Effective-mononuclear cell (E-MNC) therapy alleviates salivary gland damage by suppressing lymphocyte infiltration in Sjögren-like disease

Abstract

Introduction: Sjögren syndrome (SS) is an autoimmune disease characterized by salivary gland (SG) destruction leading to loss of secretory function. A hallmark of the disease is the presence of focal lymphocyte infiltration in SGs, which is predominantly composed of T cells. Currently, there are no effective therapies for SS. Recently, we demonstrated that a newly developed therapy using effective-mononuclear cells (E-MNCs) improved the function of radiation-injured SGs due to anti-inflammatory and regenerative effects. In this study, we investigated whether E-MNCs could ameliorate disease development in non-obese diabetic (NOD) mice as a model for primary SS. Methods: E-MNCs were obtained from peripheral blood mononuclear cells (PBMNCs) cultured for 7 days in serum-free medium supplemented with five specific recombinant proteins (5G culture). The anti-inflammatory characteristics of E-MNCs were then analyzed using a co-culture system with CD3/CD28-stimulated PBMNCs. To evaluate the therapeutic efficacy of E-MNCs against SS onset, E-MNCs were transplanted into SGs of NOD mice. Subsequently, saliva secretion, histological, and gene expression analyses of harvested SG were performed to investigate if E-MNCs therapy delays disease development. Results: First, we characterized that both human and mouse E-MNCs exhibited induction of CD11b/CD206-positive cells (M2 macrophages) and that human E-MNCs could inhibit inflammatory gene expressions in CD3/CD28- stimulated PBMNCs. Further analyses revealed that Msr1-and galectin3-positive macrophages (immunomodulatory M2c phenotype) were specifically induced in E-MNCs of both NOD and MHC class I-matched mice. Transplanted E-MNCs induced M2 macrophages and reduced the expression of T cell-derived chemokine-related and inflammatory genes in SG tissue of NOD mice at SS-onset. Then, E-MNCs suppressed the infiltration of CD4-positive T cells and facilitated the maintenance of saliva secretion for up to 12 weeks after E-MNC administration. Discussion: Thus, the immunomodulatory actions of E-MNCs could be part of a therapeutic strategy targeting the early stage of primary SS.

Keywords: Sjögren syndrome; cell therapy; macrophage; peripheral blood mononuclear cell; xerostomia.

FIGURE 1

Schematic diagram describing 5G-culture. PBMNCs were isolated from buffy coat and cultured for 6–7 days in serum-free medium supplemented with five recombinant proteins, TPO, VEGF, SCF, Flt-3 ligand, and IL-6. After cultivation, effective-mononuclear cells (E-MNCs) were obtained.

FIGURE 2

Characteristics of human E-MNCs. (A) Phase-contrast imaging of human PBMNCs (at day 0) and E-MNCs (at day 6). White boxed areas in the upper images (scale bar, 100 µm) were magnified in the lower images (scale bar, 100 µm). (B) Flow cytometric analysis of FSC/SSC gated cells, CD11b⁺/CD206⁻ [Monocytes and/or naïve (Mono-naïve) macrophages] and CD11b⁺/CD206⁺ (M2) macrophages, among PBMNCs (at day 0) and E-MNCs (at day 6). (C) Percentages of Mono-naïve (CD11b⁺/CD206⁻) and M2 (CD11b⁺/CD206⁺) macrophage fractions and their ratios (Mono-naïve/M2) among PBMNCs and E-MNCs (**p < 0.01). For statistical analysis, the Student’s t-test was performed to determine the significance of differences among PBMNCs and E-MNCs.

FIGURE 3

Characteristics of mouse E-MNCs. (A) Phase-contrast imaging of CB6F1 mouse PBMNCs (at day 0) and E-MNCs (at day 7). White boxed areas in the upper images (scale bar, 100 µm) were magnified in the lower images (scale bar, 100 µm). (B) Flow cytometric analysis of FSC/SSC gated cells, CD11b⁺/CD206⁻ (Mono-naïve) and CD11b⁺/CD206⁺ (M2) macrophages, among PBMNCs (at day 0) and E-MNCs (at day 7). (C) Percentages of Mono-naïve (CD11b⁺/CD206⁻) and M2 (CD11b⁺/CD206⁺) macrophage fractions and their ratios (Mono-naïve/M2) among PBMNCs and E-MNCs (*p < 0.05). (D) Flow cytometric analysis of CD11b⁺/Msr1⁺ (M2c; left panels) macrophages and CCR2/galectin3⁺ (right panels) CD11b-positive macrophages among PBMNCs (at day 0) and E-MNCs (at day 7). (E) Flow cytometric analysis of CCR6⁻/CCR4⁺ Th2 cells in CD3⁺/CD4⁺ gated cells of PBMNCs (at day 0) and E-MNCs (at day 7). (F) Expression of pro-inflammatory mRNAs (il-1β, ifn-γ, and tnf-α) in PBMNCs (at day 0) and E-MNCs (at day 7) (*p < 0.05, **p < 0.01). For statistical analysis, the Student’s t-test was performed to determine the significance of differences among PBMNCs and E-MNCs.

FIGURE 4

mRNA expression in CD3/CD28-stimulated PBMNCs after co-culture with E-MNCs. (A) Schematic diagram describing the experimental design for co-culture of E-MNCs and T-cell–activated PBMNCs. Anti-CD3 and -CD28 antibodies were added to the wells of a 24-well plate and incubated overnight. PBMNCs were then seeded in the wells and cultured at 37°C for 1 h. Subsequently, E-MNCs were seeded in the upper chamber and co-cultured with stimulated PBMNCs for 1 or 3 h (B and C) mRNA expression of tnf-α, ifn-γ, il-1β, il-6, il-4, and il-10 in PBMNCs and CD3/CD28-stimulated PBMNCs with/without E-MNCs for 1 h (B) or 3 h (C) (*p < 0.05, **p < 0.01). For statistical analysis, one-way ANOVA with post hoc Tukey’s multiple comparisons were performed for multiple groups.

FIGURE 5

Transplantation of E-MNCs into NOD mice at the onset of SS-like disease. (A) Schematic diagram describing the experimental design for E-MNC transplantation. E-MNCs were injected into the submandibular glands directly, and then saliva and saliva glands were harvested at 4, 8, and 12 weeks. (B) Changes in body weight and blood glucose of non-treated mice (Ctrl) and E-MNC–treated mice (E-MNC). (C) Change in saliva production (salivary flow rate; SFR) at 4, 8, and 12 weeks post-transplantation (**p < 0.01). (D) EGF concentration in saliva of non-treated mice (Ctrl) and E-MNC–treated mice (E-MNC) at 12 weeks post-transplantation (*p < 0.05). For statistical analysis, the Student’s t-test was performed to determine the significance of differences among Ctrl- and E-MNC-group.

FIGURE 6

Lymphocyte infiltration after E-MNC treatment. (A) CD4 staining of submandibular glands in non-treated mice (Ctrl) and E-MNC–treated mice (E-MNC) at 4 and 8 weeks post-transplantation (scale bar, 1,000 μm). Cells positive for CD4 (T-helper cells) appear brown in the foci. (B) Number of CD4-positive cells in lymphocyte infiltrated areas at 4 and 8 weeks (*p < 0.05). (C) H&E staining of submandibular glands in non-treated mice (Ctrl) and E-MNC–treated mice (E-MNC) at 4 and 8 weeks post-transplantation (scale bar, 200 µm). White dotted lines indicate the portion used to determine the focus score. (D) Focus score and area in non-treated mice (Ctrl) and E-MNC–treated mice (E-MNC) (*p < 0.05). For statistical analysis, the Student’s t-test was performed to determine the significance of differences among Ctrl- and E-MNC-group.

FIGURE 7

Detection of transplanted E-MNCs and M2 macrophages in submandibular glands. (A) At 3 days post-transplantation, PKH26-expressing E-MNCs (red) were detected in the interlobular space (I) (yellow dotted circle) and parenchyma (P) of submandibular glands (white dotted lines, boundary between interlobular area and parenchyma; red, PKH26-labeled cells; blue, DAPI; scale bar, 100 μm). (B) At 3 days post-transplantation, PKH26-expressing E-MNCs (red) were detected in the parenchyma (P) of submandibular glands. White dotted square area was magnified in the lower right panel (red, PKH26-labeled cells; blue, DAPI; scale bar, 100 μm). (C) At 7 days post-transplantation, PKH26-expressing E-MNCs (red) migrated to the deeper interlobular space (I) and penetrated into the parenchyma (P) (yellow dotted circle) in submandibular glands. White dotted square area was magnified in the lower right panel (white dotted lines, boundary between interlobular and parenchyma; red, PKH26-labeled cells; blue, DAPI; scale bar, 100 μm). (D) At 1 day post-transplantation, PKH26-expressing E-MNCs and host M2 macrophages expressed F4/80 (white) and CD206 (green) in the parenchyma of the submandibular glands (blue, DAPI; scale bar, 100 μm). (E) Detection of transplanted E-MNCs (red) and CD206-positive cells (green) in E-MNC-treated glands (left image) and non-treated glands (right image) (blue, DAPI; scale bar, 50 μm). (F) Number of CD206-positive cells/field (×200) in the parenchyma of submandibular gland tissues in E-MNC-treated glands (E-MNC) and non-treated glands (Ctrl) at 7 days post-transplantation (**p < 0.01). For statistical analysis, the Student’s t-test was used to analyze differences between E-MNC-group and Ctrl-group.

FIGURE 8

Gene expression in submandibular glands in the initial post-transplantation stage. (A,B) mRNA expression of the inflammatory chemokines ccl5, ccl6, ccl8, ccl19, and cxcl9 in non-treated glands (Ctrl) and E-MNC–treated glands (E-MNC) at 1 day (A) and 3 days (B) post-transplantation (**p < 0.01). (C,D) Expression of the pro-inflammatory genes tnf-α, inf-γ, and il-1β in non-treated glands (Ctrl) and E-MNC–treated glands (E-MNC) at 1 day (C) and 3 days (D) post-transplantation (*p < 0.05, **p < 0.01). For statistical analysis, the Student’s t-test was used to analyze differences between E-MNC-group and Ctrl-group.

FIGURE 9

Diagram of the cellular mechanism of E-MNC transplantation at the onset of SS in NOD mice. E-MNCs function in a paracrine manner to inhibit chemotaxis of pathogenic T cells and improve the tissue microenvironment via the immunomodulatory effects of M2 macrophages and/or Th2 cells among E-MNCs during the initial stage after transplantation.