Generation and characterization of regulatory macrophages for xenotransplantation

Abstract

The outstanding role of regulatory macrophages (Mregs) in promoting immunomodulation allowed the clinical application of Mregs-based therapies for controlling unwanted immune responses, especially in the field of transplantation. The major obstacle known to prevent pig-to-human xenotransplantation is the interaction between the human natural anti-Gal antibody and the -Gal epitope (Gal1-3Gal1-4GlcNAc-R). A promising strategy to eliminate the interaction between human immune cells and porcine transplanted organs is to generate Mregs, which is tolerant to -Gal epitope. We established a protocol to generate and characterize Mregs from human monocytes THP-1 cells, specifically resistant to porcine antigen -Gal. The cells were stimulated with a series of stimulants including GM-CSF, IFN-, dexamethasone, vitamin D3, and porcine-specific antigen -Gal. The generated Mregs displayed fiber-like morphology and high levels of CD16, CD163, CD206, Mer-TK, DHRS9, increased mitochondrial fusion and induced histone H3 lysine 4 monomethylation (H2K27me1). Importantly, Mregs showed suppressed inflammatory mediators gene expression and decreased tissue factor-coagulation signaling under co-culture with pig endothelial cells. Our results illustrate a feasible approach for generating functional Mregs which would be an effective and safe tool for pig-to-human xenotransplantation.

PDMS Micropatterns Coated with PDA and RGD Induce a Regulatory Macrophage-like Phenotype

“…As shown in Figure 6 A, cells grown on PDA+RGD-20/20 micropatterns exhibited the highest levels of DHRS9, whereas cells grown on PDA+RGD-30/20 micropatterns exhibited the second-highest levels of DHRS9. To compare the levels of this important Mreg stability marker between Mreg generated by our method and those generated by conventional methods, we generated Mreg using serial treatment with chemicals, such as granulocyte-macrophage colony-stimulating factor, interferon (IFN)-γ, dexamethasone, and vitamin D 3 [ 34 ]. The levels of DHRS9 were assessed by fluorescence microscopy ( Figure 6 B,C).…”

“…In a chemical-induced protocol, THP-1 cells were treated with 10 ng/mL granulocyte-macrophage colony-stimulating factor for 2 days, followed by treatment with 10 ng/mL interferon-γ for 2 days. The cells were then treated with 10 nM dexamethasone for 1 day prior to treatment with 10 nM Vitamin D 3 for 2 days [ 34 ]. In other groups, THP-1 cells were differentiated using 10 ng/mL PMA for 2 days and then seeded on micropatterns for 2 days.…”