Rieko Sakai

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Suppression of human macrophage-mediated cytotoxicity by transgenic swine endothelial cell expression of HLA-G.

BACKGROUND Xenotransplantation is an appealing alternative to human allotransplantation because of a worldwide shortage of organs. One of the obstacles for xenografts is cellular rejection by the innate immune system, comprised of NK cells, monocytes, and macrophages. In this study the inhibitory function of HLA-G1, a MHC Ib molecule, on macrophage-mediated cytotoxicity was examined. Furthermore, this study also evaluates the suppressive effect of cytokine production by macrophages. METHODS The expression of inhibitory receptors that interact with HLA-G1, immunoglobulin-like transcript 2 (ILT2), ILT4 and KIR2DL4 (CD158d) on in vitro generated macrophages were examined by flow cytometry. Complementary DNA (cDNA) of HLA-G1, HLA-E and human β2-microglobulin (hβ2m) were prepared and transfected into swine endothelial cells (SECs). The expression of the transgenic genes was evaluated by flow cytometry, and macrophage-mediated SEC cytolysis was assessed using the macrophages. RESULTS In vitro generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on SECs indicated significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. Furthermore, the results on real time PCR and ELISA revealed that transgenic HLA-G1 induces the anti-inflammatory cytokines, such as IL-10 and TGF-β, and suppresses iNOS mRNA expression, indicating that transgenic HLA-G1 has suppressive effects in a broad range of transplant rejection. CONCLUSION These results indicate that generating HLA-G1 transgenic pigs can protect porcine grafts from macrophage-mediated cytotoxicity.

Monocytic MDSCs regulate macrophage-mediated xenogenic cytotoxicity.

BACKGROUND Xenotransplantation is considered to be one of the most attractive strategies for overcoming the worldwide shortage of organs. However, many obstructions need to be overcome before it will achieve clinical use in patients. One such obstacle is the development of an effective immunosuppressive strategy. We previously reported that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of progenitor and immature myeloid cells, suppress xenogenic CTL-mediated cytotoxicity. Because of their heterogeneous nature, MDSC can function via several suppressive mechanisms that disrupt both innate and adaptive immunity. Since macrophages play a pivotal role in the rejection of a xenograft, in this study, we evaluated the suppressive effects of MDSC against macrophage-mediated xenogenic rejection. MATERIALS AND METHODS To evaluate the effect of monocyte-derived MDSCs on xenogenic immune reactions, a CFSE(carboxyfluorescein diacetate, succinimidyl ester)assay was employed to assess cytotoxicity. RESULTS While, in the absence of activation, primed MDSCs had no detectable effect on macrophage-induced cytotoxicity against SEC cells, LPS-activated MDSCs were found to significantly suppress xenogenic cytotoxicity. A CFSE cytotoxicity assay revealed that MDSCs significantly suppressed macrophage-induced cytotoxicity. Furthermore, an indoleamine 2,3 dioxygenase (IDO) inhibitor, 1-methyl tryptophan (1-MT), abolished the MDSC-induced suppression of macrophage-mediated xeno-rejection, indicating that MDSCs may suppress macrophage-mediated cytotoxicity in an IDO-dependent manner. CONCLUSION These findings indicate that MDSCs have great potential for immunosuppressing macrophage-mediated xeno-rejection.

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis

PurposeVarious strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages.MethodsNaïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry.ResultsWhile CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis.ConclusionsOur findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R+ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.

Correction to: Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis

In the original publication, the fifth author name was erroneously published as “Patmika Jiaravuthiasan”. The correct author name should read as, “Patmika Jiaravuthisan”. The original article was corrected.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components

BACKGROUND On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

Human CD31 on porcine cells suppress xenogeneic neutrophil-mediated cytotoxicity via the inhibition of NETosis

Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil‐targeted therapies may have the potential for increased graft survival in xenotransplantation.

