Isolation and identification of Wip1−/− MSCs from murine compact bones
To investigate the roles of Wip1 in the immunoregulatory function of MSCs for treating T1DM, MSCs were isolated from Wip1+/+ and Wip1−/− mice by cultivating the digested compact bone. Fibroblast-like cells sprouted from the bone fragments and adhered to the flask 48 h (Fig. 1A,Fig.S1). These adherent cells could be readily expanded in vitro by successive cycles of trypsinization, seeding, and culture every 3 days without visible morphologic alteration (Fig. 1B and 1C).
We then determined the adipogenic and osteogenic differentiation of Wip1−/− cells in vitro. As shown in Fig. 1D and 1E, many Oil Red-O-positive lipid droplets were available in murine Wip1−/− MSCs. To further determine the effects of Wip1 on MSCs adipogenic differentiation, the mRNA levels of adipogenic markers, including C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) were examined by qPCR. Consistent with histochemical staining results, the mRNA expression of C/EBPα and PPARγ in Wip1−/− MSCs showed elevation (Fig. 1F). Under osteogenic conditions for 2 weeks, the adherent cells from Wip1−/− mice displayed alkaline phosphatase activity (Fig. 1G and 1H). In line with the results of differentiation assays, qPCR demonstrated transcriptional expression of Osteocalcin and Runx2 under specific osteogenic cultures in adherent cells (Fig. 1I).
The immunophenotype of the Wip1−/− adherent cells was assessed by flow cytometry. The culture-expanded adherent cells were positive for CD90, CD29, and Scal-1 but were negative for CD31, MHCII, CD11b, CD34, and CD45 (Fig. 1J).
The morphologic, immunophenotypic, and differentiation assays strongly indicated that the adherent cells isolated from Wip1−/− mice were MSCs. In addition, Wip1+/+ MSCs showed similar biological characteristics (data not shown).
Therapeutic effect of Wip1−/− MSCs in T1DM mice
To assess the therapeutic effect of Wip1−/−MSCs in T1DM mice, Wip1+/+MSCs (5 × 105) and Wip1−/−MSCs(5 × 105) were intravenously administered to STZ-induced T1DM mice on day 1 and 14. The general conditions of mice including body size, body weight, and blood glucose level were monitored. As shown in Fig. 2A, the body size in Wip1−/−MSCs mice was smaller compared with Wip1+/+ MSCs mice. In T1DM mice that received MSCs, the blood glucose levels were lower in Wip1−/−MSCs group compared with those of Wip1+/+ MSCs group (Fig. 2B). Additionally, the slow gain in weight caused by hyperglycemia was controlled by MSCs infusion. The average body weight of the Wip1+/+ MSCs group was larger than that of the Wip1−/− MSCs group (Fig. 2C).
To further assess whether Wip1−/− MSCs infusion could reduce the pathological damages in T1DM mice, the histological examination of islet tissues was performed by HE staining and immunohischemical staining on day 28. As shown in Fig. 2D and 2E, islets of Wip1−/− MSCs group exhibited mild inflammation. Additionally, there were a few of islet areas with preserved islet morphology, indicating that Wip1−/− MSCs infusion could prevent the destruction of islets in T1DM. Moreover, the islet area and the mean density of insulin-positive cells in pancreatic tissues treated with Wip1+/+ MSCs were larger than that of Wip1−/− MSCs group, despite that the effects of Wip1−/− MSCs were better than T1DM mice (Fig. 2F and 2G). These findings indicated the therapeutic effects of Wip1−/− MSCs were weaker than Wip1+/+ MSCs in T1DM mice.
Wip1 −/− MSCs failed to reduce the inflammatory response in T1DM mice
Autoimmune diabetes is characterized by a Th1 immune response. Previous studies indicated that the immunoregulation of MSCs was closely involved in the inflammatory processes in T1DM[15, 16]. To investigate whether Wip1 gene regulated the immunomodulatory function of MSCs in T1DM, flow cytometry was used to detect the intracellular inflammatory factor IFN-γ in Th1 cells (CD4+INF-γ+ T cells) of spleen lymphocytes in each group. As shown in Fig. 3A and 3B, the percentage of Th1 cells (CD4+INF-γ+ T cells) in T1DM group was significantly higher than that in other groups (P < 0.01). Wip1+/+ MSCs transfusion significantly decreased the percentage of Th1 cells (P < 0.05). After treating with Wip1−/− MSCs, the percentage of Th1 (CD4+INF-γ+ T cells) cells was lower than that of T1DM group, and was higher than that of Wip1+/+ MSCs group (P < 0.05).
Subsequently, we investigated whether Wip1 could affect the expression of lymphocyte-derived immune factors mediated by MSCs infusion. Compared with Wip1+/+ MSCs group, serum IFN-γ and IL-17A showed significant increase and the IL-4 expression showed significant decline after Wip1−/− MSCs infusion (Fig. 3C). These data indicated that Wip1 gene may involve in the immunomodulatory activity of MSCs in T1DM mice.
Wip1 interacted with BST2
To investigate the involvement of Wip1 in MSCs immunomodulatory activity, we then determined the gene expression profiles in Wip1+/+ MSCs and Wip1−/− MSCs by microarray analysis. Bioinformatics methods were used to analyze the microarray results, which predicted that 23 genes were associated with immunomodulatory responses. Among the up-regulated genes in Wip1−/− MSCs, BST2 was identified to be related to inflammatory processes, and highly expressed in Wip1−/− MSCs by qPCR assay (Fig. 4A and B). To further explore whether BST2 was up-regulated in Wip1−/− MSCs, we examined the expression of Wip1 in various genotype mice. As shown in Fig. 4C and 4D, the protein expression level of Wip1 in Wip1−/− MSCs was lower than that of Wip1+/+ MSCs. Then we analyzed the BST2 protein expression in Wip1−/− MSCs, which showed that the protein expression of BST2 was significantly up-regulated in Wip1−/− MSCs (Fig. 4E and 4F).
