Lung transplantation (LTx) is the only viable treatment for end-stage lung disease (Chen-Yoshikawa et al., 2020). However, due to early primary graft dysfunction (PGD) and late chronic lung allograft dysfunction (CLAD) post-LTx, the 1- and 5-year survival rates of recipients are only 85% and 59%, respectively (Chambers et al., 2017; Chambers et al., 2021). PGD is not only the leading cause of mortality in the perioperative period but also a risk factor for CLAD (Chacon-Alberty et al., 2023). PGD arises predominantly from allograft lung ischemia-reperfusion injury (ALIRI), which can promote chronic rejection and drive CLAD (Watanabe et al., 2019). Therefore, exploring the mechanism by which ALIRI reduces the morbidity of PGD and CLAD remains a key research focus. Allograft lungs are inevitably subject to ALIRI during organ harvesting, cold preservation, and transplantation. Recent studies have shown that cell death and inflammatory responses are the two major mechanisms underlying ALIRI (Wong and Liu, 2021; Capuzzimati et al., 2022).

Cuproptosis, a new form of programmed cell death (PCD), differs from previously reported forms of cell death, such as apoptosis, ferroptosis, and necroptosis. In this process, intracellular copper-ion overload can target and bind to lipoylated components of the tricarboxylic acid (TCA) cycle, resulting in the aggregation of lipoacylated proteins and the loss of iron-sulfur cluster proteins, leading to proteotoxic stress and, ultimately, cell death (Tsvetkov et al., 2022). Researchers have also identified cuproptosis-related genes (CRGs) (Tsvetkov et al., 2022). Previous research has shown that a Cu imbalance is associated with a wide range of pathological conditions, including neurodegenerative diseases, cancer, and cardiovascular disease (Chen et al., 2022). An earlier study found that increasing the cardiac copper content can enhance ischemic reperfusion injury (IRI) (Powell et al., 1991). However, a recent study also demonstrated that Cu may have protective effects against IRI in the heart and lungs of rats (Tural et al., 2020). These findings demonstrate the important role of copper in IRI; however, its effects and underlying mechanisms need to be explored further. As a newly identified form of PCD, the role of cuproptosis in the pathogenesis of ALIRI and the immune response against ALIRI remains unclear, and its potential as a therapeutic target for ALIRI is yet to be evaluated. Therefore, it is necessary to further explore CRGs that are associated with ALIRI, which may act as new targets for treating ALIRI.

To explore the role of cuproptosis in the pathogenesis of ALIRI and the immune response against ALIRI, we used the Gene Expression Omnibus (GEO) database to analyze the differentially expressed CRGs (DE-CRGs) between pre- and post-LTx human allograft lung samples. Cuproptosis-related biomarkers were screened from DE-CRGs using three machine learning (ML) algorithms, including the least absolute shrinkage and selection operator (LASSO), support vector machine recursive feature elimination (SVM-RFE), and random forest (RF) algorithms. We investigated the infiltrating immune cells in the datasets and their correlation with these biomarkers. A competing endogenous RNA (ceRNA) network was constructed using these biomarkers. Finally, the biomarkers were verified using another independent dataset, and quantitative real-time PCR (qRT-PCR) and Western blotting (WB) in allograft lung tissues from a rat LTx model. This study is the first to investigate the role of cuproptosis in ALIRI, which may provide more insights into the role of cuproptosis in ALIRI, along with the diagnosis and treatment of ALIRI. This study was conducted in accordance with the Declaration of Helsinki (revised in 2013). The workflow of this study is illustrated in Supplementary Figure S1.

In lung transplants, ALIRI is inevitable during organ ischemia, followed by reperfusion. Previous studies have reported cell death to be one of the major mechanisms underlying ALIRI (Capuzzimati et al., 2022). Cuproptosis, a novel type of cell death, is characterized by intracellular free copper overload and lipoylated protein aggregation, leading to cytotoxic stress and cell death (Tsvetkov et al., 2022). However, the role of cuproptosis in ALIRI has not yet been investigated. In the present study, four cuproptosis-related biomarker genes (NFE2L2, NLRP3, LIPT1, and MTF1) were identified in the datasets and verified in a rat LTx model. Correlation analysis between these biomarkers and immune infiltration was performed using CIBERSORT. ROC curves and LRM analysis revealed that these biomarkers had excellent diagnostic efficiency for ALIRI. Single-gene GSEA indicated that these biomarkers were closely related to pathways involved in cell death, immune-related responses, and signal transduction and interactions.

Fifteen DE-CRGs were identified in the post- and pre-LTx samples. After 2 h of reperfusion, NFE2L2, NLRP3, FDX1, MTF1, and GLS were upregulated, whereas ATP7A, ATP7B, LIAS, LIPT1, DLD, DLAT, PDHA1, PDHB, DBT, and GCSH were downregulated. FDX1, a mitochondrial matrix enzyme, is a key regulator of copper ionophore-induced cell death, and the deletion of FDX1 confers cellular resistance to cuproptosis (Tsvetkov et al., 2022). The upregulation of FDX1 enzymatic activity can facilitate elesclomol cytotoxicity by reducing Cu²⁺ to Cu¹⁺, resulting in a unique copper-dependent cell death (Tsvetkov et al., 2019). ATP7A and ATP7B are Cu¹⁺-transporting ATPases that can translocate Cu⁺ to the extracellular medium when intracellular copper levels are elevated (Tadini-Buoninsegni and Smeazzetto, 2017). In this study, we found that the FDX1 gene was upregulated and ATP7A/B was downregulated after reperfusion, which may result in intracellular free-copper accumulation, leading to cuproptosis. GO analysis indicated that these DE-CRGs were significantly enriched in protein lipoylation, the TCA cycle, pyruvate dehydrogenase (NAD+) activity, and copper-transporting ATPase activity. After KEGG analysis, we found that DE-CRGs were enriched in the TCA cycle. These biological processes are characteristic of cuproptosis and are involved in the pathophysiological mechanisms of ALIRI.

NFE2L2, also known as nuclear factor erythroid2-related factor 2 (Nrf2), is a master regulator of the redox balance (Bellezza et al., 2018). In IRI, accumulated reactive oxygen species (ROS) result in reduced Nrf2 degradation, leading to Nrf2 upregulation, which strengthens the activity of antioxidant enzymes to reduce ROS production, prevent cell death, and reduce inflammation by decreasing pro-inflammatory factors (Sadrkhanloo et al., 2022). Dong et al. (2020) found that Nrf2 inhibits ferroptosis and protects against acute lung injury (ALI) by regulating SLC7A11 and heme oxygenase-1 (HO-1) (Dong et al., 2020). In addition, Nrf2 can alleviate ALI and inflammation by modulating TLR4 and Akt signaling (Yan et al., 2018). A recent study demonstrated that copper-induced ROS can promote lipid peroxidation, reduce antioxidant enzyme activity, and activate the Nrf2/HO-1 signaling pathway in hippocampal neurons (Lu et al., 2022). A high Cu intake can lead to oxidative damage and activate the Nrf2/HO-1-mediated antioxidant pathway in the porcine myocardium (Li et al., 2022). However, some studies have also shown that administering an optimal level of copper ions has ameliorative effects against IRI in the spinal cord and liver (Tural et al., 2021; Đurašević et al., 2021). An optimal concentration of Cu²⁺ can reduce tissue damage during IR, whereas copper-ion overload can induce cell death (Chen et al., 2023). This difference may be attributed to a difference in tissues. In this study, we found that the expression of NFE2L2 was upregulated in the post-LTx group using dataset analysis, which was verified by WB and qRT-PCR in a rat LTx model. Recently, Zhao et al. also reported that both the RNA and protein expression of NFE2L2 are increased in human and mouse ALIRI samples after LTx, which further verifies our findings (Zhao et al., 2023). However, a direct relationship between over-expressed NFE2L2 and cuproptosis has not been reported. Therefore, we speculate that Cu accumulation induced by IR may further lead to oxidative damage and activate the Nrf2/HO-1-mediated antioxidant pathway. In summary, activated NFE2L2 can protect against IRI by inhibiting ferroptosis, inflammation, and possibly cuproptosis; however, further investigation is required to confirm this.

