IL-34 Inhibits Acute Rejection of Rat Liver Transplantation by Inducing Kupffer Cell M2 Polarization.

BACKGROUND: Recent studies have demonstrated that IL-34 is implicated in the regulation of macrophage functions. However, the effect of IL-34 on Kupffer cells (KCs) in acute rejection (AR) of liver transplantation remains unclear. METHODS: IL-34 expression was detected in graft and serum from allotransplantation and syngeneic transplantation groups. The adeno-associated virus-expressing IL-34 was used to assess the effect of IL-34 on AR of rat liver transplantation. The effect of IL-34 on KC polarization was evaluated by in vitro and in vivo assays. Kupffer cells in donors were depleted by clodronate treatment before transplantation, and the nontreated KCs or lipopolysaccharide-treated KCs were transferred into recipients during liver transplantation. RESULTS: IL-34 expression levels in grafts and serum were decreased in the allotransplantation group compared with the syngeneic transplantation group. Adeno-associated virus-expressing IL-34 treatment induced KC M2 polarization in vivo and inhibited the AR of rat liver transplantation. Moreover, we found that IL-34 switched the phenotype of KCs from M1 to M2 by activating the PI3K/Akt pathway in vitro. In addition, the results of KC deletion and adaptive transfer experiments showed that the AR inhibition induced by IL-34 was M2 KC-dependent. CONCLUSIONS: IL-34 plays an important role in KC M2 polarization-dependent AR inhibition of rat liver transplantation.

Kupffer Cell Transplantation in Mice for Elucidating Monocyte/Macrophage Biology and for Potential in Cell or Gene Therapy.

Kupffer cells (KC) play major roles in immunity and tissue injury or repair. Because recapitulation of KC biology and function within liver will allow superior insights into their functional repertoire, we studied the efficacy of the cell transplantation approach for this purpose. Mouse KC were isolated from donor livers, characterized, and transplanted into syngeneic recipients. To promote cell engraftment through impairments in native KC, recipients were preconditioned with gadolinium chloride. The targeting, fate, and functionality of transplanted cells were evaluated. The findings indicated that transplanted KC engrafted and survived in recipient livers throughout the study period of 3 months. Transplanted KC expressed macrophage functions, including phagocytosis and cytokine expression, with or without genetic modifications using lentiviral vectors. This permitted studies of whether transplanted KC could affect outcomes in the context of acetaminophen hepatotoxicity or hepatic ischemia-reperfusion injury. Transplanted KC exerted beneficial effects in these injury settings. The benefits resulted from cytoprotective factors including vascular endothelial growth factor. In conclusion, transplanted adult KC were successfully targeted and engrafted in the liver with retention of innate immune and tissue repair functions over the long term. This will provide excellent opportunities to address critical aspects in the biogenesis, fate, and function of KC within their native liver microenvironment and to develop the cell and gene therapy potential of KC transplantation.

Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells.

INTRODUCTION: Cell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4(+)CD25(+) regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin(-)) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice. METHODS: Allogeneic donor Lin(-) BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4(+) T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined. RESULTS: Co-transplantation of allogeneic Lin(-) BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury. CONCLUSIONS: A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

Pretransplant replacement of donor liver grafts with recipient Kupffer cells attenuates liver graft rejection in rats.

BACKGROUND AND AIM: Rejection of liver grafts is a difficult issue that has not been resolved. Preoperative replacement of liver cells in the graft with cells from the intended recipient may attenuate rejection. We investigated whether preoperative transplant of recipient bone marrow cells (BMCs) to the donor replaced liver allograft cells and attenuated rejection. METHODS: We used a rat model of allogeneic liver transplant (LT) from Dark Agouti (DA) to Lewis (LEW) rats. In BMC group, DA rats received BMC transplants from LacZ-transgenic LEW rats at 1 week before LT. In the control group, DA rats received no preoperative treatment. We evaluated graft damage at 7 days after LT and the survival of the recipient rats. RESULTS: Rats in the BMC group experienced prolonged survival that was abrogated by the administration of gadolinium chloride to donors at 24 h before LT. Serum concentrations of total bilirubin and hyaluronic acid on day 7 were significantly lower in the BMC group, and histopathological analyses revealed that rejection of the liver graft was attenuated. X-gal staining and immunohistostaining of the liver graft revealed that BMCs engrafted in the sinusoidal space differentiated into Kupffer cells. CONCLUSIONS: Preoperative transplant of recipient BMCs to LT donors replaced donor Kupffer cells and attenuated post-LT rejection, indicating that this strategy may increase the success of LT.

Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice.

Loss-of-function mutation in the heme oxygenase 1 (Hmox1) gene causes a rare and lethal disease in children, characterized by severe anemia and intravascular hemolysis, with damage to endothelia and kidneys. Previously, we found that macrophages engaged in recycling of red cells were depleted from the tissues of Hmox1(-/-) mice, which resulted in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. Here, we report that subablative bone marrow transplantation (BMT) has a curative effect for disease in Hmox1(-/-) animals as a result of restoration of heme recycling by repopulation of the tissues with wild-type macrophages. Although engraftment was transient, BMT reversed anemia, normalized blood chemistries and iron metabolism parameters, and prevented renal damage. The largest proportion of donor-derived cells was observed in the livers of transplanted animals. These cells, identified as Kupffer cells with high levels of Hmox1 expression, persisted months after transient engraftment of the donor bone marrow and were responsible for the full restoration of heme-recycling ability in Hmox1(-/-) mice and reversing Hmox1-deficient phenotype. Our findings suggest that BMT or the development of specific cell therapies to repopulate patients' tissues with wild-type or reengineered macrophages represent promising approaches for HMOX1 deficiency treatment in humans.

Effect of Kupffer cells on immune tolerance in liver transplantation.

OBJECTIVE: To observe the effect of Kupffer cells on immune tolerance in liver transplantation. METHODS: The rats were randomly divided into A, B and C groups. A group was sham operation group. The donor rats of group B had intraperitoneal injection of 1 nmol Kuppffer cells every other day for three days before liver transplantation. Rats of group C were injected with equal saline. The rat liver transplantation models were established by modified Kamada's two-cuff technique. The rats were sacrificed after 24 hours. The concentrations of ALT and AST in serum were measured with the biochemical analyzer. The level of IL-2 and TNF-α in serum were measured by ELISA method. The apoptotic indexes were detected by immunohistochemical assay. RESULTS: The concentration of ALT, AST, IL-1 and TNF-α in A, B and C groups were increased successively. The levels of group C were significantly higher than that of group B and A (P<0.05), and the levels of group B were significantly higher than that of group A (P<0.05). The apoptotic indexes of three groups were 3.40±0.37, 14.70±2.54 and 26.33±3.65, respectively, with significant difference among three groups (P<0.05). CONCLUSIONS: Pretreatment with Kupffer cells can reduce liver injury and raise liver transplantation immune tolerance.

Dynamic changes of indoleamine 2,3-dioxygenase of Kupffer cells in rat liver transplant rejection and tolerance.

AIMS: To study the dynamic changes and the immunologic role of indoleamine 2, 3-dioxygenase (IDO) in Kupffer cells (KCs) after rat liver transplantation. METHODS: Animals were randomly divided into two groups: a rejection group (REJ; LEW to BN) and a tolerance group (TOL; BN to LEW). Liver morphological changes were observed optically with hematoxylin/eosin staining. KCs were isolated from recipients. mRNA and protein expressions of IDO were detected by real-time polymerase chain reaction and Western blotting at 1, 3, 5, and 7 days after transplantation. RESULTS: The levels of IDO mRNA and protein in KCs of TOL groups were similar to those in REJ groups at day 1 posttransplantation. However, the expression of IDO mRNA and protein time-dependently increased to much higher levels in the TOL than the in REJ groups at 3, 5, and 7 days posttransplantation (P < .05). The peak was observed at 7 days. CONCLUSIONS: The IDO level of KCs was closely associated with immune tolerance induction. IDO-mediated immune modulation appears to be an attractive means to assess transplant tolerance induction.

Role of bone marrow transplantation for correcting hemophilia A in mice.

