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Acute rejection is commonly encountered for long-term survival in liver transplant (LT) recipients and may impact their long-term survival if rejection is severe or recurrent. The aim of this study is to examine the therapeutic potential of transforming growth factor (TGF-β)-overexpressing mesenchymal stem cells (MSCs) in inducing a local immunosuppression in liver grafts after transplantation. MSCs were transduced with a lentiviral vector expressing the human TGF-β1 gene; TGF-β1-overexpressing MSCs (designated as TGF/MSCs) were then transfused into the liver grafts via the portal vein of a rat LT model of acute rejection. Rejection severity was assessed by clinical and histologic analysis. The immunity suppression effects and mechanism of TGF/MSCs were tested, focusing on their ability to induce generation of regulatory T cells (Tregs) in the liver grafts. Our findings demonstrate that transfusion of TGF/MSCs prevented rejection, reduced mortality, and improved survival of rats after LT. The therapeutic effects were associated with the immunosuppressive effects of MSCs and TGF-β1. Their reciprocal effects on Tregs induction and function resulted in more CD4 + Foxp3 + Helios- induced Tregs, fewer Th17 cells, and improved immunosuppressive effects in local liver grafts. Thus, TGF/MSCs can induce a local immunosuppressive effect in liver grafts after transplantation. The immunomodulatory activity of TGF-β1 modified MSCs may be a gateway to new therapeutic approaches to prevent organ rejection in clinical transplantation. Stem Cells 2016;34:2681-2692., (© 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Background & Aims: In living-donor liver transplantation (LDLT), "small-for-size graft (SFSG) syndrome" is a complex process resulting primarily from ischemia-reperfusion injury (IRI) and portal hypertension associated with size mismatch between graft and recipient. In the early period of LDLT, molecular events related to subsequent apoptosis, necrosis, proliferation and regeneration appeared in specific protein expression patterns., Methods: We used 2D-PAGE and MALDI-TOF/TOF technology to construct a comparative proteome profile for small-for-size liver grafts (SFSGs) during the early period of LDLT in rats (ischemia 1h, and 2, 6, 24, 48 h post-reperfusion); sham-operated liver was the control. Western blotting was used to confirm the proteomics results and immunohistochemistry was carried out to explore the cellular localization of selected proteins. We further performed cluster and bioinformatics analyses of differential proteins. Lastly, we overexpressed Prdx5 in liver grafts using an adenoviral vector to evaluate its protective role., Results: We identified 314 differential protein spots corresponding to 259 different proteins. Cluster analyses revealed six expression patterns, and bioinformatics analyses revealed that each pattern was related to many specific cell processes. We also showed that Prdx5 overexpression could attenuate injury to SFSGs and increase survival in recipients., Conclusions: Taken together, these results reveal an important proteome profile that is functional in SFSGs during early period of LDLT, and provide a strong basis for further research., (Copyright 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Ischemia-reperfusion and chronic injuries associated with small-for-size liver transplantation (SFSLT) impair the regeneration of liver graft and induce liver fibrosis. Mesenchymal stem cells (MSCs) can prevent the development of liver fibrosis, and hepatocyte growth factor (HGF) can also attenuate liver cirrhosis. Our previous studies have demonstrated that higher occurrence of liver fibrosis existed in rats post-SFSLT, and that implantation of HGF/MSCs, the human HGF (hHGF)-expressing MSCs, can improve liver regeneration, reduce mortality of rats, as well as have the potent antifibrotic effect in this SFSLT model. In the present study, we implanted HGF/MSCs into liver grafts via the portal vein and investigated their role in antifibrosis effect, using a 30% SFSLT rat model. Fibrosis indexes, including laminin (LN), hyaluronic acid (HA) levels in serum and hydroxyproline (Hyp) content in the liver grafts, the expression of transforming growth factor-beta1 (TGF-beta(1)), rat HGF (rHGF), alpha-smooth muscle actin (alpha-SMA) in hepatic stellate cells (HSCs), alanine aminotransferase (ALT), total bilirubin (BIL), and albumin (ALB) levels in serum, in rats in different treatment groups were assessed at different time points. We found that HGF/MSCs significantly inhibited the formation of liver fibrosis in rats undergoing SFSLT, while MSCs and HGF had synergistic effects in the process. The antifibrosis effect of HGF/MSCs may have contributed in modulating the activation and apoptosis of HSCs, elevating the rHGF expression level, and decreasing the TGF-beta(1) secretion of activated HSCs. These studies suggest that HGF/MSCs may be a novel therapeutic option for the treatment of liver fibrosis after SFSLT.

