KD is a common cause of vasculitis in childhood, which is characterized by fever and mucocutaneous features[13]. 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[14]. At the transcriptomic level, we can differentiate into more detailed subsets of cells based on different gene expression patterns[15][16][17]. 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 diseases and the exploration of novel treatment options[18] .
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[19]. 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, neutrophile cells 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[19], 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.[20] suggested that dendritic cell as antigen presenting cells are involved in KD arterial immune process. Miyabe et al.[21] 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[22]. 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[23]. 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.
Neutrophils activation is an important step in the pathogenesis of KD[14][24]. While sustained activation of neutrophils existed in KD patients PBMC, we observed that inflammatory marker S100A8/S100A9 are highly expressed in the KD child neutrophil. S100A8/A100A9 are calcium binding proteins that can induce neutrophil adhesion which are involved in immune defense[25]. In our case, we found that these two neutrophil markers are elevated by 58 and 48 folds in KD neutrophil compared to healthy control respectively, which is a strong evidence that neutrophil induced immune disorder existed in KD patient. Neutrophil to leukocyte ratio is a severity marker for KD[26], for further study, we can explore more cases and validate the neutrophil activation pattern stability in KD.
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[27] 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.
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[28]. CD27 is one of the marker for the immune tolerance pathway[29], 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 S3), and some studies had found that some MHC class Ⅱ alleles had correlations with the probability of autoimmune diseases[30]. 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 S2). The above results suggest that KD susceptibility is correlated with DM, DP, DQ and DR locus on HLA genes. Previous reports [31] 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.