Objective: Xenotransplantation represents a promising solution to the current shortage of organ donors.  A notable instance involved pig-to-human heart xenotransplantation, where the transplant recipient successfully avoided hyperacute rejection.  Recent research underscores the significance of the innate immune response in xenotransplant rejection.  Surfactant protein-A (SP-A), a regulator of innate immunity, interacts with signal regulatory protein α (SIRPα), initiating suppressive signals via ITIM to deliver negative signals.  In this study, we aimed to create a novel molecule, a membrane-bound human SP-A, capable of suppressing not only macrophage but also neutrophil functions.  We investigated its inhibitory effects on xenograft rejection.
Methods: The cDNA for the CRD (carbohydrate recognition domain) of SP-A was switched to that of a membrane-type protein, collectin placenta 1 (CL-P1), with Flag-tag, and named the novel molecule CL-SP-A.  The gene was incorporated into an expression vector pCXN2 (b-actin promoter + CMV enhancer + neomycin resistance), and transfected into swine endothelial cells (SECs) to generate SECs expressing SP-A (SEC/CL-SP-A).  Naive SECs and SEC/CL-SP-A were then co-cultured with human macrophages and human neutrophils to evaluate the various suppressive functions of CL-SP-A.  The suppressive functions of CL-SP-A were assessed through cytotoxicity, phagocytosis, and PMA-induced NETosis assays using naive SEC as controls.  Additionally, reactive oxygen species (ROS) production was measured using CellROX Green.  Furthermore, the inhibitory effect of CL-SP-A on inflammatory cytokine production was evaluated using RT-PCR.
Result: As a initial experiment, we confirmed the expression of SIRPα on neutrophils from peripheral blood. Additionally, we evaluated the expression of CL-SP-A.  Xenogeneic cytotoxicity to SEC/CL-SP-A by human macrophages and neutrophils was significantly suppressed by CL-SP-A (p<0.05).  Furthermore, ROS production in macrophages and neutrophils was significantly inhibited by CL-SP-A (p<0.05).  CL-SP-A also significantly suppressed human macrophage function as demonstrated by phagocytosis assay (p<0.05). Additionally,  CL-SP-A suppressed PMA-induced NETosis assay (p<0.001).  Furthermore, significant suppression of TNF-α and IL-1β expression by CL-SP-A were observed in macrophages and neutrophils, co-cultured with SECs.
Conclusion: CL-SP-A suppressed human macrophage and neutrophil-mediated xenogeneic responses.  These results indicate that CL-SP-A is highly effective in xenogeneic rejection, suggesting that the development of transgenic pigs expressing CL-SP-A may improve outcomes in xenotransplantation.