To better understand cellular basis of hemophilia, cell types capable of producing FVIII need to be identified. We determined whether bone marrow (BM)-derived cells would produce cells capable of synthesizing and releasing FVIII by transplanting healthy mouse BM into hemophilia A mice. To track donor-derived cells, we used genetic reporters. Use of multiple coagulation assays demonstrated whether FVIII produced by discrete cell populations would correct hemophilia A. We found that animals receiving healthy BM cells survived bleeding challenge with correction of hemophilia, although donor BM-derived hepatocytes or endothelial cells were extremely rare, and these cells did not account for therapeutic benefits. By contrast, donor BM-derived mononuclear and mesenchymal stromal cells were more abundant and expressed FVIII mRNA as well as FVIII protein. Moreover, injection of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from BM, or of healthy BM-derived mesenchymal stromal cells, protected hemophilia A mice from bleeding challenge with appearance of FVIII in blood. Therefore, BM transplantation corrected hemophilia A through donor-derived mononuclear cells and mesenchymal stromal cells. These insights into FVIII synthesis and production in alternative cell types will advance studies of pathophysiological mechanisms and therapeutic development in hemophilia A.

New method of delivering gene-altered Kupffer cells to rat liver: studies in an ischemia-reperfusion model.

BACKGROUND & AIMS: Kupffer cells play a major role in the pathogenesis of several diseases. They release physiologically active substances that often lead to localized tissue injury. Therefore, the aim of this study was to establish a model to protect the liver through supplementation of Kupffer cells that have been transduced by recombinant adenovirus. METHODS: Optimal conditions for intravenous injection in rats were established using carbon-labeled Kupffer cells. Adenoviral-transduced Kupffer cells encoding the Cu/Zn-SOD gene (Ad.SOD1) or beta-galactosidase reporter gene (Ad.LacZ) were transplanted into recipient rats. Twenty-four hours after transplantation, 70% hepatic ischemia-reperfusion was used to induce hepatic oxidative stress, and liver injury was determined 8 or 24 hours later. RESULTS: In initial experiments, 10%-20% of the injected carbon-labeled cells were localized in the host liver after 24 hours, representing approximately 1% of the total population of Kupffer cells. Pretreatment of the recipient with a single dose of cyclosporin A maximized Kupffer cell reseeding up to 4%-10% of the total Kupffer cell population, suggesting that efficiency is limited by host immune response. Moreover, reseeded Kupffer cells were retained in host livers for up to 14 days after transplant. In livers of animals injected with Kupffer cells transduced with Ad.LacZ, transgene expression was observed, indicating Kupffer cell functional integrity. Injection of Kupffer cells transduced with Ad.SOD1 significantly blunted the increase in serum transaminases and liver injury because of ischemia-reperfusion compared with controls. CONCLUSIONS: This novel approach allows delivery of transduced Kupffer cells in rats, which can be used as an investigative tool as well as a therapeutic strategy against inflammatory liver diseases.

[Augmentation of liver immune functions by intrasplenic transplantation of IL-2 gene-modified hepatocytes].

OBJECTIVE: To investigate the effects of intrasplenically transplanted, IL-2 gene-modified hepatocytes on the liver immune response and anti-tumor activity. METHODS: Murine fetal hepatocytes BNL CL. 2 were transfected with mIL-2 gene in vitro, and intrasplenically transplanted into syngeneic mice (2 x 10(6)/mouse). After two weeks, the Kupffer cells were isolated and studied for cytotoxicity, TNF and NO production, and Ia expresson. The therapeutic effects on the mice with the metastatic liver carcinoma were also observed. RESULTS: The Kupffer's cytotoxicity to L1210, Yac-1, P815 and H22 target cells, and their production of TNF and NO were greatly augmented, even when freshly prepared. Their Ia expression also increased. When treated with IL-2 gene-modified hepatocytes, the metastatic liver carcinoma-bearing mice survived for a longer time. CONCLUSIONS: Intrasplenic transplantation of IL-2 gene-modified hepatocytes could effectively activate the liver immune response and exert potent therapeutic effects on liver carcinoma-bearing host.