Liver Dendritic Cells Research Articles

α‐Galactosylceramide activates antitumor immunity against liver tumor

α-Galactosylceramide (α-GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α-GalCer treatment in the mouse liver tumor model. BALB/c mice bearing CMS4 liver tumor (p53 peptide-expressing tumor) were treated by α-GalCer. We evaluated the activation of liver DC and immune cells after α-GalCer treatment. Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide-specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α-GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re-challenged s.c. The liver weights of α-GalCer-treated mice were significantly lighter than those of vehicle-treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α-GalCer. α-GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co-stimulatory molecules on liver DC significantly increased with the peak at 1 day after α-GalCer administration. IFN-γ ELISPOT assay demonstrated that p53 peptide-specific CTL was generated efficiently in α-GalCer-treated mice. (51) Cr-release assay revealed that CD8(+) , not CD4(+) , CTL against CMS4 cells were generated in α-GalCer-treated mice. The mice that had been protected from CMS4 liver tumor by α-GalCer injection became resistant against s.c. CMS4 re-challenge, but not against Colon26 re-challenge. These results demonstrated the therapeutic potential of α-GalCer against liver cancer through activating liver DC and immune cells in the liver.

Changes of macrophage inflammatory protein-1α in the livers of rats with Walker-256 tumors treated with radiofrequency ablation

Objective To study changes in the expression levels of OX-62, OX-6 and CD86 of mononuclear cells and the related chemotatic factor macrophage inflammatory protein-1α (MIP-1α) in the livers of rats with Walker-256 tumors treated with radiofrequency ablation (RFA) and to elucidate the influence of RFA on differentiation and migration of liver dendritic cells(DCs).Methods Walker-256 liver tumors were induced in 60 SpragueDawley rats by implanting tumor particles. These rats were randomly divided equally into three groups from which liver tissue around the local area of the tumor was sampled at 7 d and 14 d after RFA. The mononuclear liver cells were separated with Ficoll density gradient centrifugation. The expression levels of OX-62, OX-6 and CD86 in the mononuclear cells were analyzed with flow cytometry. The expression level of MIP-1α in the liver tissue was detected by enzyme-linked immunosorbent assay (ELISA). Results The average expression of OX-6 in the control rats was 15.29 ±4.59% and those 7 d and 14 d after RFA were 34.20±11.62% and 39.18±9.14% respectively. The difference between the two RFA groups and the control group was statistically significant. The average expression rate of OX-62 in the control rats was 18.91±4.58% and those 7 d and 14 d after RFA were 24.49±4.45% and 22.77 ± 3.50% respectively. The difference between the 7 d group and the control group was significant. The average expression rate of CD86 in the control rats was 66.29±17.69% and those 7 d and 14 d after RFA were 55.29±10.69% and 55.93±12.64% respectively. These differences between both RFA groups and the controls group were not significant. The average expression level of MIP-1 α around the tumors was 232.92±54.58 pg/ml in the controls and 499.38±15.14 pg/ml and 495.90±9.94 pg/ml 7d and 14 d after RFA respectively. These differences from the controls were both statistically significant. Conclusion The expression of MIP-1α around the tumors was elevated after RFA, which could promote the migration of DCs. The changes in the expression of OX-62, OX-6 and CD86 also could reflect increased DC differentiation, which could improve local antigen-presenting capacity to a certain extent and recovery of host anti-tumor immune response. Key words: Radiofrequency ablation; Rats; Liver tumors; Dendritic cells; Macrophage inflammatory protein-1α; Migration

Immunostimulatory Properties of Dendritic Cells after Leishmania donovani Infection Using an In Vitro Model of Liver Microenvironment