NLRP3 (NOD-like receptor thermal protein domain-associated protein 3), an intracellular signaling molecule, is a member of the NOD-like receptor family, and its inflammasome complex consists of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and pro-cysteinyl aspartate-specific proteinase-1 (pro-caspase-1) (Huang et al., 2021). NLRP3 is activated by a variety of pathogen- and host-derived chemical factors through complex mechanisms (Xue et al., 2019). NLRP3 is upregulated and activated in murine lung IR, and the selective inhibition of the NLRP3 inflammasome attenuates IRI (Xu et al., 2018). Additionally, rat LTx research has shown that the NLRP3 protein is increased in the grafted lung after 2 h of reperfusion, and ozone can protect against ALIRI by attenuating NLRP3-mediated inflammation and enhancing Nrf2 antioxidant activity (Zheng et al., 2021). Furthermore, NLRP3 knockout not only reduces cerebral IR injury but also alleviates the severity of cerebral IR-induced lung injury (Xu et al., 2022). Some substances, such as dexmedetomidine and corilagin, can alleviate IR-induced lung injury by inhibiting NLRP3 inflammasome activation (Chen et al., 2020; Li et al., 2022). Recent studies have shown that activation of the NLRP3 inflammasome contributes to several types of cell death, including pyroptosis, apoptosis, necroptosis, and ferroptosis (Huang et al., 2021). However, there have been no reports on the relationship between NLRP3 and cuproptosis in patients with IRI. Deigendesch et al. found that Cu is required for NLRP3 inflammasome activation and that depletion of bioavailable Cu can reduce inflammasome activation (Deigendesch et al., 2018). In our study, the protein and mRNA levels of NLRP3 were overexpressed in both the human dataset and the rat LTx experiment, and this gene was selected as a biomarker for ALIRI. Controlling Cu ions and targeting NLRP3 may be an optional therapy for ALIRI.

Acyltransferase 1, encoded by LIPT1, transfers lipoic acid to the E2 subunit of α-ketoglutarate dehydrogenase (Mayr et al., 2014). Thus, a deficiency of the LIPT1 homolog can lead to reduced lipoylation of E2 subunits (Lv et al., 2022). Lipoic acid is an essential co-factor for α-ketoglutarate dehydrogenase and the glycine cleavage system in mitochondria, which is closely related to the TCA cycle, and LIPT1 deficiency can suppress the TCA cycle (Fan et al., 2022; Solmonson et al., 2022). LIPT1 supports lipogenesis and balances oxidative and reductive glutamine metabolism (Ni et al., 2019). Our study revealed that the protein and mRNA expression levels of LIPT1 were lower in the post-LTx group than in the pre-LTx group via dataset analysis and animal experiments, which is consistent with the findings in a cerebral infarction model (Fan et al., 2022). Single-gene GSEA indicated that 13 enrichment pathways were activated in the low-expression LIPT1 group and were involved in the cell cycle, cell metabolism, and signal transduction and interaction. Therefore, we hypothesized that in ALIRI, downregulated LIPT1 could restrain the TCA cycle and result in imbalanced oxidative and reductive glutamine metabolism, leading to cell death. Cuproptosis occurs through the direct binding of copper to the lipoylated components of the TCA cycle (Tsvetkov et al., 2022). However, the regulatory mechanism of LIPT1 in cuproptosis requires further investigation.