The immunological response of pigs against human cell, included issues such as the production of natural antibodies in newborn

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mothers milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

Human CD31 Suppress Macrophage-Mediated Xenogeneic Rejection

Introduction Cellular xenogeneic rejection (CXR) is one of the important immunological obstructions that need to be overcome, if xenogeneic organs are to be used in the clinic. In particular, innate immunity by NK cells, macrophages and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies designed to suppress innate immune cells have considerable potential in practical applications of xenotransplantation. Because the macrophage is one of main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently found that human CD31 on swine endothelial cells (SEC) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed, not only on neutrophils, but also on macrophages, we hypothesized that human CD31 on SEC may suppress the macrophage-mediated cytotoxicity. Materials and Methods To validate our hypothesis, SEC and hCD31-transfected SEC (SEC/hCD31) were cocultured with macrophages and macrophage-related cytotoxicity was evaluated using a WST-8 assay. Next, peripheral blood-derived macrophages were generated by culturing peripheral blood monocytes with 100ng/ml GM-CSF for 7 days and the cytotoxicity caused by peripheral blood-derived macrophages was assessed using a WST-8 assay. Furthermore, to confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of SHP-1 was investigated by western blotting. Results While PMA-activated THP-1 cells (monocyte-like cells) induced a significant level of cytotoxicity against SEC (cytotoxicity: 44.0±5.8%), a significant reduction in cytotoxicity by THP-1 was observed in SEC/hCD31 (21.1±10.6%; p<0.001, n=6). The cytotoxicity of macrophages against SEC was also significantly suppressed by hCD31 on SEC (SEC: 43.5±14.3%, SEC/CD31: 17.2±5.7%). Western blotting analyses revealed that a significant phosphorylation of SHP-1 was induced in macrophages and THP-1 that had been cocultured with SEC/hCD31. Conclusion Taken together, we conclude that human CD31 on porcine cells might suppress, not only neutrophils, but also macrophage-mediated cytotoxicity in a CD31 homophilic ligation-dependent manner. Our findings suggest that the generation of hCD31 transgenic pigs for use in xenografts is very attractive in terms of preventing xenogeneic rejection.

PQA-18, a Novel Immunosuppressant, Suppresses Macrophage Differentiation and Macrophage-Mediated Xenogeneic Cytotoxicity

Introduction A number of studies have confirmed that innate cellular immunity has a major role in xenograft rejection. Innate cellular rejection by NK cells, macrophages and neutrophils has been reported to cause severe rejection in xenotransplantation. However, nearly all of the currently used immunosuppressive drugs focus on acquired immunity. Therefore, the development of a new immunosuppressive drug to prevent innate immunity would be highly desirable. Prenylated quinolinecarboxylic acid (PQA) -18, a unique PAK-2 inhibitor, was recently reported to have an immunosuppressive function in vivo and in vitro. In this study, we focused on the effect of this new immunosuppressant on macrophages. Materials and Methods To evaluate the effect of PQA-18 in xenogeneic macrophage-mediated cytotoxicity, swine endothelial cells (SEC) were co-cultured with PMA-treated THP-1 (a human monocyte-like cell line) in the presence or absence of 5&mgr;M PQA-18. The cytotoxicity of THP-1 to SEC was measured by means of a WST-8 assay. PQA-18 was found to significantly suppress THP-1 induced cytotoxicity. Furthermore, we assayed the effect of PQA-18 on macrophage differentiation. Peripheral blood monocytes were obtained by density gradient centrifugation. Peripheral blood monocytes were cultured with 100ng/ml GM-CSF in the presence or absence of PQA-18 and their differentiation into macrophages was assessed by flow cytometry. Results Fiestly, we confirmed that 5&mgr;M PQA-18 have no cytotoxicity against SEC and THP-1 by fluorescence staining of propidium iodide and annexinV. PQA-18 demonstrated a significant suppression of THP-1-induced cytotoxicity against SEC in a WST-8 assay (%Cytotoxicity. Control: 37.1±10.4%, 5&mgr;M PQA-18: 25.1±6.6%). In addition, PQA-18 completely suppressed the differentiation of monocytes into macrophages. While a significant upregulation of HLA-DR expression was observed in GM-CSF generated macrophages, macrophages which were generated in the presence of PQA-18 did not express HLA-DR (Figure 1). Conclusion These findings suggest that PQA-18 suppress macrophage-mediated xenogeneic rejection and that PQA-18 have the potential to suppress antigen presentation by macrophages. Furthermore, our data indicate that the PAK-2 signaling pathway play an essential role in macrophage differentiation. PQA-18 will be useful in the development of novel immunosuppressive strategies not only in xenotransplantation but also in allogeneic transplantation. Figure. No caption available.