Differentiation and immunosuppressive function of CD19+CD24hiCD27+ regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway.

BACKGROUND: Regulatory B cells (Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs (miRNAs), miR-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and miR-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs (mBregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. METHODS: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce miR-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. RESULTS: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of mBregs in the circulating blood were significantly impaired. miR-29a-3p was found to be a regulator of mBregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5 (NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of mBregs. The inhibition of miR-29a-3p in CD19+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into mBregs. In addition, the observed enhancement of differentiation and immunosuppressive function of mBregs upon miR-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. CONCLUSIONS: miR-29a-3p was found to be a crucial regulator for mBregs differentiation and immunosuppressive function. Silencing miR-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

Post-transplant inflammatory cytokine signature adds value for predicting tumor recurrence after liver transplantation for hepatocellular carcinoma.

BACKGROUND: Cytokines are key regulators of post-transplant inflammation responses which reconstitute post-transplant hepatic and systemic environments to influence the likelihood of tumor relapse. This study investigated the prognostic value of post-transplant cytokines on tumor recurrence after liver transplantation (LT) for hepatocellular carcinoma (HCC). METHODS: A retrospective analysis was conducted in prospectively collected 150 adult HCC patients who received liver transplantation from 1997 to 2015. The post-transplant 41 inflammatory cytokines were quantified by multiplexing analysis and determined their prognostic value for predicting post-LT tumor recurrence by receiver operative characteristic analysis. A prediction model for post-LT tumor recurrence was generated by the logistic regression and internally validated Bootstrapping and compared with external prediction models. RESULTS: Post-transplant circulating CCL11, IFNα2, and IL17A cytokines were identified to be significant predictors of post-LT tumor recurrence and survival. A prediction score composed of the post-transplant 3-cytokine (P3C) signature, UCSF criteria, and pre-LT AFP was established. The P3C-UCSF-AFP score significantly predicted post-LT tumor recurrence and poor survival both in deceased donor liver transplantation (DDLT) and living donor liver transplantation (LDLT). The P3C-UCSF-AFP score was validated to significantly predict post-LT 2-year and 5-year tumor recurrence, outperforming the RETREAT score, French AFP model, up-to-seven, UCSF criteria, and Milan criteria. Importantly, the P3C-UCSF-AFP score could cost-effectively stratify high-risk recipients subjected to a refinement of post-recurrence survival. CONCLUSION: The integrated P3C-UCSF-AFP score not only compensated for the pre-LT unpredictability and predicted post-LT tumor recurrence accurately, but also guided the clinical refinements of post-LT surveillance and therapeutic strategies in transplant oncology.

Glutathione S-transferase A2 promotes hepatocellular carcinoma recurrence after liver transplantation through modulating reactive oxygen species metabolism.

Hepatocellular carcinoma (HCC) recurrence after liver transplantation remains a significant clinical problem. Ischemia-reperfusion injury (IRI) occurred inevitably at the early phase after liver transplantation (LT) spawns a significant risk of HCC recurrence. However, their linkage and IRI-derived risk factors for HCC recurrence remain exclusive. Understanding the mechanism of post-transplantation hepatic injury could provide new strategies to prevent the later event of HCC recurrence. We demonstrated that glutathione S-transferase A2 (GSTA2) expression was significantly associated with early phase hepatic and systemic injury and ROS level after liver transplantation. Early phase circulating GSTA2 (EPCGSTA2) protein was a significant predictor of HCC recurrence and survival. Heterogeneous single nucleotide polymorphism at G335C of GSTA2 was significantly associated with poor survival of HCC recipients. Enhancement of GSTA2 could protect HCC cells against H2O2-induced cell death by compensating for the elevated ROS stress. We also demonstrated that GSTA2 played crucial roles in regulating the ROS-associated JNK and AKT signaling pathways and ROS metabolism in HCCs in responding to a dynamic ROS environment. Functionally, endogenous or exogenous upregulation of GSTA2 could promote HCC growth and invasion through activating the epithelial-mesenchymal-transition process. Targeted inhibition of GSTA2 could suppress HCC growth and metastasis. In conclusion, GSTA2 could be a novel prognostic and therapeutic target to combat HCC recurrence after liver transplantation.

Monocytic MDSC mobilization promotes tumor recurrence after liver transplantation via CXCL10/TLR4/MMP14 signaling.

