TRANSCRIPTOMIC ANALYSIS OF XENOGENEIC IMMUNE REJECTION IN HYDRA

To investigate the molecular mechanisms of xenogeneic immune rejection in Hydra, we developed a novel grafting method using elongated conical toothbrush bristles as carriers to connect two polyp body segments. Successful chimeras were generated through homografts (between head-removed aposymbiotic and tail-removed symbiotic strain of Hydra sinensis) and xenografts (between head-removed Hydra vulgaris and tail-removed symbiotic strain of Hydra sinensis). Post-transplantation monitoring revealed significant immune rejection in xenograft chimeras: over 60% maintained structural integrity at 12h, ~40% at 24h, <20% at 36h, and none by 48h. Contrarily, at 96h post-transplant surgery, ~85% of homograft chimeras achieved complete tissue fusion at graft sites and survived long-term, indicating minimal immune rejection. Transcriptome analysis comparing xenograft with homograft chimeras at 24h post-operation identified 24125 differentially expressed genes (DEGs: 17707 upregulated, 6418 downregulated). KEGG enrichment analysis revealed 26 significantly enriched pathways (P<0.05), including 3 innate immunity pathways, 2 ferroptosis pathways, 2 apoptosis pathways, and 15 metabolic pathways related to immune cell metabolic reprogramming. Among 33 representative DEGs directly associated with innate immunity, we identified 12 immune response genes, 4 apoptosis-related genes, 6 protein ubiquitination genes, 4 protein folding genes, 4 signal transduction genes, and 3 material transport genes. Integrated analysis delineates the molecular roadmap of xenogeneic immune rejection in Hydra: Xenotransplantation activates innate immunity, ferroptosis, and apoptosis, with the latter two processes modulating the intensity of innate immune responses. Activated innate immunity triggers metabolic reprogramming in immune cells and initiates antigen processing and presentation, suggesting activation of a primordial acquired immune mechanism. However, no evidence supported direct involvement of an acquired immune system in xenogeneic rejection. Thus, xenogeneic immune rejection in Hydra primarily results from innate immune responses and metabolic reprogramming of immune cells. This study provides fundamental data for understanding cellular and molecular mechanisms of xenogeneic immune rejection in animals.