in mice.
- Phosphorylation of Fabp3 at S122 by Pak4 disrupts fatty acid oxidation, leading to HO formation.

## Abstract

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-Mlklem1Cd1679/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-Pak4em1Cflox/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 Fabp3S122D, 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.