Coronary atherosclerosis is a complex pathological process that begins at an early stage of human life. At first, it may be due to some minor vascular abnormalities, such as endothelial injury, arterial calcification, lipid adhesion, etc. However, it develops and deteriorates over several years to decades and finally reaches a life-threatening level. In this long process, many pathophysiological factors affect the outcome of the disease. For over one century, it has been believed that circulating lipid disorders was the key reason to induce atherosclerosis-based cardiovascular disease. 20 years ago, Professor Peter Libby put forward the view that inflammation mediated atherosclerosis, which triggered a heated discussion on immune-mediated CAD and been confirmed by several subsequent studies . Today, no one doubts that the immune system impacts the entire process of CAD. Several important examples include: allogeneic vascular transplantation still developed atherosclerotic lesions without lipid metabolism disorder background ; the cardiovascular disease treatment strategy targeting inflammation effectively reduces the incidence of acute cardiovascular events . Meanwhile, gene characteristic changes in the circulatory system of CAD patients were reported to be significantly correlated with the degree of coronary artery stenosis , and the study also showed that 12 genes related to toll-like receptors were related to coronary artery lesions . However, these projects' current sample scale and scope are still limited. We used the online dataset for re-analysis to screen the circulatory system's immune-related differential genes and immune cell subgroups. We further established a diagnostic model based on this discovery and evaluated its diagnostic efficiency. In this study, we obtained high-throughput mRNA data from 199 CAD patients and 218 healthy people samples online, then screened and established 14 immune-related genes as essential immune characteristic genes in the peripheral blood of CAD patients, including CCR9, CER1, CSF2, IL13RA1, INSL5, MBL2, MMP9, MSR1, NTS and TNFRSF19 gene were high expressed, while CXCL2, HTR3C, IL1A and NR4A2 gene were low expressed.
Some of these genes have been reported to play a protective role in CAD. The colony-stimulating factor (CSF2) promotes the colonization of granulocytes and macrophages. They are released into the blood from ischemic cardiomyocytes, promoting the homing of cells, including cardiac mesenchymal stem cells, to the heart and playing the function of damage repair . Macrophage scavenger receptor 1 (MSR1) inhibits TNFs by promoting the expression of IL-10α, MMP-9 expression to regulate immunity, which is beneficial to the stability and regression of AS and myocardial repair after infarction . Interleukin-13 receptor α1(IL-13Rα1) is one of the primary receptors of anti-inflammatory type 2 cytokine IL-13, which, as reported, has the effects of regulating myocardial homeostasis and anti-inflammatory. In previous studies, il-13Rα1 in myocardial tissue was down-regulated, probably because of the decrease of collagen deposition in the myocardial . However, in this study, the mRNA expression of IL-13RA1 in the blood of patients with CAD was up-regulated. We speculated that the decrease of this receptor in the target organ might cause the accumulation of IL-13 in the circulation and increase the compensatory expression of this gene in the circulating nucleated cells, which needs to be further confirmed. NR4A2 has been reported as a transcription factor to protect cardiomyocytes by transcriptionally inhibiting CCR5 and promoting macrophage polarization to M2 type , therefore seemed to play a positive role in anti-inflammation.
Another group of genes exacerbated the progress of CAD. The C-C motif chemokine receptor 9(CCR9) was previously reported to be related to the process of inflammatory expression and myocardial remodeling after myocardial infarction. After the gene was knocked out, it reduced cardiac-related inflammatory factors such as IL-6 and IL-1β, TNF-α and so on, which may be mainly through NF-κB and MAPK signaling pathways to regulate the production of myocardial hypertrophy . Matrix metallopeptidase 9(MMP9) regulation is closely related to multiple signaling ways of cardiovascular disease, atherosclerotic plaque instability, and myocardial tissue repair after infarction. Previous studies have shown that the change of MMP9 level in circulation is an independent predictor of atherosclerosis . Circulating Neurotensin (NTS) is a previously reported risk factor for cardiovascular disease. Its up-regulation increased the risk of type I diabetes and multiple atherosclerotic diseases by promoting lipid absorption .
Some of the genes only showed a correlation with CAD. Previous studies have shown that MBL2 is high expressed in peripheral blood of patients with CAD , related to various heart risks, and has led to the critical value of early diagnosis. CXCL2 and IL-1α were also found up-regulated in the atherosclerotic mouse model [39–40], but the results of this study were the opposite, which might be the impact of treatment. They are both typical pro-inflammatory genes and participate in CAD-related inflammatory responses. However, CXCL2 had no significant difference in our animal validation, while up-regulation of IL-1α was displayed in the CAD group. TNF receptor superfamily member 19 (TNFRSF19) is a serum biomarker of chronic inflammation. Previous studies have reported the increased expression of TNFRSF19 in CAD , but these studies were very preliminary.
