Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses

Abstract

Aim: To investigate the potential therapeutic effects of mesenchymal stem cells (MSCs) in inflammatory bowel disease (IBD), we transplanted MSCs into an experimental model of IBD.

Methods: A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg body weight) was administered to female BALB/c mice. Bone marrow mesenchymal stem cells (BMSCs) were derived from male green fluorescent protein (GFP) transgenic mice and were transplanted intravenously into the experimental animals after disease onset. Clinical activity scores and histological changes were evaluated. GFP and Sex determining region Y gene (SRY) expression were used for cell tracking. Ki67 positive cells and Lgr5-expressing cells were determined to measure proliferative activity. Inflammatory response was determined by measuring the levels of different inflammatory mediators in the colon and serum. The inflammatory cytokines included tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-17, IL-4, IL-10, and transforming growth factor (TGF-β). Master regulators of Th1 cells (T-box expressed in T cells, T-bet), Th17 cells (retinoid related orphan receptor gamma(t), RORγt), Th2 cells (GATA family of transcription factors 3, GATA3) and regulatory T cells (forkhead box P3, Foxp3) were also determined.

Results: Systemic infusion of GFP-BMSCs ameliorated the clinical and histopathologic severity of colitis, including body weight loss, diarrhea and inflammation, and increased survival (P < 0.05). The cell tracking study showed that MSCs homed to the injured colon. MSCs promoted proliferation of intestinal epithelial cells and differentiation of intestinal stem cells (P < 0.01). This therapeutic effect was mainly mediated by down-regulation of both Th1-Th17-driven autoimmune and inflammatory responses (IL-2, TNF-α, IFN-γ, T-bet; IL-6, IL-17, RORγt), and by up-regulation of Th2 activities (IL-4, IL-10, GATA-3) (P < 0.05). MSCs also induced activated CD4(+)CD25(+)Foxp3(+) regulatory T cells (TGF-β, IL-10, Foxp3) with a suppressive capacity on Th1-Th17 effecter responses and promoted Th2 differentiation in vivo (P < 0.05).

Conclusion: MSCs are key regulators of immune and inflammatory responses and may be an attractive candidate for cell-based therapy of IBD.

Keywords: Immunomodulation; Inflammatory bowel disease; Inflammatory response; Mesenchymal stem cells; Therapy; Transplantation; Trinitrobenzene sulfonic acid colitis.

Figure 1

Morphological features, immunophenotypic characterization and differentiation assays of compact bone-derived mouse mesenchymal stem cells. A: At 72 h after initial culture, the cells migrate out from the enzyme-digested bone chips (× 100); B: Green fluorescent protein -bone marrow mesenchymal stem cells (BMSCs) arranged in a bunched or radiated shape without weakening of the green fluorescence after passages (× 400); C: Osteoplastic differentiation of BMSCs (× 400); D: Apoplastic differentiation of BMSCs (× 400); E-H: BMSCs’ phenotypes detected by flow cytometry.

Figure 2

Localization of transplanted green fluorescent protein-bone marrow mesenchymal stem cells. A: Fluorescence in the lamina propria of colon using fluorescence microscopy (× 100); B: Sry gene expression (1188 bp)in the colons of 3 groups by polymerase chain reaction. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 3

Therapeutic efficacy of bone marrow mesenchymal stem cells treatment. A: Colon length at day 3; B: Macroscopic colonic damage at day 3; C: Histopathologic colonic damage by hematoxylin and eosin staining at day 3 (× 100); D-F: Clinical evolutions were monitored by body weight changes, colitis score and survival; G) Colon length changes; H: Histopathology score changes. ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 4

Immunohistochemistry of Ki67 and Lgr5 in colon tissues (× 200). A-C: Ki67; D-F: Lgr5; G, H: Five days after MSCs transplantation, Ki67 and Lgr5 expressions were significantly increased (vs control, ^bP < 0.01; vs TNBS-PBS, ^dP < 0.01). Ki67 staining was evaluated by positivity, and Lgr5 expression was represented by mean optical density. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 5

Levels of 7 cytokines were measured simultaneously from mouse serum using Fluorescence Activated Cell Sorting. A-E: TNBS-induced colitis exhibits heightened Th1-Th17 response of a complex, dynamic cytokine network [interleukin (IL)-2, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17], and MSCs maintain the balance of T-lymphocytes. F, G: In the TNBS-MSC group, transplanted MSCs promote T-lymphocytes to secrete cytokines (IL-4, IL-10) which regulate their functions. ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 6

Changes in mRNA expression and colonic protein levels of Th1-related inflammatory mediators following treatment with bone marrow mesenchymal stem cells. A-D: Real-time polymerase chain reaction. Acute TNBS colitis demonstrated a cytotoxic and chemotactic profile with significantly elevated mRNA expressions of interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and T-bet when compared to controls. Preventive treatment with BMSCs reduced the levels of the above cytokines; E-G: Flow cytometry. The changes in colonic protein levels of IL-2, IFN-γ and TNF-α followed the same trend; H: Western blotting. The changes in colonic protein levels of T-bet followed the same trend. ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 7

Changes in mRNA expression and colonic protein levels of Th2-related inflammatory mediators following treatment with bone marrow mesenchymal stem cells. A-C: Real-time polymerase chain reaction. No significant differences in mRNA expressions of interleukin (IL)-4, IL-10 and GATA family of transcription factors 3 (GATA-3) were observed when compared to controls (P > 0.05). After treatment with bone marrow mesenchymal stem cells, the mRNA expressions of IL-4, IL-10 and GATA-3 were elevated. D, E: Flow cytometry. Colonic protein levels of IL-4 and IL-10 followed the same trend; F: Western blotting. Colonic protein level of GATA-3 followed the same trend. ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 8

Changes in mRNA expression and colonic protein levels of Th17-related inflammatory mediators following treatment with bone marrow mesenchymal stem cells. A-C: Real-time polymerase chain reaction. The results showed a significant increase in mRNA expressions of interleukin (IL)-6, IL-17A and retinoid related orphan receptor gamma(t) (RORγt) when compared to controls (P < 0.01). After treatment with bone marrow mesenchymal stem cells, the levels of the above cytokines were reduced (P < 0.01); D, E: Flow cytometry. The same changes in colonic protein levels of IL-6 and IL-17A were further confirmed; F: Western blotting. The same changes in colonic protein levels of RORγt were further confirmed. ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.

Figure 9

Changes in mRNA expression and colonic protein levels of Tregs-related inflammatory mediators following treatment with bone marrow mesenchymal stem cells. A, B: Real-time polymerase chain reaction. No significant changes in mRNA expressions of transforming growth factor (TGF)-β and Foxp3 were observed when compared to controls (P > 0.05). While the mRNA expressions of TGF-β and Foxp3 increased after BMSCs transplantation (P < 0.01); C, D: Western blotting. The colonic protein level of TGF-β and Foxp3 were confirmed to show the same results as real-time polymerase chain reaction. ^cP < 0.05, ^dP < 0.01 vs TNBS-PBS. TNBS-MSC: Trinitrobenzene sulfonic acid-mesenchymal stem cells; TNBS-PBS: Trinitrobenzene sulfonic acid-phosphate-buffered saline.