IL1A regulates the inflammation in gout through the Toll-like receptors pathway

Abstract

Objective: Gout is a dangerous metabolic condition related to monosodium urate (MSU). Our aim is to study the molecular mechanisms underlying gout and to identify potential clinical biomarkers by bioinformatics analysis and experimental validation. Methods: In this study, we retrieved the overlapping genes between GSE199950-Differential Expressed Genes (DEGs) dataset and key module in Weighted Gene Co-Expression Network Analysis (WGCNA) on GSE199950. These genes were then analyzed by protein-protein interaction (PPI) network, expression and Gene Set Enrichment Analysis to identify the hub gene related to gout. Then, the gene was investigated by peripheral blood mononuclear cells (PBMCs), immunoassay and cell experiments like western blotting to uncover its underlying mechanism in gout cells. Results: From the turquoise module and 83 DEGs, we identified 62 overlapping genes, only 11 genes had mutual interactions in PPI network and these genes were highly expressed in MSU-treated samples. Then, it was found that the IL1A (interleukin 1 alpha) was the only one gene related to Toll-like receptor signaling pathway that was associated with the occurrence of gout. Thus, IL1A was determined as the hub gene in this study. In immunoassay, IL1A was significantly positively correlated with B cells and negatively correlated with macrophages. Moreover, IL1A is highly expressed in gout patients,it has a good clinical diagnostic value. Finally, the results of in vitro experiments showed that after knocking down IL1A, the expressions of pro-inflammatory cytokines and Toll-like receptor signaling pathway-related proteins (TLR2, TLR4, MyD88) were all reduced. Conclusion: It is confirmed that IL1A is a promoting gene in gout with a good diagnostic value, and specifically it affects the inflammation in gout through Toll-like receptor pathway. Our research offers fresh perspectives on the pathophysiology of gout and valuable directions for future diagnosis and treatment.

Keywords: GSE199950; Gout; IL1A; Monosodium urate; Toll like receptor signaling pathway; WGCNA.

Figure 1

The turquoise module was the key module in the gene co-expression network. (A) The above figure is a reference for the optimal soft threshold, and the red line represents the subjectively selected scale-free fitting index value. The graph below shows the mean connectivity at different soft thresholds. (B) Eigengene adjacency heatmap. The top is a dendrogram of different color modules, and the bottom is a clustering heat map of genes in different modules. The redder the color, the higher the correlation. (C) Cluster dendrogram of gene modules, a total of 18 modules were clustered after merge. (D) Heatmap of the relationship between gene modules-features. Columns represent different samples of GSDE199950 and rows represent gene modules.

Figure 2

Enrichment analysis and PPI network of the turquoise module. (A) Histogram of GO enrichment analysis of all genes in the turquoise module. (B) Bubble plot of KEGG pathway enrichment analysis of all genes in the turquoise module. The abscissa indicates the gene ratio, and the ordinate indicates the enriched KEGG pathway. (C) PPI network of genes in the turquoise module constructed based on Cytoscape. (D) Volcano plot of GSE199950-DEGs, yellow scatter on the left indicates down-regulated DEGs, and green scatter on the right indicates up-regulated DEGs. (E) Heatmap of GSE199950-DEGs, green column indicates MSU group and yellow column indicates PBS group. (F) GSEA-KEGG enrichment analysis of GSE199950-DEGs.

Figure 3

Functional enrichment analysis on GSE199950-DEGs. (A) Venn diagram, 62 genes overlap between the genes in the turquoise module and GSE199950-DEG. (B) PPI network constructed from the STRING database, where nodes represent genes and edges represent the interconnections between genes. (C-M) Expression verification of the genes corresponding to 11 nodes in the control and case groups of GSE199960, respectively RGS16, CCL20, CD300LB, CDH5, CSF2, IL1RN, JAM2, MMP3, RGS8, IL1A, SIGLEC15. Control group:lymphatic endothelial cells (LECs) with Phosphate Buffered Saline (PBS); Case group:LECs treated with 300μg/ml MSU. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

GSEA-KEGG enrichment analysis of 11 genes. (A) CCL20. (B) CD300LB. (C) CDH5. (D) CSF2. (E) IL1A. (F) IL1RN. (G) JAM2. (H) MMP3. (I) RGS8. (J) RGS16. (K) SIGLEC15. The different colored lines correspond to the signaling pathways below as well as positive and negative correlations and significance.

Figure 5

Immunoassay and ROC curve analysis of IL1A. (A) Heat map, turquoise column indicates PBS-treated group, ginger yellow column indicates MSU-treated group, each row indicates one immune cell. (B) Correlation analysis between IL1A expression and B cell infiltration level, the upper left corner shows the P value and the correlation coefficient r value. (C) Correlation analysis between IL1A expression and Macrophage infiltration level, the upper left corner shows the P value and the correlation coefficient r value. (D) The ROC curve of the prognostic value of IL1A, the abscissa is 1-Specificity (False Positive Rate; FPR), the ordinate is the sensitivity (True Positive Rate; TPR), the larger the AUC value, the higher the prediction accuracy.

Figure 6

Expression of IL1A in PBMC and MSU concentration in THP-1 cells detected by qRT-PCR. (A) IL1A mRNA level in the PBMC from patients (n = 10), and HC (n = 5) were detected by qRT-PCR. (B) qRT-PCR detection of different concentrations of MSU induced the level of IL1A mRNA in THP-1 cells. (C) The level of IL1A protein in THP-1 cells induced by different concentrations of MSU was detected by WB. (D-F) The mRNA levels of IL-1β, IL-8 and TNFα in THP-1 cells induced by different MSU concentrations were detected by qRT-PCR. *P<0.05, **P<0.01.

Figure 7

Downregulation of IL1A expression reduced the expression of pro-inflammatory cytokines. (A) qRT-PCR detection of MSU-induced knockdown efficiency of IL1A in THP-1 cells. (B) The knockdown efficiency of IL1A in THP-1 cells induced by MSU was detected by WB. (C-E) qRT-PCR was used to detect the mRNA expressions of IL-1β, IL-8, and TNFα in MSU-induced THP-1 cells after knocking down IL1A. (F) qRT-PCR was used to detect the mRNA expressions of MSU, MSU+si-NC, MSU+si-IL1A. (G) The protein expressions of MSU, MSU+si-NC, MSU+si-IL1A were detected by WB. *P<0.05, **P<0.01.

Figure 8

IL1A regulates the inflammatory response in gout via Toll-like receptor signaling pathway. (A, C, E) qRT-PCR was used to detect the mRNA expression of TLR2, TLR4 and MyD88 after IL1A knockdown in THP-1 cells induced by MSU. (B, D, F) WB assay for protein expression of TLR2, TLR4 and MyD88 after knockdown of IL1A in MSU-induced THP-1 cells. *P<0.05.