Melatonin Synergizes With Mesenchymal Stromal Cells Attenuates Chronic Allograft Vasculopathy

Abstract

Background: Chronic rejection characterized by chronic allograft vasculopathy (CAV) remains a major obstacle to long-term graft survival. Due to multiple complicated mechanisms involved, a novel therapy for CAV remains exploration. Although mesenchymal stromal cells (MSCs) have been ubiquitously applied to various refractory immune-related diseases, rare research makes a thorough inquiry in CAV. Meanwhile, melatonin (MT), a wide spectrum of immunomodulator, plays a non-negligible role in transplantation immunity. Here, we have investigated the synergistic effects of MT in combination with MSCs in attenuation of CAV.

Methods: C57BL/6 (B6) mouse recipients receiving BALB/c mouse donor aorta transplantation have been treated with MT and/or adipose-derived MSCs. Graft pathological changes, intragraft immunocyte infiltration, splenic immune cell populations, circulating donor-specific antibodies levels, cytokine profiles were detected on post-operative day 40. The proliferation capacity of CD4⁺ and CD8⁺ T cells, populations of Th1, Th17, and Tregs were also assessed in vitro.

Results: Grafts in untreated recipients developed a typical pathological feature of CAV characterized by intimal thickening 40 days after transplantation. Compared to untreated and monotherapy groups, MT in combination with MSCs effectively ameliorated pathological changes of aorta grafts indicated by markedly decreased levels of intimal hyperplasia and the infiltration of CD4⁺ cells, CD8⁺ cells, and macrophages, but elevated infiltration of Foxp3⁺ cells. MT either alone or in combination with MSCs effectively inhibited the proliferation of T cells, decreased populations of Th1 and Th17 cells, but increased the proportion of Tregs in vitro. MT synergized with MSCs displayed much fewer splenic populations of CD4⁺ and CD8⁺ T cells, Th1 cells, Th17 cells, CD4⁺ central memory T cells (Tcm), as well as effector memory T cells (Tem) in aorta transplant recipients. In addition, the percentage of splenic Tregs was substantially increased in the combination therapy group. Furthermore, MT combined with MSCs markedly reduced serum levels of circulating allospecific IgG and IgM, as well as decreased the levels of pro-inflammatory IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, and MCP-1, but increased the level of IL-10 in the recipients.

Conclusions: These data suggest that MT has synergy with MSCs to markedly attenuate CAV and provide a novel therapeutic strategy to improve the long-term allograft acceptance in transplant recipients.

Keywords: aorta transplantation; chronic allograft vasculopathy; melatonin; mesenchymal stromal cells; mice.

Figure 1

Characterization of adipose derived MSCs. (A) Morphology of p3 passage MSCs. The section is displayed at 100× magnification. The MSCs display a fibroblast-like or spindle-shaped morphology. (B) The expression of cell markers on the surface of MSCs measured by flow cytometry analysis. MSCs are positive for CD29 (100%), SCA-1 (99.2%), CD44 (91.3%), while negative for CD34 (0.2%) and CD45 (1.26%). MSCs, mesenchymal stromal cells.

Figure 2

MT either alone or combined with MSCs suppresses T cell proliferation in vitro. The splenocytes derived from B6 mice are co-cultured with MT, MSCs, and MT+MSCs respectively in the presence of the stimulators of anti-CD3 Abs, anti-CD28 Abs and IL-2 in vitro. (A) The pseudocolor of CD4⁺ and CD8⁺ T cells in vitro; (B) Contour plot of CD4⁺Ki67⁺ and CD8⁺Ki67⁺ T cells in vitro; (C) The percentage of CD4⁺ T cells; (D) The percentage of CD8⁺ T cells; (E) The percentage of CD4⁺Ki67⁺ T cells; (F) The percentage of CD8⁺Ki67⁺ T cells. Statistical analysis is performed by one-way analysis of variance (ANOVA), ^** p <.01 and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stromal cells; Sp, splenocytes; St, stimulators. The assay was conducted three times with three replicates each time.

