The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Soft/elastic nanopatterned biointerfaces in the service of cell biology.

Engineering of biomimetic interfaces has become a valuable tool for guiding cellular processes such as adhesion, spreading, motility, as well as proliferation, differentiation, and apoptosis. The interaction of cells with the extracellular matrix (ECM) or with other cells is involved in nearly every cellular response in vivo. Recent wide-ranging evidence shows that crosstalk between different environmental stimuli can have a tremendous impact on various cell functions. Therefore, the defined control of these stimuli in vitro can contribute to the understanding of the mechanisms underlying the ability of cells to perform "intelligent" missions like acquiring, processing, and responding to environmental information. This chapter summarizes recently developed nanopatterned biomimetic systems that allow independent control of different stimuli and illustrates their applications in cellular studies. Particular attention is devoted to nanopatterned 2D and 3D artificial ECM systems based on poly(ethylene glycol) materials. These allow independent control over the material elasticity and the nanoscale distribution of bioligands on the surface. In the case of engineering artificial cellular interfaces, additional attention has to be devoted to the critical functions of protein transport regulators, namely the cell membrane and the dynamic actin cytoskeleton; both are essential for the signaling activity of individual proteins and the entire cell.