BackgroundRecent advances demonstrated that liver dendritic cells (DCs) promote immunologic hyporesponsiveness that may contribute to hepatic tolerance. Although there has been significant work on the phenotypic and functional roles of such DCs, the impact of liver microenvironment on the immune properties of infected DC is still poorly explored, probably because of the limitations of modelization.Methodology/Principal FindingsHere, we hypothesized that DC tolerogenic properties have an impact on the antimicrobial response, particularly during the infection by the protozoan parasite Leishmania donovani. Indeed, a lymphocytic Th2 environment was reported to favour the growth and proliferation of L. donovani. We first modelized an adequate monocyte-differentiated DC model, either in rat liver epithelial cell- or in a human hepatic non-parenchymal cell-conditioned medium in order to infect them further. We established that DCs differentiated in a hepatic microenvironment displayed a CD14+/CD16+/CD123+ phenotype, secreted low IL-12p70 and had an impaired capacity to stimulate allogeneic T lymphocyte proliferation and IFNγ secretion. We then infected DCs with L. donovani in the in vitro-defined hepatic microenvironment. The infection of hepatic DCs restored their capacity to stimulate allogeneic T-cell proliferation and to induce lymphocytic secretion of IFNγ. Such characteristics were recently shown to favour granuloma formation in mice liver.Conclusions/SignificanceOur results suggest that the specific immunostimulatory properties of infected hepatic DCs might amplify the granuloma maturation, which warrants the effective control of infection in the liver during visceral leishmaniasis.

Liver DC modulate Foxp3 expression in allogeneic CD4+ T cells via IL-27 (145.3)

Abstract Liver dendritic cells (DC) possess immunoregulatory properties that influence the nature of T cell responses. We investigated the impact of IL-27 on CD4+ T cell responses by liver DC. Both plasmacytoid DC (mPDCA-1+) and myeloid DC (CD11c+ mPDCA-1-) were immunobead-purified from livers and spleens of Flt3L-treated C57BL/6 mice. DC phenotype was analyzed by flow cytometry and cytokine levels were quantified by ELISA. Ebi-3 (a component of the IL-27 molecule) protein expression was determined by Western blot. T cell allostimulatory capacity in MLR was quantified by CFSE dilution analysis, and Foxp3+ T cells quantified by intracellular flow cytometric analysis. Liver DC exhibited reduced expression of antigen (Ag)-presenting and costimulatory molecules and induced inferior T cell proliferation compared to splenic DC. They also produced elevated levels of IL-27p28, IL-10, and IL-6, but reduced IL-12p40 levels compared to splenic DC. Liver DC expressed higher levels of Ebi-3 protein compared to spleen DC. Blockade of IL-27 in allogeneic MLR resulted in greater levels of Foxp3+ T cells when liver but not spleen DC were used as stimulators. Thus, liver DC exhibit lower Ag-presenting and costimulatory molecule expression but produce greater levels of immunoregulatory cytokines than splenic DC, which may contribute to their poor CD4+ T cell allostimulatory capacity. Interestingly, IL-27 produced by liver DC may inhibit their ability to induce Foxp3+ regulatory T cells.

The alterations of chemokines related to dendritic cells in rats with hepatic carcinoma treated by radiofrequency ablation

Objective To investigate the changes of chemokines related to dendritic cells in liver and spleen in rats with hepatic carcinoma treated by radiofrequency ablation (RFA),and to explore the mechanism of anti-tumor responses to RFA.Methods Forty healthy SD rats with established animal model of hepatic carcinoma were randomly divided into control group (n=10),RFA 7d group (n=16) and RFA 14d group (n=14).The rats of control group were killed without treatment.The other rats were killed in 7d and 14d after RFA treatment respectively.Spleen and liver tissue around the ablation area or around the tumor were taken out.The expressions of macrophage inflammatory protein (MIP)-1a in liver tissue and MIP-3β in spleen were analyzed by ELISA.Results The expression of MIP-1a in liver tissue was (232.92±54.5B)ng/L in control group,which enhanced to (499.38±15.14)ng/L and (495.90±9.94)ng/L in RFA 7d and 14d groups respectively.There were significant differences between control and RFA 7d group,control and RFA 14d group(P＜0.05).The expression of MIP-3βin spleen was (70.08±2.67) ng/L in control group,which enhanced to (151.57±48.48)ng/L and (154.57±18.25)ng/L in RFA 7 d and 14 d groups respectively.There were significant differences between control and RFA 7 d group,control and RFA 14 d group (P＜0.05).Conclusions The expressions of MIP-1a in liver tissue and MIP-3β in spleen increase significantly after RFA.These changes will promote recruitment and migration of dendritic cells and may contribute to the anti-tumor responses after RFA. Key words: Ultrasonography; Catheter ablation; Liver neoplasm; experimental

