Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

Roles of heterogenous hepatic macrophages in the progression of liver diseases

Hepatic macrophages are key immune cells associated with the broad ranges of liver diseases including steatosis, inflammation and fibrosis. Hepatic macrophages interact with other immune cells and orchestrate hepatic immune circumstances. Recently, the heterogenous populations of hepatic macrophages have been discovered termed residential Kupffer cells and monocyte-derived macrophages, and identified their distinct population dynamics during the progression of various liver diseases. Liver injury lead to Kupffer cells activation with induction of inflammatory cytokines and chemokines, which triggers recruitment of inflammatory monocyte-derived macrophages. To understand liver pathology, the functions of different subtypes of liver macrophages should be regarded with different perspectives. In this review, we summarize recent advances in the roles of hepatic macrophages under liver damages and suggest hepatic macrophages as promising therapeutic targets for treating liver diseases.     

Pattern of cytokine expression by rat liver epithelial cells supporting long-term culture of human CD34(+)umbilical cord blood cells

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

Liver stem cells

The concept of a liver stem cell or progenitor cell has not been widely accepted until the last decade. Studies investigating liver regeneration under conditions which totally or substantially preclude hepatocyte proliferation report the proliferation of a subpopulation of small, oval-shaped cells, which are first observed in the portal triad, adjacent to the terminal ducts. These cells, termed liver progenitor oval cells (LPCs) are shown to participate in liver regeneration in a variety of rodent models of chronic liver damage. They express markers common to hepatocytes and cholangiocytes suggesting they are a common precursor of both liver cell lineages. Supporting evidence for liver stem cells has also come from cell tracing studies which show transdifferentiation of bone marrow cells into hepatocytes in both human and animal models. Another important issue is the link between LPCs and hepatocellular carcinoma (HCC). The widening liver donor-recipient gap; a consequence of poor donation rates coupled with increasing incidence of liver disease highlights the importance of establishing the utility of cell transplant as an alternative to treat liver disease. In this regard, liver stem cells and progenitor cells may have a significant role to play. To successfully utilize liver stem cells or LPCs for cell therapy, we have to first develop methods for maintaining and differentiating them in culture. This technology must be based on a thorough understanding of conditions which regulate their behaviour in vitro. In particular, we need to know which growth factors and cytokines affect them and their mechanism of action. Since they are a potential source of HCC, it is also necessary to understand the mechanisms which underlie their transformation to cancer.     

诱导性多能干细胞与肝样细胞分化

肝脏疾病正逐渐成为全球棘手的医疗问题。肝细胞是肝脏生理活动的主要承担者,在肝脏疾病的研究以及药物的研发和测试方面有着举足轻重的作用。然而,体外分离培养的原代肝细胞面临在体外不能无限增殖和稳定表达肝脏特异基因等问题。有强大的自我更新能力和三胚层分化潜能的诱导性多能肝细胞(iPSCs)能被诱导因子、外源基因和小分子化合物等定向诱导分化为功能性肝细胞。同时,还避免了伦理、宗教以及免疫排斥等诸多问题。本文简要综述了从不同策略诱导iPSCs成为功能性肝细胞的研究方法和成果,并对该领域进行小结和展望。     

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.     

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.     

Unique sensitivity to alpha-galactosylceramide of NKT cells in the uterus

It was previously reported that NKT cells, which are mainly present in the liver of mice, are also present in the uterus and that these uterine NKT cells are associated with abortion under overactivated conditions. In this study, we further examined their phenotypic and functional properties. In parallel with the progression of pregnancy, the number of uterine lymphocytes increased. This increase accompanied an increase in the number of TCRalphabeta(+) T cells and NKT cells in the uterus, although the number of NKT cells decreased at late pregnancy. Approximately one-third of the TCRalphabeta(+) cells were NKT cells at the early pregnant stage, while this ratio decreased toward late pregnancy. These uterine NKT cells were found to respond to alpha-galactosylceramide (alpha-GalCer) differently in comparison with liver NKT cells. In contrast to the apoptotic response of liver NKT cells on day 1 after alpha-GalCer injection, uterine NKT cells expanded prominently without such apoptosis. The majority of liver NKT cells were CD4(+). In contrast, almost all of the uterine NKT cells were double negative CD4(-)8(-). However, similar to liver NKT cells, uterine NKT cells used an invariant chain of Valpha14Jalpha281 gene for TCRalpha. The resistance against apoptosis might be due to the high expression of bcl-2 on uterine NKT cells after alpha-GalCer activation. Other evidence was that macrophages which existed in the pregnant uterus carried an activation marker, CD69, and could potentially produce many cytokines by their activation. The present results suggest that uterine NKT cells and surrounding macrophages are quite different in function from those in the liver.     

