Liver Mononuclear Cells Research Articles

Protein S exacerbates alcoholic hepatitis by stimulating liver natural killer T cells

Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown. This study investigated the role of PS in acute alcoholic hepatitis. A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol. The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls. The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells.

Liver sulfatide-reactive type II NKT cells recognize endogenous phospholipids

NKT cells are a unique population of T cells that recognize lipid antigens presented by a nonclassical MHC-like molecule CD1d. There are two types of NKT cells, type I and type II. Our group previously showed that type I NKT cells enhance and type II NKT cells suppress anti-tumor responses, and that these two types of NKT cells cross-regulate each other. One of the defined antigens for type I NKT cells is alpha-galactosylceramide (aGC), and aGC-loaded CD1d tetramers are widely used to study them. Unlike conventional T cells, each subset of NKT cells recognizes distinct antigens. Sulfatide (3-o-sulfo-beta-D-galactosylceramide), an endogenous lipid, is the only lipid proven to be recognized by type II NKT cells in vivo. In addition, recently phosphatidylglycerol (PG) and phosphatidylinositol (PI), also endogenous lipids, were reported to be recognized by type II NKT cell hybridomas. So far, type II NKT cells and their antigens are much less well characterized than type I due to lack of widely available tools to study them. Our laboratory had developed a new method to create sulfatide-loaded CD1d tetramers, which could identify type II NKT cells. In this study, we compared phospholipid-reactive type II NKT cells and sulfatide-reactive type II NKT cells. PG-or PI-loaded CD1d-tetramer-reactive NKT cells were observed in liver mononuclear cells, and they were distinct populations from aGC-loaded CD1d-tetramer-reactive cells, indicating that there were PG- and PI-reactive type II NKT cells. Simultaneous staining with PG-, PI-, and sulfatide-loaded CD1d-tetramers indicated, surprisingly, that these three populations were largely identical, even though the phospholipids have structures quite distinct from that of sulfatide. For functional studies, the effect of these lipid antigens in cytokine production and regulating anti-tumor immunity was tested. IFN-gamma and IL-4 production by spleen cells was detected after stimulation with sulfatide in vitro, but not with PG or PI. In the murine colon cancer cell line CT26 lung metastases model, the anti-tumor response induced by aGC was reduced by simultaneous injection of sulfatide. In contrast, PG injection didn't affect the anti-tumor response induced by aGC. These findings suggest that although PG and PI, when loaded onto CD1d, bind the same cells as do sulfatide-CD1d tetramers, their function in vitro and in vivo was distinct. Now we are further examining the activities of these phospholipids on type II NKT cells.

Development of a novel mouse model of amodiaquine-induced liver injury with a delayed onset

Amodiaquine (AQ) treatment is associated with a high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. Evidence suggests that AQ-induced IDILI is immune mediated. A significant impediment to mechanistic studies of IDILI is the lack of valid animal models. This study reports the first animal model of IDILI with characteristics similar to mild IDILI in humans. Treatment of female C57BL/6 mice with AQ led to liver injury with delayed onset, which resolved despite continued treatment. Covalent binding of AQ was detected in the liver, which was greater in female than in male mice, and higher in the liver than in other organs. Covalent binding in the liver was maximal by Day 3, which did not explain the delayed onset of alanine aminotransferase (ALT) elevation. However, coincident with the elevated serum ALT, infiltration of liver and splenic mononuclear cells and activation of CD8 T-cells within the liver were identified. By Week 7, when ALT levels had returned close to normal, down-regulation of several inflammatory cytokines and up-regulation of PD-1 on T-cells suggested induction of immune tolerance. Treatment of Rag1−/− mice with AQ resulted in higher ALT activities than C57BL/6 mice, which suggested that the adaptive immune response was responsible for immune tolerance. In contrast, depletion of NK cells significantly attenuated the increase in ALT, which implied a role for NK cells in mild AQ-induced IDILI. This is the first example of a delayed-onset animal model of IDILI that appears to be immune-mediated.

Influence of Adoptive Immunotherapy Employing Allograft Liver-Resident Lymphocytes On Alloimmune Responses.