NOD2 Ligation Subverts IFN-α Production by Liver Plasmacytoid Dendritic Cells and Inhibits Their T Cell Allostimulatory Activity via B7-H1 Up-Regulation

The nucleotide-binding oligomerization domain (NOD)2/CARD15 protein, which senses muramyl dipeptide (MDP), a product of bacterial peptidoglycan, appears to play an important role in regulating intestinal immunity. Although the liver is exposed to gut-derived MDP, the influence of NOD2 ligation on hepatic APC, in particular dendritic cells (DC), is unknown. Freshly isolated mouse liver and spleen plasmacytoid (p)DC expressed higher levels of NOD2 message than conventional myeloid (m)DC. Following MDP stimulation in vivo, liver pDC, but not mDC, up-regulated expression of IFN regulatory factor 4 (IRF-4), a negative regulator of TLR signaling, and induced less allogeneic T cell proliferation and IFN-gamma production. The adoptive transfer of liver pDC from MDP-treated mice failed to prime allogeneic T cells in vivo. By contrast, splenic DC IRF-4 levels and T cell stimulatory activity remained unchanged. Liver pDC from MDP-stimulated mice also displayed greater IkappaBalpha, cell surface B7-H1, and B7-H1 relative to CD86 than control liver pDC. No similar effects were observed for liver mDC or spleen DC. Absence of B7-H1 on liver pDC reversed the inhibitory effect of MDP. After ex vivo stimulation with LPS or CpG, liver pDC but not mDC from MDP-treated animals secreted less IL-12p70, IL-6, and TNF-alpha and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN-alpha than their splenic counterparts, and systemic levels of IFN-alpha were reduced in MDP-treated animals after CpG administration. These findings suggest that differential effects of NOD2 ligation on liver pDC may play a role in regulating hepatic innate and adaptive immunity.

Molecular Regulation of Hepatic Dendritic Cell Function and Its Relation to Liver Transplant Outcome

Studies on liver interstitial dendritic cells (DC) indicate that the maturation and function of these important antigen-presenting cells may be suppressed by continual exposure to microbial products from the gut, in particular, bacterial lipopolysaccharide. New evidence is emerging for a role of specific intracellular regulators of signal transduction and of cytokines in the hepatic microenvironment, which may contribute to a hyporesponsive state in liver DC. Analysis of signaling molecule expression within DC in liver transplant tissue is likely to uncover its relation to allograft outcome.

NFATc1 Mediates Toll-Like Receptor-Independent Innate Immune Responses during Trypanosoma cruzi Infection

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88 −/− Trif −/− mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-γ was normally induced in T. cruzi-infected Myd88 −/− Trif −/− innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca2+ level. Furthermore, T. cruzi-induced IFN-γ expression was blocked by inhibition of Ca2+ signaling. NFATc1, which plays a pivotal role in Ca2+ signaling in lymphocytes, was activated in T. cruzi-infected Myd88−/−Trif−/− innate immune cells. T. cruzi-infected Nfatc1 −/− fetal liver DCs were impaired in IFN-γ production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.