It has been shown that rat liver allografts between certain inbred major histocompatibility complex (MHC) disparate strains are accepted spontaneously, and regulatory T cells (Tregs) have been suggested to play a role in the spontaneous liver tolerance. CD8(+)CD103(+) T cells bear the phenotypes of effector cells, and they are implicated in allograft destruction. Here we provide evidence that CD8(+)CD103(+) T cells possess regulatory function and may contribute to prevent liver allograft rejection. We show that the expression of CD103 in the CD8(+) T cells was increased in spontaneous liver grafts tolerant recipients. We further show that CD8(+)CD103(-) T cells can also upregulate the expression of CD103 and Foxp3 after stimulation with alloantigen or TGF-beta in vitro, and the CD8(+)CD103(+) T cells acquired regulatory properties. The suppressive function of the alloantigen or TGF-beta conditioned CD8(+)CD103(+) T cells was cell-cell contact dependent. These results imply that liver-specific factor(s) would be involved in the generation of CD8(+)CD103(+) Tregs that contribute to spontaneous liver allografts tolerance.

Objective: Graft size is one of the major risk factors in adult-to-adult living donor liver transplantation and rapid regeneration is an essential post-operative requirement. Ischemic preconditioning (IPC) has been shown to be an effective strategy in the reduction of hepatic ischemia-reperfusion injury and stimulation of liver regeneration. This study was designed to evaluate the effects of IPC on liver regeneration in small-for-size liver grafts., Methods: We employed a rat orthotopic liver transplantation model using small-for-size (30%) grafts, in the presence or absence (control) of IPC (10 min of ischemia followed by 15 min of reperfusion). Survival rate, graft injury, hepatocellular proliferation, cell cycle progression, Stat3 activation, as well as TNF-alpha and IL-6 expression were assessed., Results: IPC significantly enhanced the extent of graft injury and hindered hepatic regeneration in small-for-size liver grafts. The 7-day survival rate was also reduced by IPC, but failed to reach statistical significance. IPC did not affect TNF-alpha levels, but significantly decreased the elevation of IL-6 after reperfusion. These findings were correlated with down-regulation of cyclin E and cyclin D1, and decreased numbers of PCNA-positive nuclei in IPC grafts. These results were inconsistent with Stat3 activation, as P-Stat3 exhibited a stronger and prolonged pattern of expression in the IPC group, compared to controls., Conclusions: Ischemic preconditioning may impair liver regeneration in small-for-size liver grafts by decreasing IL-6 and blunting cell cycle progression, through a mechanism at least partially independent of Stat3.

The biological features of intrahepatic CD4+ CD25+ T regulatory cells in the naturally tolerance of rat liver transplantation were explored. Orthotopic liver transplantation was performed in two allogeneic rat strain combinations, one with fatal immunosuppression despite a complete major histocompatibility complex mismatch. The subjects were divided into three groups according to different donors and recipients [Tolerance group: LEW-to-DA; Rejection group: DA-to-LEW; Syngegnic group (control group): DA-to-DA]. The proportion of intrahepatic CD4+CD25+ T cells from three groups was determined by flow cytometry (FCM) in different time. The intrahepatic CD4+CD25+ T cells were isolated by magnetic activated cell sorting (MACS) method and identified by FCM. The Foxp3 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). And their suppression on the proliferation of CD4+CD25− T effector cells was analyzed by cell proliferation assay in vitro. Beginning immediately after transplantation, the proportion of Treg cells increased over time in both allogeneic groups but was significantly greater in the Rejection group. The proportion of Treg cells declined after day 5, and such reduction was more dramatic in the Rejection group than in the Tolerance group. Animals in the Tolerance group showed a second increase in the proportion after day 14. Intrahepatic CD4+ CD25+ T cells isolated from spontaneous tolerance models inhibited the proliferation of mixed lymphocyte reaction. The purity of CD4+CD25+ T cells sorted by MACS was 86%–93%. The CD4+CD25+ T cells could specifically express the Foxp3 gene compared with CD4+CD25− T cells. In vitro, the spleen cells from LEW rats can irritate the proliferation of CD4+CD25+ T cells more obviously than the syngegnic spleen cells. CD4+CD25+ Tr cells could suppress the proliferation of CD4+CD25− T cells, but the inhibition was reversed by exogenous IL-2 (200 U/mL). The CD4+CD25+ T regulatory cells specifically express the Foxp3 gene, which may play an important role in the induction of liver transplantation tolerance by suppressing the reaction of effective T cells. [ABSTRACT FROM AUTHOR]

