Introduction: There are three types of HO: HO-1, -2, and -3, and HO-3 is presumed to be a pseudogene. HO-2 is constitutively expressed at low concentrations, whereas HO-1 is induced by various stresses, including oxidative stress. HO-1 breaks down heme into bilirubin, CO, and Fe2+. CO has anti-inflammatory effects. Therefore, human HO-1 (hHO-1) is thought to be effective for maintaining xenografts and has already been introduced into many gene-edited pigs, but most of the reports so far have been fragmentary analyses. In this study, to compare the functions of hHO-1 and sus scrofa HO-1 (sHO-1) in pig cells, we introduced hHO-1 or sHO-1 genes into swine endothelial cells (SECs) and compared their anti-apoptotic functions against attacks by the human innate immune system.
Methods: First, the sequence differences between the two HO-1 clones were examined. Next, cDNAs of the sas and homozygous HO-1 genes were prepared, linked to the green fluorescent protein (GFP) gene using the 2A system, and cloned into the expression vector pCXN2 (chicken β-actin promoter). These genes were introduced into SECs to establish each clone. The results were also confirmed by flow cytometry. First, the changes in α-Gal expression and overall antigenic changes of each HO-1 clone were examined using GSI-B4 and NHS, respectively. 
Next, normal human pooled serum (NHS), THP-1 (monocyte/macrophage line), peripheral blood neutrophils, and peripheral blood mononuclear cells (PBMCs) were prepared, and the inhibitory function in the cytotoxic activity of the established HO-1 clones was evaluated in comparison with naive SECs. NHS were used for complement-dependent cell lysis. NK cytotoxicity assays were performed using PBMCs co-cultured for 4 hours.
Results: The difference in the amino acid sequence between the two was 17.36%, showing high homology. As for clones, sus and homo HO-1 clones were established. In both HO-1 clones, almost no change was observed in α-Gal expression and antigenicity.     
First, neither HO-1 clone showed a protective effect against human serum, i.e., complement attack. On the other hand, both sHO-1 and hHO-1 showed significantly reduced THP-1-mediated cytotoxicity compared to naive SEC (naive: 67.0±4.8, sus: 53.7±2.2, homo: 51.7±6.1). Next, in the neutrophil-mediated cytotoxicity, both clones were significantly less cytotoxic than naive SECs in the 4-hour PMA-treated (naive: 26.5±2.5, sus: 18.0±1.8, homo: 16.7±3.3) and 24-hour PMA-free (naive: 42.4±4.5, sus: 26.7±3.8, homo: 19.2±2.5) experiments, but no significant difference was observed between the two. In the NK cell-mediated cytotoxicity, both HO-1 cell lines also suppressed cell death compared to naive SECs.
Conclusions: These results suggest that although neither sHO-1 nor hHO-1 was resistant to complement attack, they were significantly resistant to cytotoxicity by human innate immune cells.
We are now investigating the changes in endogenous sHO-1 and both introduced HO-1 mRNA in these assays.