Distinct response of liver myeloid dendritic cells to endotoxin is mediated by IL-27

The liver lies downstream of the gut, and is constantly exposed to bacteria. Liver dendritic cells (DC) are known to possess properties of tolerance, and respond to LPS differently when compared to conventional DC, but the underlying mechanisms are not completely understood. The aim of this study was to evaluate liver DC response to LPS stimulation. Liver or spleen derived DC were isoloated from mice treated with plasmid-GM-CSF hydrodynamic injection. The surface molecules and TLR4 expression on DC and cytokine productions of LPS stimulated DC were determinded by FACS analysis, ELISA and qPCR. The ability of DC to elicit T cell responses and differentiation were examined by MLR/CTL assay and qPCR for molecular markers related to Th1/Th2/Treg. In this study, we demonstrated that the threshold of LPS stimulation for liver DC was markedly higher than spleen DC, even though the expression of TLR4 on both DCs was comparable. In contrast to spleen DC that produced high levels of IL-12 and induced Th1 response upon LPS stimulation, LPS-liver DC preferentially produced IL-10 and IL-27, instead of IL-12. In addition, liver DC induced T cell hyporesponsiveness, associated with selective expansion of CD4(+)Foxp3(+)T regulatory cells. Addition of exogenous IL-12 only slightly enhanced liver DC-induced T cell response. Interestingly, abrogation of IL-27 ligation by using IL-27R(-/-) T cells synergistically augmented the effect of IL-12, suggesting that IL-27 produced by liver DC plays a crucial role in induction of T cell hyporesponsiveness. Liver DC respond distinctly to LPS stimulation by secreting IL-27 which synergizes with silencing of bioactive IL-12 activity leading to profound T cell inhibition.

Influence of radiofrequency ablation on dendritic cells in rats with liver carcinoma

Objective To investigate the change of dendritic cells (DCs) in rats with hepatic carcinoma treated by radiofrequency ablation (RFA),and to explore the mechanisms of anti-tumor immune response to RFA. Methods Forty healthy SD rats with established animal model of hepatic carcinoma were randomly divided into control group (n = 10) ,RFA 7 d group (n = 16) and RFA 14 d group (n = 14). The rats of control group were killed without treatment. The other rats were killed in 7 d and 14 d after RFA treatment respectively. Peripheral blood, liver tissue around the ablation area and spleen were taken out. The OX62,OX6,CD86 of DCs were analyzed with flowcytometry. Results ①OX62 cells accounted for (0.45 ± 0.19)% of mononuelear cells in peripheral blood in control group. The account of OX62 cells increased to (0.78 ± 0.30)% and (1.53 % 0.80)% in RFA 7 d and 14 d groups respectively. There were significant differences between control and RFA 7 d group, control and RFA 14 d group (P<0.05). ②OX62 cells accounted for (18.91 ± 4.58)% of mononuclear cells in liver tissue around the tumor in control group. The account of OX62 cells increased to (24.49 ± 4.59)% in RFA 7 d group (P<0.05). The expression of OX6 on DCs in liver tissue was (15.29 ± 4.59)% and increased to (34.2 ± 11.62)% and (39.18 ± 9.14)% in RFA 7 d and RFA 14 d group respectively (P<0.05). ③OX62 cells accounted for (11.69 ± 4.39)% of mononuclear cells in spleen in control group which increased to (15.10±2.37)% in RFA 14 d group (P<0.05). Conclusions The precursor DCs in peripheral blood and DCs in liver and spleen increased significantly after RFA. The expressions of OX6 on DCs in liver and spleen increased after RFA. RFA can promote the differentiation and maturation of DC. The increased function of antigen presenting may contribute to the anti-tumor responses after RFA. Key words: Catheter ablation; Liver neoplasms,experimental; Dendritic cells

The PD-L1 Signal is Important to Liver Dendritic Cells in Induction of Foxp3+CD4+CD25+ Treg and Liver Transplant Tolerance (141.33)