Subsequently, we further investigated whether Wip1 had direct interaction with BST2. The 293T cells were transfected with Wip1-EGFP for 48 h, followed by determining the expression of Wip1 and BST2. GFP expression indicated high transduction efficiency, which showed a transfection efficiency of 61 ± 10.81% and 69 ± 7.93% for the EGFP group and Wip1-EGFP group, respectively (Fig. 4G and 4H). As shown in Fig. 4I, 293T cells transduced with Wip1-EGFP exhibited higher Wip1 expression and lower BST2 expression. Here, our data revealed that there was interaction between Wip1 and BST2.
High expression of BST2 can significantly promote the expression of IFN–α in Wip1−/− MSC
BST2 was reported to activate plasma cell-derived dendritic cells (pDC) and promote IFN-α expression[17]. As an important inflammatory response factor, IFN-α involved in the regulation of the biological characteristics of MSCs and promoted the inflammatory response of T1DM[18–20]. Therefore, we explored whether BST2 could affect the regulation of Wip1−/− MSCs on the inflammatory process in T1DM mice via IFN-α. As shown in Fig. 5A, the mRNA expression of IFN-α in Wip1−/− MSCs was significantly higher than that of Wip1+/+ MSCs. Compared with Wip1+/+ MSCs, the protein expression of IFN-α in Wip1−/− MSCs showed a dramatic increase (Fig. 5B). To investigate whether IFN-α was dependent on the BST2 expression in Wip1−/− MSCs, we transfected Wip1−/− MSCs with BST2 siRNAs or negative control. The results showed that BST2 knockdown in Wip1−/− MSCs could significantly down-regulate the expression of IFN-α (P < 0.05, Fig. 5C). This implied that knockdown of BST2 could effectively inhibit the expression of IFN-α. On this basis, we demonstrated that BST2 was a key gene for regulating IFN-α expression in Wip1−/− MSCs.
IFN-α secreted by Wip1−/− MSCs aggravated inflammatory response in the pancreatic microenvironment
Several studies had shown that MSCs preferentially homed to the sites of tissue damages, where they enhanced wound healing, and involved in modulating the balance of inflammatory response[21]. To determine whether Wip1−/− MSCs could migrate to the damage sides in pancreas, red fluorescence stained Wip1−/− MSCs and Wip1+/+ MSCs were administrated into T1DM mice. After staining for 72 h, red fluorescence was occurred around blood vessels in pancreatic tissues (Fig. 6A), which indicated that Wip1−/− MSCs could home to the pancreatic tissues in T1DM mice.
IFN-α has been well acknowledged to regulate the innate immune response and promote the adaptive immune response. Thus, it played important roles in the regulation of immune disorders. To explore whether IFN-α was secreted by Wip1−/− MSCs, the expression of IFN-α in the pancreatic supernatant of T1DM mice in each group was detected by ELISA. The expression of IFN-α in the pancreatic supernatant of Wip1−/− MSCs group showed significant increase (2,103.14 ± 797.1 pg/mL) compared with the control group and Wip1+/+ MSCs group (P < 0.05). Compared with the T1DM model group, there was no significant difference. These data demonstrated that Wip1−/−MSCs could promote IFN-α secretion after homing to pancreatic tissue (Fig. 6B). Besides, we further examined the expression of IFN-α, IFN-β and IFN-γ in splenic lymphocytes. The expression of IFN-α, IFN-β and IFN-γ in Wip1−/− MSCs group was higher than that of Wip1+/+ MSCs group, which indicated that Wip1−/− MSCs also could promote the expression of IFN-α, IFN-β and IFN-γ in spleen lymphocytes, and further aggravate the inflammatory response(Fig.S2).
The expression of inflammatory factors (e.g. TNF-α, IL-17A, IL-4 and IL-10) in the pancreas supernatant in T1DM mice was measure by ELSIA. The expression of TNF-α (1,027.87 ± 82.9 pg/ml) and IL-17A (519.02 ± 47.01 pg/ml) in T1DM group were significantly higher than those in other groups (P < 0.05). After treating with Wip1+/+ MSCs, the expression of TNF-α (827.03 ± 102.08 pg/ml) and IL-17A (283.53 ± 48.26 pg/ml) showed significant decline compared with those in T1DM group (P < 0.05), while the expression of TNF-α (995.24 ± 562.61 pg/ml) and IL-17A (413.33 ± 42.98 pg/ml) in Wip1−/− MSCs treatment group was significantly higher than those in Wip1+/+ MSCs group (P < 0.05). However, their levels were still lower than those in T1DM group. The expression of IL-4 (50.25 ± 28.14 pg/ml) and IL-10 (1575 ± 579.38 pg/ml) in the pancreas of the Wip1+/+ MSCs group was significantly higher than those of other groups (P < 0.05). IL-4 (7.35 ± 3.5 pg/ml) and IL-10 (447.5 ± 160.17 pg/ml) expression in the Wip1−/− MSCs group was significantly lower than those in the Wip1+/+ MSCs group (P < 0.05). These data indicated that IFN-α secreted by Wip1−/− MSCs could aggravate the inflammatory response in the pancreatic microenvironment (Fig. 6C).