We have shown that an adoptive immunotherapy by using IL-2 activated lymphocytes extracted from allograft liver perfusate, which contains includes abundant IFN-γ producing CD56+ cells, mount an anti-HCC response in liver transplantation (LT) recipients. In this study, we investigated the immunological properties of liver-resident lymphocytes and the influence of immunotherapy with those cells on alloimmune-responses after LT. The liver mononuclear cells (LMCs) extracted from allograft liver perfusate included higher proportion of CD3-CD56+ NK and CD3+CD56+ NKT cells (43.5 ± 10.0 % and 14.7 ± 8.7 %, respectively, n = 5) than the peripheral blood mononuclear cells (PBMCs) from same donors (9.4 ± 2.9% and 2.7 ± 1.2 %, respectively, n = 5). One-way mixed lymphocyte reaction (MLR) assay revealed that the stimulation with the irradiated LMCs lead to the proliferation of allogeneic T cells as well as that with the PBMC from same donors, indicating that the LMCs contain the antigen presenting cells (n = 5 in each). We successfully performed immunotherapy with IL-2-activated LMCs 3 days after living donor LT in 27 recipients with HCC. In those patients and control 25 LT recipients without immunotherapy, MLR assay was performed at 1, 2, 4 and 12 weeks after LT to monitor immune status. In this assay, CFSE-labeled PBMCs from recipients were used as responders. Irradiated autologous, donor, and third-party PBMCs were used as stimulators. After coculture, the responder cells were stained with CD4 or CD8 mAbs, followed by FCM analyses. The proliferation of CD4+ and CD8+ T cell subsets in response to anti-donor and anti-third-party stimuli were analyzed using MLR. Both stimulation indexes of anti-donor CD4+ and CD8+ T cells did not differ between LT recipients treated with immunotherapy and those who did not received immunotherapy during observation period. Acute rejection episode occurred in 2 of the patients who received the immunotherapy (the incidence of rejection = 6.7%), whereas that occurred in 4 of the patients who did not receive the immunotherapy (14.8%). Thus, adoptive immunotherapy with allograft liver-resident lymphocytes did not inflict an acceleration of alloimmune responses/rejection reactions.

Adoptive Immunotherapy With Deceased Donor Derived-Liver Natural Killer (NK) Cells Is Well Tolerated and Has the Potential to Become a Powerful Therapeutic Tool for Preventing Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

Introduction: Liver transplantation (LT) is currently one of the main treatment options for hepatocellular carcinoma (HCC). In spite of careful patient selection using the Milan Criteria (MC) which is the accepted standard, the recurrence rate of HCC following LT is 10-20%. Although the outcome of these patients with recurrence is poor, there is no established treatment to prevent HCC recurrence. In this situation, there is an urgent need for alternative therapies for these patients. Liver is a unique organ for NK cells. About 30- 50 % of lymphocytes in the liver are NK cells. IL-2-stimulated donor liver mononuclear cells (LMNC) contain extremely high amounts of TNF-related apoptosis inducing ligand (TRAIL)-expressing NK cells and exert potent cytotoxicity to hepatoma cells. Based on these results, we performed a phase I clinical study of adoptive immunotherapy with deceased donor derived-liver NK cells in patients with HCC from July 2010 to April 2013. Method: LMNC extracted from deceased donor were cultured in X-VIVO15 medium with IL-2 for 3-5 days. Anti-CD3 mAb was added one day before the cell administration in order to prevent Graft versus Host Disease (GVHD). The final product was administered to the recipients through IV infusion. We retrospectively compared 18 recipients who received adoptive immunotherapy (NK therapy group) and historical control in our center. Result: Eighteen LT recipients with HCC were enrolled in this study. Eight patients exceeded MC in the pathology of the explanted liver. We had completed administration of the activated liver NK cells without safety issues. No study patients suffered from GVHD. No severe post-operative adverse events related with this therapy were seen. All HCC patients in NK cell therapy group are alive and without evidence of recurrence (Median follow-up; 21 months), except for one patient (pathological diagnosis was embryonal sarcoma, which was suspected for HCC preoperatively). Focus on the recipients exceeding the MC in the pathology of the explanted liver, the recurrence free survival rate was better in NK cell therapy group when compared with historical control. Conclusion: We can feasibly and safely perform this therapy. This therapy has the potential to become a powerful therapeutic tool for preventing HCC recurrence after LT.

