A Strategy for Suppressing Macrophage-mediated Rejection in Xenotransplantation.

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.

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

BACKGROUND: 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. METHODS: A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. RESULTS: A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1. CONCLUSION: These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.

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-1ß 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.

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

PURPOSE: Various 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. METHODS: Naïve 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. RESULTS: While 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. CONCLUSIONS: Our 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.