Identifying patterns of immune related cells and genes in the peripheral blood of acute myocardial infarction patients using a small cohort

Coronary artery disease (CAD) is a common chronic heart disease worldwide. The accumulation of a large number of lipids under the intima of the coronary artery leads to the formation of atherosclerotic plaque, which gradually leads to the narrowing of the vascular lumen, finally resulting in impaired blood perfusion of the myocardium [1]. CAD usually presents with a variety of different symptoms, including ischemic cardiomyopathy, stable and unstable angina, acute myocardial infarction (AMI), and even sudden accidental death [2]. Emergency percutaneous coronary intervention (PCI) can quickly restore cardiac perfusion and makes a great contribution in improving the prognosis of AMI patients. Nevertheless, AMI has becomea main cause of hospitalization and mortality in patients, especially in China, and its incidence is increasing annually [3]. Previous studies have shown that AMI is a complex disease that is influenced by multiple factors, such as inflammation responses [4], immune mechanisms [5], hypertension, hyperglycaemia, smoking, obesity and dyslipidemia [6]. Accumulating evidence also shows that total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) exert a synergistic effect on the immune inflammatory response, which can increase oxidative stress and vascular inflammation, leading to reduced bioavailability of nitric oxide (NO), and ultimately the formation of atherosclerotic plaque [7]. Several researches have indicated that atorvastatin therapy can effectively reduce levels of LDL-C, interleukin (IL)-1, tumour necrosis factor-alpha (TNF-α), C-reactive protein (CRP), and IL-6 in patients with high cholesterol, compared with dietary control alone [8]. At present, lipid-lowering therapy has become the cornerstone of drug therapy for CAD or AMI. We can effectively reduce the occurrence of major adverse cardiovascular events (MACEs) by downregulating LDL-C level. However, even if the level of LDL-C is reduced significantly, even until levels close to that at birth, MACEs cannot be completely eliminated. Immune inflammatory responses may partially account for this residual risk. Clear inflammatory intervention can be expected to effectively further improve the prognosis of patients, compared with only a reduction in LDL-C levels. Recently, Fernandez et al. provided the first overview of the human immune cell landscape during atherosclerosis and provided insights into the identity of immune cells that reside in the plaque and described their different activation states, which has opened the door for the study of atherosclerosis caused by autoimmune response [9]. Furthermore, Yang et al. suggested that the activation of signal cointegrator 1 complex subunit 2 (ASCC2), solute carrier family 25 member 37 (SLC25A37), and leucine rich repeat containing 18 (LRRC18), can be used as diagnostic markers of CAD, while immune cell infiltration plays a crucial role in the occurrence and development of CAD [10]. However, the pattern of immune cell infiltration in AMI has not been fully elucidated. Therefore, clarifying immune infiltration in AMI and identifying the key genes associated with immune cells may provide a novel perspective on the prevention and treatment of AMI.

Along with increased popularization and application of gene chip gene-chip sequencing technology, microarray analysis has become a practical and novel method of identifying susceptive genes correlated with AMI [11], thus helping clinicians gain a deeper understanding of the relationship between genes and disease [12, 13]. However, the sensitivity and reproducibility of microarray analysis based on differentially expressed genes may be limited [14, 15]. Weighted gene co-expression network analysis (WGCNA) is used increasingly widely to analyse a large number of gene expression data and is a powerful systematic biological approach to analyse network relationships and molecular mechanisms [16]. WGCNA is often used to identify co-expressed gene modules that are of specific biological significance and explore the association between gene modules and interesting sample characteristics [17].

For the past few years, an increased number of studies have indicated that immune cell infiltration may play a critical role in the pathogenesis and progression of CAD. Yang et al. have suggested that there is an increased in the infiltration of monocytes coupled with the decreased infiltration of CD8 + T cells in patients with CAD [10]. However, immune cell infiltration in AMI has not been fully elucidated. CIBERSORT, is an analysis tool that is used widely to explore the infiltration ratio of 22 immune cells in the samples based on the expression profiles of microarray data or RNA-seq data [18]. At present, a few studies have combined WGCNA with CIBERSORT to identify key immune related genes involved in AMI. Therefore, to meet this demand, in this study, CIBERSORT was used to calculate the proportions of 22 types of immune cells in AMI patients, while WGCNA was used to identify key modules that are significantly associated with AMI. Thereafter, the CIBERSORT results were combined with WGCNA to identify immune-related key modules and genes in patients with AMI to help elucidate the immune related molecular mechanism of AMI and lay the foundation for the development of immunomodulatory therapy for AMI.

