Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis

Abstract

Background: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice.

Methods: Using flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals.

Results: In vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed.

Conclusion: The therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.

Figures

Fig. 1

hAECs inhibit T cell responses and allogeneic and xenogeneic proliferation. Proliferative response of human PBMCs (a) or splenocytes from naïve C57BL/6 mice (b) stimulated with anti-CD3/CD28 antibodies in the presence or absence of hAECs at different hAECs to splenocyte ratios (n = 6 performed in triplicate, *P < 0.05, ***P < 0.001 compared to T cells). c Proliferative response of 2D2 splenocytes stimulated with 20 μg MOG35-55 in the presence or absence of hAECs at different ratios (n = 6 performed in triplicate, **P < 0.01, ***P < 0.001 compared to T cells). d Cytokine secretion profile in supernatant from co-cultures of hAECs and 2D2 splenocytes at 1:5 ratio (n = 5, **P < 0.01 ***P < 0.001 compared to T cells). e Proliferative response of C57BL/6 splenocytes co-cultured with hAECs (n = 4 performed in triplicate, ***P < 0.0001 compared to B6 + Bc). f Proliferative response of C57BL/6 splenocytes co-cultured with irradiated Balb/c splenocytes in the absence or presence of hAECs at hAEC to C57BL/6 T cell ratio of 1:5 (n = 4 preformed in triplicate, ***P < 0.0001 compared to B6 + Bc). g Cytokine secretion profile in supernatant from MLR cultures with hAECs. (n = 4, *P < 0.05,**P < 0.01, ***P < 0.001 compared to B6 + Bc)

Fig. 2

hAECs inhibit upregulation of T cell activation markers and retain T cells in a naïve phenotype. Naïve CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of hAECs for 5 days and then analyzed by flow cytometry. a, b Representative profiles of CD25 expression on stimulated CD4+ T cells. c, d Representative profiles of CD44 and CD62L expression on stimulated CD4 + CD25+ T cells. CD62LhiCD44lo denotes naïve T cells, CD62LloCD44hi denotes memory cells. Data are representative of two independent experiments with n = 3 mice

Fig. 3

Administration of hAECs on day 8, 10, and 12 suppresses the development of RR-EAE. a Clinical scores of NOD/Lt mice injected i.p. with one or five million hAECs or PBS on day 8, 10, and 12 (indicated by arrows) (n = 7, *P < 0.05 compared to PBS). Clinical scores (b) and cumulative disease score (c) of NOD/Lt mice injected i.p. with 0.1, 0.25, or 0.5 million hAECs or PBS on day 1 and 3 (indicated by arrows) (n = 6–7, **P < 0.01 compared to PBS). Representative spinal cord sections from PBS control mice stained with H&E (d), LFB (f), or Bielschowsky silver stain (h) or mice that received five million hAECs stained with H&E (e), LFB (g), or Bielschowsky silver stain (i) to assess inflammation, demyelination, and axonal damage, respectively. Magnification × 100. IBA-1+ immunoreactivity in the grey matter of spinal cord sections from PBS controls (j) or mice that received five million hAECs (k). Magnification × 400. (l) Total number of IBA-1+ cells/mm2 in different regions of the CNS in PBS controls and hAEC-treated mice (*P < 0.05 compared to PBS)

Fig. 4

T cell responses in RR-EAE mice following hAEC administration. Mononuclear cells were isolated from the spleen of NOD/Lt mice at day 37. Proliferative response of splenocytes stimulated with rMOG (a) or anti-CD3/CD28 (g). b–f Cytokine secretion profile in supernatant from rMOG stimulated splenocyte cultures. h–l Cytokine secretion profile in supernatant from anti-CD3/CD28 stimulated splenocyte cultures (n = 6 performed in triplicate, *P < 0.05 compared to PBS)

Fig. 5

Administration of hAECs increases the total number of Tregs and naïve CD4+ T cells in the periphery. Mononuclear cells were isolated from the lymph nodes and spleen at day 37, and the proportion and number of Tregs or naïve CD4+ T cells was analyzed by flow cytometry. a Total cell number in the lymph nodes. b Proportion of gated lymphocytes that are CD4 + CD25 + Foxp3+ Tregs in the lymph nodes. c Total Treg numbers in the lymph nodes. d Total cell number in the spleen. e Proportion of gated lymphocytes that are CD4 + CD25 + Foxp3+ Tregs in the spleen. f Total Treg numbers in the spleen. g Proportion of CD4+ that are naïve (CD62LhiCD44lo) T cells in the spleen. h Total number of CD4+ naïve (CD62LhiCD44lo) T cells in the spleen. (n = 6, *P < 0.05 compared to PBS)

Fig. 6

Administration of hAECs increases Th2 cells in the CNS and shifts the inflammatory profile towards an anti-inflammatory environment. Mononuclear cells were isolated from NOD/Lt mice at day 37 and the phenotype of T helper cells was assessed by flow cytometry. Th1 (CD4 + IFN-γ+), Th17 (CD4 + IL-17+), and Th2 (CD4 + IL-4+) cell proportions and the Th1 to Th2 and Th17 to Th2 ratios were determined in the spleen (a–e), lymph nodes (f–j), and CNS (k–o) (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001)