Phenotypic characteristics and differentiation of hPMSCs
These characteristics of isolated cells were consistent with those reported in our previous studies [2, 20]. Briefly, the isolated cells exhibited a typical fibroblastic morphology and could be differentiated to adipocytes and osteocytes, which were identified by performing oil red O and Alizarin red staining procedures, respectively. The FCM results showed that the isolated cells expressed CD73, CD90, and CD105 but not CD34, CD14, HLA-DR, or CD19.
hPMSCs inhibit the expression of PD-1 in CD4 + IL-10 + T cells during the development of GVHD
To analyze the effect of hPMSCs on the expression of PD-1 in CD4+IL-10+ T cells, extracellular PD-1 in gated CD4+IL-10+ T cells was detected by FCM (Fig. 1a, b). The results showed that the proportion of CD4+IL-10+ T cells decreased in GVHD patients and GVHDhigh mice (patients: P < 0.01, mouse model: P < 0.05; Fig. 1c, e), similar to the findings of our previous reports [9]. We further observed that PD-1 expression on the surface of CD4+IL-10+ T cells in both GVHD patients and in the liver and spleen in GVHDhigh mice was significantly higher than that in healthy donors and the normal mice group (P < 0.01, Fig. 1d, f). However, treatment with hPMSCs significantly downregulated the PD-1 expression on the surface of CD4+IL-10+ T cells in comparison with that of the PBS group (P < 0.05, Fig. 1f) and markedly increased the proportion of CD4+IL-10+ T cells in the liver and spleen in the GVHD mouse model (P < 0.01, Fig. 1e).
Considering that the expression of PD-1 in CD4+ T cells was positively correlated with ROS levels [7], hPMSCs reduced the ROS levels in T cells by demonstrating the disordered metabolism of GSH, which is downstream of Nrf2. Thus, the effect of Nrf2 was analyzed during the differentiation of CD4+IL-10+ T cells with or without hPMSCs or by blocking Nrf2 with the Nrf2 inhibitor-ML385. As shown in Fig. 1g, in the presence of hPMSCs, the proportion of CD4+IL-10+ T cells was significantly increased compared to that of the group lacking hPMSCs (P < 0.01); however, the expression of PD-1 on CD4+IL-10+ T cells was significantly lower than that in the group without hPMSCs (P < 0.01, Fig. 1h). After naive CD4+ T cells were pretreated with ML385, the proportion of CD4+IL-10+ T cells was significantly reduced (P < 0.05, Fig. 1g), but the expression of PD-1 in the ML385-pretreated group was increased (P < 0.01, Fig. 1h) compared to that in the non-pretreatment group. Moreover, when naive CD4+ T cells were pretreated with ML385 and cocultured with hPMSCs, the proportion of CD4+IL-10+ T cells decreased significantly (P < 0.01, Fig. 1g) and the expression of PD-1 increased markedly (P < 0.01, Fig. 1h) compared to that of the non-pretreated naive CD4+ T cells cocultured with hPMSCs.
The expression of PD-1 on T cells can induce T cell apoptosis and reduce the number of T cells. To confirm whether hPMSCs could affect the proportion of CD4+IL-10+ T cells by regulating CD4+IL-10+ T cell apoptosis via PD-1 expression, the effect of hPMSCs on the apoptosis of CD4+IL-10+ T cells was analyzed. The results showed that after naive CD4+ T cells were pretreated with ML385, CD4+IL-10+ T cell apoptosis was significantly increased (P < 0.01, Fig. 1i) compared to that of the non-pretreated group. After treatment with hPMSCs, CD4+IL-10+, T cell apoptosis was significantly decreased (P < 0.05, Fig. 1i). Further correlation analysis showed that there was a significant positive correlation between the apoptosis rate and the expression of PD-1 in CD4+IL-10+ T cells (P < 0.05, Fig. 1j).