In the past, the formation of atherosclerotic plaque as a result of dyslipidemia it was considered as a major cause of arteriosclerosis. However, during recent years further research has shown that arteriosclerosis is actually a chronic inflammatory process that induces strong immune activity [28]. Previous studies have suggested that a variety of immune cells play a key part in atherosclerosis. Dounousi et al. suggested that monocyte subsets play a crucial role in atherogenesis and inflammatory cascades in cardiovascular disease. Increasing counts and activity of monocytes are closely related to the clinical indexes of chronic kidney disease (CKD), atherosclerosis, and heart failure [29]. T lymphocytes are the most critical immune cells found in vivo. Based on their surface markers and functions, T lymphocytes can be classified as CD4 + and CD8 + cell subgroups. CD8 + T cells play a dual role in atherosclerosis. A compelling study pointed out that CD8 + T cells could secrete a variety of inflammatory cytokines, which could aggravate the inflammatory response and increase the instability of atherosclerotic plaques [30]. Inversely, cytotoxic activity that targets antigen presenting cells and regulatory CD8 + T cell subsets could effectively suppressed the progression of atherosclerosis by alleviating the immune reaction [30]. Other immune cell types, including neutrophils [31] and mast cells [32], also play a key part in the progression of cardiovascular disease. Notably, Han et al. suggested that the proportion of activated dendritic cells and Tfhs in CAD was remarkably higher and that the proportion of Tregs, resting CD4 + T cells, and γδ T cells was remarkably lower than in the control group. In addition, Yang et al. also identified an increase in the infiltration of monocytes but a decrease in the infiltration of CD8 + T cells in CAD subjects [10]. This data indicates that CAD exhibits inflammatory microenvironment patterns. On the contrary, persistent T-cell responses induced by myocardial infarction are significantly correlated with subsequent left ventricular remodelling, which ultimately leads to cardiac arrest and heart failure [33]. These results indicate that the immune system plays a very complex role in the pathophysiology of CAD. However, the pattern of immune cell infiltration in AMI has not been fully elucidated. To further explore the proportions and types of immune cells in AMI patients, the CIBERSORT package of R software was used to conduct a comprehensive assessment of 22 types of immune cell infiltration in AMI cases. We noticed that there was a decrease in the infiltration of CD8 + T cells, resting mast cells, and γδ T cells but an increase in the infiltration of neutrophils and M0 macrophages in AMI patients. These results indicate that there may be a difference in the immune cell infiltration pattern between AMI and CAD. These differences can better help us understand which immune cells play a vital part in processes from the deterioration of CAD to AMI. As previously mentioned, compared with normal samples, the proportion of neutrophils, which are involved in ischemic injury after stroke in ischemic stroke (IS) samples is generally higher. Neutrophils may be a promising target for IS therapies [34]. In addition, CD8 + T cells have pleural effects on atherosclerosis, and our study showed that the proportion of neutrophils were higher and that the proportion of CD8 + T cells were lower in the AMI group than in the control group. This indicates that neutrophils can accelerate but CD8 + T cells inhibit the occurrence and progression of AMI. Nevertheless, it is not clear whether the number of CD8 + T cells and neutrophils in peripheral blood samples reflect their infiltration into the vascular wall. Additionally, the current study also revealed that there were several different interactions between different immune cells in AMI. We noticed that neutrophils were negatively related to Tregs, CD8 + T cells, γδ T cells, and resting mast cells, while CD8 T + cells were positively related to resting mast cells. The immune cells infiltration analysis suggested a complicated network in AMI. Nevertheless, the potential mechanism of these relationship between infiltrated immune cells needs to be verified using in vivo and in vitro studies.

To further identify immune-related key genes involved in AMI, we conducted WGCNA combined with CIBERSORT to screen key modules that were remarkably associated with immune cells, and it was indicated that the salmon module was remarkably related to the memory B cells. Then, KEGG and GO enrichment analyses were conducted to further confirm that the genes in the salmon module were mainly involved in immune related signalling pathways and biological processes. A PPI network was built based on genes in the salmon module. Following the MCODE analysis, four different MCODE complexes were identified in the salmon module, and four hub genes (EEF1B2, RAC2, SPI1 and ITGAM) that were significantly correlated with AMI were identified. External validation showed that the expression levels of ITGAM and SPI1 were significantly different between AMI and the control group, while the expression levels of the EEF1B2 and RAC2 genes were not significantly different between the two groups. These results suggest that ITGAM and SPI1 genes may act as key immune-related genes involved in AMI.

