Extraction and identification of HO-1/BMMSCs
BMMSCs were isolated and cultured to the third generation as previously described [16]. BMMSCs transfected with an HO-1 adenovirus remained unchanged morphologically (Figure S2A), and had the ability to induce osteogenic (Figure S2B) and lipogenic (Figure S2C) differentiation in vitro. Flow cytometric identification of surface biomarkers revealed a positive rate of over 99% for CD29, CD90, and RT1A, and a negative rate of over 99% for CD34, CD45, and RT1B (Figure S2D − F). This demonstrated that the molecular biology of the HO-1 adenovirus-transfected BMMSCs was not affected. The immunofluorescence exhibited significantly higher red fluorescence intensity in the HO-1/BMMSCs compared to that of the BMMSCs (Figure S2G and H). qRT-PCR (Figure S2I) and Western blot (Figure S2J) results were also confirmed to exhibit significantly higher HO-1 expression in the HO-1/BMMSCs.
The NMP system preserves the donor livers in vitro and allows the colonization of HO-1/BMMSCs
The normal liver is bright red (Fig. 1A), while the DCD liver is a purplish red color with rounded edges and slight edema (Fig. 1B). After the liver had been preserved using the SCS method, the liver was congested with edema, enlarged in size, and rounded at the edges (Fig. 1C). A stable NMP system was established (Figure S1A and B) and the liver preserved by the NMP method was uniform in color, without edema and congestion (Fig. 1D). After the LT was completed using the "double cuff method", the liver was evenly congested, indicating that the model was successfully established (Fig. 1E and F). Respectively, 1 × 107 GFP/BMMSCs or HO-1/BMMSCs were perfused, and LT was completed. Our previous studies demonstrated that approximately 50% of BMMSCs could be colonized in the donor liver during the perfusion process [17]. The vivo imaging system was used to detect the liver grafts the fluorescence intensity of the livers perfused with HO-1/BMMSCs was higher than that of BMMSCs (Fig. 1G and H). The liver grafts were obtained, frozen sections were prepared, and the number of GFP+ cells in the frozen sections of the HO-1/BMMSCs-perfused liver grafts were significantly higher than that of the BMMSCs (Fig. 1I − K). This finding confirms that in the inflammatory environment after LT, HO-1/BMMSCs survived in significantly higher numbers in the transplanted livers compared to BMMSCs.
HO-1/BMMSCs significantly reduced ACR and improved the recipient prognosis
The morphological changes of the liver grafts were observed by HE staining (Fig. 2A). At 7 d post-LT, the SCS group liver grafts were observed to be severely damaged, a large number of hepatocytes disappeared around the vein, a large number of macrophages and lymphocytes had infiltrated, and focal necrosis of the hepatocytes with eosinophilic homogeneity and hemorrhage were locally observed. The liver grafts in the HMP and FK506 groups were significantly improved compared with the SCS, NMP, and BMP groups, there was no obvious necrosis in the liver, and the liver lobular structure was intact, with a moderate amount of lymphocytic infiltration surrounding the bile ducts and portal vein. No rats survived in the SCS group at 14 d post-LT, whereas the liver lobules were more intact in the HBP group, exhibiting neatly arranged hepatocyte strips and low levels of lymphocyte infiltration. The TUNEL results of the liver tissues showed that the apoptotic cells were significantly increased in the SCS and NMP groups compared with other groups, whereas there were no significant differences between the HMP and FK506 groups and both were reduced compared with the BMP group (Fig. 2B and C). The acute cellular rejection scores were not significantly different between the NMP and SCS groups, but significantly increased compared with all other transplantation groups. Moreover, the rejection scores in the HMP and FK506 groups were significantly lower than those in the BMP group (P < 0.05); however, there was no significant difference between the HMP and FK506 groups (Fig. 2D). These results indicate that HO-1/BMMSCs combined with NMP preserved the DCD livers in vitro, and HO-1/BMMSCs retained in the liver grafts after transplantation improved the pathology and inhibited ACR, similar to the level of FK506 application.