• Inhibiting kupffer cells Eva1a aggravates liver ischemia-reperfusion injury and faciliate the activation of NLRP3. • knockdown of kupffer cells Eva1a induces the activation of NLRP3 inflammatory bodies by impairing the autophagic flux of kupffer cells. • Eva1a and ATG16L1 work together to faciliate the formation of kupffer cells'autophagosomes during IR. • Knockdown of kupffer cells Eva1a inhibits the formation of autophagosomes through impairing ATG5/ATG12 complex in IR process, rather than through impairing Beclin-Vps34 pathway. Ischemia-reperfusion(IR) injury is one of the main complications of liver transplantation and partial hepatectomy. Innate immunity mediated by kupffer cells plays an important role in it. In this study, we focused on evaluating the intrinsic relationship between the autophagy induction of kupffer cells and the activation of NLRP3 inflammasomes caused by liver ischemia-reperfusion. Pre-depletion of kupffer cells can aggravate inflammation and tissue damage within 24 h after IR.Enhancing the autophagy of kupffer cells can inhibit the activation of NLRP3 caused by IR, and inhibiting autophagy can induce the secretion of IL1β dependent on NLRP3 activation.Eva1a is up-regulated by the inflammatory cascade activated by IR.Knockdown of Eva1a in vivo on the one hand will aggravate IR inflammation, increase the production of TNF-α, IL-1β and inhibit the secretion of IL-10.On the other hand, it will aggravate the liver histological damage. Knockout of Eva1a induces ASC activation and cleavage of caspase1 and IL1β in an NLRP3-dependent manner, which is closely related to the function of blocking Eva1a to promote autophagosome formation.We further found that knockdown of ATG16L1 will reverse the more formation of autophagosomes induced by overexpression of Eva1a, whereas knockdown of ATG16L1 did not further reduce the formation of autophagosomes inhibited by siEva1a. We also found that the addition of siATG7, siATG5 and siATG12 would reverse the IR autophagy of liver induced by overexpression of Eva1a, but inhibition of the Beclin1-Vps34 pathway did not significantly reverse the effect of overexpression of Eva1a.These prove that Eva1a and ATG16L1 may work together in the liver IR model to actively induce the formation of autophagosomes and be independent from the beclin1-vps34-induced autophagy pathway to limit the excessive activation of IR inflammation. Our study provides brand new insights into the mechanism of liver macrophages in the progression of inflammation in the context of liver ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Background: Allograft inflammatory factor-1 (AIF-1) plays an important role in immune response and vasculopathy in allografts. The present study investigated activation of AIF-1 in warm and cold ischemia-reperfusion (IR) injury of rat liver, and the potential inhibitory effect of FK506. Methods: We used warm IR injury, orthotopic transplantation, and allograft rejection models in this study. We assessed expression of AIF-1 mRNA and protein, as well as its inducers interferon-γ (IFN-γ) and interleukin-1β (IL-1β). The potential inhibitory effect of FK506 on AIF-1 in a rat macrophage cell line and in these three models was also assessed. Results: AIF-1 mRNA and protein, as well as its inducers IFN-γ and IL-1β, were significantly increased in the warm IR injury, orthotopic transplantation, and allograft rejection models. Real-time RT-PCR and Western blotting showed that pretreatment with low-dose FK506 partially inhibited AIF-1 activation as well as its inducers (IFN-γ and IL-1β) in these three models. Western blotting showed that IFN-γ and IL-1β activated AIF-1 in a macrophage cell line, but pretreatment with FK506 did not inhibit AIF-1 activation in vitro. Hematoxylin and eosin staining showed that edema and necrosis in the liver, as well as alanine aminotransferase (ALT) in serum, of these three groups was reduced after FK506 pretreatment. Conclusion: AIF-1 was activated in warm and cold IR injury, and pretreatment with low-dose FK506 partly inhibited AIF-1 activation and reduced warm and cold IR injury. [ABSTRACT FROM AUTHOR]