Induction of antibody-dependent cellular cytotoxicity in vivo by IFN-alpha and its antitumor efficacy against established B16 melanoma liver metastases when combined with specific anti-B16 monoclonal antibody

We have previously shown the ability of different cytokines to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells in vitro. In addition we found that the administration to mice of IL-2-induced cells which mediated ADCC and that these cells were phenotypically similar to the cells induced in vitro. In the present study we tested the ability of various cytokines, including IL-1, TNF, IFN-alpha, and IFN-gamma to induce ADCC in vivo. We found that both IFN-alpha and IFN-gamma induced ADCC in the livers and spleens of C3H/Hen-treated mice and that these cytokines together with TNF enhanced the IL-2-induced ADCC in vivo. In C57BL/6 mice which, as previously shown, exhibit relatively low ADCC activity, IFN-alpha and IFN-gamma increased the IL-2-induced ADCC only when 100,000 U of IL-2 were used for priming. The effect of IFN-alpha on ADCC was dose dependent and was optimal after the administration of 200,000 U of the cytokine given three times a day for 3 days. Similar to the cells induced in vivo by IL-2, the precursors of the cells mediating ADCC were asialo GM1+ whereas the effectors were mainly nonadherent, Thy-1+ cells. IFN-alpha-generated cells mediating ADCC in the liver and spleen and, when combined with IL-2, ADCC was induced in the thymus as well. This effect of IFN-alpha on the induction of ADCC was exploited in an immunotherapy model in which we found that IFN-alpha significantly enhanced the antibody-mediated antitumor effect on established B16 melanoma liver micrometastases. Furthermore, when IL-2 and IFN-alpha administration was combined with the administration of mAb, a significantly reduced number of established 6- to 8-day B16 melanoma liver macrometastases and prolonged survival of tumor-bearing mice were seen. These studies imply that the administration of appropriate cytokine combinations may be a useful adjunct to the administration of mAb for the treatment of cancer in humans.     

Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver.

We have previously reported that intrahepatic NK T cells activated by alpha-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-gamma produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.     

IL-10 Mediated Regulation of Liver Inflammation during Acute Murine Cytomegalovirus Infection

Various cell types in both lymphoid and non-lymphoid tissues produce the anti-inflammatory cytokine interleukin (IL)-10 during murine cytomegalovirus (MCMV) infection. The functions of IL-10 in the liver during acute infection and the cells that generate this cytokine at this site have not been extensively investigated. In this study, we demonstrate that the production of IL-10 in the liver is elevated in C57BL/6 mice during late acute MCMV infection. Using IL-10 green fluorescence protein (GFP) reporter knock-in mice, designated IL-10-internal ribosomal entry site (IRES)-GFP-enhanced reporter (tiger), NK cells are identified as major IL-10 expressing cells in the liver after infection, along with T cells and other leukocytes. In the absence of IL-10, mice exhibit marked elevations in proinflammatory cytokines and in the numbers of mononuclear cells and lymphocytes infiltrating the liver during this infection. IL-10-deficiency also enhances liver injury without improving viral clearance from this site. Collectively, the results indicate that IL-10-producing cells in the liver provide protection from collateral injury by modulating the inflammatory response associated with MCMV infection.     

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis

TNF-α, IFN-γ, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-α production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-γ, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-γ, which was slightly suppressed at 12 h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-α in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.     

Pyroptosis in Steatohepatitis and Liver Diseases

Pyroptosis is an inflammatory form of regulated cell death, which functions in the clearance of intracellularly replicating pathogens by cell lysis in order to induce further immune response. Since the discovery of the gasdermin (GSDM) family, pyroptosis has attracted attention in a wide range of inflammatory diseases such as nonalcoholic steatohepatitis and other liver diseases. Due to the cleavage of GSDMs by different caspases, the amino-terminal GSDM fragments form membrane pores essential for pyroptosis that facilitate the release of inflammatory cytokines by loss of ionic gradient and membrane rupture. In this review, we address the key molecular and cellular processes that induce pyroptosis in the liver and its significance in the pathogenesis of common liver diseases in different human and experimental mice studies.     

What fires prometheus? The link between inflammation and regeneration following chronic liver injury

Liver progenitor cells (LPCs) play a major role in the regeneration process after chronic liver damage, giving rise to hepatocytes and cholangiocytes. Thus, they provide a cell-based therapeutic alternative to organ transplant, the current treatment of choice for end-stage liver disease. In recent years, much attention has focused on unravelling the cytokines and growth factors that underlie this response. Liver regeneration following acute damage is achieved by proliferation of mature hepatocytes; yet similar cytokines, most related to the inflammatory process, are implicated in both acute and chronic liver regeneration. Thus, many recent studies represent attempts to identify LPC-specific factors. This review summarises our current understanding of LPC biology with a particular focus on the liver inflammatory response being associated with the induction of LPCs in the liver. We will describe: (i) the pathways of liver regeneration following acute and chronic damage; (ii) the similarities and differences between the two pathways; (iii) the liver inflammatory environment; (iv) the unique features of liver immunology as well as (v) the interactions between liver immune cells and LPCs. Combining data from studies on the LPC-driven regeneration process with the knowledge in the field of liver immunology will improve our understanding of the LPC response and allow us to regulate these cells in vivo and in vitro for future therapeutic strategies to treat chronic liver disease.     