hPMSCs improve redox metabolism and alleviate the symptoms of GVHD
Our previous research has shown the presence of an imbalance in redox metabolism during the development of GVHD [7, 10, 11]. To further explore the regulatory effects of hPMSCs on redox metabolism in GVHD, the MDA levels and SOD activity in the serum of GVHD patients and the levels of GSH and carbonyl and the activity of GCL and SOD were measured in the liver and spleen in the mouse model. The results showed that the MDA levels in the serum of GVHD patients was significantly increased; however, SOD activity was decreased compared to healthy donors (P < 0.01, Fig. 2a, b). Furthermore, the levels of carbonyl and GSSG were increased in the liver and spleen in the GVHDhigh group compared to those in the normal group; however, the levels of total GSH (T-GSH) and GSH and the activities of SOD and GCL were decreased (liver: carbonyl, GSSG, GSH, SOD, and GCL: P < 0.05, T-GSH: P < 0.01; spleen: carbonyl and T-GSH: P < 0.01, GSSG, GSH, SOD, and GCL: P < 0.05; Fig. 2c, d), and the GSH/GSSG ratio in the GVHDhigh group was lower than that in the normal group (P < 0.01, Fig. 2c, d). Furthermore, treatment with hPMSCs markedly decreased carbonyl and GSSG levels but increased the levels of T-GSH and GSH and the activities of SOD and GCL, compared to those in the PBS group (liver: carbonyl, GSSG, GSH, SOD and GCL: P < 0.01, T-GSH: P < 0.05; spleen: P < 0.01; Fig. 2c, d). Similarly, the ratio of GSH/GSSG also increased after treatment with hPMSCs compared to that of the PBS group (P < 0.01, Fig. 2c, d).
Moreover, the H&E staining results showed remarkable evidence that treatment with hPMSCs improved the pathological changes in the liver and skin, such as leukocyte infiltration, fibrosis, and tissue damage, compared to those of the PBS group (Fig. 2e). Weights of mice used in the established model and disease scores were also monitored daily, and the results showed that weight loss was significantly improved and clinical scores were significantly decreased in hPMSCs group compared to PBS group (P < 0.01, Fig. 2f). Additionally, the Masson staining results in the liver showed that hPMSCs significantly reduced the fibrosis of liver tissue and bile duct area compared to those of the PBS group, and PAS staining results showed that the content of liver glycogen in the hPMSCs group increased compared to that in the PBS group (Fig. 2e). Additionally, as shown in Fig. 2f, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were decreased in the plasma of the GVHDhigh group compared to the normal group; however, treatment with hPMSCs markedly increased the activities of ALT and AST in comparison with those of the PBS group (P < 0.05).
hPMSCs enhance the expression of Nrf2 but inhibit the activation of NF-κB in the liver tissue of GVHD mice
To explore the regulatory effects of hPMSCs on the Nrf2 signaling pathway in GVHD, considering the existence of the crosstalk between Nrf2 and NF-κB, the levels of Nrf2 and downstream target genes (HO-1, NQO1, GCLC, and GCLM) and the phosphorylation levels of NF-κB and I-κB were determined in the liver and spleen tissues of the mouse model.
The WB results showed that the levels of Nrf2 and HO-1, NQO1, GCLC, and GCLM were decreased in the liver and spleen tissues in the GVHDhigh group compared to those in the normal group (liver: Nrf2 and GCLM: P < 0.01, HO-1, NQO1, and GCLC: P < 0.05; spleen: Nrf2, HO-1, and GCLC: P < 0.05, NQO1 and GCLM: P < 0.01; Fig. 3a-d). However, after treatment with hPMSCs, Nrf2, HO-1, NQO1, GCLC, and GCLM levels were increased compared to those in the PBS group (liver: Nrf2 and HO-1: P < 0.01, NQO1, GCLC, and GCLM: P < 0.05; spleen: HO-1 and NQO1: P < 0.05, Nrf2, GCLM, and GCLC: P < 0.01; Fig. 3a-d).
As shown in Fig. 3e-h, the levels of p-NF-κB and p-I-κB were increased in the liver and spleen tissue in the GVHDhigh group compared to those in the normal group (liver: p-NF-κB: P < 0.01, p-I-κB: P < 0.05; spleen: P < 0.05). The treatment with hPMSCs significantly decreased the levels of p-NF-κB and p-I-κB compared to those in the PBS group (liver: p-NF-κB: P < 0.01, p-I-κB: P < 0.05; spleen: p-NF-κB: P < 0.05, p-I-κB: P < 0.01; Fig. 3e-h).
