Introduction Corneal xenotransplantation from pigs to non-human primates (NHPs) has shown promising long-term graft survival, but immune responses to xenogeneic antigens are still stronger than those to allogeneic antigens, even with immunosuppression. Thus, it is important to investigate major rejection pathways in corneal xenotransplantation. Although the alterations of proteomic pathways in allotransplant rejection have been investigated, to date, no analysis of such pathways in xenotransplant rejection has been reported. Therefore, this study aimed to investigate interactive immune pathways and their evolution with the progression of rejection by analyzing proteomics changes in rejected or rejection-ongoing corneal xenografts and aqueous humors in pig-to-NHP corneal xenotransplantation, even with immunosuppressive agents.

Methods Wild-type (WT) or α-1,3-galactosyltransferase gene-knockout (GTKO) porcine corneas were used as donor corneas. The NHPs with porcine corneal transplantation were divided into three to four groups depending on the permanent rejection, rejection-ongoing, survival, or donor types. The proteomes of corneal tissues and aqueous humor were analyzed with liquid chromatography-mass spectrometry. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathways and protein–protein interaction network analysis were applied to estimate the biological functions of differentially expressed proteins (DEPs).

Results Among the 416 DEPs identified in the cornea, NF-κB1 and proteosomes (PSMD7, PSMA5, and PSMD3) were found to be related to xenogeneic graft rejection. In addition, oxidative phosphorylation and leukocyte activation-related pathways were found to play a role in rejection. Among the 66 DEPs identified in the aqueous humor, complement proteins (C3, C5, and C9) and cholesterol metabolic proteins (APOA1 and APOA2) were revealed to be related with xenogeneic rejection as biomarkers. Alternative pathways of the aqueous complement system were identified to be important in graft rejected or rejection-ongoing subjects. In the subgroup analysis, a few DEPs between WT and GTKO grafted groups were found, and Ig-like domain-containing proteins were the common DEPs.

Conclusion This analysis suggests that the key roles of NF-κB1, proteosomes, oxidative phosphorylation, and leukocyte activation-related inflammation in the graft, and the clinical relevance of aqueous complement pathways as a predictive biomarker of xenogeneic rejection in pig-to-NHP corneal xenotransplantation.