The median survival time after transplantation in the Sham, HMP, and FK506 groups was > 60 d. All animals survived long-term and no statistical differences were observed regarding a comparison in the survival rate between the groups (Fig. 2E). In the survival analysis conducted using a Log-rank (Mantel-Cox) test, rats in the HBP and FK506 groups had the longest survival time (median survival time: 60 > d) compared with the SCS group (median survival time: 1.5 d), NMP group (median survival time: 16.5 d), BMP group (median survival time: 21.5 d), and SCS group. The survival time of the rats in the SCS group was significantly lower than that of the other groups (P < 0.05), and the survival time of rats in the BMP group was longer than that of the NMP group but shorter than that of the HBP and FK506 groups (P < 0.05). This finding indicates that HO-1/BMMSCs perfused via portal vein could significantly prolong the survival time of the recipient rats after LT, and the application of HO-1/BMMSCs was more effective than BMMSCs.
ALT, AST, ALP, and GGT reflected the level of liver cell injury, and the levels of ALT, AST, and ALP in the HBP group were significantly lower than those in the SCS, NMP, and BMP groups at 7 d post LT (P < 0.05). The difference with the FK506 group was not statistically significant (P > 0.05). At 14 d post-LT, no rats in the SCS group survived, and the levels of ALT, AST, and ALP in the HBP group were significantly lower than those in the SCS, NMP, and BMP groups (P < 0.05). The levels of ALB responding to hepatic synthetic function were only observed to be significantly lower in the SCS group compared to that of the Sham and HBP groups at 7 d post-LT (P < 0.05). TBil can respond to the excretory function of the liver and to hepatocyte damage. The level of TBIL in the HBP group was higher than that in the Sham group at 7 d post-LT but significantly lower than that of all the other groups (P < 0.05). The level of TBIL in the SCS group was significantly higher than that in all of the other groups. The level of TBIL in the BMP group was lower than that in the NMP group, but higher than that in the HBP group. These findings indicated that HO-1/BMMSCs significantly improved the condition of the recipients’ liver function (Fig. 2F).
Gene chip analysis of the differential gene expression in the liver grafts of each group
We detected the level of mRNA expression in the liver grafts of each group, and analyzed the differential gene expression using gene chip technology, focusing on the NMP, BMP, and HMP groups (Table S1 − 3). The combination of the GO analysis and KEGG analysis revealed that the interaction between cytokines and cytokine receptors, as well as the level of gene expression of Th1- and Th2-related cytokines were significantly different (Fig. 3A − C and S3A − D). We compared the gene expression of cytokines related to ACR (Fig. 3D) and found that the gene expression of Th1 cytokines (e.g., IFN-γ, TNFα, and IL-2) related to ACR was significantly lower in the HMP and BMP groups compared with that of the NMP group. The level of gene expression in the above cytokines was lower in the HMP group compared to that of the BMP group. In combination with the analysis of the interaction of cytokine genes associated with ACR performed in a String database (Fig. 3D), we focused on the role of IFN-γ gene expression in HO-1/BMMSCs in attenuating ACR. The effect of IFN-γ on NKT cells, NK- and Th1- and Th2-related cytokines was assessed.
HO-1/BMMSCs perfused by NMP reduced the proportion of NKT cells and inhibited IFN-γ expression following LT
Under the guidance of the gene chip analysis results, we next sought to determine the cellular source of IFN-γ. IFN-γ is primarily secreted by NKT and NK cells. Simultaneously, a large number of NKT cells in the liver of rats and NKT cells could secrete IFN-γ in large quantities within a few hours of stimulation. In light of the fact that IFN-γ can significantly promote ACR, we used flow cytometry to detect the proportion of NKT cells and IFN-γ expression in the different recipient tissues. The results showed that following LT, the proportion of NKT cells in the spleen of the NMP group was increased compared with that of the Sham group (P < 0.05); however, no significant difference was observed in the other groups (P > 0.05). While the proportion of NKT cells in the blood of the NMP group was significantly higher than that of the other groups (P < 0.05), no significant difference was observed between the BMP and the HMP groups (P < 0.05). The proportion of NKT cells in the liver of the NMP group was significantly higher than that of the other groups and the HMP group was significantly lower than that of the BMP group (P < 0.05) (Fig. 4A). Intracellular staining was used to detect the changes in IFN-γ levels in the NKT cells (Fig. 4B). The level of IFN-γ in the NKT cells of the NMP group was significantly higher than that of the BMP and HMP groups, and the HMP group was significantly lower than that of the BMP group (P < 0.05). These results demonstrated that the DCD donor liver was preserved by NMP combined with HO-1/BMMSCs. The proportion of NKT cells and level of IFN-γ in the recipients was significantly reduced following LT. HO-1/BMMSCs in the donor livers exhibit a stronger regulatory effect on the NKT cells than BMMSCs.
HO-1/BMMSCs perfusion by NMP can reduce the proportion of NK and CD8 + T cells in the liver grafts, inhibit their function, and reduce the level of inflammatory factors in the recipients
Flow cytometry analysis of the proportion of NK cells in the liver grafts revealed that the proportion of the HMP and BMP groups was significantly lower than that of the NMP group, and the HMP group was significantly lower than that of the BMP group (P < 0.05) (Fig. 5A). Intracellular IFN-γ staining revealed that the level of IFN-γ expression in the NK cells of the liver grafts of the HMP group and BMP group was significantly lower than that of the NMP group. The HMP group was significantly lower than that of the BMP group (P < 0.05) (Fig. 5B). The HO-1/BMMSCs in the DCD donor liver could reduce the proportion of NK cells in the liver grafts after LT, and the inhibitory effect of HO-1/BMMSCs on NK cells was greater than that of the BMMSCs.
Since IFN-γ can activate CD8+ T cells, we detected the proportion of T cells in the liver by flow cytometry. The proportion of CD8+ T cells in the liver grafts of the HMP and BMP groups was significantly lower than that of the NMP group, and the proportion of CD8+ T cells in the HMP group was significantly lower than that in the BMP group (P < 0.05) (Fig. 5C). The level of granzyme B (GZMB) expression reflected the intensity of the toxic effect of CD8+ T cells, and the level of GZMB expression in CD8+ T cells was significantly lower in the liver grafts of the HMP group and BMP group compared with the NMP group, and it was significantly lower in the HMP group compared with that of the BMP group (P < 0.05) (Fig. 5D).
An ELISA was used to detect the ACR-related cytokines, IFN-γ, TNF-α, and IL-2, and the results showed that the cytokines in the HMP and BMP groups were significantly lower than those of the NMP group, and the HMP group was significantly lower than that in the BMP group (P < 0.05) (Fig. 5E). These results suggest that HO-1/BMMSCs can reduce the level of inflammatory factors related to ACR after LT and the effect of HO-1/BMMSCs is greater than that of BMMSCs.
Excessive activation of NKT cells aggravate ACR
To clarify the role of NKT in the ACR of LT, the rats in the NMP and HMP group the relative specific stimulator of NKT cells were administered α-GalCer (50 µg/kg) by intraperitoneal injection. Our findings showed severe ACR in the transplanted livers of the NMP group, and the application of α-GalCer had no significant effect on the NMP group (Fig. 6A and B). The histopathology of the liver in the HMP group demonstrated that ACR was significantly reduced (Fig. 6A), the ACR score was reduced (Fig. 6B), and the degree of liver apoptosis was significantly improved (Fig. 6C). However, when α-GalCer was used to activate NKT cells, the level of acute liver rejection was more severe than that in the HMP group. The liver function (Fig. 6D) analysis showed that the level of liver enzymes and TBil were significantly increased under the administration of α-GalCer.
Further detection of NKT cells in the liver grafts revealed that the use of α-GalCer could affect the inhibitory effect of HO-1/BMMSCs on NKT cells and significantly increase the level of IFN-γ expression in the NKT cells (Fig. 7A). In the event that α-GalCer induces NKT cell activation, we tested NK cells and CD8+ T cells closely related to ACR. We found that the level of IFN-γ in the NK cells and level of GZMB in CD8+ T cells were significantly increased (Fig. 7B and C). In addition, compared to the HMP group, α-GalCer caused a significant increase in ACR-related cytokines (e.g., IFN-γ, TNFα, and IL-2) (Fig. 7D). Our results indicate that the inhibitory effect of HO-1/BMMSCs on NKT regulates ACR. This also suggests that α-GalCer has no effect on ACR after LT; however, in the case of HO-1/BMMSCs-induced transplantation tolerance, NKT cell activation can cause aggravation of ACR.