Abstract Our previous studies have demonstrated that Foxp3+CD25+CD4+ regulatory T cells (Treg) play an important role in the induction of murine liver spontaneous transplant tolerance. How Treg are induced and what their mechanisms are in the regulation of liver transplant tolerance remain undefined. Here, we investigated the role of liver dendritic cells (DCs) on the induction of Treg and liver graft tolerance in vivo and in vitro. Fms-like tyrosine kinase 3 ligand (Flt3L) mutation mice, which have severe reduction in all types of DCs, and PD-L1 deficient mice were used to examine the role of liver DCs and the PD-L1 signal in Treg induction and liver transplant tolerance. Our results showed that liver DCs, which expressed a high number of PD-L1 molecules, induced more Foxp3+CD25+CD4+ Treg in vitro compared to spleen DCs. However, DCs from PD-L1 deficient mice failed to expand Foxp3+CD25+CD4+ Treg in vitro. Adoptive transfer of Foxp3+CD25+CD4+ Treg expanded from liver DCs prolonged heart allograft survival significantly more than in the CD4 treated controls. The liver grafts from Flt3L-/- and PD-L1-/- mice were rejected acutely in the C3H recipients. Foxp3+ cells were reduced, but IL-2, IL-10, and IFN-γ producing cells were significantly increased in the liver graft and recipient spleen sections of Flt3L-/- and PD-L1-/- donors. Thus, liver DCs play a critical role in the induction of Treg, which underpin spontaneous acceptance of liver allografts. The function of DCs on Treg induction appears to depend on the PD-L1 signal.

The role of CD11c+ hepatic dendritic cells in immue tolerance to hepatocellular carcinoma (41.48)

Abstract Immune tolerance is a major mechanism involved in the development of hepatocellular carcinoma (HCC). We have previously showed, in a mouse model of spontaneous HCC, that T cell response can be induced in mice with small HCC, but not in mice with large HCC, indicating that immune tolerance occurs when tumors grow to a certain size. The mechanisms responsible for this phenomenon were not clear. In this study, we compared freshly isolated liver dendritic cell (DC) subsets in mice with no tumor, small HCC, and large HCC. We found that total numbers of CD11c+ hepatic DC were comparable in these 3 groups. However, CD11cint B220+ subset of 'plasmacytoid' DC (pDC) significantly decreased in both small and large HCC-bearing mice. Importantly, CD11chi B220- subset of mature DC significantly increased in mice with small HCC. In contrast, CD11chi B220- subset of mature DC remains same in mice with large HCC, the CD11cint B220- subset of immature DC increased instead in these mice. The surface phenotype of each subset of DCs were examined and demonstrated higher level of CD86 and CD40 expression by CD11chi subset of mature DC comparing with CD11cint subsets. These findings indicate that modulating subset distribution of liver DC, such as inhibiting the expansion of CD11chi B220- subset of mature DC, may play a role in immune tolerance to HCC.

Activated liver dendritic cells generate strong acquired immunity in α-galactosylceramide treatment

Alpha-galactosylceramide (alpha-GalCer) presented by dendritic cells (DCs) activates NKT cells that in turn drive DC maturation. However, the potential of generating acquired immunity of liver DCs in alpha-GalCer treatment remains unclear. We examined the activation of acquired immunity in the alpha-GalCer treatment against liver or spleen tumor and the ability of liver and spleen DCs in the generation of acquired immunity. Administration of alpha-GalCer resulted in generation of p53 peptide-specific cytotoxic T lymphocytes (CTLs) in mice bearing liver CMS4 tumor, aberrantly expressing p53, but not in mice bearing spleen CMS4 tumor. The growth of rechallenged CMS4 subcutaneous tumor was inhibited in alpha-GalCer-treated mice against liver CMS4 tumor, but not in alpha-GalCer-treated mice against CMS4 spleen tumor. The antigen presenting related functions of liver DCs were significantly higher than those of spleen DCs in alpha-GalCer-treated mice. Vaccination of normal mice with p53 peptide pulsed liver DCs isolated from alpha-GalCer treated mice resulted in generation of p53 peptide-specific CTLs, but that with p53 peptide pulsed spleen DCs did not. These results demonstrated that alpha-GalCer treatment induced unique immunologic activation of liver DCs in comparison with spleen DCs, which might be favorable to generate liver acquired immunity.

