Alterations of mesenchymal stem cells on regulating Th17 and Treg differentiation in severe aplastic anemia

Abstract

Immune-mediated hematopoietic destruction is a key factor in idiopathic severe aplastic anemia (SAA). With great immunomodulatory functions, mesenchymal stem cells (MSCs) are important for bone marrow niche. While the underlying etiology of immunologic changes in SAA bone marrow remains unknown, dysfunctional MSCs are implicated as a major cause. To provide evidence for their defects in immunomodulation, alterations of SAA MSCs in regulating T cell differentiation were determined. During differentiation from CD4+ T cells into T helper 17 (Th17) cells under polarization conditions, impaired inhibition on IL-17 and IL-1β production was noted when cocultured with SAA MSCs compared to control MSCs (P < 0.05). After stimulation of Th17 activation, the percentage of IL-17-secreting cells was significantly increased in the SAA group (9.1 ± 1.5% vs 6.6 ± 0.4%, P < 0.01). Under regulatory T (Treg) polarization, a higher percentage of CD4+CD25+FoxP3+ Treg cells was detected when cocultured with SAA MSCs compared to control MSCs (8.1 ± 0.5% vs 5.8 ± 0.8%, P < 0.01). Inconsistently, transforming growth factor-β (TGF-β) concentrations in the culture supernatant were decreased and IL-1β concentrations were elevated in the SAA group. Our data indicated impaired inhibition of SAA MSCs on Th17 activation and aberrant regulation of SAA MSCs on Treg differentiation. Increased IL-17 and IL-1β levels with decreased TGF-β levels in the supernatant suggested the potential of SAA MSCs for triggering a hyperinflammatory environment. Dysfunctional MSCs could contribute to the lack of immunoprotection in the bone marrow, which may be associated with SAA.

Keywords: aplastic anemia; bone marrow failure; immunomodulation; mesenchymal stem cells.

Figure 1

Characterization of MSCs. (A) In vitro culture, MSCs in the control and SAA groups shared similar growth patterns and morphologies (×100; scale bar = 100 μm). (B) Using flow cytometry, these cells were negative for CD34 and CD45, and positive for CD44 and CD105. The red dashed line indicates the isotype control; the black area indicates the stained cells. (C) There was no significant difference in the expressed MFI of CD34-PE, CD45-FITC, CD44-APC, and CD105-PE between the SAA group and the control group. (D) Osteogenic differentiation was demonstrated by mineralized deposits stainable with von Kossa stain (×100; scale bar = 100 μm). (E) Adipogenic differentiation was confirmed by intracellular accumulation of lipid droplets stainable with oil red O (×100; scale bar = 100 μm). MFI: Mean fluorescence intensity; ns: not significant; SAA: Severe aplastic anemia.

Figure 2

IL-17 and IL-1β concentrations in the culture supernatant after 5-days Th17 differentiation induction. IL-17 and IL-1β levels were lower when CD4+ T cells cocultured with control MSCs. The inhibitory effects were significantly decreased when cocultured with SAA MSCs. Data are presented as the mean ± SD. n = 5 in each group.

Figure 3

Assessment of Th17 activation after 5-days Th17 differentiation. (A) Activation of Th17 cells was confirmed by intracellular staining for IL-17 with flow cytometry. (B) The percentage of IL-17-secreting cells were lower in the control group, compared to the basic group (6.6 ± 0.4% vs 12.9 ± 1.2%, P < 0.01). The percentage of IL-17-secreting cells were significant higher in the SAA group, compared to the control group (9.1 ± 1.5% vs 6.6 ± 0.4%, P < 0.01). Data are presented as the mean ± SD. n = 5 in each group.

Figure 4

Evaluation of Treg differentiation after 5-days differentiation induction. (A) Differentiation of Treg cells was confirmed by intracellular staining for FoxP3 with flow cytometry. (B) The percentage of CD4+CD25+ FoxP3+ Treg cells was higher in the control group, compared to the basic group (5.8 ± 0.8% vs 4.3 ± 0.8%, P < 0.05). The percentage of CD4+CD25+ FoxP3+ Treg cells was significantly increased in the SAA group, compared to the control groups (8.1 ± 0.5% vs 5.8 ± 0.8%, P < 0.01). (C) In the SAA groups, TGF-β concentrations in the culture supernatant were decreased and IL-1β levels were increased, compared to the control group (P < 0.01). Data are presented as the mean ± SD. n = 5 in each group.