MTF1 (metal-regulatory transcription factor 1) is a conserved metal-binding transcription factor that can bind metal-responsive elements (MREs) to drive the transcription of target genes that maintain metal homeostasis, including Zn and copper homeostasis (Chen et al., 2020). When the intracellular copper ion concentration is elevated, MTF1 activates metallothioneins to protect the cell, while the transporter ATP7 removes excess copper from the cell by changing its subcellular localization. Under low copper conditions, MTF-1 activates the copper importer Ctr1B, and copper is taken up (Balamurugan and Schaffner, 2006). MTF1 can also regulate ATP7B expression by binding to the MRE of the ATP7B promoter (Stalke et al., 2020), which may help regulate the intracellular Cu balance. A recent study showed that Cu recruits MTF-1 to the MRE of the Nrf2 promoter and promotes Nrf2 over-expression (Zhong et al., 2023). In our study, the expression of NFE2L2 (Nrf2) and MTF1 was upregulated post-LTx, and NFE2L2 had a strong positive correlation with MTF1 (r = 0.5). Therefore, we speculate that Cu could also promote NFE2L2 upregulation via MTF1 binding to the MRE site of the Nrf2 promoter in ALIRI. In summary, MTF1 may be a protective factor that maintains the intracellular copper ion balance, induces anti-oxidative damage, and prevents cell death by upregulating NFE2L2.

Based on differences in immune infiltration, we found that in post-LTx patients, neutrophils, activated MCs, resting NK cells, and activated DCs increased, but the infiltration of plasma cells, resting memory CD4 T cells, M2 macrophages, eosinophils, and resting MCs decreased. Neutrophil infiltration is a hallmark of ALIRI and promotes allograft injury (Scozzi et al., 2017). In mouse LTx, neutrophils can be rapidly recruited to the allograft depending on the number of monocytes after reperfusion (Kreisel et al., 2010), and TLR4 expression in vascular endothelial cells regulates neutrophil recruitment after IRI (Li et al., 2022). Activated MCs can release a number of mediators, including interleukin (IL)-2, IL-7, IL-3, IL-6, IL-9, IL-10, tumor necrosis factor (TNF)-α, and chemokines, which are involved in graft injury and rejection (Mortaz et al., 2018). In mouse lungs, MCs can promote IRI by increasing the production of prostaglandin D2, TNF-a, and IL-6, which, in turn, is responsible for recruiting neutrophils (Wingard et al., 2011). Using MC stabilizers, lung IRI can be improved by inhibiting the expression of MC-derived ICAM-1 (Vural et al., 2000). Therefore, activated MCs may serve as a therapeutic target in ALIRI. A previous study indicated that NK cells were increased in the lung tissue of an IRI mouse model and seven clinical LTx patients with PGD, and IRI was attenuated in NK cell-deficient mice but restored after the adoptive transfer of NK cells (Calabrese et al., 2021). After LTx, most macrophages in the graft are donor-derived (Kopecky et al., 2020). Macrophages play a fundamental role in ALIRI and are mainly activated during cold preservation and the early (30 min) phases of reperfusion injury, and contribute to local and subsequent inflammation via the induction of cytokines (Fiser et al., 2001; Kopecky et al., 2020). Under different stimuli, macrophages can be polarized into M1 (classical) and M2 (alternative) phenotypes; the former are responsible for pro-inflammatory reactions, whereas the latter play an important role in anti-inflammatory reactions (Sica and Mantovani, 2012). In ALI, the M1 phenotype is involved in the acute phase, whereas M2 is mainly related to the recovery phase of inflammation (Johnston et al., 2012), which is consistent with our finding suggesting that the M2 phenotype was significantly lower after 2 h of reperfusion. We also found that M2 macrophages were negatively correlated with NLRP3 (r = −0.31). M2 macrophage-derived exosomes can mitigate myocardial IRI by inactivating the TLR4/NF-κB/NLRP3 inflammasome signaling pathway (Dai et al., 2020). Therefore, we speculate that the upregulation of M2 macrophages may improve ALIRI by inhibiting the NLRP3 inflammasome, and this may provide new directions for research on the treatment of ALIRI. However, some studies also discovered that M2 macrophages may promote the differentiation of lung fibroblasts and cause pulmonary fibrosis; however, the underlying mechanism was found to be complicated and unclear (Cheng et al., 2021). Thus, the role of macrophages in ALIRI needs to be further explored. A rat LTx study discovered that recipient T cells were activated and infiltrated the graft lung rapidly during the early phase of reperfusion (de Perrot et al., 2003). In contrast, in our analysis, we found that the proportion of resting memory CD4 T cells was reduced, and no obvious changes were observed in other types of T cells, which may be attributed to the extensive use of immunosuppressive agents during the perioperative period. In addition, the correlation analysis of immune cells and biomarkers revealed that MTF1 had a positive correlation with activated MCs (r = 0.35) and a negative correlation with resting memory CD4 T cells (r = −0.37) and M2 (−0.34); however, the underlying mechanism needs to be further explored. We also found that NFE2L2 had a positive correlation with activated memory CD4 T cells (r = 0.40). Zagorski et al. demonstrated that NFE2L2 activation inhibits the secretion of the Th1 cytokine IFNγ and promotes Th2 differentiation, thereby initiating an anti-inflammatory response (Zagorski et al., 2018). Furthermore, Noel et al. discovered that T-cell-specific activation of NFE2L2 can protect from IR-induced AKI in a mouse model (Noel et al., 2015). Therefore, we hypothesize that the upregulation of NFE2L2 may direct memory CD4 T cells toward Th2 differentiation and induce an anti-inflammatory response in ALIRI. This may be another mechanism through which NFE2L2 protects against ALIRI. Although most of the aforementioned studies were conducted in animals, our findings were confirmed in clinical LTx patients.

