title: The potentiating effect of hTFPI in the presence of hCD47 reduces the cytotoxicity of human macrophages.
abstract: In pig-to-human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3-galactosyltransferase knockout pigs. However, macrophage-mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD47 inhibits monocyte/macrophage-mediated phagocytosis by identifying the CD47 signal regulatory protein alpha (SIRP-α) as self/non-self. Tissue factor pathway inhibitor (TFPI) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD47, thrombospondin-1 (TSP-1). When TSP-1 binds to CD47, phagocytosis in macrophages is increased.
  
  
  
  The 2A peptide system was used to establish pig kidney cells (PK15) simultaneously expressing human CD47 and human TFPI, and they were cultured with activated THP-1 cells. After staining with 7-aminoactinomycin D, flow cytometry analysis was carried out. TFPI siRNA analysis and recombinant human TFPI (rhTFPI) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP-1 cells in the presence of CD47. Related inflammatory cytokines produced by activated THP-1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was analyzed using immunoprecipitation and Western blot.
  
  
  
  hCD47/hTFPI-PK15 cells survived better than hCD47-PK15, hTFPI-PK15, or normal PK15 cells on cytotoxicity tests using activated THP-1 cells. TSP-1, derived from these activated THP-1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells (PBMCs). The tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was further increased in the case of co-expression of CD47/TFPI than in individual non-expression or expression of CD47 or TFPI alone.
  
  
  
  When hCD47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP-α in activated THP-1 cells via hTSP-1 inhibition, and consequently, it might improve the effect of hCD47-SIRP-a signaling.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
authors.forename: Sung Han
authors.initials: SH
authors.lastname: Jung
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.|Korea Institute of Toxicology, Jeongeup-si, Jeollabuk-do, Korea.
authors.forename: Jeong Ho
authors.initials: JH
authors.lastname: Hwang
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
authors.forename: Sang Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
authors.forename: Kim
authors.initials: K
authors.lastname: Young Kyu
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
authors.forename: Hyo Chang
authors.initials: HC
authors.lastname: Park
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
authors.forename: Hoon Taek
authors.identifier: 0000-0002-7677-493X
authors.initials: HT
authors.lastname: Lee
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism; D051928:CD47 Antigen / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control; D006801:Humans; D008074:Lipoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology*; D010587:Phagocytosis; D034741:RNA, Small Interfering / Q000235:genetics; D011971:Receptors, Immunologic / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D013552:Swine; D019700:Thrombospondin 1 / Q000276:immunology; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D000943:Antigens, Differentiation; D051928:CD47 Antigen; C497894:CD47 protein, human; D016207:Cytokines; D008074:Lipoproteins; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C501920:SIRPA protein, human; D019700:Thrombospondin 1; C051928:lipoprotein-associated coagulation inhibitor; C000611726:thrombospondin-1, human
keywords: CD47; macrophages; synergistic effect; thrombospondin 1; tissue factor pathway inhibitor