Figure 3

MT either alone or combined with MSCs reduces Th1 and Th17 populations while promoting Treg population in vitro. The splenocytes derived from B6 mice were co-cultured with MT, MSCs, and MT+MSCs respectively in the presence of the stimulators of anti-CD3 Abs, anti-CD28 Abs, and IL-2 in vitro. (A) The pseudocolor of Th1 (CD4⁺IFN-γ⁺) cells and Th17 (CD4⁺IL-17A⁺) cells in vitro; (B) The pseudocolor of Tregs (CD4⁺CD25⁺Foxp3⁺) in vitro; (C) The percentage of Th1 cells; (D) The percentage of Th17 cells; (E) The percentage of Tregs. Statistical analysis is performed by one-way analysis of variance (ANOVA), ^* p <.05, ^** p <.01, and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stromal cells; Sp, splenocytes; St, stimulators. The assay was conducted three times with three replicates each time.

Figure 4

MT synergized with MSCs significantly ameliorates CAV. Each section is displayed at 400× magnification. “I” indicates neointima. “L” indicates lumen. (A) Histology of aorta allografts in transplant recipients; (B) Graft sections for immunohistochemical staining of CD4⁺ cells, CD8⁺ cells, Tregs and macrophages; (C) Intimal thickness of the aorta allografts in different groups; (D) Lumen occlusion of the aorta allografts in different groups; (E) Vessel score of the aorta allografts in different groups; (F) The percentage of intragraft CD4⁺ cells; (G) The percentage of intragraft CD8⁺ cells; (H) The percentage of intragraft Tregs; (I) The percentage of intragraft macrophages. Statistical analysis is performed by one-way analysis of variance (ANOVA), n = 10 per group, ^* p <.05, ^** p <.01, and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stromal cells. The experiments were repeated three times independently.

Figure 5

MT acts synergistically with MSCs to reduce splenic CD4⁺ and CD8⁺ T cells, inhibit B cell activation and DSA production. Splenocytes are collected from the B6 recipient mice of each group at postoperative day 40. (A) The pseudocolor of CD4⁺ and CD8⁺ T cells in recipient splenocytes; (B) The pseudocolor of B (CD19⁺CD86⁺) cells in vivo; (C) The percentage of CD4⁺ T cells; (D) The percentage of CD8⁺ T cells; (E) The percentage of B cells; (F) The serum level of donor-specific IgG; (G) The serum level of donor-specific IgM. Statistical analysis is performed by one-way analysis of variance (ANOVA), n = 10 per group, ^* p <.05, ^** p <.01, and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stem cells; MFI, mean fluorescence intensity. Data shown are representative of three separate experiments.

Figure 6

MT either alone or combined with MSCs reduces splenic Th1, Th17, CD4⁺ Tcm, and CD4⁺ Tem, but enhances Treg population in the aorta transplant recipients. Splenocytes are collected from the B6 recipient mice of each group at postoperative day 40. (A) The pseudocolor of Th1 (CD4⁺IFN-γ⁺) cells, Th17 (CD4⁺IL-17A⁺) cells, and CD4⁺Foxp3⁺ Tregs in vivo; (B) The contour plot of Tem (CD4⁺CD44^highCD62L^low) and Tcm (CD4⁺CD44^highCD62L^high) in vivo; (C) The percentage of Th1 cells; (D) The percentage of Th17 cells; (E) The percentage of Tregs; (F) The percentage of Tcm in vivo; (G) The percentage of Tem in vivo. Statistical analysis is performed by one-way analysis of variance (ANOVA), n = 10 per group, ^* p <.05, ^** p <.01, and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stromal cells. The experiments were independently repeated three times.

Figure 7

MT in combination with MSCs further achieves reduce levels of pro-inflammatory cytokines, but increased level of IL-10 in transplant recipients. The IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, MCP-1, and IL-10 are detected the in the recipient sera and grafts. (A) The level of serum IFN-γ among the groups; (B) The level of serum TNF-α among the groups; (C) The level of serum IL-1β among the groups; (D) The level of serum IL-6 among the groups; (E) The level of serum IL-17A among the groups; (F) The level of serum MCP-1 among the groups; (G) The level of serum IL-10 among the groups; (H) The mRNA expression levels of IFN-γ among the groups; (I) The mRNA expression level of TNF-α among the groups; (J) The mRNA expression level of IL-1β among the groups; (K) The mRNA expression level of IL-6 among the groups; (L) The mRNA expression level of IL-17A among the groups; (M) The mRNA expression level of MCP-1 among the groups; (N) The mRNA expression level of IL-10 among the groups. Statistical analysis is performed by one-way analysis of variance (ANOVA), n = 10 per group, ^* p <.05, ^** p <.01, and ^*** p <.001. Bar graphs represent mean ± SEM. MT, melatonin; MSCs, mesenchymal stromal cells. The experiments were independently repeated three times.