Evaluation of the use of common sculpin (Myoxocephalus scorpius) organ histology as bioindicator for element exposure in the fjord of the mining area Maarmorilik, West Greenland

The former Black Angel lead–zinc mine in Maarmorilik, West Greenland, is a historic example of how mining activity may result in a significant impact on the surrounding fjord system in terms of elevated concentrations of especially lead (Pb) and zinc (Zn) in seawater, sediments and surrounding biota. In order to shed light on the present contamination and possible effects in the fjord we initiated a range of studies including a pilot study on gill and liver morphology of common sculpins (Myoxocephalus scorpius) around Maarmorilik. Sculpins were caught and sampled at five different stations known to represent a gradient of Pb concentrations. Fish livers from all specimens were analyzed for relevant elements in the area: Fe, Zn, As, Cu, Se, Cd, Pb, Ag, Hg, Co and Ni. Lead, As and Hg showed significant differences among the five stations. For 20% of the sculpins, Hg concentrations were in the range of lowest observed effect dose (LOED) of 0.1–0.5μg/gww for toxic threshold on reproduction and subclinical endpoints. Likewise LOEDs for tissue lesions, LOEDs for biochemistry, growth, survival and reproduction were exceeded for Cd (0.42–1.8μg/gww) and for As (11.6μg/gww) in 28% and 85% of the sculpins, respectively. Similar to this, the no observed effect dose (NOED) for biochemistry was exceeded for Pb (0.32μg/gww) and for growth, mortality and reproduction for Zn (60–68μg/gww) in 33% and 24% of the sculpins, respectively. For all sculpins, females were significantly larger than males and for five of the elements (Fe, Co, Ni, Cu, Se) females had higher concentrations. The chronic lesions observed in liver (mononuclear cell infiltrates, necrosis, vacuolar hepatocytes, portal fibrosis, bile duct hyperplasia, active melanomacrophage centers) and gills (fusion and edema of secondary lamellae, laminar telangiectasis, mononuclear cell infiltrates, blebs) were similar to those in the literature studies for both wild and laboratory exposed sculpins and other fish species carrying similar or higher Hg concentrations. Ignoring sex and size, specimens with hepatic cell infiltrates had the highest concentrations of most elements, a relation that was also found for gill telangiectasis and Hg (all p<0.05). When controlling for sex and size, the prevalence of vacuolar hepatocytes and endoparasites was significantly highest at the three most contaminated stations and similar differences were found for liver necrosis. We suggest that beside exposure to mining-related elements, other environmental factors, such as parasites, might be co-factors in the development of the observed liver and gill lesions. Therefore, sculpin liver and gill pathology are likely to be suitable health indicators when biomonitoring gradients of mining and other element related activity effects; while a larger study is required to fully evaluate the relationships.

Depletion of host hematopoietic stem cells (HSCs) in utero improves engraftment after liver mononuclear cell transplant in fetuses

Depletion of host hematopoietic stem cells (HSCs) in utero improves engraftment after liver mononuclear cell transplant in fetuses

SOCS3 expression correlates with severity of inflammation in mouse hepatitis virus strain 3-induced acute liver failure and HBV-ACLF.

Recently, suppressor of cytokine signaling-3 (SOCS3) has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure (HBV-ACLF) have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 (MHV-3)-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells (PBMCs) of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immunohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B (CHB). Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.

Virological characteristics of occult hepatitis B virus in a North American cohort of human immunodeficiency virus type 1-positive patients on dual active anti-HBV/HIV therapy

