Procollagen C-Endopeptidase Enhancer 2 Secreted by Tonsil-Derived Mesenchymal Stem Cells Increases the Oxidative Burst of Promyelocytic HL-60 Cells

SIMPLE SUMMARY: Tonsil-derived mesenchymal stem cells (TMSCs) improved the reactive oxygen species (ROS) production in human promyelocytic leukaemia cells (HL-60) differentiated into neutrophil-like cells (dHL-60). TMSC-induced enhancement of ROS generation in dHL-60 cells was different depending on the TMSC donor. Comparison of RNA-sequencing data between high and low potentiating TMSC groups for ROS generation in dHL-60 cells showed elevated expressions of four genes: secreted frizzled-related protein 4, mesenteric estrogen-dependent adipogenesis, microfibrillar associated protein 5, and procollagen C-endopeptidase enhancer 2 (PCOLCE2). Real-time PCR and Western blotting confirmed high levels of PCOLCE2 in the high potentiating TMSC group for ROS generation in dHL-60 cells. In addition, knockdown of PCOLCE2 in TMSCs reduced the enhancing efficacy of TMSCs regarding ROS generation in dHL-60 cells. Finally, treatment of recombinant PCOLCE2 protein augmented ROS production in dHL-60 cells with concomitant increases of NADPH oxidase (NOX) 3, NOX4, NOX5, and dual oxidase 2. Taken together, this study showed that PCOLCE2 levels in TMSCs could be used to select TMSCs with the high potentiating ability for ROS generation in neutrophils, and both TMSCs and PCOLCE2 may have the potential to enhance a frontline defence by increasing the efficiency of ROS generation in neutrophils. ABSTRACT: Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.