Previous research has revealed that ITGAM is a member of the β2 integrin family of adhesion molecules, and adhesion molecules play an indispensable role in the recruitment and activation of neutrophils, macrophages, and monocytes during the process of inflammation [35]. Zirlik et al. proved that ITGAM plays a key role in inflammatory processes, such as the neutrophils and monocytes adhesion to injured endothelial cells and trans-endothelial migration, and is also involved in CD40 ligand-mediated atherosclerotic inflammation [36]. Previous studies have shown that the transcription profiles of monocytes following AMI in mice and human share common biological characteristics. ITGAM is one of the most common inflammation-related genes, has been shown to play a key role in monocyte inflammation, intercellular signal transduction, and cell proliferation [37]. On the other hand, Wang et al. found that ITGAM expression was correlated with various immune cells, including Tregs, M2 Macrophages, and that ITGAM plays an important role in acute myeloid leukaemia (AML) related immune regulation. Elevated ITGAM expression levels could predict poor overall survival and poor initial treatment response in patients with AML [38]. In addition, Ayari et al. found that ITGAM was significantly overexpressed in human carotid plaque [39]. Similarly, in our previous study, it was found that the expression levels of ITGAM were significantly upregulated in patients with CAD, and that high expression levels of ITGAM showed high diagnostic efficiency for the recognition ability of CAD [40]. However, to our knowledge, no reports have been published on the relationship between ITGAM and the immune microenvironment in AMI. In the current study, we noticed that ITGAM is positively correlated with neutrophils and negatively correlated with CD8 + T cells and resting mast cells. Meanwhile, we also noted that ITGAM was significantly overexpressed in patients with AMI. Based on these results, we speculated that the level of ITGAM overexpression is significantly correlated with the occurrence and development of AMI, and that ITGAM is expected to be a novel immune-related target for the prevention and treatment of AMI.

SPI1 encodes an ETS-domain transcription factor, PU.1, which is essential for the development of myeloid cells and B lymphocytes, and is the primary regulator of cell-to-cell communication in the immune system [41]. Pulugulla et al. noticed that the expression level of SPI1 mRNA is upregulated in activated T cells, and it may play a role in regulating the expression of interleukin 1 beta (IL1B) following the activation of CD4 T cells [42]. Yashiro et al. found that SPI1 could activate the C–C motif chemokine ligand 22 (CCL22) gene in dendritic cells and macrophages by directly binding to two key elements in the promoter, thereby mediating the migration of different subsets of leukocytes during the immune response [43]. The continuous overexpression of SPI1 in hematopoietic cells leads to the differentiation of macrophages, and SPI1 is an important regulatory factor for all states of tumour-associated macrophages (TAMs). Inhibition of the expression of SPI1 can effectively reduce the maturation and polarization of TAMs to play an anti-tumour role [44]. Previous studies have suggested that SPI1 acts as a key transcription factor that regulates the expression of several inflammatory genes, and has been found to be significantly overexpressed in advanced plaques. High expression levels of SPI1 showed modest efficiency in distinguishing the capacity of CAD [45]. Similarly, Qiao found that SPI1 plays a key role in the occurrence and development of ischemic cardiomyopathy and dilated cardiomyopathy by regulating apoptosis- and inflammation-related genes [46]. In addition, SPI1 has been predicted to regulate the expression of key genes that lead to heart failure following AMI [47]. However, the correlation between SPI1 and the immune microenvironment of AMI has not been reported on. Fortunately, in this research study, we noted that SPI1 is positively correlated with ITGAM and neutrophils but negatively correlated with Tregs, CD8 + T cells, and resting mast cells. Meanwhile, the gene expression level of SPI1 in AMI patients was also significantly higher than those in the control group. This suggests that SPI1 may be a novel potential molecular target for the diagnosis and treatment of AMI.

The current research study has several limitations. First, the validation samples included in this study were recruited from a single centre and had a small sample size. It is not clear whether the findings of this study are similar among individuals in other regions and ethnic groups. Therefore, the validity of the results of this study need to be further tested using multi-centre and larger samples. Second, it is not clear whether SPI1 acts as a transcription factor to regulate the expression of ITGAM. Third, further in vivo and in vitro research is needed to clarify the underlying mechanism of the correlations between ITGAM and SPI1expression levels and the infiltration of immune cells in AMI.

Immune cell infiltration plays a crucial role in the occurrence and development of AMI. ITGAM and SPI1 are the key immune-related genes that have the potential to become targets for the prevention and treatment of AMI.