In this study, we screened a total of eighty DEGs between DCM patients and non-DCM patients using two DCM gene expression profiles from GSE3585 and GSE84796. We performed enrichment analysis to explore the potential mechanisms underlying DCM. We also conducted immune infiltration analysis for DCM patients. Then, PPI network was constructed and a total of four algorithms were used within Cytoscape to identify the hub genes based on PPI network. Correlation analysis between hub genes and infiltrated immune cells to identify potential immune-related genes. The expression analysis and diagnosis value analysis of potential immune-related genes were performed. Finally, the expression analysis with validation set (GSE57338) and relationship analysis with CTD were performed for identification of the key immune-related genes in DCM. The main findings are that: 1) Three key immune-related genes (COL1A2, COL3A1, and POSTN) were identified between DCM patients and non-DCM patients; 2) The three up-regulated key genes might be implicated in the immune-related pathophysiological process in DCM. However, further studies are needed for illustrating the underlying mechanisms and providing potential therapeutic targets for DCM.
DCM, one of main types of cardiomyopathies, is characterized by the left ventricular or/and biventricular dilatation, ventricular wall thinner, and impaired myocardial systolic dysfunction, which ultimately leads to systolic heart failure. It has reported that several cardiac risk factors and cardiovascular diseases may be involved in the DCM, including family history of cardiomyopathy, myocarditis, and toxic effects from illicit drugs, alcohol, or medications. Multiple physiopathology mechanisms had been reported to be implicated in the development of DCM, such as oxidative stress, immune responses abnormal, inflammation, fibrosis and genetic changes. However, the key genes and precise molecular mechanisms underlying DCM, especially immune response abnormal, remain elusive. Interestingly, accumulated evidence had revealed that innate and adaptive immune responses abnormal may play an essential role in the initiation and progression of DCM.
Preliminary studies had suggested that multiple immune cells, including cytotoxic CD8(+) T cells, B cells, and macrophages, were infiltrated in various types of DCM (such as Chronic Chagas cardiomyopathy and idiopathic dilated cardiomyopathies), in which T cells related differentiation might play an important role on the progression of abnormal immune and inflammatory response in DCM[6]. A recent study related to T cell subpopulations analysis, which based on the in silico approach and unsupervised machine learning methods, suggested a robust systemic inflammation and highly activated CD8(+) T cells were displayed in DCM patients[7]. Similarly, Fonseca et al.[8] also revealed that the predominance and maintenance of CD8(+) T cell may be the key cell type in the tissue damage of Chagas disease induced DCM. T follicular helper cells, a specialized subset of CD4(+) T cells, have been demonstrated to provide help for B cells in the secondary lymphoid organs, which is significantly associated with multiple pathogenesis of diseases, including infectious diseases, allergies, and autoimmune diseases[9]. MHCII proteins, widely expressed in several immune cells (such as B cells, macrophages, and dendritic cells), could bind self or foreign proteins and participate the activation and regulation of adaptive immune response[10]. Increasing studies have indicated that MHC might sever as a marker for the abnormal immune response in multiple myocardial damage, including DCM[11, 12].
Reportedly, cardiac fibrosis, an essential feature in the progression of DCM, has been demonstrated to be associated with multiple immune cells, such as macrophages and T cells, which could directly or indirectly activate the cardiac fibroblasts[13]. Meanwhile, more and more studies had revealed that immune response abnormal could lead to inflammatory activation and fibrosis, especially in term of cardiovascular diseases[14-16]. A study comprising a total of 182 biopsy samples derived from consecutive DCM patients suggested that M2 macrophages infiltration was significantly related to the worse outcome (hazard ratio: 1.77, P<0.05), and might be an independent determinant with collagen area fraction in DCM (P<0.05)[17]. Also, nuclear receptors, a family of lipid- and hormone- activated transcription factors, including retinoic acid receptor, PPARγ, glucocorticoid receptor and, vitamin D receptor, were significantly associated with immunological recognition, immune regulation, and inflammatory immunity[18].
