Next, we asked whether the microbiota can affect BMMSC immunomodulation. It has been reported that BMMSCs cannot suppress immune reactions, unless they were preactivated by certain combinations of the inflammatory environment [

17

,

18

]. The microbiota played a critical role in the maturation of immune system and tolerance [

19

,

20

]. Thus, we reasoned that the immunomodulatory capacities of BMMSCs may be affected by the microbiota. We first cocultured BMMSCs from different groups with pan-T cells and examined their abilities to induce T-cell apoptosis. Flow cytometry analysis showed that SPF-derived and ConvD-derived BMMSCs significantly induced more T-cell apoptosis when compared with BMMSCs from germ-free mice (Fig. 

3a

). In addition, cytokine array analysis showed that GF-derived BMMSCs secreted increased proinflammatory factors interleukin-23 (IL-23) and chemokine C–C motif ligand 5 (CCL5) (Fig. 

3b

) which contributed to inflammatory disease. To further investigate the ability for immunomodulation of BMMSCs, the GF, SPF, and ConvD BMMSCs were systemically infused into a 3% dextran sulfate sodium (DSS)-induced experimental colitis mouse model [

21

] at 3 days post DSS induction (Fig. 

3c

). The colitis mice had significantly reduced body weight compared to C57BL6 control mice from days 5 to 9 post DSS induction (Fig. 

3c

). After infusion of either SPF-derived or ConvD-derived BMMSCs, but not GF BMMSCs, the body weight of the colitis mice was partially rescued at 9 days post DSS induction (Fig. 

3c

). The disease activity index (DAI), including body weight loss, diarrhea, and bleeding, was significantly elevated in the colitis mice compared to the control group. After infusion of SPF and ConvD BMMSCs, the DAI in the colitis mice was obviously decreased at day 9, while GF BMMSC infusion failed to reduce the DAI (Fig. 

3d

). BMMSCs from microorganism-exposed mice such as SPF and ConvD significantly relieved the disease activity more than twofold compared to the GF BMMSC and PBS treated groups (3.36 ± 0.48, 3.28 ± 0.61 vs 11.29 ± 0.43, 9.43 ± 0.67; Fig. 

3d

). In addition, H&E staining and the quantitative inflammatory score showed that the SPF-derived and ConvD-derived BMMSCs greatly decreased the colitis inflammation (inflammation scores 3.67 ± 0.33 and 2.33 ± 0.88) compared with PBS and GF BMMSC treated groups (inflammation scores 6.0 ± 0.58 and 5.33 ± 0.88; Fig. 

3e

).

Fig. 3

Microbiota is required for BMMSC immunomodulation. a Flow cytometry analysis shows that BMMSCs induced T-cell apoptosis by detecting Annexin V-positive cells in a coculture system. SPF-derived and ConvD-derived BMMSCs induced more T-cell apoptosis. b Cytokine array shows that GF-derived BMMSCs expressed more IL-23 and CCL5 when compared to the other two groups exposed to the microbiota. c Infusion of SPF and ConvD BMMSCs rescue weight loss in DSS-induced colitis mice, while GF BMMSCs had comparable weight loss to the PBS control. d Disease activity index shows that BMMSCs from GF mice did not decrease the disease activity, while the other two groups of BMMSCs decreased the disease activity index. e PBS and GF BMMSC treated colitis mice show severe epithelial disruption and inflammatory cell infiltration compared to ConvD and SPF BMMSC treated colitis mice (bar = 500 μm). All experimental data verified in at least three independent experiments. Error bars represent the SEM from the mean values. ***P < 0.005; **P < 0.001; *P < 0.05. BMMSC bone marrow mesenchymal stem cell, CCL5 chemokine C–C motif ligand 5, ConvD conventionalized, DSS 3% dextran sulfate sodium, GF germ free, IL interleukin, PBS phosphate-buffered saline, SPF specific pathogen free

The role of the microbiota in regulating immune cell polarization and human diseases is receiving increasing attention [22, 23]. BMMSCs reside in the skeleton to maintain osteoblastic lineage cell function and serve as a niche for hematopoietic stem cells, which involves several physiological regulations and interplays with the immune system. Our data indicated that in the unique niche of both mesenchymal stem cells and hematopoietic stem cells, the microbiota also regulated the BMMSCs, which may be related to the maturation or apoptosis of hematopoietic cell lines.