Liver transplantation is a proven treatment for end-stage liver disease. However, TCMR substantially risks graft failure and elevates patient mortality, highlighting the imperative for investigating its molecular determinants 13. Such inquiries are fundamental to enhancing early detection and therapeutic interventions. While the original study utilized the dataset to develop a diagnostic system for rejection, this study delves deeper into the molecular mechanisms of TCMR and identifies potential drug targets, thus providing a valuable complement to the original research 14.
In the initial phase of our research, We identified DEGs between the TCMR and NR groups from the GSE145780 dataset and combined a WGCNA network to select LTR-DEGs. GO and KEGG enrichment analyses connected these LTR-DEGs with immune functions such as cytokine activation and antigen processing. Following this, we constructed a PPI network and used 4 algorithms to further identify the candidate genes. Machine learning further validated the reliability of our findings, pinpointing five key genes (ITGB2, FCER1G, IL-18, GBP1, and CD53) linked to TCMR. Further interrogation using scRNA-seq confirmed that the five key genes were upregulated in both T cells and myeloid cells within the TCMR group.
ITGB2/CD18 encodes an integrin beta chain protein, which is specifically expressed by leukocytes and can form corresponding β2 integrin heterodimers with four different known alpha chains, participating in cell adhesion and cell surface-mediated signal transduction 15. Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18), as members of the β2 integrin family, play critical roles in mediating leukocyte adhesion to target structures and other immune cells 16. Research found that their interaction with ICAM-1 facilitates adhesion, crucial for lymphocyte regulation and inflammation control during early liver transplant rejection 17; this finding is consistent with our research results. Additionally, in mucosal biopsies from lung transplant recipients, ITGB2 has been identified as a key gene associated with lung transplant rejection18. And in a rabbit model of heterotopic heart transplantation, antibody therapy targeting ITGB2 has shown significant efficacy in preventing transplant rejection19.
Leukocyte surface antigen CD53, a member of the tetraspanin family, is a transmembrane signaling and pro-inflammatory protein widely expressed in various types of immune cells 20,21. Literature reports that CD53 can induce homotypic cell adhesion by activating β2 integrin LFA-1 in NK, T, and B cells, promoting adhesion and migration of immune cells 22,23. Additionally, studies have shown that CD53 stimulation enhances T cell proliferation. It drives the transition of naive T cells to effector/memory phenotypes. These effects have been observed in vitro in human T cells and in genetically modified mice. Further investigation revealed CD53's role in regulating migration rate and stability of CD45RO on T cell surfaces. This regulation impacts TCR signal transduction. The absence of CD53 results in a loss of CD45RO expression on T cells. Consequently, it alters the expression of CD45 isoforms and diminishes T cell activation 24. In a transcriptomic analysis of post-kidney transplant recipients, CD53 was identified as a biomarker for acute rejection in kidney transplantation25.
FCER1G encodes the γ subunit of the high-affinity immunoglobulin E (IgE) Fc receptor, widely expressed in various types of immune cells. The FCER1G gene is involved in various biological processes, including neutrophil activation, T cell differentiation, immunoglobulin-mediated immune responses, and Fc receptor-mediated signaling pathways 26–28. Similar to this study, FCER1G has been proven to be an upregulated gene highly representative of acute rejection in heart, liver, and kidney transplants in a transcriptome data analysis of various solid organ transplants 29.
GBP1 is a GTPase of the dynamin superfamily, involved in the regulation of membrane, cytoskeleton, and cell cycle progression dynamics 30. Furthermore, GBP1 is key to host cell immunity and antibacterial protection, recognizing infection and inhibiting bacterial proliferation by activating inflammasomes and regulating pyroptosis 31. GBP1 is highly expressed in macrophages, endothelial cells, and epithelial cells, with continued high expression levels in T cells after IFN-γ stimulation induced by interferon-γ (IFN-γ) 32. In several studies of renal transplant rejection, researchers have confirmed through transcriptomic analysis and tissue biopsies that GBP1 may serve as a biological characteristic and predictive model for acute rejection33–35. Recent research have found that GBP1 binds to lipopolysaccharide (LPS) during bacterial infection, mediating the recruitment and activation of inflammatory caspase-4 through cleavage of GSDMD to induce pyroptosis 36, 37.
