Immunomodulatory Macrophages Enable E-MNC Therapy for Radiation-Induced Salivary Gland Hypofunction

Abstract

A newly developed therapy using effective-mononuclear cells (E-MNCs) is reportedly effective against radiation-damaged salivary glands (SGs) due to anti-inflammatory and revascularization effects. However, the cellular working mechanism of E-MNC therapy in SGs remains to be elucidated. In this study, E-MNCs were induced from peripheral blood mononuclear cells (PBMNCs) by culture for 5-7 days in medium supplemented with five specific recombinant proteins (5G-culture). We analyzed the anti-inflammatory characteristics of macrophage fraction of E-MNCs using a co-culture model with CD3/CD28-stimulated PBMNCs. To test therapeutic efficacy in vivo, either E-MNCs or E-MNCs depleted of CD11b-positive cells were transplanted intraglandularly into mice with radiation-damaged SGs. Following transplantation, SG function recovery and immunohistochemical analyses of harvested SGs were assessed to determine if CD11b-positive macrophages contributed to tissue regeneration. The results indicated that CD11b/CD206-positive (M2-like) macrophages were specifically induced in E-MNCs during 5G-culture, and Msr1- and galectin3-positive cells (immunomodulatory macrophages) were predominant. CD11b-positive fraction of E-MNCs significantly inhibited the expression of inflammation-related genes in CD3/CD28-stimulated PBMNCs. Transplanted E-MNCs exhibited a therapeutic effect on saliva secretion and reduced tissue fibrosis in radiation-damaged SGs, whereas E-MNCs depleted of CD11b-positive cells and radiated controls did not. Immunohistochemical analyses revealed HMGB1 phagocytosis and IGF1 secretion by CD11b/Msr1-positive macrophages from both transplanted E-MNCs and host M2-macrophages. Thus, the anti-inflammatory and tissue-regenerative effects observed in E-MNC therapy against radiation-damaged SGs can be partly explained by the immunomodulatory effect of M2-dominant macrophage fraction.

Keywords: cell therapy; macrophage; peripheral blood mononuclear cells; radiation-induced damage; salivary gland.

Figure 1

Schematic diagram of 5G-culture. Cultured PBMNCs were enriched in a population of CD11b-positive cells (M1- and specifically induced M2-macrophage-like cells).

Figure 2

Characteristics of human E-MNCs. (A) Phase contrast images of MNCs. White arrows indicate round-shaped macrophage-like cells. Scale bar: 200 µm. (B) Analysis with flow cytometry. FSC/SSC gated cells, and M1; CD11b⁺/CD206⁻, M2; CD11b⁺/CD206⁺ macrophages among PBMNCs (day 0) and E-MNCs (day 7). (C) Flow cytometric analysis of FSC/SSC gated cells, and M1; CD11b⁺/CD206⁻, M2; CD11b⁺/CD206⁺ macrophages among CD11b-positive and -negative cell fractions after sorting CD11b-postive cells of E-MNCs with MACS. (D) mRNA expression in CD3/CD28-stimulated PBMNCs after co-culture with E-MNCs and CD11b-positive cells and CD11b-negative cells (* p < 0.05, ** p < 0.01). (E) Phagocytosis assay with fluorescent beads. Flow cytometric analysis of FITC-expressing cells at 0, 1, 2, and 3 h after addition of fluorescent beads (right panel) in FSC/SSC gated cells of E-MNCs (left panel). (F) Phagocytosis assay with fluorescent beads. Phase-contrast and fluorescence microscopic images of macrophage-like round- and spindle-shaped cells among E-MNCs at 1 h. Green: fluorescent beads; blue: DAPI. Scale bar: 20 µm.

Figure 3

Characteristics of mouse E-MNCs. (A) Analysis of flow cytometry. M2; CD11b⁺/CD206⁺, M2c; CD11b⁺/Msr1⁺, and M2c; CCR2⁻/galectin3⁺ macrophages among PBMNCs and E-MNCs. (B) Analysis of the characters of CD11b-positive E-MNCs. Double immunofluorescence staining of CD11b cells (green) for CD206, Msr1, or IGF1 (red). Blue: DAPI; negative Ctrl (control), no primary antibody. Scale bar: 50 µm.

Figure 4

Transplantation of E-MNCs or CD11b-negative E-MNCs into a prevention model. (A) Analysis of flow cytometry. M1; CD11b⁺/CD206⁻) and M2; CD11b⁺/CD206⁺ macrophage fractions among PBMNCs, E-MNCs, CD11b-positive cells (red box area in E-MNCs), and CD11b-negative cells (yellow box area in E-MNCs), respectively. (B) Changes in saliva production (salivary flow rate; SFR) at 1, 2, 5, 9, and 13 weeks post-IR in each group. Lower graphs are scatter diagrams of SFR at specific time point (5, 9, and 13 weeks post-IR) for all groups (* p < 0.05, ** p < 0.01). (C) Concentrations of salivary EGF and HGF at 9 and 13 weeks post-IR (* p < 0.05, ** p < 0.01).

