To date, surgery has been the primary treatment option for TOF. However, there's an increasing trend of patients requiring subsequent surgeries, with some having a lifelong dependency on surgical intervention [23]. Therefore, it is important to explore effective biomarkers that can help explore better treatment options for TOF. Inflammation might be a factor that correlates heart failure in CHD with its complications. However, it has not yet received sufficient attention in clinical diagnosis or routine treatment.
With both LASSO and RF machine learning algorithms, five biomarkers associated with inflammation (CXCL6, F3, MARCO, SLC7A1, and SLC7A2) were obtained for the diagnosis of TOF. CXCL6, also known as granulocyte chemotactic protein-2, is primarily expressed in the lungs, heart, liver, pancreas, brain, kidneys, and placenta, and initiates chemotaxis by binding to chemokine receptors CXCR1 and CXCR2 [24]. Waehre et al. performed Affymetrix analysis of the right ventricle in mice with right ventricular pressure overload. The results showed upregulated expression of chemokines CXCL10, CXCL6, CX3CL1, CCL5, CXCL16, and CCL2, while CXCL16, CX3CL1, and CCL5 regulated SLRP expression in cardiac fibroblasts and post-translational modifications, suggesting that inflammatory mechanisms are associated with the development of right ventricular dysfunction [25]. Wang et al. showed that CXCL6 and Sirt3 are downstream of HIF-1α and that CXCL6 regulates Sirt3 expression through AKT/FOXO3a activation, which in turn regulates permeability, proliferation, and apoptosis in HBMEC after ischemia-reperfusion injury [26]. F3, as the gene encoding coagulation factor ⅲ, is a cell surface glycoprotein. Patients infected with SARS-CoV-2 showed elevated levels of F3 transcripts and raised circulating extracellular vesicles, which may contribute to disease-associated coagulation, thrombosis, and increased mortality [27]. MARCO, a macrophage receptor with collagenous structure is found on the surface of mucosal plasma membranes. Its function involves the incorporation of various extracellular substances into cells through macro-pinocytosis and endocytic pathways [28]. It is closely associated with the prognosis of several tumors, such as bladder cancer, breast cancer, and lung squamous cell carcinoma [29]. The cationic amino acid transporter proteins SLC7A1 (CAT1) and SLC7A2 (CAT2) are important arginine transporter proteins for T cells. They are members of the SLC7 family, which, together with members of the SLC1 family that are glutamate transporters, function in a variety of situations involving immune and inflammatory responses (such as viral infections, chronic intestinal inflammation, and tumor immunotherapy) by regulating astrocyte, macrophage, and T cell functions [30]. In our study, we found for the first time that CXCL6, F3, MARCO, SLC7A1, and SLC7A2 are associated with the diagnosis of TOF, laying the foundation for subsequent studies on the mechanisms of TOF.
GSEA enrichment analysis showed that biomarkers were enriched to immune-related pathways, such as 'activation of immune response', 'adaptive immune response', 'cytokine-cytokine receptor interaction', etc. Chemokines can be categorized into four subfamilies, CXC, CC, XC, and CX3C, based on the arrangement of their cysteine residues near the amino terminus. The receptors corresponding to these chemokines are designated as CXCR, CCR, XCR1, and CX3CR1, respectively. Sauce et al. showed that in pediatric patients with CHD, the level of serum CCL5 (RANTES) and MIF were elevated, while the concentrations of angiogenic chemokine GROα, which is associated with impaired lung function, were decreased. Moreover, the plasma of adult patients with CHD exhibited higher levels of IL-1β, IL-8, and eotaxin, an eosinophilic protein of the CC chemokine subfamily [31]. Wienecke et al. suggested that since CXCR4 or fractalkine and its receptor CX3CR1 regulate monocyte recruitment, platelet activation, and inflammation in cardiovascular disease, their study could be further developed in CHD regarding inflammation [12]. However, overall, the current research in CHD regarding chemokines and their receptors is still not widely studied.
The current study showed that activated dendritic cells, immature dendritic cells, central memory CD4 T cells, central memory CD8 T cells, neutrophil, monocyte, natural killer T cells, mast cells, regulatory T cells (type 1), and bone marrow-derived suppressor cells (MDSC) showed significant differences in the abundance of immune infiltration between TOF and controls. These differences also showed a significant correlation with biomarkers. Neutrophils are the first responders to the inflammatory signals released by cell and tissue injury, and the interaction between neutrophils and cardiomyocytes results in an elevated neutrophil-lymphocyte ratio (NLR) in patients with CHD before surgery [15, 32–35]. A study by Manuel et al. showed that for pediatric patients with CHD undergoing extracorporeal circulation, patients with cyanotic CHD had higher preoperative NLR levels [36]. For patients with TOF undergoing postoperative repair, higher preoperative NLR is associated with longer ICU and hospital stays [37]. Human monocytes are divided into three subpopulations, CD14 + + CD16- (Mon1, classical), CD14 + CD16+ (Mon2, intermediate), and CD14 + CD16++ (Mon3, non-classical), among which Mon2 and Mon3 are considered closely associated with inflammation. Their levels were found to be elevated in adult patients with CHD, and they increase in response to escalating severity of heart failure [38]. Hamada et al. showed that increased expression of mast cell chymotrypsin was associated with early pulmonary vascular disease in the lung tissue of patients with CHD. However, the exact biological mechanism is still unknown [39]. There are fewer studies on TOF and CHD from an immunological perspective, and there is a need for further research to enhance our understanding in this area.
In addition, it was predicted that lncRNAs, miRNAs, and TFs have regulatory roles with biomarkers through online databases. Zhang et al. showed that FGD5-AS1 is a pivotal lncRNA in the TOF cardiac ceRNA network and might play a repressive role in cardiac development by regulating the transcription of genes associated with CHD [40]. Liang et al. showed that reducing miRNA-940 expression affects the proliferation and migration of progenitor cells in the secondary heart field by targeting JARID2, leading to the development of TOF [41]. Yue et al. showed that hsa-miR-16 and hsa-miR-124 are key miRNAs for TOF and mainly regulate the expression of NT5DC1, ECHDC1, HSDL2, FCHO2, and ACAA2, which are involved in the rate of ATP conversion and fatty acid metabolism in mitochondria [42] However, to learn more about how the lncRNA-miRNA-mRNA and TF-mRNA connection pairs with regulatory effects discovered in our work are regulated by one another, further research is required. Previous studies have shown that miRNA-related single nucleotide polymorphisms (SNPs) may affect disease susceptibility and phenotype in an SNP genotype-dependent manner by altering the regulation of miRNAs [43]. In the present study, a miRNA-SNP-mRNA network was established. The findings propose that nine SNPs could potentially function as intermediaries between miRNAs and mRNAs. However, the exact mechanisms through which they operate require a more comprehensive study. Moreover, potential drugs were identified using biomarkers as targets, but there are fewer studies on the correlation between the predicted drugs and TOF, and further in-depth studies are needed.
However, this study still has several limitations. First, further experimental studies should be conducted to elaborate the molecular mechanism of TOF, focusing on inflammation. Second, more TOF samples need to be collected and studied to elaborate the role of biomarkers and their associated pathways in the mechanism of TOF.