BackgroundOccult hepatitis B virus infection (OBI) is defined as low-level HBV DNA presence in serum, liver and/or peripheral blood mononuclear cells (PBMC) in individuals that lack serum hepatitis B virus surface antigen (HBsAg). HIV+ patients with OBI may be at risk for HBV reactivation, and often receive dual active anti-HBV/HIV therapy, such as lamivudine (LMV). ObjectivesTo determine the presence of OBI in a North American cohort of HIV-1-positive patients. Study design/methods45 HIV-1-positive, serum HBsAg-negative patients, reactive for antibodies to HBV core antigen (anti-HBc), were tested for HBV DNA in plasma and for HBV DNA and covalently closed circular DNA (cccDNA) in PBMC. Ten patients were re-tested after ∼5–10 years, including genotyping and clonal sequence analysis of the HBV polymerase (P) gene and overlapping HBV surface (S) gene from 8 PBMC samples. ResultsOverall, 42% (19/45) tested HBV DNA positive, especially in PBMC (18/45), including 3/18 that were reactive for HBV cccDNA, compared to 17% (8/45) that were HBV DNA reactive in plasma. In 8 patients on LMV, sequence analysis in PBMC showed that all were HBV genotype C or D. Several carried HBV P region variants at residues associated with anti-HBV drug resistance and overlapping S gene region within the major HBsAg “a determinant”. ConclusionOBI is common in HIV-positive, anti-HBc reactive patients on anti-HBV/HIV therapy, particularly in PBMC. HBV sequence analysis revealed that all had HBV genotype C or D and often had P/overlapping S gene variants possibly associated with dual-active anti-HIV/HBV therapy.

Liver sulfatide-reactive type II NKT cells recognize endogenous phospholipids. (INC9P.445)

Abstract NKT cells are T cells recognizing lipids presented by CD1d. Type I and II subsets recognize distinct antigens. Type I NKT cells recognize alpha-galactosylceramide (aGC). aGC-loaded CD1d tetramers are widely used to study them. Type II NKT cells and their antigens are much less well characterized than type I due to lack of widely available tools to study them. Sulfatide (3-o-sulfo-beta-D-galactosylceramide), an endogenous lipid, is the only lipid proven to be recognized by type II NKT cells in vivo. Recently, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were reported to be recognized by type II NKT cell hybridomas. Here, PG-or PI-loaded CD1d-tetramer-reactive cells were detected in liver mononuclear cells as a distinct population from aGC-loaded CD1d-tetramer-reactive type I NKT cells, consistent with type II NKT cells. Simultaneous staining with PG-, PI-, and sulfatide-loaded CD1d-tetramers indicated, surprisingly, that these three populations were largely identical, even though the phospholipids have structures quite distinct from that of sulfatide. IFN-gamma and IL-4 production by spleen cells was detected after stimulation with sulfatide, but not with PG or PI. These findings suggest that although PG and PI, when loaded onto CD1d, bind the same cells as do sulfatide-CD1d tetramers, their ability to induce cytokine production was distinct. Whether these phospholipids are weak agonists or antagonists or induce other functional activities is under investigation.

Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/Macrophages in CCl4-Induced Acute Hepatic Injury

We previously reported that F4/80+ Kupffer cells are subclassified into CD68+ Kupffer cells with phagocytic and ROS producing capacity, and CD11b+ Kupffer cells with cytokine-producing capacity. Carbon tetrachloride (CCl4)-induced hepatic injury is a well-known chemical-induced hepatocyte injury. In the present study, we investigated the immunological role of Kupffer cells/macrophages in CCl4-induced hepatitis in mice. The immunohistochemical analysis of the liver and the flow cytometry of the liver mononuclear cells showed that clodronate liposome (c-lipo) treatment greatly decreased the spindle-shaped F4/80+ or CD68+ cells, while the oval-shaped F4/80+ CD11b+ cells increased. Notably, severe hepatic injury induced by CCl4 was further aggravated by c-lipo-pretreatment. The population of CD11b+ Kupffer cells/macrophages dramatically increased 24 hour (h) after CCl4 administration, especially in c-lipo-pretreated mice. The CD11b+ Kupffer cells expressed intracellular TNF and surface Fas-ligand (FasL). Furthermore, anti-TNF Ab pretreatment (which decreased the FasL expression of CD11b+ Kupffer cells), anti-FasL Ab pretreatment or gld/gld mice attenuated the liver injury induced by CCl4. CD1d−/− mouse and cell depletion experiments showed that NKT cells and NK cells were not involved in the hepatic injury. The adoptive transfer and cytotoxic assay against primary cultured hepatocytes confirmed the role of CD11b+ Kupffer cells in CCl4-induced hepatitis. Interestingly, the serum MCP-1 level rapidly increased and peaked at six h after c-lipo pretreatment, suggesting that the MCP-1 produced by c-lipo-phagocytized CD68+ Kupffer cells may recruit CD11b+ macrophages from the periphery and bone marrow. The CD11b+ Kupffer cells producing TNF and FasL thus play a pivotal role in CCl4-induced acute hepatic injury.

Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNγ treatment in mouse tumor models

Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55-deficient mice. In previous work we demonstrated that P55+/- mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNγ) remain fully functional in these mice. Hence, a high dose of TNF/IFNγ has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNγ treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNγ antitumor therapy.

In Utero Depletion Of Fetal Host Hematopoietic Stem Cells Improves Engraftment Following Neonatal Transplantation In Mice

IntroductionIn utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital disorders as the fetal host can potentially be tolerized to transplanted cells early in gestation. However, levels of engraftment have been low and fetal host conditioning strategies to increase space in hematopoietic niches have not been widely explored. We hypothesized that depletion of fetal host hematopoietic stem cells (HSC) using an antibody against the c-kit receptor (ACK2), a strategy which selectively depletes HSC by disrupting stem cell factor (SCF) signaling, would improve engraftment after HSC transplantation. MethodsFetal C57B6.CD45.2 (B6) mice were injected with increasing doses of ACK2 (2.5-50 µg/fetus) or isotype control antibody on E14.5 and surviving pups were transplanted with congenic B6.CD45.1 fetal liver mononuclear cells (2.5×106 cells/pup) on day of life 1 (P1, 7 days after in utero injection), allowing post-transplantation host monitoring. Host HSC depletion and residual serum ACK2 concentration were examined on P1. Peripheral blood chimerism, defined as donor/(donor+host) CD45 cells, as well as the lineage distribution of chimeric cells, were determined beginning 4 weeks after transplantation. ResultsSurvival to birth among fetuses injected with 2.5, 5, or 10 µg of ACK2 was similar to controls (control: 74%; 2.5 µg: 80%; 5 µg: 71%; 10 µg: 60%, p=0.2 by chi-square test, n≥45/group) but was significantly lower at higher concentrations (20 µg: 37%; 50 µg: 31%, p<0.001 vs. control, n≥70/group). Transient anemia and leukopenia were observed on P1 with doses ≥ 5 µg which resolved by P7 (n=17). Four of 19 pups previously treated with ACK2 (2.5-10 µg) and observed long-term had patchy coat discoloration, possibly a manifestation of disruption of C-kit+ melanocyte migration.In utero ACK2 treatment resulted in significant and dose-dependent depletion of host HSCs (defined as Lin-Sca-1+C-kit+, KLS) in the bone marrow of treated animals by P1 (Figure 1A). There was no depletion of KLS cells in the liver. Residual ACK2 antibody was undetectable in the serum by P1, validating our strategy of in utero depletion and neonatal transplantation. [Display omitted] In animals receiving neonatal transplantation, ACK2 depletion resulted in a significant increase in levels of engraftment 4 weeks after transplantation compared to controls (control: 3.3±0.3%; 2.5 µg: 13±1.4%; 5 µg: 10±2.4%; 10 µg: 11±2.0%, p<0.05 for each dose vs control by ANOVA). Accordingly, we detected an increased number total bone marrow KLS cells 7 days after transplantation in ACK2 treated animals compared to controls (412±45.9 vs. 933±112 cells, p=0.01, n≥3/group). Moreover, levels of chimerism increased over time in treated animals (Figure 1B; 12 weeks: 2.5 µg: 190%; 5 µg: 170%; 10 µg: 160%) while they remained unchanged in controls. Overall, levels of chimerism achieved with ACK2 treatment were significantly higher than that observed in animals that received in utero transplantation without ACK2 depletion.Lineage analysis of peripheral blood for granulocytes, B cells, and T cells indicated an equal increase in all lineages, suggesting ACK2 depletes true HSCs and not committed progenitors. Interestingly, ACK2 depletion at doses 2.5-10 µg did not result in engraftment of allogeneic BALB/c cells (n=11), indicating that allogeneic neonatal transplantation, unlike in utero transplantation, is limited by a host immune response which is unaffected by ACK2. ConclusionWe have demonstrated that fetal HSC depletion using ACK2 can lead to clinically relevant levels of donor cell engraftment with minimal toxicity. In previous studies with this antibody, host HSC depletion required either immunodeficient animals or concurrent irradiation, whereas we achieved depletion in wild-type fetal hosts, suggesting differences in fetal vs. adult HSC sensitivity to SCF signaling. Future studies should explore this strategy to improve engraftment in large animals models of IUHCTx. Disclosures:Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Characterizing the Lymphopoietic Kinetics and Features of Hematopoietic Progenitors Contained in the Adult Murine Liver In Vivo

The appearance of donor-derived lymphocytes in liver transplant patients suggests that adult livers may contain cells capable of lymphopoiesis. However, only a few published studies have addressed the lymphopoietic capacity of adult liver cells, and its kinetics and features remain unclear. Herein, we investigated the lymphopoietic capacity of adult liver mononuclear cells (MNCs) and purified liver hematopoietic progenitor cells (HPCs) in vivo. Similar to bone-marrow transplantation (BMT), transplantation of liver MNCs alone was able to rescue survival of lethally irradiated mice. In terms of kinetics, liver MNC-derived myeloid lineage cells reconstituted more slowly than those from BMT. Liver MNC-derived lymphocyte lineage cells in the blood, spleen and BM also reconstituted more slowly than BMT, but lymphocytes in the liver recovered at a similar rate. Interestingly, liver MNCs predominantly gave rise to CD3+CD19− T cells in both irradiated WT and non-irradiated lymphocyte-deficient Rag-1−/−Il2rg−/− recipients. To define the lymphopoietic potential of various cell populations within liver MNCs, we transplanted purified lineage-negative (Lin−) liver HPCs into recipient mice. Unlike total liver MNCs, liver HPCs reconstituted T and B cells in similar frequencies to BMT. We further determined that the predominance of T cells observed after transplanting total liver MNCs likely originated from mature T cells, as purified donor liver T cells proliferated in the recipients and gave rise to CD8+ T cells. Thus, the capacity of donor adult liver cells to reconstitute lymphocytes in recipients derives from both HPCs and mature T cells contained in the liver MNC population.

Development of acute megakaryoblastic leukemia in Down syndrome is associated with sequential epigenetic changes

AbstractAcute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

Natural killer cell‐mediated contact sensitivity develops rapidly and depends on interferon‐α, interferon‐γ and interleukin‐12

Natural killer (NK) cell-mediated contact sensitivity was recently described in mice. Here, we confirm NK cell-mediated contact sensitivity (CS) in SCID and RAG1(-/-) mice but not in SCIDbeige mice, which have non-functional NK cells that lack NK cell granules. NK cell-mediated CS was transferred by liver mononuclear cells and the DX5(+) fraction of liver cells, confirming that NK cells mediate CS in the absence of T and B cells. Participation of NKT cells and B-1 cells was ruled out using Jα18(-/-) and JH(-/-) mice, respectively. Remarkably, NK cell-mediated CS was observed just 1hr after immunization and was detectable as early as 30min after challenge. Further, we examined cytokine requirements for NK cell-mediated CS, and found that liver mononuclear cells from interleukin-12(-/-) , interferon-γ(-/-) and interferon-α receptor(-/-) donors fail to transfer NK cell-mediated CS to naive hosts. Our studies clearly show that dinitrofluorobenzene sensitized NK cells mediate very rapid, antigen-specific cell-mediated immunity, with features of both innate and acquired immune responses.