Furthermore, 5-hydroxytryptamine receptor 3C (HTR3C), Cerberus 1(CER1) and insulin-like 5(INSL5) were screened out for the first time and might have the potential to participate in the CAD process. As far as we know, they have never been reported to be directly related to CAD before, but some have been confirmed to affect the changes of immune targets in circulation. For example, INSL5, as a peptide hormone, after intraperitoneal injection, leads to significant changes in inflammatory factors such as IL-5, IL-7, M-CSF, IL-15 and IL-27 , and these inflammatory signals typically play a regulatory role in CAD. PCR experiments based on animal models partially confirmed the results of the above screening (Fig. 7).
With the popularization of single-cell sequencing technology, we can further describe the immune system's participation in the CAD pathological process [43, 44] on the single-cell levels. The understanding of the landscape of immune cells has therefore changed significantly. Investigating the distribution characteristics of immune cell subsets in patients with coronary heart disease has been revealing enormous value in CAD studies , however, more studies focused on the aorta and plaque. By constantly observing, the pathological landscape world of the lesion was mapped , but insufficient attention was paid to the changes in immune cell subsets in peripheral blood. In many diseases, including CAD, the state change of circulatory system content is the decisive factor of aorta local pathological risk. In atherosclerotic plaque, many inflammatory cells with chemotaxis participate in plate and lipid infiltration . It is of great significance to clarify the peripheral immune subsets for precise drug use. In particular, when the distribution of cell subsets changes, immune-related genes change accordingly; this will provide critical clues to clarify the causal relationship of the complex immune world. Our results showed that the number of mast cells, neutrophils, activated dendritic cells, MDSC, type 17 T helper cells in the CAD group increased sharply. The neutrophil is a crucial cell to promote the progress of atherosclerosis. It can increase the area and instability of plaque by releasing a variety of cytokines and adhesion factors, which would promote macrophage phagocytosis of lipids and inhibit MMP-9 inactivation. In this study, the number of neutrophils in patients with CAD was associated with the increasing high expression of MMP-9 (Fig. 6C), which further proved its critical role in the development of CAD. Dendritic cells proliferated and activated in the presence of GM-CSF produced by endothelial cells , which is consistent with the findings of this study. Generally, B1 lymphocytes were considered to have a solid ability to resist atherosclerosis plaque formation and play an essential role in reducing foam cells . In this study, the number of activated B cells was significantly reduced compared with those in the healthy group, suggesting that the adaptive immune function of the patients was decreased to a certain extent. Natural killer cell-deficient mice were found to have elevated serum cholesterol levels and increased plaque area in the former studies . Consistently, in this study, CD56dim natural killer Cells of the CAD group showed a downward trend. However, although differentially expressed in this study, the role of mast cells, MDSC, type 17 T helper cells in CAD is still unclear. In fact, the effects of most immune cells in immune-related diseases remain complex and two-sided, which makes the identification of their role ambiguous. Therefore, it is quite difficult to treat them as reliable targets. Anyway, our study showed that this existing phenomenon could be used as a mean of early identification and screening of CAD and a starting point for further confirmation of these cell functions.
GO analysis showed that the differential genes were mainly concentrated in cell proliferation, intercellular communication and mutual regulation, cell adhesion and migration, which were closely related to the progress of CAD key pathological. KEGG pathway enrichment analysis suggested that the IL-17 signaling pathway and cytokine-cytokine receptor interaction were still the main enrichment pathways, which was previously emphasized in a similar study . The results reaffirmed that the genes related to these two pathways are significantly enriched in CAD patients, reminding us that continuous attention to them is necessary to be taken. Moreover, the pathways were also associated with nervous system diseases relevant pathways, indicating that they might be related to the simultaneous neurodegenerative changes of patients (such as Alzheimer's disease Parkinson's syndrome).
Notably, in this study, some traditionally considered risk genes and cell subsets of CAD patients did not enter our candidate team, such as CRP, MCP-1, monocytes, etc. We speculated that these patients might be related to statins or other therapeutic drugs, which have proved to be significantly changed the immune characteristics in peripheral blood .
The potential of serum immune biomarkers in the diagnosis and typing of CAD has attracted more and more attention. Because of its accessibility and non-invasive, circulating mRNA detection and immune cell subsets detection have attracted more and more attention in the early screening of diseases. Our study suggested that the diagnostic model constructed by combining multiple immune-related genes had high sensitivity and specificity for identifying CAD and healthy individuals. Therefore, it might play a role in clinical application in the future. At the same time, some newly discovered immune characteristics may be used as the starting point for further research. However, the results of this study still have some limitations. First, to control the variables associated with CAD as much as possible, we used model animals for verification. Although we know that the above genes are highly conserved between mice and humans, the results may still differ from human samples; Second, due to the lack of evaluation of the individual information from the sample source, the impact of inter-individual treatment schemes and primary diseases on the changes of these immune indicators cannot be evaluated and removed. These need to be verified by large-scale randomized clinical trials.