In this study, similar to previous studies[19, 20], our enrichment analysis for DEGs revealed that multiple enrichment items were significantly involved in immune response. Regulation of memory T cell differentiation, including regulation of T cell mediated cytotoxicity, immunological memory formation, and so forth, has been mainly enriched in biological process. MHC class II protein complex and fibrillar collagen trimer were mainly enriched in cellular component. MHC class II receptor activity, nuclear receptor activity, and collagen binding were mainly enriched in molecular function. Importantly, enriched immune system processes for GO were mainly involved in the regulation of memory T cell differentiation and positive regulation of T cell mediated immunity, which further indicated that DEGs were associated with immunity. Cell adhesion and cell-cell interactions have been revealed as the crucial mediators for the activation of immune cells[21]. Interestingly, our KEGG analysis showed that the mainly enriched item was the cell adhesion molecules, which indicated an immunology biological pathway involved in DEGs. Moreover, immune infiltration analysis between DCM and non-DCM patients was performed. Consistent with previous studies[7, 9, 17], we found that a higher proportion of CD8(+) T cell, T follicular helper cells, and M2 macrophages was shown in DCM patients, indicating that a potential abnormal immune regulation and response, as well as cardiac fibrosis, in DCM. In summary, DCM-related DEGs were involved in immune-related pathological process, highlighting a significant role of immunity during the development of DCM.
COL1A2 (collagen type I alpha 2 chain) could encode the pro-alpha2 chain of type I collagen, which is widely distributed in most connective tissues, such as cornea, tendon, bone, and dermis. Preliminary studies showed that, except for accelerating the increase of extracellular collagen fibers, the up-regulated expression of COL1A2 was significantly associated with immune cells infiltration in multiple diseases, including gastric cancer, radiation-induced lung injury, and Chagas disease cardiomyopathy[22-24]. Consistent with previous studies, our study showed was COL1A2 also significantly up-regulated in DCM patients. Importantly, the results of correlation analysis of COL1A2 and infiltrated immune cells revealed the up-regulated expression of COL1A2 was positively correlated with immune activation related cells (e.g., CD8(+) T cells and M2 macrophages) and negatively correlated with immune homeostasis related cells (e.g., eosinophils, memory B cells, and resting CD4(+) memory T cells). These results indicated that the up-regulated COL1A2 expression may promote the activation of immune system and aggravate the immune-dependent injury, thus facilitating the development of DCM.
In DCM, the cardiac fibrosis, an important pathological characteristic, could be activated by multiple humoral and cellular factors, leading to the increased cardiac rigidity, decreased myocardial performance, and enhanced sudden death risk. Accumulated studies had revealed various immune cells (e.g., macrophages, CD8(+) T cells, and activated B cells) were involved in the development of cardiac fibrosis via directly activating cardiac fibroblasts or/and indirectly releasing pro-fibrotic cytokine[13, 25]. COL3A1, also named collagen type III alpha 1 chain, plays a significant role in encoding fibrillar collagen, contributing to the progression of fibrosis in extensible connective tissues such as lung, skin and the vascular system. Consistent with our study, multiple researches had indicated an elevated expression of COL3A1 in DCM patients compared with non-DCM patients. Guo et al.[26] established coxsackie virus B3 induced DCM mice model, and the results showed the up-regulated COL3A1 expression companied with immune cells infiltration (e.g., T helper cells) and myocardial fibrosis. Similarly, our results showed the elevated COL3A1 expression was positively correlated with CD8(+) T cells, M2 macrophages, and T follicular helper cells, while also negatively correlated with eosinophils, resting CD4(+) memory T cells, and memory B cells, which suggested COL3A1 may sever as a key immune-related gene playing a central role of immune mechanism in the progression of DCM.
POSTN is also named periostin, and encodes a secreted extracellular matrix protein, playing a significant role in tissue development and regeneration, including wound healing, and ventricular remodeling post myocardial infarction. An increasing number of researches revealed POSTN was significantly associated with immune response abnormality, inflammation, and fibrosis[27-29]. Similarly, our study showed the elevated POSTN expression was positively correlated with T follicular helper cells, CD8(+) T cells, and M2 macrophages while negatively correlated with resting CD4(+) memory T cells and eosinophils. These results suggested POSTN may be a key immune-related gene in DCM.