A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration

Components of innate immunity have recently been implicated in the regulation of developmental processes. Most strikingly, complement factors appear to be involved in limb regeneration in certain urodele species. Prompted by these observations and anticipating a conserved role of complement in mammalian regeneration, we have now investigated the involvement of complement component C5 in liver regeneration, using a murine model of CCl(4)-induced liver toxicity and mice genetically deficient in C5. C5-deficient mice showed severely defective liver regeneration and persistent parenchymal necrosis after exposure to CCl(4.) In addition, these mice showed a marked delay in the re-entry of hepatocytes into the cell cycle (S phase) and diminished mitotic activity, as demonstrated, respectively, by the absence of 5-bromo-2'-deoxyuridine incorporation in hepatocytes, and the rare occurrence of mitoses in the liver parenchyma. Reconstitution of C5-deficient mice with murine C5 or C5a significantly restored hepatocyte regeneration after toxic injury. Furthermore, blockade of the C5a receptor (C5aR) abrogated the ability of hepatocytes to proliferate in response to liver injury, providing a mechanism by which C5 exerts its function, and establishing a critical role for C5aR signaling in the early events leading to hepatocyte proliferation. These results support a novel role for C5 in liver regeneration and strongly implicate the complement system as an important immunoregulatory component of hepatic homeostasis.     

Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration

Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.     

The Cdk inhibitor p21 is required for necrosis,but it inhibits apoptosis following toxin-induced liver injury

Liver injury and repair were examined in wild type, p21Waf1/Cip1, and p27Kip1-deficient mice following carbon tetrachloride (CCl4) administration. In wild type liver, p21 expression is induced in a biphasic manner following injection of CCl4, with an early peak of p21 expression occurring in pericentral hepatocytes at 6 h, prior to evidence of injury, and a second peak succeeding regenerative proliferation. In contrast, p27 is present throughout the quiescent liver, but its expression decreases following CCl4 injection. Surprisingly, p21-deficient animals were resistant to CCl4-induced necrotic injury, indicating that rapid induction of p21 in pericentral hepatocytes following CCl4 injection contributes to subsequent necrosis. Expression of cytochrome P450 2E1, which plays an essential role in CCl4-induced necrotic injury, was not affected in p21-deficient mice. Although they had the least injury, p21-deficient mice had the highest levels of hepatic proliferation that correlated with increases in hyperphosphorylated retinoblastoma protein and Cyclin A gene expression. Increased replication in p21-deficient livers was counteracted by an increase in hepatocyte apoptosis as detected by caspase-3 activation. p21 plays distinct and opposing roles regulating hepatocyte survival during injury and subsequent repair, with early induction of p21 contributing to necrotic injury and later expression to cessation of proliferation and hepatocyte survival.     

Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S1P receptor 2 (S1P(2)) in hepatocytes in rats in vitro. A potential role of S1P and S1P(2) in liver regeneration and fibrosis was examined in S1P(2)-deficient mice. Nuclear 5-bromo-2'-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S1P(2)-deficient mice after a single carbon tetrachloride (CCl(4)) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S1P(2)-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl(4)- and DMN-induced liver injury, suggesting that S1P(2) inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl(4) administration, fibrosis was less apparent, with reduced expression of smooth-muscle alpha-actin-positive cells in the livers of S1P(2)-deficient mice, suggesting that S1P(2) inactivation ameliorated CCl(4)-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S1P plays a significant role in regeneration and fibrosis after liver injury via S1P(2).     

The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Background

Liver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).

Methods/Principal Findings

Mice received CCl₄ injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl₄ injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl₄-injuries.

Conclusions/Significance

These findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.     

Early growth response (EGR)-1 is required for timely cell-cycle entry and progression in hepatocytes after acute carbon tetrachloride exposure in mice

Cell-cycle induction in hepatocytes protects from prolonged tissue damage after toxic liver injury. Early growth response (Egr)-1(-/-) mice exhibit increased liver injury after carbon tetrachloride (CCl(4)) exposure and reduced TNF-α production. Because TNF-α is required for prompt cell-cycle induction after liver injury, here, we tested the hypothesis that Egr-1 is required for timely hepatocyte entry into the cell cycle after CCl(4)-induced liver injury. Acute liver injury was induced by a single injection of CCl(4). Assays were employed to assess indices of the cell cycle in liver after CCl(4) exposure. Bromodeoxyuridine incorporation peaked in wild-type mice at 48 h after CCl(4) but was reduced by 80% in Egr-1(-/-) mice. Proliferating-cell nuclear-antigen immunohistochemistry revealed blocks in cell-cycle entry and progression to DNA synthesis in Egr-1-deficient mice 48 h after CCl(4). Cyclin D, important for G0/G1 progression, was reduced at baseline and 36 h after CCl(4). Cyclin E1, required for G1/S-phase transition, was reduced in Egr-1(-/-) mice 24 and 48 h after CCl(4) exposure and was associated with reduced phosphorylation of the retinoblastoma protein. Proliferation in Egr-1(-/-) mice was delayed, rather than blocked, because indices of cell-cycle progression were restored 72 h after CCl(4) exposure. We concluded that Egr-1 was required for prompt cell-cycle entry (G0- to G1-phase) and G1/S-phase transition after toxic liver injury. These data support the hypothesis that Egr-1 provides hepatoprotection in the CCl(4)-injured liver, attributable, in part, to timely cell-cycle induction and progression.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

LPS/Bcl3/YAP1 signaling promotes Sox9+HNF4α+ hepatocyte-mediated liver regeneration after hepatectomy

Recent reports have demonstrated that Sox9⁺HNF4α⁺ hepatocytes are involved in liver regeneration after chronic liver injury; however, little is known about the origin of Sox9⁺HNF4α⁺ hepatocytes and the regulatory mechanism. Employing a combination of chimeric lineage tracing, immunofluorescence, and immunohistochemistry, we demonstrate that Sox9⁺HNF4α⁺ hepatocytes, generated by transition from mature hepatocytes, play an important role in the initial phase after partial hepatectomy (PHx). Additionally, knocking down the expression of Sox9 suppresses hepatocyte proliferation and blocks the recovery of lost hepatic tissue. In vitro and in vivo assays demonstrated that Bcl3, activated by LPS, promotes hepatocyte conversion and liver regeneration. Mechanistically, Bcl3 forms a complex with and deubiquitinates YAP1 and further induces YAP1 to translocate into the nucleus, resulting in Sox9 upregulation and mature hepatocyte conversion. We demonstrate that Bcl3 promotes Sox9⁺HNF4α⁺ hepatocytes to participate in liver regeneration, and might therefore be a potential target for enhancing regeneration after liver injury.Subject terms: Ubiquitylation, Transdifferentiation, NF-kappaB, Regeneration, Stem-cell research     

Generation of C5a in the absence of C3: a new complement activation pathway

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.     

Overexpression of suppressor of cytokine signaling-3 in T cells exacerbates acetaminophen-induced hepatotoxicity

Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4(+) T cells into T and B cell-deficient RAG-2(-/-) mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-gamma and TNF-alpha in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-gamma and TNF-alpha were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-gamma and TNF-alpha.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Plasmin decreases the BH3-only protein BimEL via the ERK1/2 signaling pathway in hepatocytes

Since the signal transduction mechanisms responsible for liver regeneration mediated by the plasminogen/plasmin system remain largely undetermined, we have investigated whether plasmin regulates the pro-apoptotic protein Bim(EL) in primary hepatocytes. Plasmin bound to hepatocytes in part via its lysine binding sites (LBS). Plasmin also triggered phosphorylation of ERK1/2 without cell detachment. The plasmin-induced phosphorylation of ERK1/2 was inhibited by the LBS inhibitor epsilon-aminocaproic acid (EACA), the serine protease inhibitor aprotinin, and the MEK inhibitor PD98059. DFP-inactivated plasmin failed to phosphorylate ERK1/2. Plasmin temporally decreased the starvation-induced expression of Bim(EL) and activation of caspase-3 via the ERK1/2 signaling pathway, resulting in an enhancement of cell survival. The amount of mRNA for Bim increased 1 day after the injection of CCl(4) in livers of plasminogen knockout (Plg-KO) and the wild-type (WT) mice. The increase in Bim(EL) protein persisted for at least 7 days post-injection in livers of Plg-KO mice, whereas WT mice showed an increase in Bim(EL) protein 1 day after the injection. Plg-KO and WT mice showed notable phosphorylation of ERK1/2 7 and 3 days after the injection of CCl(4), respectively. Our data suggest that the plasminogen/plasmin system could decrease Bim(EL) expression via the ERK1/2 signaling pathway during liver regeneration.     

Increased hepatic fibrosis and JNK2-dependent liver injury in mice exhibiting hepatocyte-specific deletion of cFLIP

Chronic liver disease promotes hepatocellular injury involving apoptosis and triggers compensatory regeneration that leads to the activation of quiescent stellate cells in the liver. The deposition of extracellular matrix from activated myofibroblasts promotes hepatic fibrosis and the progression to cirrhosis with deleterious effects on liver physiology. The role of apoptosis signaling pathways in the development of fibrosis remains undefined. The aim of the current study was to determine the involvement of the caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) during the initiation and progression of fibrosis. Liver injury and fibrosis from carbon tetrachloride (CCl(4)) and thioacetamide (TAA) were examined in mice exhibiting a hepatocyte-specific deletion of cFLIP (flip(-/-)). Acute liver injury from CCl(4) and TAA were enhanced in flip(-/-) mice. This was accompanied by increased activation of caspase-3 and -9, pronounced phosphorylation of JNK, and decreased phosphorylation of Erk. Deletion of the cJun NH(2)-terminal kinase 2 (JNK2) in flip(-/-) mice protected from injury. Hepatic fibrosis was increased at baseline in 12-wk-old flip(-/-) mice, and progression of fibrosis from TAA was accelerated compared with the wild type. In conclusion, deletion of cFLIP in hepatocytes leads to increased fibrosis and accelerated fibrosis progression. This is accompanied by increased injury involving the activation of caspases and JNK2. Thus predisposition to liver injury involving increased hepatocellular apoptosis is a critical mediator of accelerated fibrogenesis, and prevention of liver injury will be a most important measure for patients with chronic liver disease.