Baicalein Selectively Induces Apoptosis in Activated Lymphocytes and Ameliorates Concanavalin A-Induced Hepatitis in Mice

BackgroundInsufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Baicalein (BE), a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, possesses anti-inflammatory properties. However, whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied.Methodology/Principal FindingsThe pro-apoptotic properties of BE were evaluated in vitro on different types of immune cells, and in vivo effects of BE were examined in a murine model of Concanavalin A (Con A)-induced hepatitis. In vitro treatment with BE resulted in a higher increase in the level of apoptosis in Con A-stimulated murine splenocytes, Con A-stimulated CD3+ splenocytes, lipopolysaccharide (LPS)-stimulated CD19+ splenocytes, and phorbol 12-myristate 13-acetate/ionomycin-stimulated Jurkat T cells, compared with that in unstimulated naïve ones. Murine bone marrow-derived dentritic cells, peritoneal macrophages, and RAW264.7 cells, either stimulated with LPS or unstimulated, were all insensitive to the BE-induced apoptosis. BE treatment also led to a loss of mitochondrial membrane potential, an increase of cytochrome c release from mitochondria to the cytosol, a decrease in the ratio of Bcl-2/Bax, and activation of caspase-9,-3 in Con A-stimulated CD3+ splenocytes and LPS-stimulated CD19+ splenocytes, while showing no impact on Fas/FasL expressions and caspase-8 activation. In vivo administration of BE alleviated Con A-induced liver injury, suppressed serum level of TNF-α and IFN-γ, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, BE treatment increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3+ and CD19+ splenocytes. When liver MNCs and splenocytes from BE-treated mice were cultured in vitro for 24 h, they exhibited marked increase in apoptosis compared to vehicle-treated control.Conclusions/SignificanceThe present study demonstrates the ability of BE to promote apoptosis in activated lymphocytes through mitochondrial pathway and its potential use in the treatment of AIH.

Glucocorticoid receptor-dependent immunomodulatory effect of ursodeoxycholic acid on liver lymphocytes in mice

Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (wt/vol) UDCA for 4 wk. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN-γ production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it increased mortality of mice following Escherichia coli infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induce IFN-γ production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN-γ levels after E. coli, LPS, or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN-γ production. Furthermore, RU486, a GR antagonist, restored the serum IFN-γ level in UDCA-fed mice after E. coli, LPS, or Con-A challenge. Taken together, these results suggest that IFN-γ-reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18-independent manner.

Comparative Analysis of T-Cell Depletion Method for Clinical Immunotherapy—Anti–Hepatitis C Effects of Natural Killer Cells Via Interferon-γ Production

Liver transplantation (LT) is a life-saving treatment for liver cirrhosis patients with hepatocellular carcinoma (HCC). However, 10%–20% HCC recurrence rate after LT is due to the immunosuppression inducing tumor growth. We recently reported a novel immunotherapy with donor liver natural killer (NK) cells to prevent HCC and hepatitis C virus (HCV) recurrence after LT. In this cell processing procedure, Muromonab-CD3 (Orthoclone OKT3, an anti-CD3 antibody) was added to the culture medium to deplete CD3+ T cells to prevent graft-versus-host disease. However, the manufacture of OKT3 was discontinued in 2010, when other treatments with similar efficacy and fewer side effects became available. In this study, we examined alternative reagents for T-cell depletion-MACS GMP CD3 pure (GMP CD3), antithymocyte globulin, and alemtuzumab-for NK cell immunotherapy in the allogeneic setting. We observed that GMP CD3 showed exactly the same effects on liver mononuclear cells as OKT3, including activation of NK cells and depletion of T cells. Interestingly, binding of T-cell depletion antibodies to NK cells led to an anti-HCV effect via interferon-γ production. These results with the use of in vitro culture systems suggested that antibodies which produce T-cell depletion affected NK cell function.

The combination therapy of α‐galactosylceramide and 5‐fluorouracil showed antitumor effect synergistically against liver tumor in mice

α-Galactosylceramide (α-GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo-immunotherapy using α-GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α-GalCer and 5-fluorouracil (5-FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor-bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5-FU or α-GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α-GalCer treatment induced significant activation of liver NK cells in vivo, but 5-FU treatment did not. 5-FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae-1 and H60) and DNAM-1 ligands (CD112 and CD155) on MC38 cells, but α-GalCer did not. The cytolytic activity of α-GalCer-activated liver mononuclear cells against 5-FU-treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5-FU partially depended on NKG2D-Rae-1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5-FU against MC38 liver tumors, which suggested that the antitumor effect of 5-FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α-GalCer and 5-FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo-immunotherapy against metastatic liver cancer.