Figure 5

Fibrosis in submandibular glands after irradiation. (A) Histological analysis of submandibular glands at 9 weeks post-IR. Each panel shows representative images. Upper panels: HE staining; lower panels: Masson’s trichrome staining in each group. Fibrosis areas are stained blue. Scale bar: 200 µm. (B) Fibrosis area per field (%) in submandibular glands at 9 weeks (* p < 0.05, ** p < 0.01). (C) Histological analysis of submandibular glands at 13 weeks post-IR. Each panel shows representative images. Upper panels: HE staining; lower panels: Masson’s trichrome staining in each group in each group. Fibrosis areas are recognized as blue. Scale bar: 200 µm. (D) Percentage (%) of fibrosis area per field in submandibular glands at 13 weeks (* p < 0.05).

Figure 6

Gene expression in submandibular glands. (A,B) Expression of mRNAs of genes associated with proinflammation (IL-1β, IL-6, TLR2, TLR4), anti-inflammation (IL-10, CD206), tissue regeneration (IGF1), and fibrosis (TGFβ) in submandibular glands of nontreated (IR), E-MNC-treated, and CD11b(−)-treated mice at 10 days (A) and 2 weeks (B) of IR (* p < 0.05, ** p < 0.01).

Figure 7

Detection of M2-macrophages in submandibular glands at initial stages after transplantation. (A,B) Double-stained images of F4/80 (pink) and CD206 (red) (upper panel) and single-stained images of CD206 (red) (lower panel) in tissues at 10 days (A) and 2 weeks (B) in each group. White arrow: F4/80⁺/CD206⁺ M2-macrophages. Green: transplanted E-MNCs or CD11b(−) cells. Blue: DAPI. Scale bar: 50 µm. (C) Graphs show the number of F4/80- and CD206-expressing cells in submandibular glands at 10 days and 2 weeks post-IR (* p < 0.05, ** p < 0.01).

Figure 8

Detection of HMGB1 clearance in submandibular glands in the initial stages after transplantation. ((A,C) upper panel) Detection of released HMGB1 (red) in submandibular glands at 10 days (A) and 2 weeks (C) post-IR. Green: transplanted E-MNCs or CD11b(−) cells. Blue: DAPI. Scale bar: 50 µm. ((A,C) lower panel) Detection of Msr1-positive cells (M2c-macrophages) (pink) in submandibular glands at 10 days (A) and 2 weeks (C) post-IR. White arrow: Msr1-positive donor (EGFP⁺) cells. Green: transplanted E-MNCs or CD11b(−) cells. Blue: DAPI. Scale bar: 50 µm. (B,D) Merged images of HMGB1 (red) and Msr1 (pink) staining (yellow box areas in (A,C)) in E-MNC-injected SGs at 10 days (B) and 2 weeks (D) post-transplantation. White arrows: HMGB1⁺/Msr1⁺/EGFP⁺ E-MNCs. Blue: DAPI. Scale bar: 50 µm. (E) Graphs show the number of Msr1-positive cells in submandibular glands at 10 days or 2 weeks post-IR (* p < 0.05, ** p < 0.01). (F) Graph shows the concentration of HMGB1 in glands at 5 weeks (* p < 0.05, ** p < 0.01).

Figure 9

IGF1 production in phagocytic M2-macrophages in the initial stages after transplantation. (A,B) Detection of IGF1—(red) and Msr1 (pink)—expressing cells in gland tissues at 10 days (A) and 2 weeks (B) post-IR. Green: transplanted E-MNCs or CD11b(−) cells. White arrows: IGF1⁺/Msr1⁺/EGFP⁺ E-MNCs. Blue: DAPI. Scale bar: 50 µm. (C) Graphs show the number of IGF1-expressing cells in submandibular glands at 10 days or 2 weeks (* p < 0.05, ** p < 0.01).

Figure 10

C-Kit/Sca-1 expressions in gland tissues at 2 weeks of IR. (A) c-Kit (green)/Sca-1 (red) expressions in each group. White arrows: c-Kit⁺/Sca-1⁺ (ductal stem/progenitor) cells. Blue: DAPI. Scale bar: 50 µm. (B) Number of c-Kit/Sca-1-expressing cells in each group (* p < 0.05, ** p < 0.01).

Figure 11

Transplantation of E-MNCs or CD11b-negative E-MNCs into an established model of radiation injury to salivary glands. (A) Changes in saliva production (SFR) at 2, 5, 9, and 13 weeks post-IR in each group. Lower figures, scatter diagram of SFR of each specimen in all groups at 9 and 13 weeks (* p < 0.05, ** p < 0.01). (B,C) Histological analysis of submandibular glands at 9 weeks (B) and 13 weeks (C) post-IR. Each panel shows representative images. Upper panels: HE staining; and lower panels: Masson’s trichrome staining in each group. Areas of fibrosis are stained blue. Scale bar: 200 µm. (D) Percentage of fibrosis area per field in gland tissues at 9 weeks after IR (* p < 0.05, ** p < 0.01). (E) Percentage of fibrosis area per field in gland tissues at 13 weeks of IR (* p < 0.05).

Figure 12

Schematic diagram of the suggested mechanism of E-MNC treatment. CD11b-positive cells among E-MNCs might contribute to convert the condition of damaged tissue from a proinflammation to an anti-inflammation and promote tissue regeneration by mediating DAMP clearance and IGF1 production in cooperation with host macrophages.