Gut Homing Receptors on CD8 T Cells Are Retinoic Acid Dependent and Not Maintained by Liver Dendritic or Stellate Cells

Lymphocytes primed by intestinal dendritic cells (DC) express the gut-homing receptors CCR9 and alpha4beta7, which recognize CCL25 and mucosal addressin cell-adhesion molecule-1 in the intestine promoting the development of regional immunity. In mice, imprinting of CCR9 and alpha4beta7 is dependent on retinoic acid during T-cell activation. Tissue specificity is lost in primary sclerosing cholangitis (PSC), an extraintestinal manifestation of inflammatory bowel disease, when ectopic expression of mucosal addressin cell-adhesion molecule-1 and CCL25 in the liver promotes recruitment of CCR9+alpha4beta7+ T cells to the liver. We investigated the processes that control enterohepatic T-cell migration and whether the ability to imprint CCR9 and alpha4beta7 is restricted to intestinal DCs or can under some circumstances be acquired by hepatic DCs in diseases such as PSC. Human and murine DCs from gut, liver, or portal lymph nodes and hepatic stellate cells were used to activate CD8 T cells. Imprinting of CCR9 and alpha4beta7 and functional migration responses were determined. Crossover activation protocols assessed plasticity of gut homing. Activation by gut DCs imprinted high levels of functional CCR9 and alpha4beta7 on naïve CD8 T cells, whereas hepatic DCs and stellate cells proved inferior. Imprinting was RA dependent and demonstrated plasticity. Imprinting and plasticity of gut-homing human CD8 T cells requires primary activation or reactivation by gut DCs and is retinoic acid dependent. The inability of liver DCs to imprint gut tropism implies that alpha4beta7+CCR9+ T cell that infiltrate the liver in PSC are primed in the gut.

Human Liver Dendritic Cells Promote T Cell Hyporesponsiveness

The liver is believed to promote tolerance, which may be beneficial due to its constant exposure to foreign Ags from the portal circulation. Although dendritic cells (DCs) are critical mediators of immune responses, little is known about human liver DCs. We compared freshly purified liver DCs from surgical specimens with autologous blood DCs. Liver and blood DCs were equally immature, but had distinct subset compositions. BDCA-1(+) DCs represented the most prevalent liver DC subset, whereas the majority of peripheral blood DCs were CD16(+). Upon TLR4 ligation, blood DCs secreted multiple proinflammatory cytokines, whereas liver DCs produced substantial amounts of IL-10. Liver DCs induced less proliferation of allogeneic T cells both in a primary MLR and after restimulation. Similarly, Ag-specific CD4(+) T cells were less responsive to restimulation when initially stimulated by autologous liver DCs rather than blood DCs. In addition, liver DCs generated more suppressive CD4(+)CD25(+)FoxP3(+) T regulatory cells and IL-4-producing Th2 cells via an IL-10-dependent mechanism. Our findings are critical to understanding hepatic immunity and demonstrate that human liver DCs promote immunologic hyporesponsiveness that may contribute to hepatic tolerance.

QS475. In States of Hepatic Cirrhosis, Liver Dendritic Cells Control the Hepatic Cytokine Milieu and Gain Enhanced Capacity to Stimulate Innate and Adaptive Immunity

QS475. In States of Hepatic Cirrhosis, Liver Dendritic Cells Control the Hepatic Cytokine Milieu and Gain Enhanced Capacity to Stimulate Innate and Adaptive Immunity

Receptor for advanced glycation end product (RAGE)-dependent modulation of early growth response-1 in hepatic ischemia/reperfusion injury

We previously showed that blockade of RAGE significantly attenuates hepatic ischemia/reperfusion (I/R) injury in mice. Here, we identify that early growth response-1 (Egr-1) is a downstream target of RAGE in hepatic I/R injury. Hepatic I/R was induced in male mice. Liver remnants were analyzed for induction of Egr-1 and cytokines, as well as regulation of apoptotic pathways after reperfusion. Egr-1 was upregulated in the liver remnants after hepatic I/R injury and was suppressed by administration of soluble RAGE or deletion of the RAGE gene. RAGE-mediated increased expression of Egr-1 upregulates a central downstream gene, MIP2. In contrast, RAGE-stimulated Egr-1-independent pathways regulate TNF-alpha production and apoptosis in response to I/R. Consistent with these findings, phospho-p44/42 and phospho-JNK MAPK and c-Jun were strikingly suppressed in RAGE(-/-) versus WT mice, but not in Egr-1(-/-) mice. RAGE ligand HMGB1 was upregulated after I/R in the liver remnants. In vitro, incubation of RAGE-expressing liver dendritic cells (DCs) with recombinant HMGB-1 resulted in increased Egr-1 transcripts, in a manner suppressed by RAGE gene deletion, soluble RAGE and inhibitors of p44/p42 or JNK MAP kinase. Suppression of Egr-1 may contribute to the protective mechanisms underlying the beneficial impact of RAGE blockade or deletion.

The Liver Mediates Apoptotic Cell‐Induced Immune Regulation

Allogeneic apoptotic cells have been demonstrated to induce allograft tolerance, but the mechanisms for this remain unclear. The study presented here investigates organs in which the tolerogenic immune responses may occur. Distribution of live or apoptotic CFSE(+) splenocytes in recipients' organs and phagocytosis by liver antigen-presenting cells (APC) were investigated by fluorescence microscopy and flow cytometry, and cytokine expression was analysed by Multiplex and ELISA. It was found that allogeneic or autogenic apoptotic cells preferentially accumulated in the liver within 30 min, peaked at 60 min, and disappeared at 12 h after infusion, whereas these cells scarcely appeared in the spleen. The accumulation in the liver was apoptotic cell-specific as both allogeneic and autogenic live splenocytes were completely deposited in the spleen. Liver phagocytes, including Kupffer cells, liver sinusoidal endothelial cells and dendritic cells, all efficiently phagocytized apoptotic cells in vitro and in vivo. Although a Th1 cytokine profile found both in the spleen and liver in the recipients of allogeneic apoptotic cells, a rapid and consistent Th2 cytokine profile specifically was initiated in the liver. From this, we conclude that liver APC phagocytize donor apoptotic cells and induce liver-specific Th2 cytokines, which may contribute to the mechanisms of allograft tolerance induced by donor apoptotic cells.

Decreased expressions of CD1d molecule on liver dendritic cells in subcutaneous tumor bearing mice

Alpha-Galactosylceramide (alpha-GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. However, the activation of liver innate immunity by alpha-GalCer should be examined because clinical trials of alpha-GalCer resulted in limited clinical responses. We examined the activation of liver innate immunity by alpha-GalCer in subcutaneous Colon26 tumor bearing-mice (C26s.c.TB-mice). The expressions of CD1d molecule on liver dendritic cells (DCs) were significantly lower in C26s.c.TB-mice than those in tumor-unbearing normal mice. Although liver NK cells and NKT cells activated in normal mice after alpha-GalCer treatment, the activation of these cells were significantly inhibited in C26s.c.TB-mice. Alpha-GalCer treatment resulted in significant antitumor effect against Colon26 metastatic liver tumor in normal mice, but not in C26s.c.TB-mice. The serum levels of TGF-beta, known to suppress the CD1d expressions on DCs, in C26s.c.TB-mice were significantly higher than those in normal mice. Surgical subcutaneous tumor mass reduction resulted in the reduction of serum TGF-beta, the recovery of CD1d expressions on liver DCs and the improvement of antitumor effect of alpha-GalCer against metastatic liver tumor. These results suggested that tumor burden reduces CD1d expressions on liver DCs, thus impeding alpha-GalCer-mediated NK cell activation and antitumor activity in the liver.

Blockade of PD-1/B7-H1 Interaction Restores Effector CD8+ T Cell Responses in a Hepatitis C Virus Core Murine Model

The impaired function of CD8(+) T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8(+) T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8(+) T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8(+) T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-gamma, TNF-alpha, and granzyme B production by CD8(+) T cells. In addition, the impaired CD8(+) T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8(+) T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8(+) T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection.