AF is the most prevalent cardiac arrhythmia, however, the specific molecular mechanisms of AF still remain unclear. Despite the great improvements has been gained in the field of diagnosis and treatment, AF remains a leading cause of mortality and disability . Drugs for rate control, oral anticoagulants for stroke prevention, antiarrhythmic drug and catheter ablation for conversion are main treatments for AF patients . But efficacy and safety of these treatment measures are still not well understood. Meanwhile, many asymptomatic patients, especially with paroxysmal AF, are difficult to find. This is the first study to identify biomarkers of AF associated with immune cells infiltration. Two gene expression datasets from the GEO were merged and conducted an integrated analysis. 129 DEGs were detected using “limma” package. Enrichment analysis showed that these 129 DEGs were significantly correlated with immune and inflammatory responses. The relationship between inflammatory response and AF has been widely studied in the past decades and various regular anti-inflammatory biomarkers were found be related to AF. It has been reported that patients with AF had increased IL-6, IL-8 and TNF-α, meanwhile, these inflammatory markers can predict the outcome of AF ablation [33–37]. High-sensitive C-reactive protein (hsCRP) is also associated with increased risk of AF recurrence following successful electrical cardioversion and catheter ablation . Accumulating studies have demonstrated that the increase of TGFβ1 in AF patients promote atrial fibrosis, which plays a pivotal role in atrial structural remodeling in AF [39, 40]. Additionally, the critical role of various immune cells in the pathogenesis of AF has attracted more and more attention, including infiltrating the atrium and secreting several chemokines and cytokines to regulate the microenvironment of the heart . Our GSEA results are also in general agreement with the previous findings that immune cells infiltration participates in the pathogenesis of AF [8–9]. Medicine is the earliest application field of AI. In the past few decades, AI technology, especially machine learning, has made great progress in the diagnosis and treatment of a variety of diseases, including cardiovascular diseases, nervous system diseases and genetic diseases . Therefore, for the first time, we sought to screen out key DEGs between AF and SR patients as biomarkers based on machine learning methods and explore its relationship with immune cells infiltration in AF. Overlapping the results from two machine learning algorithms, CYBB, CXCR2 and S100A4 were identified as key DEGs and biomarkers of AF.
CYBB, also known as NOX2, has been implicated in oxidative stress in various cardiovascular diseases . Pignatelli et al. reported that serum NOX2 can be used as one of the important indicators to predict vascular embolism events in . In animal models, numerous studies have demonstrated that inhibition of NOX2 mediated reactive oxygen species (ROS) prevents atrial remodeling [45, 46]. In addition, atrial electrical remodeling can also be alleviated by inhibiting NOX2 and oxidative stress . In human AF, NOX2 has also been demonstrated to participate in the atrial structural remodeling and electrophysiological remodeling, and up-regulation of NOX2 is associated with an enhanced risk of AF [48, 49].
The chemokine receptor CXCR2, encoding by CXCR2, belongs to chemokines family, mediates cellular migration of immune cells . The expression level of CXCR2 are tightly regulated during infection and inflammation. CXCR2 is key stimulant of immune cells infiltration and recruitment, especially of neutrophils. Our results of evaluation of 22 subtypes immune cells infiltration showed that neutrophils are significantly elevated in AF patients compared with SR . It is also reported that CXCR2 was involved in atrial monocytes infiltration, which accelerates atrial fibrosis and promotes atrial remodeling. Therefore, blocking CXCR2 may serve as a new therapeutic strategy for AF patients . Moreover, CXCR2 is also a crucial regulator of hypertension. In spontaneously hypertensive rats, Zhang et al. identified that inhibition of CXCR2 could prevent the occurrence of AF . In angiotensin II-induced cardiac atrial fibrillation animal model, CXCR2 has also been proved to participate in immune cells infiltration and mediates cardiac hypertrophy and remodeling through regulation of monocyte .
S100A4, also known as fibroblast specific protein 1 (FSP1), is involved in numerous biological processes including cell survival, cell motility, and cell differentiation . Numerous studies have revealed roles of S100A4 in cancer progression, particularly the ability of enhancing metastasis. Nevertheless, S100A4 has been linked to various diseases besides tumor, such as cardiac fibrosis and hypertrophy, kidney fibrosis and pulmonary disease. All of these diseases are involved in inflammatory processes and greatly dependent on tissue remodeling . S100A4 expressed in normal human heart and increased in hypertrophic left ventricles [56–58]. In addition, S100A4 is a key regulator of endothelial mesenchymal transformation (EMT), which is related to immune cell infiltration, making epithelial cells present mesenchymal cell phenotype and ultimately resulting in enhanced migration ability, enhanced anti apoptosis ability and a large number of extracellular matrix components. Recently, studies demonstrated that EMT occurs in the atrium of AF patients and contribute to fibroblast accumulation. Meanwhile, S100A4 also had significant correlations with left atrial dimension in AF patients .
We used CIBERSORT to evaluate the degree of infiltrating immune cells in the present study. We found reduced infiltration of T cells follicular helper, as well as increased neutrophils, mast cells resting and T cells gamma delta in AF. Neutrophils represent activated nonspecific inflammation and have been found as markers of inflammation in various diseases. Correlation between inflammatory markers and cardiovascular diseases has been studied widely and the relationship between neutrophils and cardiovascular diseases has been confirmed in the past. It is reported that the level of neutrophils is an independent predictor for the prognosis of acute coronary syndrome . Moreover, accumulating studies have reported that increased neutrophil/lymphocyte ratio is related to the increased risk of AF occurrence [61, 62]. Mast cells, tissue-specific innate immune cells, present in virtually all body tissues including the heart. Liao et al. reported that mast cells stabilization associated with rescued atrial fibrosis and AF in animal models . Numerous inflammatory mediators secreted by mast cells including IL-1β, IL-6 and TGF-β1 also participate in atrial structural remodeling and development of AF . T follicular helper cells has also been reported to function in AF, which might be activated by Toll-like receptor 2 (TLR2) and TLR4 [65,66]. We also studied the correlation between CYBB, CXCR2, S100A4 and infiltrating immune cells. Based on the correlation analysis results, CYBB, CXCR2 and S100A4 appear to play key roles in regulation of immune cells infiltration.