Tumor recurrence is the major obstacle for pushing the envelope of liver transplantation for hepatocellular carcinoma (HCC) patients. The inflammatory cascades activated by acute liver graft injury promote tumor recurrence. We aimed to explore the role and mechanism of myeloid-derived suppressor cell (MDSC) mobilization induced by liver graft injury on tumor recurrence. By analyzing 331 HCC patients who received liver transplantation, the patients with graft weight ratio (GWR, the weight of liver graft divided by the estimated standard liver weight of recipient) <60% had higher tumor recurrence than GWR ≥60% ones. MDSCs and CXCL10/TLR4 levels were significantly increased in patients with GWR <60% or tumor recurrence. These findings were further validated in our rat orthotopic liver transplantation model. In CXCL10-/- and TLR4-/- mice of hepatic ischemia/reperfusion injury plus major hepatectomy (IRH) model, monocytic MDSCs, instead of granulocytic MDSCs, were significantly decreased. Importantly, CXCL10 deficiency reduced the accumulation of TLR4+ monocytic MDSCs, and CXCL10 increased MDSC mobilization in the presence of TLR4. Moreover, MMP14 was identified as the key molecule bridging CXCL10/TLR4 signaling and MDSC mobilization. Knockout or inhibition of CXCL10/TLR4 signaling significantly reduced the tumor growth with decreased monocytic MDSCs and MMP14 in the mouse tumor recurrent model. Our data indicated that monocytic MDSCs were mobilized and recruited to liver graft during acute phase injury, and to promote HCC recurrence after transplantation. Targeting MDSC mobilization via CXCL10/TLR4/MMP14 signaling may represent the therapeutic potential in decreasing post-transplant liver tumor recurrence.

Inhibition of Carnitine Palmitoyltransferase 1A Aggravates Fatty Liver Graft Injury via Promoting Mitochondrial Permeability Transition.

BACKGROUND: Hepatic steatosis is a major risk factor for graft failure due to increased susceptibility of fatty liver to ischemia-reperfusion injury (IRI) during transplantation. Here, we aimed to investigate the role of carnitine palmitoyltransferase 1A (CPT1A) in fatty liver graft injury and to explore the underlying mechanism and therapeutic potential on attenuating hepatic IRI. METHODS: Intragraft CPT1A expression profile and the association with fatty graft injury were investigated in human and rat liver transplantation samples. The underlying mechanism and therapeutic potential of CPT1A activator against IRI were also explored in mouse hepatic ischemia-reperfusion plus major hepatectomy model and in in vitro. RESULTS: CPT1A expression was significantly reduced (P = 0.0019; n = 96) in human fatty liver graft compared with normal one at early phase after transplantation. Low expression of CPT1A was significantly associated with high serum alanine aminotransferase (P = 0.0144) and aspartate aminotransferase (P = 0.0060) levels. The inhibited CPT1A and poor liver function were consistently observed in rat and mouse models with fatty livers. Furthermore, inhibition of CPT1A significantly promoted the translocation of chloride intracellular channel 1 to form chloride ion channel. The dysregulation of chloride ion channel activity subsequently triggered mitochondrial permeability transition (MPT) pore opening, exacerbated cellular oxidative stress, and energy depletion. Importantly, our intravital confocal imaging showed that CPT1A activation attenuated hepatic injury through preventing MPT after reperfusion in fatty mice. CONCLUSIONS: CPT1A inhibition triggered MPT contributed to severe IRI in fatty liver graft. CPT1A restoration may offer therapeutic potential on attenuating hepatic IRI.

FTY720 Suppresses Liver Tumor Growth and Metastasis by Reducing Circulating Regulating T Cells and Enhancing the Anti-Tumor Effect of Rapamycin.

BACKGROUND: In this study, we aimed to study the effect of FTY720 treatment in reducing circulating Tregs level and then suppressing liver tumor metastasis after hepatectomy and I/R injury in animal models. Furthermore, we also investigated the synergistic anti-tumor effect of FTY720 combined with rapamycin on hepatocellular carcinoma. METHODS: The effect of FTY720 on suppressing Tregs mobilization and tumor metastasis after hepatectomy was investigated in an orthotopic liver tumor rat model with hepatectomy and hepatic ischemia/reperfusion (I/R) injury. The synergistic anti-tumor effect of FTY720 combined with rapamycin was further explored both in in vitro functional study and in orthotopic liver tumor mouse model. RESULTS: In rat model, hepatic I/R promoted tumor metastasis and increased circulating Tregs after hepatectomy. The treatment of FTY720 reduced liver tumor metastasis and the number of circulating Tregs. Furthermore, FTY720 enhanced the anti-tumor capacity of rapamycin by inhibiting tumor cell proliferation and migration in vitro and reducing tumor growth in vivo through suppressing hepatic stellate cell activation and tumor angiogenesis. CONCLUSION: FTY720 suppressed liver tumor growth and metastasis by reducing the population of circulating Tregs and enhancing the anti-tumor effect of rapamycin. It was suggested that FTY720 single or combined with rapamycin might provide novel insight for suppressing tumor growth and metastasis for HCC patients.

ApoA-1 accelerates regeneration of small-for-size fatty liver graft after transplantation.

OBJECTIVES: Apolipoprotein A-1 (ApoA-1) is involved in regulating both lipid and energy metabolism, which may play important roles in liver regeneration, especially for the liver with steatosis. We here intended to investigate the role of ApoA-1 in regeneration of small-for-size fatty liver graft and to explore the underlying mechanism. METHODS: The association of ApoA-1 expression with liver regeneration was studied in rat liver transplantation models using small-for-size normal graft or small-for-size fatty graft. The direct role of ApoA-1 in liver regeneration was studied in mouse hepatectomy model in vivo and hepatocytes in vitro. RESULTS: Compared to small-for-size normal graft, decreased expression of ApoA-1 associated with delayed regeneration were detected in small-for-size fatty liver graft after transplantation. In functional study, the expression of ApoA-1 was decreased in hepatocytes with steatosis and was inversely associated with the concentration of oleic acid. The ApoA-1 administration effectively attenuated hepatocytes steatosis and accelerated hepatocytes proliferation. In mouse model, ApoA-1 treatment promoted liver regeneration at day 2 after major hepatectomy. In addition, the treatment of ApoA-1 increased the expressions of PGC-1α and its target genes Tfam, Ucp2 and SDHB. CONCLUSIONS: ApoA-1 may accelerate regeneration of small-for-size fatty liver graft at day 2 after transplantation through regulating mitochondrial function. ApoA-1 may be the potential new therapy of promoting liver regeneration.

Glutathione Peroxidase 3 Delivered by hiPSC-MSCs Ameliorated Hepatic IR Injury via Inhibition of Hepatic Senescence.

Background and Aims: Down-regulation of GPx3 accelerated hepatic senescence, which further caused overwhelming inflammation and severe liver graft injury. MSCs derived from human induced pluripotent stem cells (hiPSC-MSCs) have been developed as more efficient delivery vehicle with the property of injury tropism. Here, we aimed to explore the suppressive role of GPx3 in hepatic IR injury using novel delivery system of hiPSC-MSCs. Methods: The mice IR injury model with partial hepatectomy was established. The engineered hiPSC-MSCs delivering GPx3 was constructed. All the mice were segregated into three groups. hiPSC-MSC-GPx3, hiPSC-MSC-pCDH (vector control) or PBS were injected via portal vein after reperfusion. Liver injury was evaluated by histological and serological test. Hepatic apoptosis was detected by Tunel staining and remnant liver regeneration was assessed by Ki67 staining. The role of hepatic senescence in liver graft injury was evaluated in rat orthotopic liver transplantation model. The suppressive effect of GPx3 on hepatic senescence was examined in mice IR injury model and confirmed in vitro. Hepatic senescence was detected by SA-ß-Gal and P16/ink4a staining. Results: GPx3 can be successfully delivered by hiPSC-MSCs into liver tissues. Histological examination showed that hiPSC-MSC-GPx3 treatment significantly ameliorated hepatic IR injury post-operation. Significantly lower LDH (891.43±98.45 mU/mL, P<0.05) and AST (305.77±36.22 IU/L, P<0.01) were observed in hiPSC-MSC-GPx3 group compared with control groups. Less apoptotic hepatocytes were observed and the remnant liver regeneration was more active in hiPSC-MSC-GPx3 group. In rat orthotopic liver transplantation model, more senescent hepatocytes were observed in small-for-size liver graft, in which GPx3 expression was significantly compromised. In mice IR injury model, hiPSC-MSC-GPx3 significantly suppressed hepatic senescence. In addition, rGPx3 inhibited cellular senescence of liver cells in a dose dependent manner. Four candidate genes (CD44, Nox4, IFNG, SERPERINB2) were identified to be responsible for suppressive effect of GPx3 on hepatic senescence. Conclusion: Engineered hiPSC-MSCs delivering GPx3 ameliorated hepatic IR injury via inhibition of hepatic senescence.

