Background
Kawasaki disease (KD), also known as cutaneous mucous lymph node syndrome, is an autoimmune disease with systemic diffuse vasculitis as the main symptom, which tends to occur in children under 5 years old [
1]. As the most common cause of acquired heart disease in developed countries, KD is prone to cause coronary artery damage, including coronary dilatation and coronary aneurysms, and may even lead to myocardial ischemia, myocardial infarction and sudden death [
2]. In recent years, many studies have suggested that the pathogenesis of KD is significantly correlated with infection, genetic susceptibility and immune response, and there are on-going studies focusing on its treatment based on different pathogenesis hypothesis. According to previous report, the acute phase of KD occurred with severe immune dysfunction, the activation of the immune system and inflammation factor of the cascade amplification effect is considered to be the main characteristics of KD. A certain number of researchers believe that KD is caused by pathogenic bacteria infection, leading to abnormal activation of the immune system and causing cascade release of inflammatory factors [
3]. Recent studies have shown that the regulatory T cell is an important marker in determining the severity and susceptibility of KD [
4]. In addition, lymphocyte subsets and immunoglobulin level are existed laboratory markers to differentiate KD from other febrile infectious diseases and healthy children [
3].
Advances in next-generation sequencing technologies have recently made it possible to study the immune system at single cell level. Tang [
5] et al. first published single cell transcriptome sequencing (scRNA-seq) technology in 2009, which has enabled high resolution mapping of cellular heterogeneity, development, and activation states in diverse systems. scRNA-seq is to amplify the trace transcriptome RNA of isolated single cells for high-throughput sequencing to obtain the expression profile of the complete transcriptome in the single cell level, thus revealing the molecular regulatory mechanisms of specific biological processes and disease processes [
6,
7]. This technique is of great significance for the discovery of new therapeutic targets for cardiovascular diseases. At present, there are few reports on scRNA-seq of peripheral blood mononuclear cells (PBMC) in KD.
In this study, scRNA-seq was applied to PBMC of a KD patient and a healthy control, and bioinformatics analysis were performed to explore the cell cluster differences and DEGs that may affect the development of KD, so as to provide new targets for KD treatment.
Discussion
KD is a common cause of vasculitis in childhood, which is characterized by fever and mucocutaneous features [
14] . Current studies suggest that the regulation of T cell activation determines the susceptibility of KD and the severity of coronary artery lesions. However, despite emerging treatment options, the precise immune process of KD has remained unclear. Transcriptome is an important pathway linking the genome to the proteome and can provide new methods for the diagnosis, treatment and prognosis of diseases [
15] . In previous study, a 13-transcript blood gene expression signature distinguished KD from other febrile conditions, including S100P, CD163 and RTN1, etc. [
16]. And deep RNA sequencing was performed to reveal 1074 differentially expressed RNAs to provide direction for future etiology studies. At the transcriptomic level, we can further differentiate into more detailed subsets of cells based on different gene expression patterns [
17‐
19]. scRNA-seq would differentiate the cell types in a complex population combination, promote the recognition of new cell types, and contribute to the understanding of the physiological processes of KD and the exploration of novel treatment options [
20] .
Our study introduced a typical child PBMC example for single cell transcriptome study. We found that in healthy child, the dominant cell clusters are NKT cell, CD4+ cytotoxic T cell, T helper cell, CD8+ memory T cell and naive CD8+ T cell. These dominant cell clusters in healthy child were different from
healthy adults, of which the dominant cell clusters were T cell, B cell, CD14+ monocyte, CD16+ monocyte and natural killer cell [
21]. Back to the KD child, though no significant new cluster shown in the KD child compared to the healthy child, we found that the proportion of main cell clusters shifts in the KD child, with more NKT cells, plasmacytoid dendritic cell and less CD8+ T cells, T helper cells and B cells. We also observed that multilymphoid progenitor cells tends to decrease in KD child. Multilymphoid progenitor cells were believed to differentiate into multiple lymphoid cells including T cells and B cells. Multilymphoid progenitor cells were normally present in umbilical cord blood [
21], and upper category progenitor cell was also found in PBMC (CellMarker Database).
