Identifying the pattern of immune related cells and genes in the peripheral blood of ischemic stroke

Abstract

Background: Ischemic stroke (IS) is the second leading cause of death worldwide which is a serious hazard to human health. Evidence suggests that the immune system plays a key role in the pathophysiology of IS. However, the precisely immune related mechanisms were still not been systematically understood.

Methods: In this study, we aim to identify the immune related modules and genes that might play vital role in the occurrence and development of IS by using the weighted gene co-expression network analysis (WGCNA). Meanwhile, we applied a kind of deconvolution algorithm to reveal the proportions of 22 subsets of immune cells in the blood samples.

Results: There were total 128 IS patients and 67 healthy control samples in the three Gene Expression Omnibus (GEO) datasets. Under the screening criteria, 1082 DEGs (894 up-regulated and 188 down-regulated) were chosen for further analysis. A total of 11 clinically significant modules were identified, from which immune-related hub modules and hub genes were further explored. Finally, 16 genes were selected as real hub genes for further validation analysis. Furthermore, these CIBERSORT results suggest that detailed analysis of the immune subtype distribution pattern has the potential to enhance clinical prediction and to identify candidates for immunotherapy. More specifically, we identified that neutrophil emerge as a promising target for IS therapies.

Conclusions: In the present study, we investigated the immune related gene expression modules, in which the SLAMF1, IL7R and NCF4 may be novel therapeutic targets to promote functional and histological recovery after ischemic stroke. Furthermore, these hub genes and neutrophils may become important biological targets in the drug screening and drug designing.

Keywords: Bioinformatics analysis; Immune cell subtype distribution pattern; Ischemic stroke; Pathological process; Weighted gene co-expression network analysis.

Fig. 1

Data Preprocessing and identification of DEGs. a PCA for stroke and healthy control samples before batch correction with ComBat. b PCA for stroke and healthy control samples after batch correction with ComBat. c The volcano plot of DEGs. d The heatmap of DEGs

Fig. 2

Cluster dendrogram and module-trait relationship plot. a Dendrogram of all differentially expressed genes clustered based on a dissimilarity measure (1-TOM). b Heatmap of the correlation between module eigengenes and ischemic stroke. Scatter diagrams for module membership vs. gene significance of disease state. c Turquoise module. d Black module. e Blue module. f Purple module. g Green module. h Yellow module. i Pink module. j Venn plot of hub genes in the co-expression and PPI networks

Fig. 3

Verification using GSE37587 dataset. Green: healthy control samples; red: ischemic stroke samples. Statistically significant was considered as the p-value < 0.05. a–p Verification of the expression of IL-7R, ITGAM, NCF4, PAK1, PTEN, MYD88, FGR, SLAMF1,TLR8, ATG7, MAPK1, IFNAR1, CCR7, TIMP2, CANT1 and WAS, respectively, in the HC and IS group of validation cohort

Fig. 4

The profiles of immune cell subtype distribution pattern in GSE58294 cohort. a The bar plot visualizing the relative percent of 22 immune cell in each sample. b Heatmap of the 22 immune cell proportions in each sample. c Correlation heatmap of all 22 immune cells. d Violin plot of all 22 immune cells differentially infiltrated fraction

Fig. 5

Correlation analysis of hub genes expression and the proportion of neutrophils. Red bar: samples with a high proportion of neutrophils; green bar: samples with a low proportion of neutrophils. Statistically significant was considered as the p-value < 0.05. a–p Correlation analysis of the proportion of neutrophils and expression of IL-7R, ITGAM, NCF4, PAK1, PTEN, MYD88, FGR, SLAMF1, TLR8, ATG7, MAPK1, IFNAR1, CCR7, TIMP2, CANT1 and WAS, respectively

Fig. 6

Verification in the clinical samples. a–l Verification using qRT-PCR analysis. m Venn plot of validated hub genes. Red: upregulated genes; blue: downregulated genes. n Neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO) and basophils (BA) percentage in the clinical samples. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group