Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

Background: Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes. Methods: Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Results: Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Conclusion: Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA.