Cell-free secretome of CD56(bright)CD16(bright) directly reprogrammed NK cells enhances wound healing via CCL3/4/5-CCR5 signaling.

Rationale: Natural killer (NK) cells are emerging as a promising source of immunomodulatory secretomes with regenerative potential. However, heterogeneity in primary NK cell populations limits the reproducibility of NK-derived cell-free therapies. To address this, we developed directly reprogrammed NK (drNK) cells with a stable CD56(bright)CD16(bright) phenotype and investigated the therapeutic potential of their conditioned medium (drNK-CM) in wound healing, focusing on underlying molecular mechanisms such as chemokine signaling and angiogenesis. Methods: drNK cells were generated by transcription factor-mediated reprogramming (OCT4, SOX2, KLF4, MYC) and characterized via flow cytometry and RNA-seq. The secretome profile of drNK-CM was evaluated using proteomic analysis. Human epidermal keratinocytes (HEKs), dermal fibroblasts (HDFs), and endothelial cells (HUVECs) were treated with drNK-CM to assess proliferation, migration, and extracellular matrix (ECM) remodeling. Chemokine receptor involvement was evaluated using CCR1, CCR3, and CCR5 antagonists. In vivo efficacy was tested in mouse excisional wound models, with histological and immunofluorescence evaluation of angiogenesis, re-epithelialization, and collagen deposition. Results: drNK-CM significantly promoted proliferation and migration of HEKs, HDFs, and HUVECs, accompanied by enhanced expression of Type I/III collagen, VEGF, and MMPs. Transcriptomic profiling revealed that drNKs uniquely upregulated genes associated with ECM remodeling, chemokine signaling (CCL3/4/5), and angiogenesis. Notably, CCR5 inhibition by maraviroc abrogated drNK-CM-induced cell migration and delayed wound closure in vivo, highlighting the central role of the CCL3/4/5-CCR5 axis. Furthermore, drNK-CM activated AKT and ERK pathways and promoted anti-inflammatory macrophage polarization. In vivo application of drNK-CM accelerated wound closure, improved neovascularization, and supported organized tissue regeneration compared to controls. Conclusion: This study demonstrates that drNK-CM enhances wound healing through coordinated actions on epithelial, stromal, and endothelial compartments. The reparative effects are primarily mediated via the CCL3/4/5-CCR5 signaling axis and pro-angiogenic cascades. Given their consistent phenotype and reproducible secretome, drNKs represent a scalable and safe source for cell-free regenerative therapeutics.