Nestin+ Mesenchymal Precursors Generate Distinct Spleen Stromal Cell Subsets and Have Immunomodulatory Function

Abstract

Mesenchymal stromal cells (MSCs) are known to be widespread in many tissues and possess a broad spectrum of immunoregulatory properties. They have been used in the treatment of a variety of inflammatory diseases; however, the therapeutic effects are still inconsistent owing to their heterogeneity. Spleen stromal cells have evolved to regulate the immune response at many levels as they are bathed in a complex inflammatory milieu during infection. Therefore, it is unknown whether they have stronger immunomodulatory effects than their counterparts derived from other tissues. Here, using a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, Nes-GFP⁺ cells from bone marrow and spleen were collected. Artificial lymphoid reconstruction in vivo was performed. Cell phenotype, inhibition of T cell inflammatory cytokines, and in vivo therapeutic effects were assessed. We observed Nes-GFP⁺ cells colocalized with splenic stromal cells and further demonstrated that these Nes-GFP⁺ cells had the ability to establish ectopic lymphoid-like structures in vivo. Moreover, we showed that the Nes-GFP⁺ cells possessed the characteristics of MSCs. Spleen-derived Nes-GFP⁺ cells exhibited greater immunomodulatory ability in vitro and more remarkable therapeutic efficacy in inflammatory diseases, especially inflammatory bowel disease (IBD) than bone marrow-derived Nes-GFP⁺ cells. Overall, our data showed that Nes-GFP⁺ cells contributed to subsets of spleen stromal populations and possessed the biological characteristics of MSCs with a stronger immunoregulatory function and therapeutic potential than bone marrow-derived Nes-GFP⁺ cells.

Keywords: Nestin; immunoregulation; inflammatory bowel diseases; mesenchymal stromal cells; spleen.

Figure 1

Characterization of Nestin expression in the spleen: (A) Nestin (green) expression in the spleens of Nes-GFP transgenic mice. Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm; (B) qRT-PCR analysis of Nestin gene expression in the spleen of normal mice on days 7, 14, 28, 90 after birth. The relative mRNA expression levels of Nestin were normalized to reference GAPDH controls. n = 3. (C–E) Immunohistochemical analyses showed that the Nes-GFP⁺ cells in the spleen expressed markers of FRCs, FDCs, and MRCs; (C) ER-TR7 (FRCs); (D) CD35 (FDCs); (E) MadCAM-1 (MRCs). Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. White Pulp, WP; Germinal Center, GC; (F) The co-expression area of Nes-GFP⁺ cells with ER-TR7, CD35, and MadCAM-1 was counted. n = 7.Data represent mean values ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Isolation and characterization of Nes-GFP⁺ cells from the spleen. (A,B) Flow cytometry was used to isolate Nes-GFP⁺ cells from the spleens of Nes-GFP transgenic mice. Cells from wild-type C57BL/6 mice were isolated as a control. To detect the percentage and number of Nes-GFP⁺ cells in cell sorting more precisely, we added the counting beads (both positive in PE and GFP channel) in the cell suspension of the spleen. (C) Representative images showing the clonal sphere growth of single Nes-GFP⁺ cells were observed using a bright field (left panel) or fluorescence (middle panel). Scale bars, 50 μm. (D) Representative stained images showing that mouse spleen-derived Nes-GFP⁺ cells could differentiate into osteocytes (Alizarin red), adipocytes (Oil red O), and chondrocytes (toluidine blue). Scale bars, 100 μm. (E) The expression of cell surface markers on Nes-GFP⁺ cells was detected by flow cytometry.

Figure 3

Spleen-derived Nes-GFP⁺ cells could form secondary lymphoid organs. (A) Spleen CD45^-Nes-GFP⁻ and CD45^-Nes-GFP⁺ cells of Nes-GFP transgenic mice with collagen scaffolds were transplanted into the kidney capsule of wild-type mice. Scale bars, 100 μm. (B,C) Representative stained images of transplants showed that Nes-GFP⁺ cells, instead of Nes-GFP^- cells, could form lymphoid tissue-like organoids. Scale bars, 100 μm. (D) Representative stained images of lymph node. Scale bars, 100 μm.

Figure 4

Immunosuppressive properties of Nes-GFP⁺ cells derived from the spleen (cytokine assay). (A,B) Nes-GFP⁺ cells were cocultured with sorted CD3⁺ T cells, and the pro-inflammatory cytokine TNF-α secretion of T cells was evaluated by flow cytometry. Representative plots of TNF-α production by T cells at different ratios. n = 3. (C,D) Nes-GFP⁺ cells were cocultured with sorted CD3⁺ T cells, and the pro-inflammatory cytokine IFN-γ secretion of T cells was evaluated by flow cytometry. Representative plots of IFN-γ production by T cells at different ratios. n = 3. (E) Bar graphs showed the inhibition rate of IFN-γ- and TNF-α-producing CD3⁺ T cells after coculture with Nes-GFP⁺ cells. Data represent mean values ± SD of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 5

Immunosuppressive properties of Nes-GFP⁺ cells (T cells proliferation and activation suppression assay): (A) Gating strategy for the evaluation of T cells proliferation; (B) Nes-GFP⁺ cells were cocultured with sorted CD3⁺ T cells, and the percentage of T cells proliferation was evaluated by flow cytometry. Representative plots of three independent experiments. (C) Gating strategy for the evaluation of T cells activation markers (CD69 and CD25); (D,E) Nes-GFP⁺ cells were cocultured with sorted CD3⁺ T cells, and the percentage of CD69 and CD25 was evaluated by flow cytometry. Representative plots of three independent experiments; (F–H) Bar graphs showed the inhibition rate of T cells proliferation and activation after coculture with Nes-GFP⁺ cells. Data represent mean values ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Spleen-derived Nes-GFP⁺ cells as a potential source for the treatment of inflammatory diseases: (A) Therapeutic efficacy evaluation of TNBS-induced IBD model, bodyweight change percentage of each group. n = 3; (B) Disease activity index (DAI) were measured. n = 5; (C) Representative colonic length of mice; (D) Quantification of the colonic length of mice. n = 5; (E,F) H&E staining and corresponding histological scoring of each group. n = 5. Scale bars, 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.