Concentrated growth factor regulates the macrophage-mediated immune response

Abstract

Concentrated growth factor (CGF) is a promising regenerative material that serves as a scaffold and adjunct growth factor for tissue engineering. The host immune response, particularly macrophage activity, plays a critical role in injury repair and tissue regeneration. However, the biological effect of CGF on the immune response is not clear. To enrich the theoretical groundwork for clinical application, the present study examined the immunoregulatory role of CGF in macrophage functional activities in vitro. The CGF scaffold appeared as a dense fibrin network with multiple embedded leukocytes and platelets, and it was biocompatible with macrophages. Concentrated bioactive factors in the CGF extract enhanced THP-1 monocyte recruitment and promoted the maturation of suspended monocytes into adherent macrophages. CGF extract also promoted THP-1 macrophage polarization toward the M2 phenotype with upregulated CD163 expression, as detected by cell morphology and surface marker expression. A cytokine antibody array showed that CGF extract exerted a regulatory effect on macrophage functional activities by reducing secretion of the inflammatory factor interleukin-1β while inducing expression of the chemokine regulated on activation, normal T cell expressed and secreted. Mechanistically, the AKT signaling pathway was activated, and an AKT inhibitor partially suppressed the immunomodulatory effect of CGF. Our findings reveal that CGF induces a favorable immune response mediated by macrophages, which represents a promising strategy for functional tissue regeneration.

Keywords: concentrated growth factor; immune response; macrophage; signaling pathway.

Figure 1.

Morphological features and cytocompatibility of the CGF scaffold. (A) Ten milliliters of whole blood after centrifugation. (B) A buffy coat of CGF gel separated from the blood sample. (C) The fibrin network membrane of the CGF scaffold after compression. (D) Histological features of the upper part of the CGF scaffolds evaluated using H & E staining. (E) Leukocytes and red blood cells embedded in the lower portion. (F) The surface ultrastructure of the CGF scaffold observed by scanning electron microscopy. (G) Platelets (yellow arrow) and leukocytes (red arrow) embedded in CGF scaffolds. (H) Macrophages (white arrow) seeded on the CGF scaffolds were fully stretched on the network surface.

Figure 2.

CGF extract induced monocyte migration and maturation. (A) THP-1 monocyte migration increased significantly in the presence of CGF-conditioned medium (CCM), as determined by a Transwell assay (n = 3). (B) CCM significantly induced fusion and adherence of THP-1 monocytes (n = 5). (C) The number of adherent PMA-induced THP-1 macrophages was significantly increased with CCM treatment (n = 5). The data are presented as the mean ± SD. Significance was determined using one-way analysis of variance (ANOVA) and post hoc Dunnett tests: *P < 0.05, **P < 0.01 and ***P < 0.001. CGF-conditioned medium (100% CCM) was diluted with medium to 50, 20 and 10% CCM, and medium lacking CCM served as the control (CTR).

Figure 3.

CGF extract promoted macrophage polarization toward the M2 phenotype. (A) Morphological features of CCM-treated THP-1 macrophages were observed under an inverted microscope. CCM groups showed a smaller nuclear-to-cytoplasmic ratio and an elongated cell shape. (B) The M2 (CD163+/CD206+) and M1 (CD80+/CD86+) macrophage phenotypes were analyzed using flow cytometry. (C) The percentage of M2 macrophages increased after CCM treatment, but no variation was observed in the percentage of M1 macrophages. (D) The expression of CD163 mRNA was upregulated, and CD80 mRNA was downregulated in THP-1 macrophages cultured with CCM. (E) Green fluorescent staining of CD163 was increased in THP-1 macrophages treated with CCM. DAPI staining of the nucleus is shown in blue. The quantitative data are presented as the mean ± SD (n = 3), and significance was determined using one-way analysis of variance (ANOVA) and post hoc Dunnett tests: *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4.

CGF extract modulated macrophage cytokine secretion. (A) The supernatants of CCM-treated macrophages were screened using a human cytokine antibody array. (B) Representative cytokines are presented. (C) A GO analysis of differentially expressed cytokines enriched in BP terms after 20% CCM treatment. (D) Enriched MF categories. (E) The mRNA expression of differentially expressed cytokines (IL-1β, IL-7, RANTES and MCP-1) in CCM-treated macrophages was analyzed using q-PCR. (F) The secretion of IL-1β and RANTES by CCM-treated macrophages was further confirmed using ELISAs. The quantitative data are presented as the mean ± SD (n = 3) and were analyzed using one-way analysis of variance (ANOVA) and post hoc Dunnett tests: *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

CGF extract regulated the macrophage-mediated immune response via the AKT pathway. (A) A western blot analysis of the proteins involved in the PI3K/AKT (p-AKT, AKT and PI3K) and JAK/STAT (p-JAK, JAK, p-STAT3 and STAT3) signaling pathways in CCM-treated THP-1 macrophages. AKT phosphorylation was increased, but no significant differences were observed in the JAK/STAT pathway (n = 3). (B) The effect of an AKT inhibitor on AKT pathway inhibition was analyzed using western blotting (n = 3). (C) Representative images of adherent THP-1 macrophages cultured in CCM with or without AKT inhibitor were analyzed using ImageJ software (n = 5). (D) The secretion of IL-1β and RANTES by THP-1 macrophages were assayed using ELISAs (n = 3). (E) Expression of the M2 subtype marker CD163 was detected using immunofluorescence staining (n = 3). The data are presented as the mean ± SD and were analyzed using one-way analysis of variance (ANOVA) and post hoc tests: *P < 0.05, **P < 0.01 and ***P < 0.001.