Tasuku Kodama

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.

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.

Postoperative Platelet Count can Predict Splenic Volume Expansion after Living Donor Liver Transplantation in Pediatric Patients with Biliary Atresia

Introduction Although spleen volume (SV) has been thought to decrease after living donor liver transplantation (LDLT), there are a few cases in which SV gradually increases and the expansion can lead to life-threatening complications. However, it is unclear which patients have such a risk. This study was conducted to investigate factors associated with SV expansion after LDLT. Materials Patients with liver cirrhosis due to biliary atresia (BA), who had LDLT between March 2008 and July 2017 in our institute, were included in this study. CT scan was performed approximately 3 months (3±1 months) after LDLT. Those patients, who had splenectomy with LDLT simultaneously and who died within 3 months after LDLT, were excluded. Methods Age, sex, standard liver volume (SLV) for each patient, transplanted graft volume (GV), SV before and approximately 3 months after LDLT were evaluated. The major, minor and longitudinal diameter of spleen were measured on the cross-sectional surface with splenic hilar. SV was calculated by multiplying these values and evaluated as SV/body surface area (BSA) to compare patients with variable ages. Patients were divided into SV/BSA ≥1,000ml and <1,000ml. The volume reduction rate was calculated using the formula of [((Preoperative SV) – (Postoperative SV))/Preoperative SV]×100. Using this rate, patients were divided into two groups; SV reduction and SV expansion. Factors which could affect postoperative SV were evaluated using the univariable regression analysis. The impact of postoperative Plt on SV expansion was measured with the logistic regression analysis. Results 33 patients (11 males and 22 females) were enrolled.The mean age of these patients was 3.7±5.4 years old. The mean GV/SLV and GV/body weight were 76.8±22.1% and 2.47±0.94%, respectively. The mean SV/BSA before and after LDLT, and SV reduction rate were 780.8±287.0ml/m2, 569.2±254.7ml/m2 and 23.4±31.0%, respectively. There were five patients whose SV increased postoperatively. The mean age and postoperative Plt of patients with preoperative SV/BSA ≥1,000ml was significantly older and lower than those with <1,000ml (p=0.0016 and 0.039, respectively). All laboratory values except Plt were significantly improved after LDLT, and there was a significant difference in postoperative Plt between SV reduction and SV expansion groups (19.0±7.8 and 10.7±5.6 (×104/&mgr;l), respectively (p=0.031)). In the univariable regression analysis, postoperative Plt had a significant effect on SV reduction rate (regression coefficient, 1.52 (CI 0.23-2.82; p=0.022)). In the logistic regression analysis, there was a significant impact of postoperative Plt on SV expansion after LDLT (OR 0.808 (CI 0.661-0.988; p=0.038)). Figure. No caption available. Figure. No caption available. Figure. No caption available. Discussion/Conclusion Remaining low Plt after LDLT could indicate the poor SV reduction and may predict SV expansion. Adolescent patients with preoperative SV/BSA ≥1,000ml should undergo splenectomy with LDLT.

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.

A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells

OBJECTIVE Surfactant protein D (SP-D), which is secreted mainly in the lung, is an oligometric C type lectin that promotes phagocytosis by binding to carbohydrates on microbial surfaces. SP-D can also bind SIRPα, leading to a decrease in cytokine production by monocytes/macrophages. In the present study, we examined the possibility that SP-D suppresses macrophage-mediated xenogeneic cytotoxicity, by creating a membrane-type SP-D. METHODS The cDNA for the carbohydrate recognition domain (CRD) of human SP-D was switched to that of a membrane-type protein, collectin placenta 1 (CL-P1), with a Flag-tag. The cDNA of CD47 was prepared as a control. The suppressive function of the membrane-type protein of the hybrid molecule, CL-SP-D, to monocytes/macrophages was then studied and the results compared with that for CD47. RESULTS The expression of Flag-tagged CL-SP-D on the transfected SECs and the SIRPα on monocyte-like cells, THP-1 cells, was confirmed by FACS using anti-Flag Ab and anti-CD172a, respectively. The molecular size of the hybrid protein was next assessed by western blot. While significant cytotoxicity against SEC was induced in differentiated THP-1 cells, CL-SP-D significantly reduced THP-1-mediated cytotoxicity. In addition, phosphorylated SHP-1 was clearly detected in SEC/CL-SP-D in western blots. Moreover, IL-10 production was upregulated and IL-1β production was suppressed in the case of THP-1 and SEC/CL-SP-D, compared with naïve SEC. Next, the cytotoxicity caused by the in vitro generated macrophage was assessed under the same conditions as were used for THP-1. CL-SP-D also showed the significant down-regulation on the macrophage. In addition, changes in IL-10 production by the macrophage confirmed the results. CONCLUSIONS These findings indicate that the membrane-type SP-D serve as an effective therapeutic strategy for inhibiting macrophage-mediated xenograft rejection in xenotransplantation.