应用骨髓干细胞治疗肝硬化研究进展

随着干细胞研究的深入和技术的发展,再生医学的干细胞疗法治疗肝脏疾病已成为研究热点。骨髓来源造血干细胞和间充质干细胞等在肝脏疾病治疗方面有巨大潜力。骨髓干细胞参与肝纤维化与肝硬化修复主要包括迁移、归巢与转化等过程,并需要多种细胞因子和趋化因子的协同作用促进肝细胞再生与减轻肝纤维化。本文拟对骨髓干细胞治疗肝硬化的最新研究进展进行综述。     

Synemin down-regulation in human hepatocellular carcinoma does not destabilize cytoskeletons in vivo

Synemin is a large intermediate filament protein that has been identified in all types of muscle cells. It plays a role in human muscle diseases; however, the role of synemin in tumor cell transformation has rarely been investigated. Because hepatocellular carcinoma cells are morphologically different from normal human hepatocytes, we hypothesized that altered synemin expression and cytoskeletal disorganization might underlie this pleomorphic transformation. To test this hypothesis, we studied synemin expression in hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblotting. In addition, we analyzed the expression level and organization of all cytoskeletal elements after synemin knock-down in human Chang liver cells. Previously we found that plectin knock-down in human Chang liver cells causes a reduction in cytokeratin 18 expression with effects on intermediate filament disorganization and altered cellular morphology. In this study we also compared the effects of synemin knock-down and plectin knock-down on the cytoskeleton expression and organization. The results revealed that synemin expression was down-regulated in human hepatocellular carcinoma compared with normal liver, which is similar to the plectin expression. Surprisingly, the expression of cytoskeletal elements (cytokeratin 18, actin and tubulin) was not influenced by synemin knock-down in human Chang liver cells. The organization of cytoskeletal networks was also unaltered after synemin knock-down. In conclusion, both plectin and synemin are down-regulated in human hepatocellular carcinoma in vivo and transformed human liver cell in vitro. However, the mechanism of cell transformation caused by synemin knock-down is different from that of plectin knock-down. Plectin, but not synemin, knock-down provoked liver cell transformation via suppressing cytokeratin 18 expression and disrupting intermediate filament networks. Synemin knock-down did not influence the cytoskeleton expression and organization of human Chang liver cells.     

Cyst formation, increased anti-inflammatory cytokines and expression of chemokines support for Clonorchis sinensis infection in FVB mice

To verify the hypothesis that different pathology of Clonorchis sinensis infection by mouse strains is determined by different responses of cytokines and chemokines, we compared those responses of FVB with those of BALB/c mice. All of FVB mice infected with 30 metacercariae of C. sinensis showed cystic dilatation in the liver, whereas infected BALB/c mice did not. Mature worms were recovered from 19 of 20 liver sections of FVB mice while only one of 20 sections of BALB/c mice revealed a mature worm. In both strains the proportion of CD4⁺ T cells was lower in C. sinensis-infected than in the uninfected group. However, the proportion of CD8⁺ T cells was elevated in C. sinensis-infected from both strains compared to uninfected mice. The Th2-associated anti-inflammatory cytokines such as IL-4, IL-5 and IL-13, IL-10 and TGF-β, were significantly more produced by the lymphocytes of FVB than by those of BALB/c mice. Especially, the 2 anti-inflammatory cytokines, IL-10 and TGF-β, were presumably related with susceptibility and the development of worms in the liver. C. sinensis infected FVB mice also produced more chemokines such as RANTES and MIP-1α in the liver lymphocytes than BALB/c mice. In conclusion, the FVB mice provide the favorable niche for C. sinensis by cyst formation in the bile duct, increased production of Th2-associated anti-inflammatory cytokines and upregulation of chemokines.     

Strategies to improve the efficiency of mesenchymal stem cell transplantation for reversal of liver fibrosis

End‐stage liver fibrosis frequently progresses to portal vein thrombosis, formation of oesophageal varices, hepatic encephalopathy, ascites, hepatocellular carcinoma and liver failure. Mesenchymal stem cells (MSCs), when transplanted in vivo, migrate into fibrogenic livers and then differentiate into hepatocyte‐like cells or fuse with hepatocytes to protect liver function. Moreover, they can produce various growth factors and cytokines with anti‐inflammatory effects to reverse the fibrotic state of the liver. In addition, only a small number of MSCs migrate to the injured tissue after cell transplantation; consequently, multiple studies have investigated effective strategies to improve the survival rate and activity of MSCs for the treatment of liver fibrosis. In this review, we intend to arrange and analyse the current evidence related to MSC transplantation in liver fibrosis, to summarize the detailed mechanisms of MSC transplantation for the reversal of liver fibrosis and to discuss new strategies for this treatment. Finally, and most importantly, we will identify the current problems with MSC‐based therapies to repair liver fibrosis that must be addressed in order to develop safer and more effective routes for MSC transplantation. In this way, it will soon be possible to significantly improve the therapeutic effects of MSC transplantation for liver regeneration, as well as enhance the quality of life and prolong the survival time of patients with liver fibrosis.