IL-18 is a member of the IL-1 family of pro-inflammatory cytokines, originally identified in the cytoplasm of macrophages as an inactive precursor (pro-IL-18). This precursor is transformed into an active mature form by proteolytic cleavage 38. Recent proteomic studies have revealed IL-18 plays a significant biological role in regulating innate and adaptive immunity 39. It also effectively induces IFN-γ production, exerting various immunoregulatory functions in the presence of different cytokines. These functions include regulating the transformation of T cells to regulatory T cells, modulating Th1 and Th2 responses, and participating in Th17 responses 40–42. It has been documented in existing literature that IL-18 plays a significant role in the rejection reactions of various solid organs. In a rat orthotopic liver transplantation model, blocking the binding of IL-18 to its receptor significantly alleviated graft rejection43. Elevated levels of IL-18 have also been observed in acute rejection reactions following kidney and heart transplantation44,45.Historically, the maturation of IL-18 has been ascribed primarily to Caspase-1-mediated cleavage within the NLRP3 inflammasome complex 46,47. However, recent studies indicate that in Caspase-4, as part of the non-canonical pyroptosis pathway, can recognize and cleave the same site on pro-IL-18, leading to its activation. This interaction establishes a Caspase-4–IL-18 axis that links non-canonical pyroptosis to adaptive immunity 48. Our findings lend support to the hypothesis that both GBP1 and IL-18 levels increase substantially in liver tissues following TCMR. This suggests that post-transplantation dysbiosis and the aberrant translocation of lipopolysaccharide (LPS) into the liver through the gut-liver axis might provoke non-canonical pyroptotic pathways in liver macrophages and other cells, mediated by GBP1. This cascade potentially triggers TCMR via cytokine release subsequent to cell death.
TCMR is characterized by a complex, multifactorial immune response, with extant experimental research underscoring the pivotal role of various immune cells in this process 49. In our study, immune infiltration analysis of two groups of data revealed a significant increase in the majority of immune cells in TCMR tissues, with scRNA-seq clustering analysis further showing the most significant upregulation in T and myeloid cells. Additionally, correlation analysis of the five selected key genes with 28 types of immune cells showed strong correlations with T cells, B cells, myeloid cells, etc. scRNA-seq revealed that the expression of the five key genes in the TCMR group was significantly increased in T and myeloid cell clusters compared to the NR group. Integrating bulk RNA-seq and scRNA-seq analyses, these key genes appear to be pivotal in immune activation during TCMR, significantly influencing antigen presentation and lymphocyte activation between Antigen-Presenting Cell (APC) and T cells.
To deepen the understanding of the specific mechanisms of TCMR, we explored differences in miRNA and lncRNA levels based on mRNA differences and constructed an entire ceRNA network, which may be involved in important biological pathways related to TCMR. Currently, some studies have found that these miRNAs affect the progression of certain immune-related diseases by regulating corresponding mRNAs. This holds important guiding significance for transplant rejection, which also involves aberrant activation and dysfunction of immune cells. Research has found that miR-346 expression is present in synovial cells activated by LPS induction of pattern recognition receptors (PRRs) in rheumatoid arthritis patients. This miRNA negatively regulates the IL-18 response in fibroblast-like synoviocytes by inhibiting the transcription of Bruton's tyrosine kinase. This mechanism has been validated in THP-1 cells, demonstrating miR-346's inhibitory effect on IL-1850. Researchers have found through dual-luciferase reporter analysis that miR-130a negatively regulates IL-18, thereby participating in the regulation of primary immune thrombocytopenia51. The miR-124-3p, which regulates GBP1, acts as a suppressor in various cancers and also plays a role in cell apoptosis and inflammatory lesions52. This approach potentially offers a new perspective for researching TCMR mechanisms, discovering novel therapeutic targets, and developing biomarkers. Moreover, drug prediction for the five key genes identified 26 therapeutic drugs corresponding to three key genes, with tacrolimus, mycophenolate mofetil, methylprednisolone, and cyclosporine already widely used in the treatment of liver transplant rejection 49,53,54. Previous research has confirmed that colchicine inhibits T cell proliferation by blocking the expression of the IL-2R gene, damaging the antigen recognition process 55. Lifitegrast, an integrin antagonist, has been proven in dry eye disease research to block the antigen transfer process from APC to T cells 56. These targeted drugs may provide new insights for the prevention and treatment of TCMR after liver transplantation.
Finally, immunohistochemistry was used to validate the upregulated expression of key genes in human liver biopsy tissues. The significant upregulation of four key genes: GBP1, IL-18, CD53, FCER1G, in the TCMR group further supports their potential as predictive indicators and therapeutic targets for liver transplant TCMR. This finding also provides a theoretical basis for further exploration of their molecular mechanisms. However, we acknowledge that our study has limitations, primarily due to a scarcity of post-transplant liver biopsy samples, which has led to insufficient validation. Our data were derived from public databases, which may introduce an element of heterogeneity. Consequently, the specific functions and underlying mechanisms of key genes in liver transplant rejection require further investigation. The predictions for miRNA, lncRNA, and targeted drugs are also based on public databases and have not been experimentally validated. To address this, we plan to conduct additional experimental and clinical research in subsequent studies. An increase in sample size may improve the reliability and significance of the results. Validation of the current study using data from only three patients and three controls may limit the ability to generalize the results. We will improve this in subsequent studies.