CXCL10/CXCR3 signaling mobilized-regulatory T cells promote liver tumor recurrence after transplantation.

BACKGROUND & AIMS: Liver graft injury and tumor recurrence are the major challenges of liver transplantation for the patients with hepatocellular carcinoma (HCC). Here, we aimed to explore the role and mechanism of liver graft injury mobilizing regulatory T cells (Tregs), which lead to late phase tumor recurrence after liver transplantation. METHODS: The correlation among tumor recurrence, liver graft injury and Tregs mobilization were studied in 257 liver transplant recipients with HCC and orthotopic rat liver transplantation models. The direct roles of CXCL10/CXCR3 signaling on Tregs mobilization and tumor recurrence were investigated in CXCL10-/- and CXCR3-/- mice models with hepatic IR injury. RESULTS: Clinically, patients received the graft with graft weight ratio (GWR) <60% had higher HCC recurrence after liver transplantation than the recipients with GWR ⩾60% graft. More circulating Tregs and higher intragraft TLR4/CXCL10/CXCR3 levels were detected in recipients with GWR <60% graft. These results were further validated in rat transplantation model. Foxp3+ cells and expressions of TLR4, CXCL10, TGFß, CTLA-4 and CD274 were increased in rat liver tumor tissues from small-for-size graft group. In mouse model, the mobilization and recruitment of Tregs were decreased in TLR4-/-, CXCL10-/- and CXCR3-/- mice compared to wild-type mice. Moreover, less CXCR3+ Tregs were recruited into liver in CXCL10-/- mice after hepatic IR injury. The knockout of CXCL10 and depletion of Tregs inhibited tumor recurrence after hepatic IR injury. CONCLUSION: CXCL10/CXCR3 signaling upregulated at liver graft injury directly induced the mobilization and intragraft recruitment of Tregs, which further promoted HCC recurrence after transplantation. LAY SUMMARY: There were positive correlation among tumor recurrence, circulating Tregs and liver graft injury after human transplantation for HCC patients. The knockout of CXCL10 decreased hepatic recruitment of CXCR3+ Tregs and late phase tumor recurrence after hepatic IR injury.

Repressor and activator protein accelerates hepatic ischemia reperfusion injury by promoting neutrophil inflammatory response.

Repressor and activator protein (Rap1) directly regulates nuclear factor-κB (NF-κB) dependent signaling, which contributes to hepatic IRI. We here intended to investigate the effect of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying mechanisms. The association of Rap1 expression with hepatic inflammatory response were investigated in both human and rat liver transplantation. The effect of Rap1 in hepatic IRI was studied in Rap1 knockout mice IRI model in vivo and primary cells in vitro. Our results showed that over expression of Rap1 was associated with severe liver graft inflammatory response, especially in living donor liver transplantation. The results were also validated in rat liver transplantation model. In mice hepatic IRI model, the knockout of Rap1 reduced hepatic damage and hepatic inflammatory response. In primary cells, the knockout of Rap1 suppressed neutrophils migration activity and adhesion in response to liver sinusoidal endothelial cells through down-regulating neutrophils F-Actin expression and CXCL2/CXCR2 pathway. In addition, the knockout of Rap1 also decreased production of pro-inflammatory cytokines/chemokines in primary neutrophils and neutrophils-induced hepatocyte damage. In conclusion, Rap1 may induce hepatic IRI through promoting neutrophils inflammatory response. Rap1 may be the potential therapeutic target of attenuating hepatic IRI.

FAM83D activates the MEK/ERK signaling pathway and promotes cell proliferation in hepatocellular carcinoma.

Publicly available microarray data suggests that the expression of FAM83D (Family with sequence similarity 83, member D) is elevated in a wide variety of tumor types, including hepatocellular carcinoma (HCC). However, its role in the pathogenesis of HCC has not been elucidated. Here, we showed that FAM83D was frequently up-regulated in HCC samples. Forced FAM83D expression in HCC cell lines significantly promoted their proliferation and colony formation while FAM83D knockdown resulted in the opposite effects. Mechanistic analyses indicated that FAM83D was able to activate the MEK/ERK signaling pathway and promote the entry into S phase of cell cycle progression. Taken together, these results demonstrate that FAM83D is a novel oncogene in HCC development and may constitute a potential therapeutic target in HCC.