Dendritic cell is the professional antigen presenting cell with the strongest function in the body. It can efficiently absorb, process and present antigens, and is the central link in initiating, regulating and maintaining the immune response. Cameron et al. [
22] suggested that dendritic cell as antigen presenting cells are involved in KD arterial immune process. Miyabe et al. [
23] demonstrate that Dectin-2–mediated induction of CCL2 production by macrophages resident in coronary arteries initiates vascular inflammation in a model of KD, suggesting the participation of innate immune system in initiating vasculitis. Another study suggests that mature arterial myeloid dendritic cell might be activating T cells and may be a significant factor in the pathogenesis of coronary arteritis in KD [
24] . In addition, T cell regulation appears to be important at the tissue level for the resolution of inflammation in KD and evidence suggests that the Fc stimulates immature myeloid dendritic cell to expand the regulatory T cells [
25] . With the study of dendritic cell at the molecular level, it has been found that it has great therapeutic potential in various autoimmune diseases, but the specific mechanism in KD need further exploration.
Furthermore, with the analysis of DEGs of immune cells in the KD and healthy control, we identified IL7R, CD3D and CD27 as the common DEGs that shared by three T cell clusters. IL7R encodes the receptor of interleukin 7, which is a crucial marker for T cell development and play essential role in immune competence. CD3D is associated with immune checkpoints [
26] and may offer an possible new therapy for KD. CD27 is a marker of memory B cells and also detected on normal plasma cells, it is believed that CD 27+ memory B cells contributed to the pathogenesis of KD inflammation. In addition, we defined distinct subsets of NK T cells characterized by GNLY and GZMB. GNLY is found in cytotoxic granules in both CTL and NK cells, which is a member of the saposin-like protein family. It has been associated with a variety of infectious diseases and involved in the removal of virus such as varicella zoster and Epstein-Barr virus (EBV). One case report demonstrated that EBV infection could be considered as a suspected causative agent because of the potential effect on the immune system in KD [
27]. GZMB represents a serine proteinase and plays a central role in killing human tumor cell lines, which can induce cell death, apoptosis [
28]. A previous study found that GZMB gene silencing acts to inhibit MAPK signaling pathway through regulating the expressions of inflammatory factors, thus relieving the injury brought by Rheumatoid arthritis [
29]. Moreover, studies have found that inhibiting MAPK signaling pathway activation can reduce the occurrence of the inflammatory response [
30]. Collectively, we speculate that GNLY and GZMB may be potential targets for the treatment in KD.
After KEGG and GO annotation, we observed that the DEGs of T cells and B cells are specified enriched in Cell activation, Lymphocyte activation and positive regulation of immune system process. All the three shared biological process indicated that there existed abnormal activation of immune cells in molecular pathway level in the KD patient. Based on the pathogen theory of KD, the immune system is activated to eliminate the pathogen with stimulation of multiple T cells. When the pathogen replication is not well controlled, this continuous stimulation would lead to contraction of effector cells and cause their exhaustion [
31]. CD27 is one of the marker for the immune tolerance pathway [
32], the activation of CD27 and the enriched pathways indicated that immune checkpoint can be a possible new target for KD treatment.
Besides, MHC class II protein complex was significantly enriched only in B cell (Supplemental Table S
3), and some studies had found that some MHC class II alleles had correlations with the probability of autoimmune diseases [
33]. In addition, the DEGs of MHC class II has 10 genes in KD child B cells, including
HLA-DMA,
HLA-DMB,
HLA-DPA1,
HLA-DPB1,
HLA-DRA,
HLA-DQA1,
HLA-DQA2,
HLA-DRB1,
HLA-DRB5 and
HLA-DQB1 (Supplemental Table S
2). The above results suggest that KD susceptibility is correlated with DM, DP, DQ and DR locus on HLA genes. Previous reports [
34] have shown that HLA locus may play a potential role in the pathogenesis of disease in the process of antigen processing and presentation in MHC region, and it is believed that the expression of class HLA-I alleles is related to KD. These studies confirmed the HLA genes and MHC class II molecules are involved in the pathogenesis of KD, providing a new direction for the diagnosis and treatment of KD.
In summary, this study preliminarily explored the immune mechanism of KD by scRNA-seq technology, and explored relevant molecular markers and major enrichment function through bioinformatics analysis. These markers may be important targets for future treatment of KD. The sample size of this experiment is limited for solid conclusion, so it is necessary to further expand the sample size and proceed the study for further experimental research.
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