hPMSCs enhance the expression of Nrf2 but inhibit the activation of NF-κB in mononuclear cells of GVHD mice
Considering that hPMSCs can alleviate the inflammatory response elicited in GVHD by regulating the GSH and GST levels in T cells [7], based on the above-mentioned changes of Nrf2 and NF-κB levels in tissues, the Nrf2 and NF-κB levels in mononuclear cells in the liver and spleen tissues of the mouse model were also analyzed. As illustrated in Fig. 4, the Nrf2, HO-1, NQO1, GCLC, and GCLM levels were decreased in liver and spleen mononuclear cells in the GVHDhigh group compared to those in the normal group (liver: Nrf2 and HO-1: P < 0.01, NQO1, GCLC, and GCLM: P < 0.05; spleen: HO-1, NQO1: P < 0.01, Nrf2, GCLC, and GCLM: P < 0.01; Fig. 4a-d). However, the levels of Nrf2, HO-1, NQO1, GCLC, and GCLM in mononuclear cells were increased after hPMSC intervention compared to those in the PBS group (liver: HO-1 and GCLC: P < 0.05, Nrf2, NQO1, and GCLM: P < 0.01; spleen: Nrf2, HO-1, GCLM, and GCLC: P < 0.05, NQO1: P < 0.01; Fig. 4a-d). In contrast, the levels of p-NF-κB and p-I-κB were increased in liver and spleen mononuclear cells in the GVHDhigh group compared to those in the normal group (liver: p-NF-κB: P < 0.01, p-I-κB: P < 0.05; spleen: P < 0.05, Fig. 4e-h). The p-NF-κB and p-I-κB levels in mononuclear cells were decreased after hPMSC intervention compared to those in the PBS group (liver: p-NF-κB: P < 0.01, p-I-κB: P < 0.05; spleen: p-NF-κB: P < 0.05, p-I-κB: P < 0.01; Fig. 4e-h).
hPMSCs induce the differentiation of CD4 + IL-10 + T cells by controlling the activation of the Nrf2 and NF-κB signaling pathways
To further confirm the role of the Nrf2 signaling pathway in the generation of CD4+IL-10+ T cells, expression of Nrf2, its downstream region, and expression of NF-κB were also analyzed during the formation of CD4+IL-10+ T cells from naive CD4+T cells. As demonstrated by the results obtained, when hPMSCs were present, the levels of Nrf2, HO-1, NQO1, GCLC, and GCLM were significantly increased compared to those in the hPMSCs-free group (WB: Nrf2, NQO1, and GCLM: P < 0.05, HO-1 and GCLC: P < 0.01; RT-PCR: P < 0.01; Fig. 5a, b). After naive CD4+ T cells pretreated with ML385 were cocultured with hPMSCs, the levels of Nrf2, HO-1, NQO1, GCLC, and GCLM were decreased significantly compared to those of the untreated naive CD4+ T cells cocultured with hPMSCs (WB: Nrf2, NQO1, and GCLC: P < 0.01, HO-1 and GCLM: P < 0.05; RT-PCR: NQO1: P < 0.01, Nrf2, HO-1, GCLC, and GCLM: P < 0.05; Fig. 5a, b). Moreover, the results showed that when hPMSCs were present, the levels of p-NF-κB and p-I-κB were significantly decreased compared to those in the group where hPMSCs were absent (P < 0.01, Fig. 5c). In the presence of hPMSCs, the levels of p-NF-κB and p-I-κB increased significantly in ML385-pretreated naive CD4+ T cells compared to those observed in the untreated naive CD4+ T cells (P < 0.01, Fig. 5c).
hPMSCs upregulate the expression of Nrf2 by inhibiting the levels of NF-κB in the nucleus of CD4 + IL-10 + T cells
Previous studies have indicated that Nrf2 and NF-κB can competitively translocate into the nucleus [22]; thus, the levels of Nrf2 and NF-κB in the nucleus of CD4+IL-10+T cells were further determined during the formation of CD4+IL-10+ T cells from naive CD4+ T cells. As shown in Fig. 6a and 6b, the Nrf2 levels in the nucleus of CD4+IL-10+ T cells were higher in the presence of hPMSCs than those observed in the absence of hPMSCs (P < 0.05), but the levels of NF-κB were lower (IF: P < 0.05; WB: P < 0.01). Further analysis showed that when ML385-pretreated naive CD4+ T cells were cocultured with hPMSCs, the Nrf2 levels in the nucleus of CD4+IL-10+ T cells markedly decreased (IF: P < 0.05; WB: P < 0.01), while the levels of NF-κB increased compared to those observed in untreated naive CD4+ T cells cocultured with hPMSCs (IF: P < 0.05; WB: P < 0.01).