Co-culture experiments observed the effect of HO-1/BMMSCs on IFN-γ expression in NKT cells
We co-cultured HO-1/BMMSCs and liver MNCs in vitro to verify whether it could regulate the function of NKT cells activated by α-GalCer. Our results showed that the level of IFN-γ expression in NKT cells was significantly increased following stimulation with α-GalCer. When co-cultured with BMMSCs or HO-1/BMMSCs, the level of IFN-γ expression was significantly reduced in NKT cells, and the HO-1/BMMSCs inhibited IFN-γ expression in NKT cells more significantly than in BMMSCs (Fig. 8A). We detected ACR-related cytokines (e.g., IFN-γ, TNF-α, and IL-2) in the culture supernatant, and found that the co-culture of HO-1/BMMSCs with NKT cells could significantly reduce the levels of these cytokines (Fig. 8B). In vitro studies have shown that HO-1/BMMSCs can inhibit IFN-γ expression in the NKT cells and reduce the level of cytokines related to ACR.
HO-1/BMMSCs in the liver grafts affects the co-inhibitory receptor expression of NKT cells
Our previous study confirmed the significant inhibitory effect of HO-1/BMMSCs on the level of IFN-γ expression in NKT cells. We further investigated the mechanism of action of HO-1/BMMSCs on NKT cells, the activation of which is triggered by linking semi-invariant TCRs, as well as a series of costimulatory and co-inhibitory receptors. We detected the co-receptor expression on NKT cells in the liver grafts of each group (Fig. 9A and B), and found that NKT cells constitutively expressed a variety of co-stimulatory receptors; however, few differences were observed between the groups. The examination of the co-inhibitory receptors revealed that the expression of NKT cell surface expression of the co-inhibitory receptors, BTLA and CD160, differed significantly in each group. In the HMP and BMP groups, BTLA and CD160 expression (proportion of positive cells and MFI) in the NKT cells were significantly higher than that of the NMP group (Fig. 9C and D) and the level of expression in the HMP group was higher than that of BMP group. This finding confirms that the HO-1/BMMSCs in the liver grafts increasing the expression of co-inhibitory receptors BTLA and CD160 on NKT cells, through which inhibitory signals are transmitted to NKT cells, reducing the level of IFN-γ secretion from NKT cells and reducing ACR after LT. Moreover, the HO-1/BMMSCs that colonized the transplanted livers had a greater regulatory effect on the NKT cells than the BMMSCs.
The regulation of NKT cell function by HO-1/BMMSCs is achieved through BTLA and CD160
To further confirm the role of BTLA and CD160 in the regulation of NKT cell function by HO-1/BMMSCs, we extracted MNCs from the normal rat livers using α-GalCer to activate the NKT cells. A blocking antibody was used to block BTLA or/and CD160 in NKT cells, and co-cultured with HO-1/BMMSCs. Our research showed that when BTLA or CD160 was blocked, the level of IFN-γ expression in the NKT cells increased to varying degrees compared with that of the control group (Fig. 10A). The levels of IFN-γ, TNF-α, and IL-2 in the culture supernatant were also significantly increased compared to that of the control group (Fig. 10B). The level of IFN-γ expression in NKT cells was further increased when BTLA and CD160 were both blocked (Fig. 10A), the concentration of IFN-γ, TNF-α, and IL-2 were also further increased (Fig. 10B). These results suggest that HO-1/BMMSCs affects the surface expression of BTLA and CD160 on NKT cells, transmitting inhibitory signals to NKT cells and regulating the levels of IFN-γ, inhibiting the ACR of LT. Thus, BTLA and CD160 may be co-inhibitory receptors with non-overlapping functions.