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

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.

Keywords: donor variation; neutrophil; oxidative burst; procollagen C-endopeptidase enhancer 2; tonsil derived mesenchymal stem cell.

Figure 1

Tonsil-derived mesenchymal stem cells (TMSCs) potentiate reactive oxygen species (ROS) generation from differentiated HL-60 cells (dHL-60). (A) The levels of differentiation markers, CD11b, CD35, and CD71, measured by flow cytometry analysis. (B) The viability of dHL-60 cells measured after coculture with TMSCs or bone marrow-derived mesenchymal stem cells (BMSCs) for 48 h. (C) ROS generation from phorbol 12-myristate 13-acetate (PMA)-stimulated dHL-60 cells cocultured with TMSCs and BMSCs. (D) Donor variation of TMSCs in the potentiating capacity of ROS generation from dHL-60 cells. (E) The average ROS generation and (F) doubling time of high and low ROS groups. Values are expressed as the mean ± SEM (n = 3–25, *** p < 0.001).

Figure 2

RNA-sequencing data analysis of tonsil-derived mesenchymal stem cells (TMSCs) distributed in the high and low reactive oxygen species (ROS) groups. (A) A dendrogram of hierarchical clustering, (B) a heat map, (C) genes significantly up- and downregulated by more than 1.5-fold, and (D) a volcano plot between the high and low ROS groups. The Gene Ontology analysis of (E) biological processes, (F) cellular component, and (G) molecular function (** p < 0.01; *** p < 0.001).

Figure 3

PCOLCE2 expression is increased in the high reactive oxygen species (ROS) group compared with the low ROS group. (A) Altered genes by RNA sequencing more than 2-fold in the high ROS group compared with the low ROS group. The (B) increased and (C) decreased genes in the high ROS group were confirmed using real-time PCR. The protein levels of PCOLCE2 were analysed with (D) cell lysates and (E) conditioned medium using Western blotting and quantified with ImageJ (Figure S2). Values are expressed as the mean ± SEM (n = 6−7; * p < 0.05; *** p < 0.001). APCDD1—adenomatosis polyposis downregulated 1; COL8A1—collagen type VIII alpha 1 chain; MEDAG—mesenteric estrogen-dependent adipogenesis; MFAP5—microfibrillar associated protein 5; PCOLCE2—procollagen C-endopeptidase enhancer 2; PDPN—podoplanin; PSG1—pregnancy-specific beta-1-glycoprotein 1; RUNX3—runt related transcription factor 3; SFRP4—Secreted frizzled-related protein 4; SNAP25—synaptosome associated protein 25.

Figure 4

PCOLCE2 promotes reactive oxygen species (ROS) generation from dHL-60 cells. (A) The siRNA-mediated PCOLCE2 knockdown confirmed with real-time PCR. (B) ROS generation from dHL-60 cells after (B) coculture with PCOLCE2 knockdown tonsil-derived mesenchymal stem cells (TMSCs) or (C) addition of PCOLCE2 protein. (D) ROS generation from dHL-60 cells treated with PCOLCE2 protein or bovine serum albumin (BSA). (E) The cell viability after PCOLCE2 treatment. Values are expressed as the mean ± SEM (n = 5; * p < 0.05; ** p < 0.01; *** p < 0.001). The siNeg-TMSC—TMSCs treated with siRNA for negative control; siPCOLCE2-TMSC—TMSCs transfected with siRNA for PCOLCE2.

Figure 5

Alteration of genes involved in neutrophil function by PCOLCE2. Expression of genes involved in (A) oxidative burst, (B) formation of neutrophil extracellular traps, and (C) phagocytosis were analysed by real-time PCR. Values are expressed as mean ± S.E.M. (n = 4; * p < 0.05; ** p < 0.01; *** p < 0.001). DUOX—dual oxidase; MAP2K2—mitogen-activated protein kinase kinase 2; MAPK3—mitogen-activated protein kinase 3; NOX—NADPH oxidase; PADI4—peptidyl arginine deiminase 4; PTK2—protein tyrosine kinase 2; RAC2—Rac family small GTPase 2; RAF1—Raf-1 proto-oncogene, serine/threonine kinase.