Finally, we analyzed the ceRNA network of biomarker genes. miRNAs play important roles in the development of IRI (Kura et al., 2020). In lung IRI, miR-18a-5p regulates cellular ROS levels and affects the nuclear localization of NFE2L2 into the nucleus (Xiao et al., 2021). Ding et al. demonstrated that the inhibition of miR-29a can ameliorate myocardial IRI by reducing oxidative stress and NLRP3-mediated pyroptosis (Ding et al., 2020). MiR-148a, derived from exosomes of M2 macrophages, can also improve myocardial IRI by inactivating the TLR4/NF-κB/NLRP3 inflammasome signaling pathway (Dai et al., 2020). In our study, five lncRNAs regulated the expression MTF1 by competitively binding with hsa-miR-148a-3p. However, whether our predicted non-coding RNA plays a role in ALIRI is unclear and requires further experimental verification.

To the best of our knowledge, this is the first study to investigate the role of cuproptosis in ALIRI. Similar to other studies, this study has some limitations. First, the number of transcriptome datasets used was limited. In this study, we analyzed only one training dataset and one validation set. Second, the datasets were derived from human samples but were validated with rat samples, and it might be more beneficial to confirm this using clinical samples in the future.

Fifteen DE-CRGs and four biomarker genes (NFE2L2, NLRP3, LIPT1, and MTF1) identified via bioinformatic analysis and three ML algorithms were closely associated with the pathogenesis of cuproptosis in ALIRI. ROC curves and LRM analysis revealed that these biomarkers exhibited excellent diagnostic efficiency for ALIRI and may serve as predictors of cuproptosis. Subsequent analysis of these genes helped uncover the potential biological pathways and mechanisms of cuproptosis in ALIRI. We performed a differential analysis of immune cell infiltration between post- and pre-LTx samples, followed by correlation analysis between biomarkers and 22 immune cells to obtain DICs. These results provide a certain reference value for basic research on cuproptosis and immune cell infiltration in ALIRI.